Towards a physical map of the Listeria chromosome: the pulsed field electrophoresis approach.

The possibility of the electrophoretic study of very large fragments of the Listeria chromosome (DNAs up to 1000 Kb) may help to the understanding of the physical organization and functional mapping of the entire genome of this organism. Several experiences were done to apply this technology to Listeria. Agarose inserts were prepared with intact cells and protoplasts, and lysis was induced in situ. Inserts with a convenient amount of DNA were cleaved in situ again by diffusing restriction enzymes into the agarose and submitted to one dimensional electric field that was periodically inverted, to cause changes of direction of the DNA fragments. We obtained a good band resolution of bands with Eco R1 and HindIII. Experiments are in progress to select other restriction enzymes leading to larger fragments. This technique must be combined with the use of blotting with known DNA sequences, such as the corresponding to the haemolysin, to arrive to a comprehensive map of the Listeria chromosome. Cloning of Listeria genes involved in several biochemical characteristics of Listeria, or at least a good collection of mutants will be quite necessary for the progress of such approach.