Despite the importance of one-carbon units in cell physiology, our understanding of the regulation of their biosynthesis and utilization is limited. The long term objective of this research project is to define the molecular mechanisms for regulation of the genes involved in their production and utilization in both E. coli and S. typhimurium. We have taken a genetic and a biochemical approach to study one-carbon metabolism. New selection procedures have been developed to facilitate the isolation and characterization of regulatory mutations. In addition, the lac operon of E. coli has been fused to the glyA and serB promoters of E. coli and the glyA, metE, metB and metJ promoters of S. typhimurium. Fusions of the S. typhimurium metH and metF promoters and the E. coli gcv promoter(s) to the lac operon will be constructed. Mutations that alter expression of the lac genes will be isolated and characterized in an effort to identify gene products (e.g., activator or repressor proteins) and DNA sequence elements (e.g., promoters, operators or activator binding sites) that regulate expression of the genes involved in one-carbon metabolism. Genetic techniques (transduction, partial diploid analysis, etc.) will be used to locate the sites of the mutations on the E. coli and S. typhimurium linkage maps and to determine the effects of the mutations in trans. Several genes involved in one-carbon metabolism have been cloned (glyA, gcv, serB, metJ, metB, metE, metH and metF), and a number of biochemical procedures have been utilized to analyze these genes at the molecular level (DNA sequencing, DNAase I footprinting, S1 nuclease mapping, etc.). In addition, a cell-free transcription-translation system will be used to examine regulation in vitro, using plasmid DNA carrying the lac fusions as templates. Ultimately, this system will provide an assay for cellular components directly involved in the regulation of these genes. Together, the genetic and biochemical analyses will allow us to determine the nature of the controls acting on genes directly involved in one-carbon production and utilization, and the importance of these pathways in cell physiology.
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