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An improved method for yeast 2 microns plasmid curing.

W Xiao1, G H Rank

  • 1Department of Biology, University of Saskatchewan, Saskatoon, Canada.

Gene
|April 16, 1990
PubMed
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This study introduces a new method for efficiently removing 2-micron DNA from yeast cells using the SMR1-410 resistance marker. This technique offers a faster and more convenient way to achieve DNA loss in wild-type cells.

Area of Science:

  • Yeast genetics
  • Molecular biology
  • Genetic engineering

Background:

  • The 2-micron DNA is a high-copy-number plasmid essential for yeast episomal plasmid (YEp) vectors.
  • Efficient methods for the loss of 2-micron DNA are crucial for various molecular biology applications.
  • Previous methods for 2-micron DNA curing were often inefficient or inconvenient.

Purpose of the Study:

  • To develop a novel and efficient protocol for the rapid loss of native 2-micron DNA from yeast cells.
  • To utilize the SMR1-410 dominant resistance marker for maintaining high-copy-number YEp vectors.
  • To establish a convenient curing methodology applicable to wild-type prototrophic yeast strains.

Main Methods:

  • Cloning the SMR1-410 dominant resistance marker into the FLP gene of 2-micron DNA to create the chimeric YEp vector pWX823B.

Related Experiment Videos

  • Applying selection pressure with high concentrations of sulfometuron methyl to maintain the pWX823 vector.
  • Monitoring the loss of native 2-micron DNA in yeast cell populations.

    • Main Results:

    • The SMR1-410 marker effectively maintained the pWX823 vector at a high copy number.
    • High concentrations of sulfometuron methyl induced rapid and efficient loss of native 2-micron DNA.
    • Approximately 15% of monitored cells exhibited loss of 2-micron DNA using this protocol.

    Conclusions:

    • The developed protocol provides a more efficient and convenient method for 2-micron DNA curing compared to existing techniques.
    • This methodology is applicable to wild-type prototrophic yeast cells, expanding its utility.
    • The SMR1-410 marker serves as a valuable tool for managing high-copy-number YEp vectors and facilitating DNA loss.