Recombination in yeast based on six base pairs of homologous sequences: Structural instability in two sets of...

Multicopy episomal yeast (Saccharomyces cerevisiae) plasmids are frequently employed in research and industrial production despite their known limited structural and segregational stability. Employing a set of six yEGFP3 model expression plasmids (identical in size but differing in the arrangement of their functional sequences, joined via six base pair SacI sequences), we used back transformation of total DNA extracted from yeast transformants into Escherichia coli to detect potential plasmid rearrangements. This approach revealed deletions, translocations, duplications, and flippings of functional sequences in our plasmids based on homologous recombination between the SacI sequences. To extend our findings, we assembled and analysed in the same way a corresponding plasmid set of six isoforms expressing the antibacterial insect peptide defensin A. In 833 individual amp^R clones (both sets combined), we traced 28 cases (3.4%) with precise structural changes. However, the frequency in one isoform in the pIFC4.13X series, pIFC4.131, was particularly high with 18.5% (15 out of 81 clones), indicating that the architecture of this plasmid is unfavourable to the host. With an increased sensitivity, a Polymerase Chain Reaction (PCR) approach revealed further structural changes in at least half of the isoforms of each set. The changes are considered the consequence of homologous recombination events involving the SacI sequences in a random fashion. The frequency of plasmid alterations is the product of selection and counterselection seemingly favouring or disfavouring certain structures. Although no sole architectural arrangement stuck out as being particularly stable, we were able to determine with our approach unfavourable sequence associations that should be avoided.