My immediate goals are: (1) to isolate clones for the rat gene encoding C1-THF synthase; (2) to use an in vitro transcription system to analyze regulatory elements of the C1-THF synthase gene in rat; and (3) to determine the extent and the control points of compartmentation of folate coenzymes and their activated one-carbon units between the cytoplasm and mitochondria. The health-relatedness of this project stems from the ubiquitous nature of folate-mediated one-carbon metabolism in all cells, and the critical role played by C1-THF synthase in folate interconversions. The experimental design and methods used to accomplish the specific aims involve (1) the use of rat C1-THF synthase cDNA clones as probes for the isolation of the rat gene encoding C1-THF synthase; (2) characterization of the C1-THF synthase gene by sequence analysis, and deletion analysis of the 5' flanking region to define promoter and other regulatory sequences; (3) identification of trans-acting transcription factors using a combination of gel mobility shift, footprinting and in vitro transcription assays; and (4) analysis of the intercompartmental pathway of folate-mediated one-carbon metabolism in yeast using a combination of biochemical, molecular genetic, and NMR techniques.
