Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1979 Sep;76(9):4627–4631. doi: 10.1073/pnas.76.9.4627

Cloned pairs of variable region genes for immunoglobulin heavy chains isolated from a clone library of the entire mouse genome.

D J Kemp, S Cory, J M Adams
PMCID: PMC411632  PMID: 116236

Abstract

To investigate the organization of immunoglobulin genes, we have constructed a clone library containing 10(6) randomly generated fragments of mouse embryo DNA, corresponding to eight equivalents of the genome. The cloning method involved methylation of embryo DNA at EcoRI recognition sites, partial digestion by EcoRI* endonclease activity, and direct ligation of the resulting large fragments to the lambda phage vector Charon 4A. The library was searched for sequences homologous to a cloned complementary DNA copy of a mu heavy chain mRNA. Nine clones bearing variable heavy chain (VH) sequences were isolated, representing at least eight distinct VH genes. Thus, multiple related VH genes are available in the genome to contribute to immunoglobulin diversity. Each of the two clones carries a pair of VH genes, one pair separated by 15 +/- 1 kilobase pairs of mouse DNA and the other by 14 +/- 2 kilobase pairs. This indicates that related VH genes are clustered and may occur in a tandem array having a repeating unit of 14--16 kilobase pairs. The large spacer sequences between VH genes cannot, however, be highly conserved.

Full text

PDF
4627

Images in this article

4628Image
on p.4628
4628Image
on p.4628
4629Image
on p.4629
4630Image
on p.4630
4630Image
on p.4630

