Manipulation of Cloned Yeast DNA

Victoria Lundblad1, Grant Hartzog2, Zarmik Moqtaderi2

1 Baylor College of Medicine, Houston, Texas, 2 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 13.10
DOI:  10.1002/0471142727.mb1310s39
Online Posting Date:  May, 2001
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Abstract

A major advantage of working with yeast is the ability to replace the wild‐type chromosomal copy of a gene with a mutant derivative that is constructed in vitro using a cloned copy of the gene. This techniqueunavailable in most other eukaryotesallows the phenotype of the mutation to be studied under accurate in vivo conditions, with the mutation present in single copy at its normal chromosomal location. In the first protocol, a plasmid harboring both a selectable marker and a cloned gene of interest is integrated at the chromosomal location of the cloned gene via homologous recombination (integrative trandformation). Four methods are described for constructing a mutation in vitro in a cloned gene and reintroducing this mutation at the correct chromosomal site. This allows assessment of the genetic consequences of a mutation, and is often used to determine whether or not a gene is essential (by determining if a complete gene deletion is viable). Two of these techniquesintegrative disruption and one‐step gene disruptiongenerate either insertion or deletion mutations. The third techniquetransplacementis more generally applicable: it can be used to introduce insertion or deletion mutations containing a selectable marker, but it can also be used to introduce nonselectable mutations, such as conditional lethal mutations in an essential gene. Protocols are also provided to allow creation of modified genes by one‐step integrative replacement, and also conditional alleles by a copper‐inducible double‐shutoff procedure.

     
 
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Table of Contents

  • Basic Protocol 1: Integrative Transformation
  • Gene Replacement Techniques
  • Basic Protocol 2: Integrative Disruption
  • Basic Protocol 3: One‐Step Gene Disruption
  • Alternate Protocol 1: PCR‐Mediated One‐Step Gene Disruption
  • Basic Protocol 4: Transplacement
  • Basic Protocol 5: Creating Modified Genes by One‐Step Integrative Replacement
  • Alternate Protocol 2: Creating Modified Genes by Transplacement
  • Basic Protocol 6: Creation of Conditional Alleles by Copper‐Inducible Double‐Shutoff Procedure
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Integrative Transformation

  Materials
  • YIp shuttle vector (unit 13.4)
  • Appropriate restriction enzyme (unit 3.1)
  • Purified DNA from gene of interest
  • Carrier DNA
  • Selective medium (unit 13.1)
  • Additional reagents and equipment for subcloning (unit 3.16), yeast transformation (unit 13.7), yeast genomic DNA isolation (unit 13.11), and Southern blotting (units 2.9 & 2.10) or PCR (unit 15.1)

Basic Protocol 2: Integrative Disruption

  Materials
  • YIp shuttle vector (unit 13.4)
  • Purified DNA from gene of interest
  • Appropriate restriction enzyme (unit 3.1)
  • Additional reagents and equipment for subcloning (unit 3.16) and integrative transformation (see protocol 1)

Basic Protocol 3: One‐Step Gene Disruption

  Materials
  • Purified DNA from gene of interest
  • Purified DNA from a selectable gene
  • Appropriate restriction enzyme (unit 3.1)
  • Appropriate yeast strain (e.g., Table 13.10.2)
  • Additional reagents and equipment for subcloning (unit 3.16), gel purification of DNA (unit 2.6), yeast transformation (unit 13.7), yeast plasmid DNA isolation (unit 13.11), Southern blotting (units 2.9 & 2.10) or PCR (unit 15.1), and tetrad analysis (unit 13.2; optional)
    Table 3.0.2   Materials   Suggested Yeast Strains for Gene Replacement b   Suggested Yeast Strains for Gene Replacement

    Strain ATCC number Genotype
    BY4700 200866 MATaura3Δ
    BY4736 200898 MATa ade2Δ0::hisG his3Δ200 met15Δ0 trp1Δ63 ura3Δ0
    BY4727 200889 MATα his3Δ200 leu2Δ0 lys2Δ0 met15Δ0 trp1Δ63 ura3Δ0

     bMany other suitable strains are available from the ATCC (strains 200866 to 200902). A recipient diploid can be made by mating BY4736 and BY4727 and selecting for Ade+ Leu+ Lys+ colonies.

