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REP3-derived yeast shuttle vector
1Central Institute of Microbiology and Experimental Therapy, Academy of Sciences of the GDR, Jena.
Abstract:
A yeast-shuttle vector using a XbaI-PstI fragment of the 2 microns DNA 1293 bp in length has been constructed. This sequence spans the REP 3 locus and the origin of replication. Besides the 2 microns DNA derived sequences the resulting yeast shuttle vector contains the yeast LEU 2 gene and the Tet' gene of pBR 322. The results demonstrate that the XbaI-PstI fragment is sufficient for proper amplification and partitioning of 2 microns DNA derived yeast-shuttle vectors in yeast. The interruption of the FRT site in this type of vector seems to prevent any recombination between endogenous 2 microns DNA and hybrid plasmid molecules. Moreover, the disturbance of the FLP-system has no influence on the copy number of the plasmid stability.
Insights
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:
- 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.
