AbstractA series of circular shuttle vectors were constructed that could replicate and transcribe in the cells of both Escherichia coli and Saccharomyces cerevisiae. 2‐D agarose gel electrophoresis run without or in the presence of different concentrations of chloroquine (CHL) revealed that bacterial plasmids were more negatively (−) supercoiled than minichromosomes isolated from budding yeast. Attempts to increase (−) supercoiling in S. cerevisiae or to reduce it in E. coli have deleterious biological consequences. These observations indicate that DNA supercoiling can vary in different species but cells are exquisitely sensitive to sudden changes in supercoiling. In E. coli, the observation that cell growth as well as ColE1 plasmid copy number decrease when DNA relaxes suggests that supercoiling could affect cell viability by regulating the initiation of both transcription and replication.