Yeast Vectors and Assays for Expression of Cloned Genes

Ann Reynolds1, Victoria Lundblad2, David Dorris3, Marie Keaveney3

1 University of Washington, Seattle, Washington, 2 Baylor College of Medicine, Houston, Texas, 3 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 13.6
DOI:  10.1002/0471142727.mb1306s39
Online Posting Date:  May, 2001
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Abstract

This unit describes some of the most commonly used yeast vectors, as well as the cloned yeast genes that form the basis for these plasmids. Yeast vectors can be grouped into five general classes, based on their mode of replication in yeast: YIp, YRp, YCp, YEp, and YLp plasmids. With the exception of the YLp plasmids (yeast linear plasmids), all of these plasmids can be maintained in E. coli as well as in S. cerevisiae and thus are referred to as shuttle vectors. The nomenclature of different classes of yeast vectors, as well as details about their mode of replication in yeast are discussed.

     
 
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Table of Contents

  • Basic Protocol 1: Construction of lacZ Fusion Vectors for Studying Yeast Gene Regulation
  • Basic Protocol 2: Assay for β‐Galactosidase in Liquid Cultures
  • Alternate Protocol 1: Screening for β‐Galactosidase‐Expressing Yeast Colonies Using a Filter Lift Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Construction of lacZ Fusion Vectors for Studying Yeast Gene Regulation

  Materials
  • YPD or other appropriate medium (unit 13.1)
  • Yeast strain containing lacZ fusion gene (see protocol 1)
  • Appropriate inducing agent (optional)
  • recipeZ buffer (see recipe)
  • 0.1% sodium dodecyl sulfate (SDS)
  • Chloroform
  • 4 mg/ml ONPG (Table 97.80.4711) in 0.1 M potassium phosphate, pH 7.0 ( appendix 22; filter sterilized and stored frozen)
  • 1 M Na 2CO 3
  • 30°C water bath

Basic Protocol 2: Assay for β‐Galactosidase in Liquid Cultures

  Materials
  • Yeast strain containing lacZ fusion gene (see protocol 1)
  • Selective medium plates (unit 13.1)
  • Liquid nitrogen
  • recipeZ buffer (see recipe)
  • 20 mg/ml Xgal (Table 97.80.4711) in dimethylformamide
  • 30°C incubator
  • Circular nitrocellulose membrane filters
  • Whatman 3MM paper
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Figures

Videos

Literature Cited

Literature Cited
   Durfee, T., Becherer, K., Chen, P.‐L., Yeh, S.‐H., Yang, Y., Kilburn, A.‐E., Lee, W.‐H., and Elledge, S.J. 1993. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes & Dev. 7:555‐569.
   Guarente, L. 1983. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 101:181‐191.
   Guarente, L., Lauer, G., Roberts, T.M., and Ptashne, M. 1980. Improved methods for maximizing expression of a cloned gene: A bacterium that synthesizes rabbit β‐globin. Cell 20:543‐553.
   Miller, J.H. 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Staudinger, J., Perry, M., Elledge, S.J., and Olson, E.N. 1993. Interaction among vertebrate helix‐loop‐helix proteins in yeast two‐hybrid system. J. Biol. Chem. 268:4608‐4611.
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