The initiation of chromosome replication is the key to the control of chromosome replication and cell proliferation. We propose to work on the mechanism of the initiation of bacterial chromosomal replication at the origin using Bacillus subtilis. Particularly interesting is our finding, by using temperature-sensitive mutants, that the replication origin is associated with the cell membrane, and this membrane association is necessary for initiation. The isolation of the membrane-DNA complex from the mutants permitted us to demonstrate specific dissociation of host origin-carrying DNA and also a plasmid DNA at a high temperature (45 degrees C) from the membrane both in vivo and in vitro. We plan to extend this research in the following areas: (1) In vitro specific dissociation and association of origin-carrying host DNA and plasmids from membrane will be further studied in more defined conditions and by using other replication mutants; (2) Membrane proteins will be analyzed to identify the product proteins of the initiation mutants and to study the interaction of these proteins with the DNA fragments carrying the replication origin; (3) In vitro initiation at the replication origin will be attempted by incorporating the membrane components; (4) physiology of initiation will be studied by using plasmids carrying the chromosomal origin and those carrying heterologous plasmid origins.