Alternate Protocol 1: PCR‐Mediated One‐Step Gene Disruption

  Materials
  • 10× PCR buffer (e.g., as supplied with Taq DNA polymerase from Boehringer Mannheim)
  • 100 mM Tris⋅Cl/15 mM MgCl 2/500 mM KCl, pH 8.3 (20°C)
  • 4dNTP mixture (unit 3.4)
  • pRS40X‐series vector (e.g., Table 13.10.1)
  • pRS Left and Right primers (see step )
  • Taq DNA polymerase
  • Appropriate yeast strain (e.g., Table 13.10.2)
  • Selective medium (unit 13.1)
  • Additional reagents and equipment for PCR (unit 15.1), yeast transformation (unit 13.7), yeast genomic DNA isolation (unit 13.11) and Southern blotting (units 2.9 & 2.10; optional)

Basic Protocol 4: Transplacement

  Materials
  • YIp shuttle vector (unit 13.4)
  • Purified DNA from gene of interest
  • Nonselective liquid medium ( YPD or uracil‐containing minimal medium; unit 13.1)
  • 5‐FOA plates (unit 13.1)
  • Additional reagents and equipment for subcloning (unit 3.16), integrative transformation (see protocol 1), yeast genomic DNA isolation (unit 13.11), and Southern blotting (units 2.9 & 2.10) or PCR (units 15.1)

Basic Protocol 5: Creating Modified Genes by One‐Step Integrative Replacement

  Materials
  • Purified DNA from gene of interest
  • Yeast integrating shuttle vector with selectable marker
  • Appropriate restriction enzyme (unit 3.1)
  • Appropriate yeast strain
  • Selective medium (unit 13.1)
  • Additional reagents and equipment for subcloning (unit 3.16), yeast transformation (unit 13.7), yeast genomic DNA isolation (unit 13.11), and Southern blotting (units 2.9 & 2.10) or PCR (unit 15.1)

Alternate Protocol 2: Creating Modified Genes by Transplacement

  Materials
  • Plasmid ZM168, containing the ANB1 promoter driving a ubiquitin‐arginine‐lacI‐HA (URLF) cassette immediately followed by a polylinker
  • Yeast integrating shuttle vector with selectable marker
  • Yeast strain ZMY60 (MATa, ade2‐101, HIS+, LEU+, trp1Δ1, ura3‐52)
  • Centromeric plasmid bearing the same marker as the integrating vector
  • Synthetic medium plates selecting for the integrated plasmid marker (unit 13.1), with and without 500 µM cupric sulfate (CuSO 4)
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Figures

Videos

Literature Cited

Literature Cited
   Baudin, A., Ozier‐Kalogeropoulos, O., Denouel, A., Lacroute, F., and Cullin, C. 1993. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucl. Acids Res. 21:3329‐3330.
   Brachmann, C.B., Davies, A., Cost, G.J., Caputo, E., Li, J., Hieter, P., and Boeke, J. 1998. Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR‐mediated gene disruption and other applications. Yeast, 14:115‐132.
   Hinnen, A., Hicks, J.B., and Fink, G.R. 1978. Transformation of yeast. Proc. Natl. Acad. Sci. U.S.A. 75:1929‐1933.
   Huismann, O., Raymond, W., Foelich, K., Errada, P., Kleckner, N., Botstein, D., and Hoyt, M.A. 1987. A Tn10‐lacZ‐kanR‐URA3 gene fusion transposon for insertion mutagenesis and fusion analysis of yeast and bacterial genes. Genetics 1116:191‐199.
   Moqtaderi, Z., Bai, Y., Poon, D., Weil, P.A., and Struhl, K. 1996. TBP‐associated factors are not generally required for transcriptional activation in yeast. Nature 383:188‐191.
   Rothstein, R.J. 1983. One‐step gene disruption in yeast. Methods Enzymol. 101:202‐210.
   Scherer, S. and Davis, R.W. 1979. Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc. Natl. Acad. Sci. U.S.A. 76:4951‐4955.
   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.
   Shortle, D., Haber, J.E., and Botstein, D. 1982. Lethal disruption of the yeast actin gene by integrative DNA transformation. Science 217:371‐373.
   Sikorski, R.S. and Hieter, P. 1989. A System of shuttle vectors and yeast host strains designed for efficient manipulation of DNA is Saccharomyces Cerevisiae. Genetics 122:19‐27.
   Varshavsky, A. 1992. The N‐end rule. Cell 69:725‐735.
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