DNA INSERTIONS IN THE SILENT REGIONS OF THE 2-MU-M PLASMID OF SACCHAROMYCES-CEREVISIAE INFLUENCE PLASMID STABILITY

The 2-mu-m plasmid of the yeast Saccharomyces cerevisiae is in principle a suitable vector for expression of foreign genes, due to its high copy number and extreme stability. However, the cloning of genes into 2-mu-m often results in a reduced copy number and/or reduced stability. One reason for this observed instability could be that the inserts in general were made in one of the several open reading frames (ORFs) of the plasmid. Therefore we studied the effect on stability of insertions in the silent regions of 2-mu-m without interrupting any known essential regions or ORFs. Using the SnaBI site, a yeast-integrating plasmid (Yip5) was introduced into the region between the ARS and STB locus in two possible orientations. The resulting plasmids could be stably maintained in the cells without the need for complementation by the wild-type 2-mu-m plasmid. However, the stability of these plasmids in a cir-degrees host was still one to two orders of magnitude lower (0.2% and 0.8% respectively) as reported for the wild-type 2-mu-m (0.01%). Removal of 2 kb of the bacterial sequences from Yip5 did not increase stability. The stability was dependent on the orientation of the insert. We found that in the less stable orientation, transcription originating from the insert was running into the STB region. DNA inserted in the XmaIII site located outside the ORFs in the REP2/FLP intergenic region influenced both stability and copy number of the plasmid. These effects are strongly dependent on the size of the insert. Insertion of a 2 kb DNA fragment increased the copy number, probably through an effect on FLP expression.