The broad, long-term objectives of this proposal are to mount a second generation' attack on eukaryotic one-carbon metabolism in order to more fully understand its role and response in normal and pathological states.
The specific aims are: (1) to isolate clones for the rat gene encoding C1- THF synthase; (2) to use an in vitro transcription system to analyze cis- and trans-acting elements responsible for basal and tissue-specific expression 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 centrality of this pathway is reflected by the large number of human disease states which involve and perturb one-carbon metabolism, such as cancer, gout, and Lesch- Nyhan syndrome. The experimental design and the 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 using an in vitro transcription system to define promoter and other regulatory sequences involved in basal and tissue-specific expression; (3) identification of transacting transcription factors important in basal and tissue-specific expression of the rat C1-THF synthase gene 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.
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