Transfer of YAC Clones to New Hosts by Karyogamy‐Deficient Mating

Forrest Spencer1, Giora Simchen2

1 Johns Hopkins University School of Medicine, Baltimore, Maryland, 2 The Hebrew University, Jerusalem, Israel
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 5.14
DOI:  10.1002/0471142905.hg0514s06
Online Posting Date:  May, 2001
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Abstract

This unit provides a protocol for moving yeast artificial chromosome (YAC) clones to new yeast hosts using basic microbial techniques and pulsed‐field gel analysis. In contrast to other methods that can be used to transfer YAC clones, this requires neither optimization to achieve high‐efficiency DNA‐mediated transformation of chromosome‐sized DNA nor specialized equipment for tetrad dissection and analysis. Instead, chromosome (YAC) transfer is selected in rare segregants (“YACductants”) from a yeast mating that is rendered incomplete in most cell pairings by the presence of a kar1 (karyogamy‐deficient) mutation in either parental strain. The in this unit details the transfer of a YAC clone from yeast strain AB1380 (host to nearly all existing YAC libraries) to YPH925, a strain with nonreverting genetic markers compatible with existing plasmid constructs useful in YAC modification.

     
 
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Table of Contents

  • Basic Protocol 1: YAC Transfer by Kar1− Mating
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: YAC Transfer by Kar1− Mating

  Materials
  • Liquid medium appropriate for recipient yeast strain: e.g., YPD (unit 5.5) for strain YPH925
  • Liquid medium appropriate for donor yeast strain: e.g., recipeSC −Ura −Trp (see recipe) for strain AB1380 + YAC
  • Donor yeast strain: e.g., AB1380 containing a pYAC4 clone
  • Recipient yeast strain: e.g., YPH925 (kar1Δ15; Table 5.14.2)
  • recipeSC −Ura −Trp +Cyh, SC −Ura +Cyh, and SC −His +Cyh plates (see recipe)
  • Low gelling/melting temperature agarose
  • 0.125 M EDTA, pH 8.0
  • ET buffer: 0.05 M EDTA (pH 8.0)/0.01 M Tris⋅Cl (pH 8.0; appendix 2D)
  • recipe20 µg/ml Zymolyase 20T solution (see recipe)
  • recipeLET buffer (see recipe)
  • recipeNDS buffer (see recipe)
  • recipeProteinase K stock solution (see recipe)
  • 0.5× TBE buffer ( appendix 2D)
  • 30°C incubator, preferably with roller drum
  • Culture tubes suitable for use with roller drum, if used
  • Spectrophotometer or microscope and hemacytometer
  • Replica platform and sterile velvets for replica plating
  • Sterile 2.1‐cm‐diameter Whatman no. 1 filter paper circles (optional)
  • Sterile pipets, micropipet tips, toothpicks, or wooden sticks
  • 45°C water bath
  • Additional reagents and equipment for pulsed‐field gel electrophoresis (unit 5.1) and Southern blot analysis of YACs (unit 5.7)
NOTE: All reagents and equipment coming into contact with live cells must be sterile.
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Figures

Videos

Literature Cited

Literature Cited
   Burke, D., Carle, G., and Olson, M. 1987. Cloning of large segments of DNA into yeast by means of artificial chromosome vectors. Science 236:806‐812.
   Campbell, C., Gulati, R., Nandi, A., Floy, K., Hieter, P., and Kucherlapati, R. 1991. Generation of a nested series of deletions in yeast artificial chromosomes carrying human DNA. Proc. Natl. Acad. Sci. U.S.A. 88:5744‐5748.
   Conde, J. and Fink, G. 1976. A mutant of Saccharomyces cerevisiae defective for nuclear fusion. Proc. Natl. Acad. Sci. U.S.A. 73:3651‐3655.
   Dutcher, S. 1981. Internuclear transfer of genetic information in kar1‐1/KAR1 heterokaryons in Saccharomyces cerevisiae. Mol. Cell. Biol. 1:245‐253.
   Hugerat, Y. and Simchen, G. 1993. Mixed segregation and recombination of chromosomes and YACs during single‐division meiosis in spo13 strains of S. cerevisiae. Genetics 135:297‐308.
   Hugerat, Y., Spencer, F., Zenvirth, D., and Simchen, G. 1994. A versatile method for efficient YAC transfer between any two strains. Genomics 22:108‐117.
   Huxley, C., Green, E., and Dunham, I. 1990. Rapid assessment of S. cerevisiae mating type by PCR. Trends Genet. 6:236.
   Ji, H., Moore, D., Blomberg, M., Braiterman, L., Voytas, D., Natsoulis, G., and Boeke, J. 1993. Hotspots for unselected Ty1 transposition events on yeast chromosome III are near tRNA genes and LTR sequences. Cell 73:1007‐1018.
   Pavan, W., Hieter, P., and Reeves, R. 1990. Generation of deletion derivatives by targeted transformation of human‐derived yeast artificial chromosomes. Proc. Natl. Acad. Sci. U.S.A. 87:1300‐1304.
   Rose, M. and Fink, G. 1987. KAR1, a gene required for function of both intranuclear and extranuclear microtubules in yeast. Cell 48:1047‐1060.
   Rose, M., Winston, F., and Hieter, P. 1990. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, New York.
   Rothstein, R. 1991. Targeting, disruption, replacement, and allele rescue: Integrative DNA transformation in yeast. Methods Enzymol. 194:281‐301.
   Schwartz, D. and Cantor, C. 1984. Separation of yeast chromosome–sized DNAs by pulsed‐field gradient gel electrophoresis. Cell 37:67‐75.
   Spencer, F., Ketner, G., Connelly, C., and Hieter, P. 1993. Targeted recombination‐based cloning and manipulation of large DNA segments in yeast. Methods 5:161‐175.
   Spencer, F., Hugerat, Y., Simchen, G., Hurko, O., Connelly, C., and Hieter, P. 1994. Yeast kar1 mutants provide an effective method for YAC transfer between hosts. Genomics 22:118‐126.
   Vallen, E., Hiller, M., Scherson, T., and Rose, M. 1992. Separate domains of KAR1 mediate distinct functions in mitosis and nuclear fusion. J. Cell Biol. 117:1277‐1287.
Key References
   Dutcher 1981. See above.
  The original observation of chromosome transfer in Kar1− matings.
   Hugerat et al., 1994. See above.
  Evaluations of the application of chromosome transfer in Kar1− matings to the transfer of YACs among yeast hosts.
   Spencer et al., 1994. See above.
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