Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1988 May 11;16(9):3863–3875. doi: 10.1093/nar/16.9.3863

Mapping genomic organization by field inversion and two-dimensional gel electrophoresis: application to the murine T-cell receptor gamma gene family.

T Woolf 1, E Lai 1, M Kronenberg 1, L Hood 1
PMCID: PMC336561  PMID: 3375073

Abstract

A new two-dimensional gel electrophoresis technique has been developed for the mapping of multigene families. Resolution in the first dimension is based on the generation of large size DNA fragments by infrequently-cutting restriction enzymes, and separation of these fragments by field inversion gel (FIG) electrophoresis. A second restriction enzyme digestion is then carried out with the separated DNA fragments in the agarose gel. Standard gel electrophoresis in the second dimension allows one to estimate the number of hybridizing genes contained in each large DNA fragment. We have also developed a novel method to increase the separation, resolution and hybridization signal in the second dimension by condensing the bands from the first dimension into spots. As an example, we have applied these techniques to determine the organization of the murine T-cell receptor gamma locus. The murine gamma gene family was found to be contained on two DNA fragments encompassing 195 kilobases of DNA. The two-dimensional gel electrophoresis method is particularly useful in the analysis of the organization of multigenic families where single copy probes are not readily available, and should extend the potential usefulness of field inversion gel electrophoresis in gene mapping.

Full text

PDF
3863

Images in this article

3868Image
on p.3868
3869Image
on p.3869
3870Image
on p.3870

Selected References

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

  1. Bank I., DePinho R. A., Brenner M. B., Cassimeris J., Alt F. W., Chess L. A functional T3 molecule associated with a novel heterodimer on the surface of immature human thymocytes. Nature. 1986 Jul 10;322(6075):179–181. doi: 10.1038/322179a0. [DOI] [PubMed] [Google Scholar]
  2. Boehm T. L., Drahovsky D. Two-dimensional restriction mapping by digestion with restriction endonucleases of DNA in agarose and polyacrylamide gels. J Biochem Biophys Methods. 1984 May;9(2):153–161. doi: 10.1016/0165-022x(84)90006-x. [DOI] [PubMed] [Google Scholar]
  3. Brenner M. B., McLean J., Dialynas D. P., Strominger J. L., Smith J. A., Owen F. L., Seidman J. G., Ip S., Rosen F., Krangel M. S. Identification of a putative second T-cell receptor. Nature. 1986 Jul 10;322(6075):145–149. doi: 10.1038/322145a0. [DOI] [PubMed] [Google Scholar]
  4. Carle G. F., Frank M., Olson M. V. Electrophoretic separations of large DNA molecules by periodic inversion of the electric field. Science. 1986 Apr 4;232(4746):65–68. doi: 10.1126/science.3952500. [DOI] [PubMed] [Google Scholar]
  5. Chen C. W., Thomas C. A., Jr A rapid two-dimensional fractionation of DNA restriction fragments. Anal Biochem. 1983 Jun;131(2):397–403. doi: 10.1016/0003-2697(83)90190-2. [DOI] [PubMed] [Google Scholar]
  6. Eisen H. N., Reilly E. B. Lambda chains and genes in inbred mice. Annu Rev Immunol. 1985;3:337–365. doi: 10.1146/annurev.iy.03.040185.002005. [DOI] [PubMed] [Google Scholar]
  7. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  8. Garman R. D., Doherty P. J., Raulet D. H. Diversity, rearrangement, and expression of murine T cell gamma genes. Cell. 1986 Jun 6;45(5):733–742. doi: 10.1016/0092-8674(86)90787-7. [DOI] [PubMed] [Google Scholar]
  9. Hayday A. C., Saito H., Gillies S. D., Kranz D. M., Tanigawa G., Eisen H. N., Tonegawa S. Structure, organization, and somatic rearrangement of T cell gamma genes. Cell. 1985 Feb;40(2):259–269. doi: 10.1016/0092-8674(85)90140-0. [DOI] [PubMed] [Google Scholar]
  10. Hinds K. R., Litman G. W. Major reorganization of immunoglobulin VH segmental elements during vertebrate evolution. Nature. 1986 Apr 10;320(6062):546–549. doi: 10.1038/320546a0. [DOI] [PubMed] [Google Scholar]
  11. Iwamoto A., Rupp F., Ohashi P. S., Walker C. L., Pircher H., Joho R., Hengartner H., Mak T. W. T cell-specific gamma genes in C57BL/10 mice. Sequence and expression of new constant and variable region genes. J Exp Med. 1986 May 1;163(5):1203–1212. doi: 10.1084/jem.163.5.1203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Koning F., Stingl G., Yokoyama W. M., Yamada H., Maloy W. L., Tschachler E., Shevach E. M., Coligan J. E. Identification of a T3-associated gamma delta T cell receptor on Thy-1+ dendritic epidermal Cell lines. Science. 1987 May 15;236(4803):834–837. doi: 10.1126/science.2883729. [DOI] [PubMed] [Google Scholar]
  13. Kranz D. M., Saito H., Heller M., Takagaki Y., Haas W., Eisen H. N., Tonegawa S. Limited diversity of the rearranged T-cell gamma gene. 1985 Feb 28-Mar 6Nature. 313(6005):752–755. doi: 10.1038/313752a0. [DOI] [PubMed] [Google Scholar]
  14. Maeda K., Nakanishi N., Rogers B. L., Haser W. G., Shitara K., Yoshida H., Takagaki Y., Augustin A. A., Tonegawa S. Expression of the T-cell receptor gamma-chain gene products on the surface of peripheral T cells and T-cell blasts generated by allogeneic mixed lymphocyte reaction. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6536–6540. doi: 10.1073/pnas.84.18.6536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Malissen M., McCoy C., Blanc D., Trucy J., Devaux C., Schmitt-Verhulst A. M., Fitch F., Hood L., Malissen B. Direct evidence for chromosomal inversion during T-cell receptor beta-gene rearrangements. Nature. 1986 Jan 2;319(6048):28–33. doi: 10.1038/319028a0. [DOI] [PubMed] [Google Scholar]
  16. Pardoll D. M., Fowlkes B. J., Bluestone J. A., Kruisbeek A., Maloy W. L., Coligan J. E., Schwartz R. H. Differential expression of two distinct T-cell receptors during thymocyte development. Nature. 1987 Mar 5;326(6108):79–81. doi: 10.1038/326079a0. [DOI] [PubMed] [Google Scholar]
  17. Pelkonen J., Traunecker A., Karjalainen K. A new mouse TCR V gamma gene that shows remarkable evolutionary conservation. EMBO J. 1987 Jul;6(7):1941–1944. doi: 10.1002/j.1460-2075.1987.tb02455.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Poddar S. K., Maniloff J. Chromosome analysis by two-dimensional fingerprinting. Gene. 1986;49(1):93–102. doi: 10.1016/0378-1119(86)90388-4. [DOI] [PubMed] [Google Scholar]
  19. Popko B., Puckett C., Lai E., Shine H. D., Readhead C., Takahashi N., Hunt S. W., 3rd, Sidman R. L., Hood L. Myelin deficient mice: expression of myelin basic protein and generation of mice with varying levels of myelin. Cell. 1987 Feb 27;48(4):713–721. doi: 10.1016/0092-8674(87)90249-2. [DOI] [PubMed] [Google Scholar]
  20. Reed K. C., Mann D. A. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 1985 Oct 25;13(20):7207–7221. doi: 10.1093/nar/13.20.7207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schwartz D. C., Cantor C. R. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell. 1984 May;37(1):67–75. doi: 10.1016/0092-8674(84)90301-5. [DOI] [PubMed] [Google Scholar]
  22. Steinmetz M., Stephan D., Fischer Lindahl K. Gene organization and recombinational hotspots in the murine major histocompatibility complex. Cell. 1986 Mar 28;44(6):895–904. doi: 10.1016/0092-8674(86)90012-7. [DOI] [PubMed] [Google Scholar]
  23. Strauss W. M., Quertermous T., Seidman J. G. Measuring the human T cell receptor gamma-chain locus. Science. 1987 Sep 4;237(4819):1217–1219. doi: 10.1126/science.3498213. [DOI] [PubMed] [Google Scholar]
  24. Traunecker A., Oliveri F., Allen N., Karjalainen K. Normal T cell development is possible without 'functional' gamma chain genes. EMBO J. 1986 Jul;5(7):1589–1593. doi: 10.1002/j.1460-2075.1986.tb04400.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Weiss A., Newton M., Crommie D. Expression of T3 in association with a molecule distinct from the T-cell antigen receptor heterodimer. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6998–7002. doi: 10.1073/pnas.83.18.6998. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES