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Fragmentation and Integrative Modification of YACs

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YAC Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 54))

Abstract

The very large cloning capacity of yeast artificial chromosomes (YACs) has facilitated the analysis of complex genomes by bridging physical and genetic maps. The large size of YAC inserts also creates some unique problems, including identification of novel genes on large stretches of uncharacterized DNA, creating physical maps of large genomic inserts in YACs, and localizing defined sequences within the YAC. The well-characterized and highly efficient system of homologous recombination in Saccharomyces cerevisiae (1) can be used to introduce additional markers (e.g., neo R cassette, polylinkers, bacterial markers) and modifications to YACs that allow these problems to be addressed. This chapter covers fragmentation and integration, two recombination-based techniques for modifying the insert DNA and vector arm sequences of YACs. Specialized vectors containing cloned repetitive elements or sequences specific to YAC vector arms can recombine with homologous sequences in the YAC (2). Repetitive sequences are ideal for recombination-based modification because they are present at a high copy number and sufficiently widespread in the genome that most YACs will contain multiple representatives of these families.

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References

  1. Orr-Weaver, T., Szostak, J., and Rothstein, R. (1983) Genetic applications of yeast transformation with linear and gapped plasmids. Methods Enzymol. 101, 228–245.

    Article  PubMed  CAS  Google Scholar 

  2. Reeves, R. H., Pavan, W. J., and Hieter, P. (1990) Modification and manipulation of mammalian DNA cloned as yeast artificial chromosomes. Genet. Anal. Technol. Appl. 7, 107–113.

    Article  CAS  Google Scholar 

  3. Vollrath, D. M., Davis, R. W., Connelly, C., and Hieter, P. (1993) Physical mapping of large DNA by chromosome fragmentation. Proc. Natl. Acad. Sci. USA 85, 6027–6031.

    Article  Google Scholar 

  4. Pavan, W. J., Hieter, P., and Reeves, R. H. (1990) Generation of deletion derivatives by targeted transformation of human-derived yeast artificial chromosomes. Proc. Natl. Acad. Sci. USA 87, 1300–1304.

    Article  PubMed  CAS  Google Scholar 

  5. Pavan, W. J., Hieter, P., Sears, D., Burlkhoff, A., and Reeves, R. H. (1991) High efficiency yeast artificial chromosome fragmentation vectors. Gene 106, 125–127.

    Article  PubMed  CAS  Google Scholar 

  6. Lewis, B. C., Shah, N. P., Braun, B. S., and Denny, C. T. (1992) Creation of a yeast artificial chromosome fragmentation vector based on lysine-2. GATA 9, 86–93.

    CAS  Google Scholar 

  7. Sikorski, R. S., and Hieter, P. (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27.

    PubMed  CAS  Google Scholar 

  8. Spencer, F., Hugerat, Y., Simchen, G., Hurko, O., Connelly, C., and Hieter, P. (1994) Yeast kar1 mutants provide an efficient method for YAC transfer between strains. Genomics 22, 118–126.

    Article  PubMed  CAS  Google Scholar 

  9. Pavan, W. J. and Reeves, R. H. (1991) Integrative selection of human chromosome-specific yeast artificial chromosomes. Proc. Natl. Acad. Sci. USA 88, 7788–7791.

    Article  PubMed  CAS  Google Scholar 

  10. Pavan, W. J., Hieter, P., and Reeves, R. H. (1990) Modification and transfer into an embryonal carcinoma cell line of a 360 kb human-derived yeast artificial chromosome. Mol. Cell Biol. 10, 4163–4169.

    PubMed  CAS  Google Scholar 

  11. Das Gupta, R., Morrow, B., Marondel, I., Parimoo, S., Goei, V., Gruen, J., et al. (1993) An integrated approach for identifying and mapping human genes. Proc. Natl. Acad. Sci. USA 90, 4364–4368.

    Article  PubMed  Google Scholar 

  12. Winston, F., Chumley, F., and Fink, G. R. (1983) Evictions and transplacement of mutant genes in yeast. Methods Enzymol. 101, 211–228.

    Article  PubMed  CAS  Google Scholar 

  13. Pachnis, V., Pevny, L., Rothstein, R., and Constantini, F. (1990) Transfer of a yeast artificial chromosome carrying human DNA from Saccharomyces cerevisiae into mammalian cells. Proc. Natl. Acad. Sci. USA 87, 5109–5113.

    Article  PubMed  CAS  Google Scholar 

  14. Hermanson, G. G., Hoekstra, M. F., McElligott, D. C., and Evans, G. A. (1991) Rescue of end fragments of yeast artificial chromosomes by homologous recombination in yeast. Nucleic Acids Res. 19, 4943–4948.

