Selective isolation of linear plasmids from Streptomyces spp. by concentration at a phenol-buffer interface
Abstract (Summary)A method has been developed to isolate linear plasmids of the protein- capped type fiom the interface of phenol extractions. This method was refined using pSCLl of Streptomyces clavuligerus,a fully sequenced plasmid, as a mode1 linear plasrnid. Extraction of ce11 lysates under a variety of conditions demonstrated that pSCLl could be concentrated at the interface, with selective removal of proteins and non-protein-linked nucleic acids. Several reagents were tested to determine the most effective agent for releasing the holoplasmid from the ethanol precipitated interface material and leaving it in solution. Urea proved the most successful for recovery of the protein-capped plasmid. Digestion of the precipitated interface material by proteinase K in the presence of SDS released the protein-free DNA efficientiy. Most of the proteins in ceIl lysates were shown to partition into the phenol phase during extraction, explaining this reagent's well known success in preparing DNA and RNA without major losses due to enzyme digestion. This method was tested on other Streptomyces species to show that it could be applied to a broader spectrum of organisms. It was also used in attempis to isolate the previously undetected plasmid SLP3 of Streptomyces lividans . The terminal protein of pSCLl still remains unidentified. The bond between the protein and DNA appears to be somewhat labile and the DNAprotein conjugate does not survive heating, as shown by SDS-polyacrylamide gel experiments. An approach to the isolation of linear plasmid terminal proteins is proposed, based on the use of the phenol method.
Source Type:Master's Thesis
Date of Publication:01/01/1998