Each cell of Salmonella typhimurium possesses a chemosensory system, which detects gradients, and a flagellum, which propels it along the gradient. The flagellum comprises a 6000 rpm rotary motor, located in the cell's inner membrane, land a propeller, which is a helically shaped filament made up of flagellin subunits. Reversal of the direction of motor rotation causes the cell to tumble and therefore to reorient, a process abetted by a change in the pitch and hand of the helical filament. The chemosensory system controls the path of the cell by modulating the frequency of motor reversal as a function of changes in concentration of chemical attractants and repellents. We are interested in seeing the three-dimensional structure of the flagellar components, especially the motor and the filament, in determining their compositions, in identifying the genes which encode for these structures, in understanding the mechanism by which these structures function, and in determining how the cells assemble such structures. We propose to (1) isolate and characterize a more complete motor complex than has heretofore been studied; (2) determine the three-dimensional structure of the motor complex to 20 Angstroms or better, using cryoelectron microscopy; (3) attempt to correlate structure and function by analyzing images of the motor complex from mutant cells; (4) determine the three-dimensional structure of the filament to 10 Angstroms resolution by cryoelectron microscopy in order to visualize the domains of the flagellin subunit; (5) compare the 10 Angstroms maps of filaments in different structural states in order to determine the mechanism by which the filament adopts and changes its shape; and (5) alter the amino acid sequence of the flagellin subunit to probe the mechanism of flagellar filament assembly. topoisomerases include the elucidation of the mechanism of excision of rDNA rings from the chromosomal cluster when the total cellular level of DNA topoisomerases is low; the idea that the supercoiling of the DNA template by transcription is the underlying cause of instability of genomic clusters of rDNA and other repetitive sequences will be tested. Genetic and biochemical approaches, including the design of sequence-specific supercoiling enzymes, will be used to test whether there are specific anchoring points that divide the chromosomes into topologically separate domains, and whether DNA topoisomerase II has a role in the organization of such loops. Experiments will also be carried out to test further the dispensability of DNA topoisomerases in the initiation of replication in eukaryotes.
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