The replication terminus of the chromosome of Escherichia coli inhibits replication forks. The terminus region contains very few genetic loci, and it has been difficult to gain further information about the functions encoded in its approximately 250 kilobases of DNA. This situation has recently changed, however, since both a genetic map and a restriction map of this region are now available. In addition, our laboratory has recently isolated a number of strains in which large parts of the teminus region are deleted. This research plan is designed to use this newly available information to study the functions encoded in the terminus region.
The specific aims of this research project are as follows: 1) Clone a large number of DNA fragments from the terminus region. These fragments are essential for all aspects of this project. 2) Obtain strains in which varying amounts of the terminus are deleted. Relationships between particular functions and regions can then be determined. 3) Study the sites at which replication forks are inhibited. This will be done in normal cells, in plasmids containing the relevant fragments, and in strains in which the primary sites of inhibition have been removed. 4) Determine in which deletion strains the partitioning of daughter chromosomes is affected. This region will then be studied further in normal cells, and in plasmids containing this region. In particular, the association of this region with the cell envelope will be studied. 5) Determine if replication of the terminus is important in controlling cell division. Deletion strains will be used to detect the absence of control. 6) Use plasmids containing the cloned fragments to re-insert fragments into deletion strains. This will allow us to determine if the fragments functio in a cis or trans fashion, and if the insertion site is important for expression. 7) Determine the proteins encoded in the cloned DNA's. These experiments will provide fundamental information about the control of bacterial growth and division, which can be used to combat bacterial diseases.
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