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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK036913-06
Application #
3235508
Study Section
Biochemistry Study Section (BIO)
Project Start
1986-04-01
Project End
1994-11-30
Budget Start
1991-12-01
Budget End
1992-11-30
Support Year
6
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Texas Austin
Department
Type
Schools of Arts and Sciences
DUNS #
City
Austin
State
TX
Country
United States
Zip Code
78712
Howard, Katherine M; Muga, Stephanie J; Zhang, Liwen et al. (2003) Characterization of the rat cytoplasmic C1-tetrahydrofolate synthase gene and analysis of its expression in liver regeneration and fetal development. Gene 319:85-97
West, M G; Barlowe, C K; Appling, D R (1993) Cloning and characterization of the Saccharomyces cerevisiae gene encoding NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase. J Biol Chem 268:153-60
Garcia-Martinez, L F; Appling, D R (1993) Characterization of the folate-dependent mitochondrial oxidation of carbon 3 of serine. Biochemistry 32:4671-6
Pasternack, L B; Laude Jr, D A; Appling, D R (1992) 13C NMR detection of folate-mediated serine and glycine synthesis in vivo in Saccharomyces cerevisiae. Biochemistry 31:8713-9
Appling, D R (1991) Compartmentation of folate-mediated one-carbon metabolism in eukaryotes. FASEB J 5:2645-51
Appling, D R; Ayo, S H; Kreisberg, J I (1990) Immunolocalization of C1-tetrahydrofolate synthase in the rat kidney. Biochem Biophys Res Commun 168:625-30
Barlowe, C K; Appling, D R (1990) Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme. Mol Cell Biol 10:5679-87
Thigpen, A E; West, M G; Appling, D R (1990) Rat C1-tetrahydrofolate synthase. cDNA isolation, tissue-specific levels of the mRNA, and expression of the protein in yeast. J Biol Chem 265:7907-13
Barlowe, C K; Appling, D R (1990) Isolation and characterization of a novel eukaryotic monofunctional NAD(+)-dependent 5,10-methylenetetrahydrofolate dehydrogenase. Biochemistry 29:7089-94
Barlowe, C K; Williams, M E; Rabinowitz, J C et al. (1989) Site-directed mutagenesis of yeast C1-tetrahydrofolate synthase: analysis of an overlapping active site in a multifunctional enzyme. Biochemistry 28:2099-106

Showing the most recent 10 out of 13 publications