    Article  PubMed  CAS  Google Scholar 

  15. Smith, D. R., Smyth, S. P., Strauss, W. M., and Moir, D. T. (1993) Incorporation of copy number control elements into yeast artificial chromosomes by targeted homologous recombination. Mammal. Gen. 4, 141–147.

    Article  CAS  Google Scholar 

  16. Davies, N. P., Rosewell, I. R., and Bruggemann, M. (1992) Targeted alterations in yeast artificial chromosomes for inter-species gene transfer. Nucleic Acids Res. 20, 2693–2698.

    Article  PubMed  CAS  Google Scholar 

  17. Markie, D., Ragoussis, J., Senger, G., Rowan, A., Sansom, D., Trowsdale, J., et al. (1993) New vector for transfer of yeast artificial chromosomes to mammalian cells. Somatic Cell Mol. Gen. 19, 161–169.

    Article  CAS  Google Scholar 

  18. Eliceiri, B., Labella, T., Hagino, Y., Srivastava, A. K., Schlessinger, D., Pilia, G., et al. (1991) Stable integration and expression in mouse cells of yeast artificial chromosmes harboring human genes. Proc. Natl. Acad. Sci. USA 88, 2179–2183.

    Article  PubMed  CAS  Google Scholar 

  19. Srivastava, A. K. and Schlessinger, D. (1991) Vectors for inserting selectable markers in vector arms and human DNA inserts of yeast artificial chromosomes (YACs). Gene 103, 53–59.

    Article  PubMed  CAS  Google Scholar 

  20. Riley, J. H., Morten, J. E. N., and Anand, R. (1992). Targeted integration of neomycin into yeast artificial chromosomes (YACs) for transfection into mammalian cells. Nucleic Acids Res. 20, 2971–2976.

    Article  PubMed  CAS  Google Scholar 

  21. Karube, I., Tamiya, E., and Matsuoka, H. (1985) Transformation of Saccharomyces cerevisiae spheroplasts by high electric pulse. FEBS Lett. 182, 90–94.

    Article  CAS  Google Scholar 

  22. Simon, J. and McEntee, K. (1989) A rapid and efficient procedure for transformation of intact Saccharomyces cerevisiae by electroporation. Biochem. Biophys. Res. Commun. 164, 1157–1164.

    Article  PubMed  CAS  Google Scholar 

  23. Hinnen, A., Hicks, J., and Fink, G. (1978) Transformation of yeast. Proc. Natl. Acad. Sci. USA 75, 1929–1933.

    Article  PubMed  CAS  Google Scholar 

  24. Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163–168.

    PubMed  CAS  Google Scholar 

  25. Jones, J. and Prakash, L. (1990) Yeast Saccharomyces cerevisiae selectable markers in pUC18_polylinkers. Yeast 6, 363–366.

    Article  PubMed  CAS  Google Scholar 

  26. Burke, D. T., Carle, G. F., and Olson, M. V. (1987) Cloning large segments of DNA into yeast by means of artificial chromosomes. Science 236, 906–912.

    Article  Google Scholar 

  27. Lamb, B. T., Sisodia, S. S., Lawler, A. M., Slunt, H. H., Kitt, C. A., Kearns, W. G., et al. (1993) Introduction and expression of the 400 kilobase amyloid precursor protein gene in transgenic mace. Nat. Gen. 5, 22–30.

    Article  CAS  Google Scholar 

  28. Cabin, D. E., Hawkins, A., Griffin, C., and Reeves, R. H. (1995) YAC transgenic mace in the study of the genetic basis of Down Syndrome. Progr. Clin. Biol. Res. (in press).

    Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Physiology, Johns Hopkins University, Baltimore, MD

    Jennifer W. McKee-Johnson & Roger H. Reeves

Authors
  1. Jennifer W. McKee-Johnson
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  2. Roger H. Reeves
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Editor information

Editors and Affiliations

  1. Paediatric Research Unit, United Medical and Dental Schools of Guy’s and St. Thomas’ Hospitals, London, UK

    David Markie

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© 1996 Humana Press Inc.

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McKee-Johnson, J.W., Reeves, R.H. (1996). Fragmentation and Integrative Modification of YACs. In: Markie, D. (eds) YAC Protocols. Methods in Molecular Biology™, vol 54. Humana Press. https://doi.org/10.1385/0-89603-313-9:167

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  • DOI: https://doi.org/10.1385/0-89603-313-9:167

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-313-9

  • Online ISBN: 978-1-59259-541-9

  • eBook Packages: Springer Protocols

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