In E. coli, the intracellular levels of cAMP differ depending on the carbon source used in the culture media. It is known that in the presence of glucose both cAMP and CRP concentrations decrease; however, the mechanism(s) by which glucose lowers cAMP and CRP levels are not fully understood. Furthermore, it is yet to be determined how the concentrations of cAMP are regulated in vivo. Thus, the long range objective of this project is to elucidate the molecular basis (control) of adenylate cyclase gene (cya) expression and cAMP production in enteric bacteria.
The specific aims are: (i) to characterize cya and crp mutants, (ii) to continue analysis of clones and subclones of the cya gene, (iii) to develop in vitro and in vivo assay systems to measure the effects of small metabolites upon the expression of the adenylate cyclase structural gene and cAMP production, (iv) to determine the entire nucleotide sequence of the cya promoter and structural gene, (v) to localize and determine binding domains within the cya promoter region, (vi) to conduct site-directed mutagenesis of the cya promoter/structural gene, (vii) to assess the effects of site-specific mutations upon the expression of the cya gene and cAMP accumulation and (viii) to elucidate the molecular mechanism(s) by which cAMP levels are regulated in E. coli. Overall, the full understanding of the molecular basis of the cya gene expression and cAMP should provide additional information on cellular regulation in organisms capable of involvement in human diseases. The following methodologies will be employed in the proposed study: recombinant DNA technology, in vitro transcription-translation protocols, S1 nuclease mapping, DNA footprinting, nick-translation blotting techniques, nucleotide sequencing, in vitro mutagenesis, and synthesis of oligonucleotide probes and primers.
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