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Related Experiment Videos

Yeast/E. coli shuttle vectors with multiple unique restriction sites.

J E Hill1, A M Myers, T J Koerner

  • 1Department of Biological Sciences, Columbia University, New York, NY 10027.

Yeast (Chichester, England)
|September 1, 1986
PubMed
Summary

New shuttle vectors, YEp351 and YEp352, enable autonomous replication in yeast and E. coli. These vectors facilitate yeast genomic library construction and DNA fragment cloning with numerous restriction sites.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Development of versatile cloning vectors is crucial for molecular biology research.
  • Shuttle vectors that function in both yeast (Saccharomyces cerevisiae) and bacteria (E. coli) offer significant advantages for genetic manipulation.
  • Existing vectors may have limitations in replication, selection markers, or restriction enzyme compatibility.

Purpose of the Study:

  • To construct and characterize novel yeast/E. coli shuttle vectors, YEp351 and YEp352.
  • To evaluate their utility for constructing yeast genomic libraries and cloning DNA fragments.
  • To develop derivative plasmids for integration into yeast chromosomal DNA.

Main Methods:

  • Construction of two shuttle vectors, YEp351 and YEp352, incorporating replication origins for both yeast and E. coli.

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  • Inclusion of the beta-lactamase gene for ampicillin resistance in E. coli and the pUC18 multiple cloning region.
  • Incorporation of yeast LEU2 and URA3 genes for auxotrophic selection.
  • Modification of vectors to create YIp351 and YIp352 derivatives for chromosomal integration.

    • Main Results:

    • YEp351 and YEp352 replicate autonomously in both Saccharomyces cerevisiae and E. coli.
    • Vectors confer ampicillin resistance and allow for blue-white screening via beta-galactosidase activity.
    • Unique restriction sites in the multiple cloning region facilitate diverse cloning strategies.
    • High-frequency retention in yeast indicates high plasmid copy number.
    • Derivative plasmids YIp351 and YIp352 enable integration into yeast chromosomes.

    Conclusions:

    • YEp351 and YEp352 are versatile shuttle vectors suitable for yeast genomic library construction and DNA fragment cloning.
    • The unique and numerous restriction sites offer flexibility in molecular cloning applications.
    • The derivative plasmids YIp351 and YIp352 provide a tool for targeted integration of sequences into the yeast genome.