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. 1988 Aug;85(16):6027–6031. doi: 10.1073/pnas.85.16.6027

Physical mapping of large DNA by chromosome fragmentation.

D Vollrath 1, R W Davis 1, C Connelly 1, P Hieter 1
PMCID: PMC281898  PMID: 3045811

Abstract

A technique is described for physically positioning any cloned DNA on a native or artificial Saccharomyces cerevisiae chromosome. The technique involves splitting a chromosome at a specific site by transformation with short linear molecules containing the cloned DNA at one end and telomeric sequences at the other. Recombination between the end of the linear molecules and homologous chromosomal sequences gives rise to chromosome fragments comprising all sequences distal or proximal to the mapping site depending on the orientation of the cloned DNA. The recombinant products are recovered by screening for stabilization of a suppressor tRNA on the linear molecules using a colony color assay. The cloned DNA is positioned relative to the chromosome ends by sizing the chromosomal fragments using alternating contour-clamped homogeneous electric field gel electrophoresis. Application of this technique to organisms other than S. cerevisiae and to the analysis of exogenous DNA cloned in yeast is discussed.

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Selected References

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

  1. Burke D. T., Carle G. F., Olson M. V. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science. 1987 May 15;236(4803):806–812. doi: 10.1126/science.3033825. [DOI] [PubMed] [Google Scholar]
  2. Carle G. F., Olson M. V. An electrophoretic karyotype for yeast. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3756–3760. doi: 10.1073/pnas.82.11.3756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carlson M., Celenza J. L., Eng F. J. Evolution of the dispersed SUC gene family of Saccharomyces by rearrangements of chromosome telomeres. Mol Cell Biol. 1985 Nov;5(11):2894–2902. doi: 10.1128/mcb.5.11.2894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chan C. S., Tye B. K. Organization of DNA sequences and replication origins at yeast telomeres. Cell. 1983 Jun;33(2):563–573. doi: 10.1016/0092-8674(83)90437-3. [DOI] [PubMed] [Google Scholar]
  5. Chu G., Vollrath D., Davis R. W. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science. 1986 Dec 19;234(4783):1582–1585. doi: 10.1126/science.3538420. [DOI] [PubMed] [Google Scholar]
  6. Dunn B., Szauter P., Pardue M. L., Szostak J. W. Transfer of yeast telomeres to linear plasmids by recombination. Cell. 1984 Nov;39(1):191–201. doi: 10.1016/0092-8674(84)90205-8. [DOI] [PubMed] [Google Scholar]
  7. Eibel H., Philippsen P. Preferential integration of yeast transposable element Ty into a promoter region. 1984 Jan 26-Feb 1Nature. 307(5949):386–388. doi: 10.1038/307386a0. [DOI] [PubMed] [Google Scholar]
  8. Garvey E. P., Santi D. V. Stable amplified DNA in drug-resistant Leishmania exists as extrachromosomal circles. Science. 1986 Aug 1;233(4763):535–540. doi: 10.1126/science.3726545. [DOI] [PubMed] [Google Scholar]
  9. Hieter P., Mann C., Snyder M., Davis R. W. Mitotic stability of yeast chromosomes: a colony color assay that measures nondisjunction and chromosome loss. Cell. 1985 Feb;40(2):381–392. doi: 10.1016/0092-8674(85)90152-7. [DOI] [PubMed] [Google Scholar]
  10. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kostriken R., Strathern J. N., Klar A. J., Hicks J. B., Heffron F. A site-specific endonuclease essential for mating-type switching in Saccharomyces cerevisiae. Cell. 1983 Nov;35(1):167–174. doi: 10.1016/0092-8674(83)90219-2. [DOI] [PubMed] [Google Scholar]
  12. Mortimer R. K., Schild D. Genetic map of Saccharomyces cerevisiae, edition 9. Microbiol Rev. 1985 Sep;49(3):181–213. doi: 10.1128/mr.49.3.181-213.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Murray A. W., Szostak J. W. Construction and behavior of circularly permuted and telocentric chromosomes in Saccharomyces cerevisiae. Mol Cell Biol. 1986 Sep;6(9):3166–3172. doi: 10.1128/mcb.6.9.3166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Orbach M. J., Vollrath D., Davis R. W., Yanofsky C. An electrophoretic karyotype of Neurospora crassa. Mol Cell Biol. 1988 Apr;8(4):1469–1473. doi: 10.1128/mcb.8.4.1469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354–6358. doi: 10.1073/pnas.78.10.6354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Snell R. G., Wilkins R. J. Separation of chromosomal DNA molecules from C.albicans by pulsed field gel electrophoresis. Nucleic Acids Res. 1986 Jun 11;14(11):4401–4406. doi: 10.1093/nar/14.11.4401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. St John T. P., Davis R. W. The organization and transcription of the galactose gene cluster of Saccharomyces. J Mol Biol. 1981 Oct 25;152(2):285–315. doi: 10.1016/0022-2836(81)90244-8. [DOI] [PubMed] [Google Scholar]
  20. Surosky R. T., Tye B. K. Construction of telocentric chromosomes in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1985 Apr;82(7):2106–2110. doi: 10.1073/pnas.82.7.2106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tschumper G., Carbon J. Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene. Gene. 1980 Jul;10(2):157–166. doi: 10.1016/0378-1119(80)90133-x. [DOI] [PubMed] [Google Scholar]
  22. Van der Ploeg L. H., Schwartz D. C., Cantor C. R., Borst P. Antigenic variation in Trypanosoma brucei analyzed by electrophoretic separation of chromosome-sized DNA molecules. Cell. 1984 May;37(1):77–84. doi: 10.1016/0092-8674(84)90302-7. [DOI] [PubMed] [Google Scholar]
  23. Van der Ploeg L. H., Smits M., Ponnudurai T., Vermeulen A., Meuwissen J. H., Langsley G. Chromosome-sized DNA molecules of Plasmodium falciparum. Science. 1985 Aug 16;229(4714):658–661. doi: 10.1126/science.3895435. [DOI] [PubMed] [Google Scholar]
  24. Vollrath D., Davis R. W. Resolution of DNA molecules greater than 5 megabases by contour-clamped homogeneous electric fields. Nucleic Acids Res. 1987 Oct 12;15(19):7865–7876. doi: 10.1093/nar/15.19.7865. [DOI] [PMC free article] [PubMed] [Google Scholar]

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