Genome‐Wide Transposon Mutagenesis in Yeast

Anuj Kumar1, Michael Snyder1

1 Yale University, New Haven, Connecticut
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 13.3
DOI:  10.1002/0471142727.mb1303s51
Online Posting Date:  May, 2001
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Abstract

This unit provides comprehensive protocols for the use of insertional libraries generated by shuttle mutagenesis. From the basic protocol, a small aliquot of insertional library DNA may be used to mutagenize yeast, producing strains containing a single transposon insertion within a transcribed and translated region of the genome. This transposon‐mutagenized bank of yeast strains may be screened for any desired mutant phenotype. Alternatively, since the transposon contains a reporter gene lacking its start codon and promoter, transposon‐tagged strains may also be screened for specific patterns of gene expression. Strains of interest may be characterized by vectorette PCR (protocol provided) in order to locate the precise genomic site of transposon insertion within each mutant. A method by which Cre/lox recombination may be used to reduce the transposon in yeast to a small insertion element encoding an epitope tag is described. This tag serves as a tool by which transposon‐mutagenized gene products may be analyzed further (e.g., localized to a discrete subcellular site).

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Generating Yeast Mutants From mTn‐Mutagenized Library DNA
  • Support Protocol 1: Vectorette Polymerase Chain Reaction
  • Support Protocol 2: Epitope Tagging of mTn‐Mutagenized Gene Products
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Generating Yeast Mutants From mTn‐Mutagenized Library DNA

  Materials
  • Transposon‐mutagenized genomic library plasmid DNA (available upon request; see )
  • 10× TE buffer, pH 8.0 ( appendix 22), sterile
  • E. coli tets, kans (e.g., DH5α)
  • 14‐cm LB plates and medium supplemented with 3 µg/ml tetracycline and 40 µg/ml kanamycin (unit 1.1)
  • LB medium (unit 1.1)
  • Glycerol, sterile
  • NotI restriction endonuclease and buffer (unit 3.1)
  • Ura3 yeast culture (Chapter )
  • recipeOne‐step buffer (see recipe)
  • 10 mg/ml denatured salmon sperm DNA (unit 14.7)
  • CM dropout plates and medium without uracil (–Ura; unit 13.1)
  • YPAD plates: YPD plates (unit 13.1) supplemented with 80 mg/liter adenine
  • Chloroform
  • Xgal plates (unit 13.1)
  • Clinical tabletop centrifuge
  • 45°C water bath or incubator
  • Sterile toothpicks
  • 3MM filter paper (Whatman)
  • 30°C incubator
  • 9‐cm and 15‐cm glass petri dishes
  • Additional reagents and equipment for culturing E. coli (units 1.1, 1.2 & 1.3), preparing and transforming competent E. coli (unit 1.8), isolating plasmid DNA by miniprep (unit 1.6) or large‐scale preparation (unit 1.7), restriction endonuclease digestion (unit 3.1), yeast culture (unit 13.2), agarose gel electrophoresis (unit 2.5), culturing yeast (units 13.1 & 13.2), transforming yeast (unit 13.7), and assaying for β‐galactosidase activity (unit 13.6)

Support Protocol 1: Vectorette Polymerase Chain Reaction

  Materials
  • Anchor bubble primers 1 and 2 (see Fig. for sequences; see units 2.11 & 2.12 for synthesis techniques)
  • 1 M MgCl 2 ( appendix 22)
  • Universal vectorette (UV) and mTn primers (see Fig. for sequences; see units 2.11 & 2.12 for synthesis techniques)
  • Transposon‐mutagenized yeast strain (see protocol 1)
  • Appropriate restriction endonucleases (e.g., AluI or DraI) and buffers (unit 3.1)
  • 10× T4 DNA ligase buffer (unit 3.4)
  • 5 mM ATP (unit 3.4)
  • 400 U/µl T4 DNA ligase (measured in cohesive end ligation units)
  • 5 U/µl Taq DNA polymerase and 10× buffer (units 3.5 & 15.1)
  • 2.5 mM 4dNTP mix (unit 15.1)
  • Heat block
  • Automated thermal cycler
  • Additional reagents and equipment for preparing yeast DNA (unit 13.11), restriction endonuclease digestion (unit 3.1), ligation of DNA fragments (unit 3.16), PCR amplification (unit 15.1), purification of DNA from agarose gels (unit 2.6), and DNA sequence analysis (unit 7.4)

