The Absence of Cruciform Structures from pA03 Plasmid DNA In vivo

Abstract

We extracted pA03 plasmid DNA from E. coli cells, having “frozen” the transitions between cruciform and double-helical conformations in DNA. The characteristic feature of the DNA isolation procedure is that all steps were carried out at temperature between 0 and 4 C and no phenol deproteinization was used, since it has been discovered that phenol destabilizes cruciform structures in pA03 DNA. Two-dimensional gel electrophoresis has revealed no cruciform structures in the pA03 DNA preparations obtained this way, although the superhelical density of DNA was sufficient for them. Cruciform structures are absent from intracellular pA03 DNA at all growth stages of the bacterial culture: stationary and logarithmic, and under the induction of pA03 DNA replication in chloramphenicol-treated cells.