Selected References

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

  1. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  2. Bernard O., Hozumi N., Tonegawa S. Sequences of mouse immunoglobulin light chain genes before and after somatic changes. Cell. 1978 Dec;15(4):1133–1144. doi: 10.1016/0092-8674(78)90041-7. [DOI] [PubMed] [Google Scholar]
  3. Blattner F. R., Williams B. G., Blechl A. E., Denniston-Thompson K., Faber H. E., Furlong L., Grunwald D. J., Kiefer D. O., Moore D. D., Schumm J. W. Charon phages: safer derivatives of bacteriophage lambda for DNA cloning. Science. 1977 Apr 8;196(4286):161–169. doi: 10.1126/science.847462. [DOI] [PubMed] [Google Scholar]
  4. Blin N., Stafford D. W. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 1976 Sep;3(9):2303–2308. doi: 10.1093/nar/3.9.2303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brack C., Hirama M., Lenhard-Schuller R., Tonegawa S. A complete immunoglobulin gene is created by somatic recombination. Cell. 1978 Sep;15(1):1–14. doi: 10.1016/0092-8674(78)90078-8. [DOI] [PubMed] [Google Scholar]
  6. Cameron J. R., Panasenko S. M., Lehman I. R., Davis R. W. In vitro construction of bacteriophage lambda carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for DNA ligase. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3416–3420. doi: 10.1073/pnas.72.9.3416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cory S., Adams J. M. A very large repeating unit of mouse DNA containing the 18S, 28S and 5.8S rRNA genes. Cell. 1977 Aug;11(4):795–805. doi: 10.1016/0092-8674(77)90292-6. [DOI] [PubMed] [Google Scholar]
  8. Dreyer W. J., Bennett J. C. The molecular basis of antibody formation: a paradox. Proc Natl Acad Sci U S A. 1965 Sep;54(3):864–869. doi: 10.1073/pnas.54.3.864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dugaiczyk A., Hedgpeth J., Boyer H. W., Goodman H. M. Physical identity of the SV40 deoxyribonucleic acid sequence recognized by the Eco RI restriction endonuclease and modification methylase. Biochemistry. 1974 Jan 29;13(3):503–512. doi: 10.1021/bi00700a016. [DOI] [PubMed] [Google Scholar]
  10. Early P. W., Davis M. M., Kaback D. B., Davidson N., Hood L. Immunoglobulin heavy chain gene organization in mice: analysis of a myeloma genomic clone containing variable and alpha constant regions. Proc Natl Acad Sci U S A. 1979 Feb;76(2):857–861. doi: 10.1073/pnas.76.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gally J. A., Edelman G. M. The genetic control of immunoglobulin synthesis. Annu Rev Genet. 1972;6:1–46. doi: 10.1146/annurev.ge.06.120172.000245. [DOI] [PubMed] [Google Scholar]
  12. Greene P. H., Poonian M. S., Nussbaum A. L., Tobias L., Garfin D. E., Boyer H. W., Goodman H. M. Restriction and modification of a self-complementary octanucleotide containing the EcoRI substrate. J Mol Biol. 1975 Dec 5;99(2):237–261. doi: 10.1016/s0022-2836(75)80143-4. [DOI] [PubMed] [Google Scholar]
  13. Gross-Bellard M., Oudet P., Chambon P. Isolation of high-molecular-weight DNA from mammalian cells. Eur J Biochem. 1973 Jul 2;36(1):32–38. doi: 10.1111/j.1432-1033.1973.tb02881.x. [DOI] [PubMed] [Google Scholar]
  14. Hedgpeth J., Goodman H. M., Boyer H. W. DNA nucleotide sequence restricted by the RI endonuclease. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3448–3452. doi: 10.1073/pnas.69.11.3448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Helling R. B., Goodman H. M., Boyer H. W. Analysis of endonuclease R-EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J Virol. 1974 Nov;14(5):1235–1244. doi: 10.1128/jvi.14.5.1235-1244.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hood L., Campbell J. H., Elgin S. C. The organization, expression, and evolution of antibody genes and other multigene families. Annu Rev Genet. 1975;9:305–353. doi: 10.1146/annurev.ge.09.120175.001513. [DOI] [PubMed] [Google Scholar]
  17. Jeffreys A. J., Flavell R. A. A physical map of the DNA regions flanking the rabbit beta-globin gene. Cell. 1977 Oct;12(2):429–439. doi: 10.1016/0092-8674(77)90119-2. [DOI] [PubMed] [Google Scholar]
  18. Laird C. D. Chromatid structure: relationship between DNA content and nucleotide sequence diversity. Chromosoma. 1971 Mar 16;32(4):378–406. doi: 10.1007/BF00285251. [DOI] [PubMed] [Google Scholar]
  19. Laskey R. A., Mills A. D. Enhanced autoradiographic detection of 32P and 125I using intensifying screens and hypersensitized film. FEBS Lett. 1977 Oct 15;82(2):314–316. doi: 10.1016/0014-5793(77)80609-1. [DOI] [PubMed] [Google Scholar]
  20. Lenhard-Schuller R., Hohn B., Brack C., Hirama M., Tonegawa S. DNA clones containing mouse immunoglobulin kappa chain genes isolated by in vitro packaging into phage lambda coats. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4709–4713. doi: 10.1073/pnas.75.10.4709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Loening U. E. The determination of the molecular weight of ribonucleic acid by polyacrylamide-gel electrophresis. The effects of changes in conformation. Biochem J. 1969 Jun;113(1):131–138. doi: 10.1042/bj1130131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. MacKenzie M. R., Gutman G. A., Warner N. L. The binding of murine IgM to Staphylococcal A protein. Scand J Immunol. 1978;7(5):367–370. doi: 10.1111/j.1365-3083.1978.tb00466.x. [DOI] [PubMed] [Google Scholar]
  23. Maniatis T., Hardison R. C., Lacy E., Lauer J., O'Connell C., Quon D., Sim G. K., Efstratiadis A. The isolation of structural genes from libraries of eucaryotic DNA. Cell. 1978 Oct;15(2):687–701. doi: 10.1016/0092-8674(78)90036-3. [DOI] [PubMed] [Google Scholar]
  24. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  25. Panasenko S. M., Alazard R. J., Lehman I. R. A simple, three-step procedure for the large scale purification of DNA ligase from a hybrid lambda lysogen constructed in vitro. J Biol Chem. 1978 Jul 10;253(13):4590–4592. [PubMed] [Google Scholar]
  26. Paterson B. M., Roberts B. E., Kuff E. L. Structural gene identification and mapping by DNA-mRNA hybrid-arrested cell-free translation. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4370–4374. doi: 10.1073/pnas.74.10.4370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Polisky B., Greene P., Garfin D. E., McCarthy B. J., Goodman H. M., Boyer H. W. Specificity of substrate recognition by the EcoRI restriction endonuclease. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3310–3314. doi: 10.1073/pnas.72.9.3310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  29. Sakano H., Rogers J. H., Hüppi K., Brack C., Traunecker A., Maki R., Wall R., Tonegawa S. Domains and the hinge region of an immunoglobulin heavy chain are encoded in separate DNA segments. Nature. 1979 Feb 22;277(5698):627–633. doi: 10.1038/277627a0. [DOI] [PubMed] [Google Scholar]
  30. Seidman J. G., Leder A., Edgell M. H., Polsky F., Tilghman S. M., Tiemeier D. C., Leder P. Multiple related immunoglobulin variable-region genes identified by cloning and sequence analysis. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3881–3885. doi: 10.1073/pnas.75.8.3881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Seidman J. G., Leder A., Nau M., Norman B., Leder P. Antibody diversity. Science. 1978 Oct 6;202(4363):11–17. doi: 10.1126/science.99815. [DOI] [PubMed] [Google Scholar]
  32. Sgaramella V. Enzymatic oligomerization of bacteriophage P22 DNA and of linear Simian virus 40 DNA. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3389–3393. doi: 10.1073/pnas.69.11.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  34. Sternberg N., Tiemeier D., Enquist L. In vitro packaging of a lambda Dam vector containing EcoRI DNA fragments of Escherichia coli and phage P1. Gene. 1977 May;1(3-4):255–280. doi: 10.1016/0378-1119(77)90049-x. [DOI] [PubMed] [Google Scholar]
  35. Tonegawa S., Brack C., Hozumi N., Schuller R. Cloning of an immunoglobulin variable region gene from mouse embryo. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3518–3522. doi: 10.1073/pnas.74.8.3518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tonegawa S., Maxam A. M., Tizard R., Bernard O., Gilbert W. Sequence of a mouse germ-line gene for a variable region of an immunoglobulin light chain. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1485–1489. doi: 10.1073/pnas.75.3.1485. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES