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. 1991 Sep 25;19(18):4943–4948. doi: 10.1093/nar/19.18.4943

Rescue of end fragments of yeast artificial chromosomes by homologous recombination in yeast.

G G Hermanson 1, M F Hoekstra 1, D L McElligott 1, G A Evans 1
PMCID: PMC328794  PMID: 1923762

Abstract

Yeast artificial chromosomes (YACs) provide a powerful tool for the isolation and mapping of large regions of mammalian chromosomes. We developed a rapid and efficient method for the isolation of DNA fragments representing the extreme ends of YAC clones by the insertion of a rescue plasmid into the YAC vector by homologous recombination. Two rescue vectors were constructed containing a yeast LYS2 selectable gene, a bacterial origin of replication, an antibiotic resistance gene, a polylinker containing multiple restriction sites, and a fragment homologous to one arm of the pYAC4 vector. The 'end-cloning' procedure involves transformation of the rescue vector into yeast cells carrying a YAC clone, followed by preparation of yeast DNA and transformation into bacterial cells. The resulting plasmids carry end-specific DNA fragments up to 20 kb in length, which are suitable for use as hybridization probes, as templates for direct DNA sequencing, and as probes for mapping by fluorescence in situ hybridization. These vectors are suitable for the rescue of end-clones from any YAC constructed using a pYAC-derived vector. We demonstrate the utility of these plasmids by rescuing YAC-end fragments from a human YAC library.

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Selected References

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

  1. Albertsen H. M., Abderrahim H., Cann H. M., Dausset J., Le Paslier D., Cohen D. Construction and characterization of a yeast artificial chromosome library containing seven haploid human genome equivalents. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4256–4260. doi: 10.1073/pnas.87.11.4256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barnes D. A., Thorner J. Genetic manipulation of Saccharomyces cerevisiae by use of the LYS2 gene. Mol Cell Biol. 1986 Aug;6(8):2828–2838. doi: 10.1128/mcb.6.8.2828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barton M. C., Hoekstra M. F., Emerson B. M. Site-directed, recombination-mediated mutagenesis of a complex gene locus. Nucleic Acids Res. 1990 Dec 25;18(24):7349–7355. doi: 10.1093/nar/18.24.7349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bronson S. K., Pei J., Taillon-Miller P., Chorney M. J., Geraghty D. E., Chaplin D. D. Isolation and characterization of yeast artificial chromosome clones linking the HLA-B and HLA-C loci. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1676–1680. doi: 10.1073/pnas.88.5.1676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brownstein B. H., Silverman G. A., Little R. D., Burke D. T., Korsmeyer S. J., Schlessinger D., Olson M. V. Isolation of single-copy human genes from a library of yeast artificial chromosome clones. Science. 1989 Jun 16;244(4910):1348–1351. doi: 10.1126/science.2544027. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Burke D. T., Olson M. V. Preparation of clone libraries in yeast artificial-chromosome vectors. Methods Enzymol. 1991;194:251–270. doi: 10.1016/0076-6879(91)94020-d. [DOI] [PubMed] [Google Scholar]
  8. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Green E. D., Olson M. V. Chromosomal region of the cystic fibrosis gene in yeast artificial chromosomes: a model for human genome mapping. Science. 1990 Oct 5;250(4977):94–98. doi: 10.1126/science.2218515. [DOI] [PubMed] [Google Scholar]
  10. Green E. D., Olson M. V. Systematic screening of yeast artificial-chromosome libraries by use of the polymerase chain reaction. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1213–1217. doi: 10.1073/pnas.87.3.1213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [PubMed] [Google Scholar]
  12. Huxley C., Hagino Y., Schlessinger D., Olson M. V. The human HPRT gene on a yeast artificial chromosome is functional when transferred to mouse cells by cell fusion. Genomics. 1991 Apr;9(4):742–750. doi: 10.1016/0888-7543(91)90369-p. [DOI] [PubMed] [Google Scholar]
  13. Lichter P., Tang C. J., Call K., Hermanson G., Evans G. A., Housman D., Ward D. C. High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science. 1990 Jan 5;247(4938):64–69. doi: 10.1126/science.2294592. [DOI] [PubMed] [Google Scholar]
  14. Mead D. A., Szczesna-Skorupa E., Kemper B. Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng. 1986 Oct-Nov;1(1):67–74. doi: 10.1093/protein/1.1.67. [DOI] [PubMed] [Google Scholar]
  15. Nelson D. L. Current methods for YAC clone characterization. Genet Anal Tech Appl. 1990 Sep;7(5):100–106. doi: 10.1016/0735-0651(90)90014-7. [DOI] [PubMed] [Google Scholar]
  16. Olson M., Hood L., Cantor C., Botstein D. A common language for physical mapping of the human genome. Science. 1989 Sep 29;245(4925):1434–1435. doi: 10.1126/science.2781285. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Riley J., Butler R., Ogilvie D., Finniear R., Jenner D., Powell S., Anand R., Smith J. C., Markham A. F. A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucleic Acids Res. 1990 May 25;18(10):2887–2890. doi: 10.1093/nar/18.10.2887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rommens J. M., Iannuzzi M. C., Kerem B., Drumm M. L., Melmer G., Dean M., Rozmahel R., Cole J. L., Kennedy D., Hidaka N. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science. 1989 Sep 8;245(4922):1059–1065. doi: 10.1126/science.2772657. [DOI] [PubMed] [Google Scholar]
  20. Seifert H. S., Chen E. Y., So M., Heffron F. Shuttle mutagenesis: a method of transposon mutagenesis for Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1986 Feb;83(3):735–739. doi: 10.1073/pnas.83.3.735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sherman F., Hicks J. Micromanipulation and dissection of asci. Methods Enzymol. 1991;194:21–37. doi: 10.1016/0076-6879(91)94005-w. [DOI] [PubMed] [Google Scholar]
  22. Silverman G. A., Ye R. D., Pollock K. M., Sadler J. E., Korsmeyer S. J. Use of yeast artificial chromosome clones for mapping and walking within human chromosome segment 18q21.3. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7485–7489. doi: 10.1073/pnas.86.19.7485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  25. Traver C. N., Klapholz S., Hyman R. W., Davis R. W. Rapid screening of a human genomic library in yeast artificial chromosomes for single-copy sequences. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5898–5902. doi: 10.1073/pnas.86.15.5898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wahl G. M., Lewis K. A., Ruiz J. C., Rothenberg B., Zhao J., Evans G. A. Cosmid vectors for rapid genomic walking, restriction mapping, and gene transfer. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2160–2164. doi: 10.1073/pnas.84.8.2160. [DOI] [PMC free article] [PubMed] [Google Scholar]

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