Skip to main content
NCBI 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
. 1984 May;81(9):2723–2727. doi: 10.1073/pnas.81.9.2723

Two-dimensional S1 nuclease heteroduplex mapping: detection of rearrangements in bacterial genomes.

T Yee, M Inouye
PMCID: PMC345142  PMID: 6326139

Abstract

A method of two-dimensional S1 nuclease heteroduplex mapping was developed to detect gene rearrangements and repeated sequences in total bacterial chromosomes. To detect DNA rearrangements between two variant bacterial strains, total chromosomal DNA preparations from the two strains are digested with four-base-recognizing restriction enzymes, mixed together, denatured, renatured, and separated on first-dimension polyacrylamide slab gels. Gel strips are cut out and soaked in a buffer containing S1 nuclease, which diffuses into the strips and digests the DNA fragments at single-stranded regions. The digested DNA is then electrophoresed in a second dimension perpendicular to the first dimension. DNA heteroduplexes that were digested by the S1 nuclease are resolved as distinct spots below a bright unresolved band of homoduplex. This report describes testing of this method on a model system consisting of two nearly isogeneic strains of Escherichia coli, and the application of this method in detecting DNA rearrangements associated with phase variation in Myxococcus xanthus.

Full text

PDF
2723

Images in this article

2724Image
on p.2724
2725Image
on p.2725
2726Image
on p.2726
2726Image
on p.2726

Selected References

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

  1. Bernards A., Van der Ploeg L. H., Frasch A. C., Borst P., Boothroyd J. C., Coleman S., Cross G. A. Activation of trypanosome surface glycoprotein genes involves a duplication-transposition leading to an altered 3' end. Cell. 1981 Dec;27(3 Pt 2):497–505. doi: 10.1016/0092-8674(81)90391-3. [DOI] [PubMed] [Google Scholar]
  2. Burchard R. P., Burchard A. C., Parish J. H. Pigmentation phenotype instability in Myxococcus xanthus. Can J Microbiol. 1977 Dec;23(12):1657–1662. doi: 10.1139/m77-238. [DOI] [PubMed] [Google Scholar]
  3. Burchard R. P., Dworkin M. Light-induced lysis and carotenogenesis in Myxococcus xanthus. J Bacteriol. 1966 Feb;91(2):535–545. doi: 10.1128/jb.91.2.535-545.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Campos J. M., Geisselsoder J., Zusman D. R. Isolation of bacteriophage MX4, a generalized transducing phage for Myxococcus xanthus. J Mol Biol. 1978 Feb 25;119(2):167–178. doi: 10.1016/0022-2836(78)90431-x. [DOI] [PubMed] [Google Scholar]
  5. Campos J. M., Zusman D. R. Regulation of development in Myxococcus xanthus: effect of 3':5'-cyclic AMP, ADP, and nutrition. Proc Natl Acad Sci U S A. 1975 Feb;72(2):518–522. doi: 10.1073/pnas.72.2.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Denhardt D. T. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966 Jun 13;23(5):641–646. doi: 10.1016/0006-291x(66)90447-5. [DOI] [PubMed] [Google Scholar]
  7. Green C., Tibbetts C. Reassociation rate limited displacement of DNA strands by branch migration. Nucleic Acids Res. 1981 Apr 24;9(8):1905–1918. doi: 10.1093/nar/9.8.1905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kaiser D., Manoil C., Dworkin M. Myxobacteria: cell interactions, genetics, and development. Annu Rev Microbiol. 1979;33:595–639. doi: 10.1146/annurev.mi.33.100179.003115. [DOI] [PubMed] [Google Scholar]
  9. Kuner J. M., Kaiser D. Introduction of transposon Tn5 into Myxococcus for analysis of developmental and other nonselectable mutants. Proc Natl Acad Sci U S A. 1981 Jan;78(1):425–429. doi: 10.1073/pnas.78.1.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. LENNOX E. S. Transduction of linked genetic characters of the host by bacteriophage P1. Virology. 1955 Jul;1(2):190–206. doi: 10.1016/0042-6822(55)90016-7. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Meyer T. F., Mlawer N., So M. Pilus expression in Neisseria gonorrhoeae involves chromosomal rearrangement. Cell. 1982 Aug;30(1):45–52. doi: 10.1016/0092-8674(82)90010-1. [DOI] [PubMed] [Google Scholar]
  13. Nasmyth K. A., Tatchell K., Hall B. D., Astell C., Smith M. A position effect in the control of transcription at yeast mating type loci. Nature. 1981 Jan 22;289(5795):244–250. doi: 10.1038/289244a0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Shenk T. E., Rhodes C., Rigby P. W., Berg P. Biochemical method for mapping mutational alterations in DNA with S1 nuclease: the location of deletions and temperature-sensitive mutations in simian virus 40. Proc Natl Acad Sci U S A. 1975 Mar;72(3):989–993. doi: 10.1073/pnas.72.3.989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Silverman M., Simon M. Phase variation: genetic analysis of switching mutants. Cell. 1980 Apr;19(4):845–854. doi: 10.1016/0092-8674(80)90075-6. [DOI] [PubMed] [Google Scholar]
  17. Simon M., Zieg J., Silverman M., Mandel G., Doolittle R. Phase variation: evolution of a controlling element. Science. 1980 Sep 19;209(4463):1370–1374. doi: 10.1126/science.6251543. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Tonegawa S., Sakano H., Make R., Traunecker A., Heinrich G., Roeder W., Kurosawa Y. Somatic reorganization of immunoglobulin genes during lymphocyte differentiation. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 2):839–858. doi: 10.1101/sqb.1981.045.01.102. [DOI] [PubMed] [Google Scholar]
  20. Wireman J. W., Dworkin M. Morphogenesis and developmental interactions in myxobacteria. Science. 1975 Aug 15;189(4202):516–523. doi: 10.1126/science.806967. [DOI] [PubMed] [Google Scholar]
  21. Yee T., Inouye M. Two-dimensional DNA electrophoresis applied to the study of DNA methylation and the analysis of genome size in Myxococcus xanthus. J Mol Biol. 1982 Jan 15;154(2):181–196. doi: 10.1016/0022-2836(82)90059-6. [DOI] [PubMed] [Google Scholar]
  22. Zieg J., Simon M. Analysis of the nucleotide sequence of an invertible controlling element. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4196–4200. doi: 10.1073/pnas.77.7.4196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. van de Putte P., Cramer S., Giphart-Gassler M. Invertible DNA determines host specificity of bacteriophage mu. Nature. 1980 Jul 17;286(5770):218–222. doi: 10.1038/286218a0. [DOI] [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