Support Protocol 2: Epitope Tagging of mTn‐Mutagenized Gene Products

  Materials
  • mTn‐mutagenized yeast strain (see protocol 1)
  • pGAL‐cre (pB227; available upon request from authors, see )
  • Raff/ CM dropout plates and medium –Leu, –Ura with 2% (w/v) raffinose (unit 13.1)
  • Gal/ CM dropout medium –Leu with 2% (w/v) galactose (unit 13.1)
  • Glc/ CM dropout medium –Leu with 2% (w/v) glucose (unit 13.1)
  • 5‐fluoroorotic acid plates (5‐FOA; unit 13.1)
  • Glycerol, sterile
  • 30°C shaker and incubator
  • Additional reagents and equipment for culture of yeast (unit 13.2) and transformation of yeast cells (unit 13.7)
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Figures

Videos

Literature Cited

Literature Cited
   Burns, N., Grimwade, B., Ross‐Macdonald, P.B., Choi, E.‐Y., Finberg, K., Roeder, G.S., and Snyder, M. 1994. Large‐scale analysis of gene expression, protein localization and gene disruption in Saccharomyces cerevisiae. Genes Dev. 8:1087‐1105.
   Chen, D.C., Yang, B.C., and Kuo, T.T. 1992. One‐step transformation of yeast in stationary phase. Curr. Genet. 21:83‐84.
   Devine, S.E. and Boeke, J.D. 1994. Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis. Nucleic Acids Res. 22:3765‐3772.
   Garfinkel, D.J. and Strathern, J.N. 1991. Ty mutagenesis in Saccharomyces cerevisiae. Methods Enzymol. 194:342‐361.
   Hoekstra, M.F., Seifert, H.S., Nickoloff, J., and Heffron, F. 1991. Shuttle mutagenesis: bacterial transposons for genetic manipulation in yeast. Methods Enzymol. 194:329‐342.
   Ji, H., Moore, D.P., Blomberg, M.A., Braiterman, L.T., Voytas, D.F., Natsoulis, G., and Boeke, J.D. 1993. Hotspots for unselected Ty1 transposition events on yeast chromosome III are near tRNA genes and LTR sequences. Cell 73:1007‐1018.
   Riley, J., Butler, R., Ogilvie, D., Finniear, R., Jenner, D., Powell, S., Anand, R., Smith, J.C., and Markham, A.F. 1990. A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucleic Acids Res. 18:2887‐2890.
   Ross‐Macdonald, P., Sheehan, A., Roeder, G.S., and Snyder, M. 1997. A multipurpose transposon system for analyzing protein production, localization, and function in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 94:190‐195.
   Ross‐Macdonald, P., Coelho, P., Roemer, T., Agarwal, S., Kumar, A., Cheung, K.‐H., Jansen, R., Sheehan, A., Symoniatis, D., Umansky, L., Nelson, K., Iwasaki, H., Hager, K., Gerstein, M., Miller, P., Roeder, G.S., and Snyder, M. 1999. Large‐scale analysis of the yeast genome by transposon tagging and gene disruption. Nature 402:413‐418.
   Seifert, H.S., Chen, E.Y., So, M., and Heffron, F. 1986. Shuttle mutagenesis: A method of transposon mutagenesis for Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 83:735‐739.
Key References
   Kumar, A., Cheung, K.‐H., Ross‐MacDonald, P., Coelho, P.S.R., Miller, P., and Snyder, M. 2000. TRIPLES: A database of gene function in S. cerevisiae. Nucleic Acids Res. 28:81‐84.
  Provides a helpful explanation of resources freely available from the authors' web site (see ).
   Seifert et al., 1986. See above.
  An early application of Tn3‐based shuttle mutagenesis to Saccharomyces cerevisiae.
   Ross‐Macdonald et al., 1997. See above.
  Provides an in‐depth description of multifunctional Tn3‐minitransposons used in this unit.
   Ross‐Macdonald et al., 1999. See above.
  Presents an extensive application of Tn3‐mediated shuttle‐mutagenesis towards functional genomics in yeast, with protocols for the genome‐wide analysis of disruption phenotypes, gene expression, and protein localization.
Internet Resources
  http://ygac.med.yale.edu
  Many strains and reagents used in this protocol (including all transposon‐insertion libraries) may be requested from this, the authors' web site.
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