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. 1990 Feb;87(4):1300–1304. doi: 10.1073/pnas.87.4.1300

Generation of deletion derivatives by targeted transformation of human-derived yeast artificial chromosomes.

W J Pavan 1, P Hieter 1, R H Reeves 1
PMCID: PMC53462  PMID: 2406718

Abstract

Mammalian DNA segments cloned as yeast artificial chromosomes (YACs) can be manipulated by DNA-mediated transformation when placed in an appropriate yeast genetic background. A "fragmenting vector" has been developed that can introduce a yeast telomere and selectable marker into human-derived YACs at specific sites by means of homologous recombination, deleting all sequences distal to the recombination site. A powerful application of the method uses a human Alu family repeat sequence to target recombination to multiple independent sites on a human-derived YAC. Sets of deletion derivatives generated by this procedure greatly facilitate restriction mapping of large genomic segments. Targeting recombination with single copy sequences, such as cDNAs, will have many additional applications. This approach establishes a paradigm for manipulation and characterization of mammalian DNA segments cloned as YACs.

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Selected References

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

  1. Botstein D., Fink G. R. Yeast: an experimental organism for modern biology. Science. 1988 Jun 10;240(4858):1439–1443. doi: 10.1126/science.3287619. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Carle G. F., Olson M. V. Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis. Nucleic Acids Res. 1984 Jul 25;12(14):5647–5664. doi: 10.1093/nar/12.14.5647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Davis R. W., Thomas M., Cameron J., St John T. P., Scherer S., Padgett R. A. Rapid DNA isolations for enzymatic and hybridization analysis. Methods Enzymol. 1980;65(1):404–411. doi: 10.1016/s0076-6879(80)65051-4. [DOI] [PubMed] [Google Scholar]
  6. Deininger P. L., Jolly D. J., Rubin C. M., Friedmann T., Schmid C. W. Base sequence studies of 300 nucleotide renatured repeated human DNA clones. J Mol Biol. 1981 Sep 5;151(1):17–33. doi: 10.1016/0022-2836(81)90219-9. [DOI] [PubMed] [Google Scholar]
  7. Deininger P. L., Slagel V. K. Recently amplified Alu family members share a common parental Alu sequence. Mol Cell Biol. 1988 Oct;8(10):4566–4569. doi: 10.1128/mcb.8.10.4566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [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. Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Moyzis R. K., Torney D. C., Meyne J., Buckingham J. M., Wu J. R., Burks C., Sirotkin K. M., Goad W. B. The distribution of interspersed repetitive DNA sequences in the human genome. Genomics. 1989 Apr;4(3):273–289. doi: 10.1016/0888-7543(89)90331-5. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Orr-Weaver T. L., Szostak J. W. Yeast recombination: the association between double-strand gap repair and crossing-over. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4417–4421. doi: 10.1073/pnas.80.14.4417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Reeves R. H., Crowley M. R., Lorenzon N., Pavan W. J., Smeyne R. J., Goldowitz D. The mouse neurological mutant weaver maps within the region of chromosome 16 that is homologous to human chromosome 21. Genomics. 1989 Oct;5(3):522–526. doi: 10.1016/0888-7543(89)90018-9. [DOI] [PubMed] [Google Scholar]
  16. Runge K. W., Zakian V. A. Introduction of extra telomeric DNA sequences into Saccharomyces cerevisiae results in telomere elongation. Mol Cell Biol. 1989 Apr;9(4):1488–1497. doi: 10.1128/mcb.9.4.1488. [DOI] [PMC free article] [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. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Struhl K. Nucleotide sequence and transcriptional mapping of the yeast pet56-his3-ded1 gene region. Nucleic Acids Res. 1985 Dec 9;13(23):8587–8601. doi: 10.1093/nar/13.23.8587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Vollrath D., Davis R. W., Connelly C., Hieter P. Physical mapping of large DNA by chromosome fragmentation. Proc Natl Acad Sci U S A. 1988 Aug;85(16):6027–6031. doi: 10.1073/pnas.85.16.6027. [DOI] [PMC free article] [PubMed] [Google Scholar]

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