ABILITY OF HIGH HYDROSTATIC-PRESSURE TREATED PLASMIDS AND CELLS OF ESCHERICHIA-COLI TO GENETICALLY TRANSFORM

The exposure of plasmid pUC18 and pBR322 DNA to high hydrostatic press ure increased the ability of plasmids to transform competent Escherich ia coli cells. For pUC18 plasmid, a pressure of 400 MPa, and for pBR32 2, a pressure of 200 MPa was found to provide the highest transformati on efficiency. The DNA duplexes of the two plasmids were found to be t he most stable for melting conditions at these pressures. At pressures higher than these, both the stability of the duplex DNA and the trans formation efficiency were affected. The stabilizing effect of high hyd rostatic pressure on the hydrogen bond may be responsible for the obse rved increase in transformation efficiency of the pressure-exposed pla smid DNA. The possibility of pressure-induced changes in the structure and conformation of DNA was studied using various techniques. In agar ose gel electrophoresis, pressure-treated plasmids (pUC18 at 400 MPa a nd pBR322 at 200 MPa) consistently showed visibly distinct higher mobi lity compared to untreated plasmids. Pressure-treated pUC18 as well as pBR322 DNA showed significant reduction in ethidium bromide binding a s is evident from the reduced intensity of fluorescence of the dye bou nd pressure-treated DNA. Spectroscopic studies using circular dichrois m and Fourier transform infrared (FTIR) spectroscopy also showed signi ficant differences in the absorption profiles of pressure-treated plas mids as compared to an untreated control. These studies revealed that the pressure-induced changes in the conformation of these DNAs may be responsible for the observed increase in the transformation ability of the plasmids. On the other hand, the exposure of competent cells of E . coil to a high hydrostatic pressure of 50 MPa not only reduced their colony-forming ability but also drastically reduced their ability to take up plasmid DNA.