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 ampR 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.

中文翻译：

---

基于六个碱基对的同源序列在酵母中进行重组：两组异构模型表达质粒中的结构不稳定。

多拷贝游离型酵母（Saccharomyces cerevisiae）质粒尽管已知结构和分离稳定性有限，但却经常用于研究和工业生产。使用一组六个yEGFP3模型表达质粒（大小相同，但功能序列排列不同，通过六个碱基对SacI序列连接），我们使用从酵母转化子中提取的总DNA反向转化到大肠杆菌中来检测潜在的质粒重排。这种方法基于SacI序列之间的同源重组，揭示了我们质粒中功能序列的缺失，易位，重复和翻转。为了扩展我们的发现，我们以相同的方式组装并分析了表达抗菌昆虫肽防御素A的六个同工型的相应质粒集。在833个单独的ampR克隆（两组）中，我们追踪了28例（3.4％）具有精确结构变化的病例。但是，pIFC4.13X系列中一种同工型的频率pIFC4.131的频率特别高，为18.5％（81个克隆中的15个），这表明该质粒的结构不利于宿主。随着灵敏度的提高，聚合酶链反应（PCR）方法揭示了每组至少一半同工型的进一步结构变化。该变化被认为是随机涉及SacI序列的同源重组事件的结果。质粒改变的频率是似乎有利或不利于某些结构的选择和反选择的产物。尽管没有唯一的建筑布置特别稳定，