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Related Experiment Videos

REP3-derived yeast shuttle vector.

U Schubert1, D Gniel, H Lang

  • 1Central Institute of Microbiology and Experimental Therapy, Academy of Sciences of the GDR, Jena.

Biomedica Biochimica Acta
|January 1, 1989
PubMed
Summary
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A new yeast shuttle vector utilizing a specific fragment of 2 microns DNA ensures stable plasmid amplification and partitioning in yeast. This design prevents recombination with endogenous DNA, maintaining plasmid stability.

Area of Science:

  • Molecular Biology
  • Yeast Genetics
  • Plasmid Engineering

Background:

  • The 2 microns DNA is a high-copy-number plasmid found in Saccharomyces cerevisiae, essential for its replication and maintenance.
  • Yeast shuttle vectors are crucial tools for genetic manipulation in yeast, enabling gene expression and cloning.
  • Understanding the functional elements of 2 microns DNA is key to developing stable and efficient yeast vectors.

Purpose of the Study:

  • To construct and characterize a novel yeast shuttle vector incorporating a specific fragment of the 2 microns DNA.
  • To determine if this fragment is sufficient for autonomous replication and stable partitioning in yeast.
  • To investigate the role of the FRT site and FLP-system in the stability and recombination of the engineered vector.

Main Methods:

Related Experiment Videos

  • Construction of a yeast shuttle vector using a 1293 bp XbaI-PstI fragment of 2 microns DNA, including the REP 3 locus and origin of replication.
  • Incorporation of the yeast LEU 2 gene and the tetracycline resistance gene (Tet') from pBR 322 into the vector.
  • Evaluation of vector amplification and partitioning in yeast.
  • Analysis of plasmid stability and recombination events in the presence and absence of functional FLP-system and with interrupted FRT sites.

Main Results:

  • The constructed XbaI-PstI fragment of 2 microns DNA is sufficient for the proper amplification and partitioning of yeast shuttle vectors.
  • Interruption of the FRT (Flippase Recombination Target) site within the vector sequence prevents recombination between endogenous 2 microns DNA and the hybrid plasmid.
  • Disruption of the FLP-system does not affect the copy number or stability of the engineered plasmid.

Conclusions:

  • A minimal fragment of 2 microns DNA containing REP 3 and the origin of replication is sufficient for stable yeast shuttle vector function.
  • Engineering the vector to interrupt the FRT site effectively blocks unwanted recombination with the host's native 2 microns DNA.
  • The developed yeast shuttle vector offers enhanced stability and controlled maintenance, making it a valuable tool for yeast molecular biology applications.