One of the major questions being pursued in developmental biology today is, """"""""How are specific genes turned on and off at the appropriate times in a developmental sequence?"""""""" The ultimate goal of the research conducted in our laboratory is to study the modes of regulation involved in differentiation in the life cycle of the dimorphic bacterium Caulobacter crescentus. Work carried out in our laboratory so far has resulted in the development of genetic techniques and the construction of a genetic map for C. crescentus. In addition, techniques for the cloning or identification of specific cloned genes have been elucidated. The work contained in this proposal will involve an analysis of the regulation of the expression of the genes involved in motility and chemotaxis. We will determine when in the cell cycle the flagellar genes are expressed and which genes must be functional for the expression of each gene. These studies will help us to understand which genes are the primary genes involved in the cell cycle dependent regulation of flagellar gene expression. Specifically we propose: 1) To determine the time in the cell cycle when cloned fla genes are expressed. 2) To examine the expression of cloned flagellar genes in strains containing various fla mutations. 3) To examine the expression of the flaF and flaG genes in an in vitro expression system. 4) To analyze the phenomenon of the excretion of unassembled flagellin monomers by flaE and flaH mutants. 5) To establish a correlation between the genetic and physical maps of C. crescentus using pulsed current electrophoresis and a collection of Tn5 insertion mutations. 6) To identify additional fla genes using pulsed-current electrophoresis to determine the map locations of previously unmapped fla mutations. 7) To characterize che mutants unable to respond to particular attractants.
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