The 2-micron plasmid as a nonselectable, stable, high copy number yeast vector.
The endogenous 2-microns plasmid of Saccharomyces cerevisiae has been used extensively for the construction of yeast cloning and expression plasmids because it is a native yeast plasmid that is able to be maintained stably in cells at high copy number. Almost invariably, these plasmid constructs, containing some or all 2-microns sequences, exhibit copy number levels lower than 2-microns and are maintained stably only under selective conditions. We were interested in determining if there was a means by which 2-microns could be utilized for vector construction, without forfeiting either copy number or nonselective stability. We identified sites in the 2-microns plasmid that could be used for the insertion of genetic sequences without disrupting 2-microns coding elements and then assessed subsequent plasmid constructs for stability and copy number in vivo. We demonstrate the utility of a previously described 2-microns recombination chimera, pBH-2L, for the manipulation and transformation of 2-microns as a pure yeast plasmid vector. We show that the HpaI site near the STB element in the 2-microns plasmid can be utilized to clone yeast DNA of at least 3.9 kb with no loss of plasmid stability. Additionally, the copy number of these constructs is as high as levels reported for the endogenous 2-microns.
Ludwig
D L
DL
Microbiology Program, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Bruschi
C V
CV
D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005822:Genetic Vectors; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic