Mammalian Fibroblasts Lacking Mitochondrial NAD+-dependent Methylenetetrahydrofolate Dehydrogenase-Cyclohydrolase Are Glycine Auxotrophs

Primary fibroblasts established from embryos of NAD-dependent mitochondrial methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) knockout mice were spontaneously immortalized or transformed with SV40 Large T antigen. Mitotracker Red CMXRos staining of the cells indicates the presence of intact mitochondria with a membrane potential. The nmdmc(-/-) cells are auxotrophic for glycine, demonstrating that NMDMC is the only methylenetetrahydrofolate dehydrogenase normally expressed in the mitochondria of these cell lines. Growth of null mutant but not wild type cells on complete medium with dialyzed serum is stimulated about 2-fold by added formate or hypoxanthine. Radiolabeling experiments demonstrated a 3–10 × enhanced incorporation of radioactivity into DNA from formate relative to serine by nmdmc(-/-) cells. The generation of one-carbon units by mitochondria in nmdmc(-/-) cells is completely blocked, and the cytoplasmic folate pathways alone are insufficient for optimal purine synthesis. The results demonstrate a metabolic role for NMDMC in supporting purine biosynthesis. Despite the recognition of these metabolic defects in the mutant cell lines, the phenotype of nmdmc(-/-) embryos that begin to die at E13.5 is not improved when pregnant dams are given a glycine-rich diet or daily injections of sodium formate. Primary fibroblasts established from embryos of NAD-dependent mitochondrial methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) knockout mice were spontaneously immortalized or transformed with SV40 Large T antigen. Mitotracker Red CMXRos staining of the cells indicates the presence of intact mitochondria with a membrane potential. The nmdmc(-/-) cells are auxotrophic for glycine, demonstrating that NMDMC is the only methylenetetrahydrofolate dehydrogenase normally expressed in the mitochondria of these cell lines. Growth of null mutant but not wild type cells on complete medium with dialyzed serum is stimulated about 2-fold by added formate or hypoxanthine. Radiolabeling experiments demonstrated a 3–10 × enhanced incorporation of radioactivity into DNA from formate relative to serine by nmdmc(-/-) cells. The generation of one-carbon units by mitochondria in nmdmc(-/-) cells is completely blocked, and the cytoplasmic folate pathways alone are insufficient for optimal purine synthesis. The results demonstrate a metabolic role for NMDMC in supporting purine biosynthesis. Despite the recognition of these metabolic defects in the mutant cell lines, the phenotype of nmdmc(-/-) embryos that begin to die at E13.5 is not improved when pregnant dams are given a glycine-rich diet or daily injections of sodium formate. Folate-dependent enzymes are found in the mitochondria as well as the cytoplasm of eukaryotic cells. Isozymes of certain folate-dependent enzymes are present in both compartments, and a number of observations demonstrated that the folate-dependent pathways in mitochondria contribute to total cellular folate metabolism. For example, serine hydroxymethyltransferase, encoded by two different nuclear genes, is located in each compartment where it catalyzes the interconversion of serine and tetrahydrofolate (THF) 1The abbreviations used are: THF, tetrahydrofolate; DC, methylenetetrahydrofolate dehydrogenase-cyclohydrolase; DCS, methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase; with glycine and methylenetetrahydrofolate. A Chinese hamster ovary cell line that is missing mitochondrial serine hydroxymethyltransferase (glyA) was shown to be a glycine auxotroph despite the fact that it retains the cytoplasmic isoform of the enzyme (1Chasin L.A. Feldman A. Konstam M. Urlaub G. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 718-722Crossref PubMed Scopus (73) Google Scholar). A similar phenotype was seen in the auxB1 mutant cell line that lacks the ability to make folate polyglutamates. Replacing the missing folylpoly-γ-glutamate synthetase in the cytoplasm of this cell line reverses the requirement for thymidine and purines, but the enzyme must be targeted to mitochondria to overcome its requirement for glycine (2Garrow T.A. Admon A. Shane B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9151-9155Crossref PubMed Scopus (83) Google Scholar, 3Lin B.F. Huang R.-F.S. Shane B. J. Biol. Chem. 1993; 268: 21674-21679Abstract Full Text PDF PubMed Google Scholar, 4Lin B.F. Shane B. J. Biol. Chem. 1994; 269: 9705-9713Abstract Full Text PDF PubMed Google Scholar, 5Chen L Qi H. Korenberg J. Garrow T.A. Choi Y.J. Shane B. J. Biol. Chem. 1996; 271: 13077-13087Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Methylenetetrahydrofolate dehydrogenase-cyclohydrolase activities are located in both cellular compartments. In yeast, both cytoplasmic and mitochondrial isoenzymes occur as trifunctional NADP-dependent dehydrogenase-cyclohydrolase-synthetase (DCS) proteins (6Shannon K.W. Rabinowitz J.C. J. Biol. Chem. 1986; 261: 12266-12271Abstract Full Text PDF PubMed Google Scholar). The D and C activities interconvert methyleneTHF and 10-formylTHF, and the S converts formate to formylTHF in an ATP-dependent reaction. Dean Appling's group (7Pasternack L.B. Laude D.A. Appling D.R. Biochemistry. 1994; 33: 74-82Crossref PubMed Scopus (50) Google Scholar, 8West M.G. Horne D.W. Appling D.R. Biochemistry. 1996; 35: 3122-3132Crossref PubMed Scopus (49) Google Scholar, 9Kastanos E.K. Woldman Y.Y. Appling D.R. Biochemistry. 1997; 36: 14956-14964Crossref PubMed Scopus (85) Google Scholar) has proposed a rational model wherein mitochondria use the synthetase activity “in reverse” to produce formate, ATP, and THF from formylTHF. The formate is then recaptured as an important source of active one-carbon units by the activities of the cytoplasmic DCS (7Pasternack L.B. Laude D.A. Appling D.R. Biochemistry. 1994; 33: 74-82Crossref PubMed Scopus (50) Google Scholar, 8West M.G. Horne D.W. Appling D.R. Biochemistry. 1996; 35: 3122-3132Crossref PubMed Scopus (49) Google Scholar, 9Kastanos E.K. Woldman Y.Y. Appling D.R. Biochemistry. 1997; 36: 14956-14964Crossref PubMed Scopus (85) Google Scholar, 10Piper M.D. Hong S.P. Ball G.E. Dawes I.W. J. Biol. Chem. 2000; 275: 30987-30995Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In mammals, the cytoplasmic DCS is ubiquitously expressed in all cells and tissues(11Mejia N.R. MacKenzie R.E. J. Biol. Chem. 1985; 260: 14616-14620Abstract Full Text PDF PubMed Google Scholar, 12Smith G.K. Banks S.D. Monaco T.J. Rigual R. Duch D.S. Mullin R.J. Huber B.E. Arch. Biochem. Biophys. 1990; 283: 367-371Crossref PubMed Scopus (20) Google Scholar, 13Peri K.G. MacKenzie R.E. FEBS Lett. 1991; 294: 113-115Crossref PubMed Scopus (14) Google Scholar, 14Thigpen A.E. West M.G. Appling D.R. J. Biol. Chem. 1990; 265: 7907-7913Abstract Full Text PDF PubMed Google Scholar),whereas a mitochondrial version(15Mejia N.R. MacKenzie R.E. Biochem. Biophys. Res. Comm. 1988; 155: 1-6Crossref PubMed Scopus (44) Google Scholar), NAD+-dependent DC (NMDMC), is expressed during embryogenesis and is detectable in all immortalized cells (11Mejia N.R. MacKenzie R.E. J. Biol. Chem. 1985; 260: 14616-14620Abstract Full Text PDF PubMed Google Scholar, 13Peri K.G. MacKenzie R.E. FEBS Lett. 1991; 294: 113-115Crossref PubMed Scopus (14) Google Scholar, 15Mejia N.R. MacKenzie R.E. Biochem. Biophys. Res. Comm. 1988; 155: 1-6Crossref PubMed Scopus (44) Google Scholar). This bifunctional enzyme was shown to have Mg2+ and inorganic phosphate-dependent methyleneTHF dehydrogenase activity (11Mejia N.R. MacKenzie R.E. J. Biol. Chem. 1985; 260: 14616-14620Abstract Full Text PDF PubMed Google Scholar, 16Mejia N.R. Rios-Orlandi E.M. MacKenzie R.E. J. Biol. Chem. 1986; 261: 9509-9513Abstract Full Text PDF PubMed Google Scholar). Based on its kinetic properties, NMDMC was proposed to have derived from an NADP+-dependent precursor (17Yang X-M. MacKenzie R.E. Biochemistry. 1993; 32: 11118-11123Crossref PubMed Scopus (37) Google Scholar, 18Pawelek P.D. MacKenzie R.E. Biochim. Biophys. Acta. 1996; 1296: 47-54Crossref PubMed Scopus (7) Google Scholar). More recent evidence comparing the nucleotide sequence of the synthetase domain of the cytoplasmic DCS with the sequence of the 3′-untranslated region of the NMDMC cDNA supported the conclusion that its putative precursor was in fact an NADP+-dependent DCS (19Patel H. Christensen K.E. Mejia N.R. MacKenzie R.E. Arch. Biochem. Biophys. 2002; 403: 145-148Crossref PubMed Scopus (18) Google Scholar). The metabolic advantage for the mitochondrial NMDMC enzyme to lose its synthetase activity is not obvious. This feature raises the question as to whether mammalian cells expressing the NMDMC enzyme export formate from their mitochondria to the cytoplasm, and if so, what is the mechanism for the generation of formate? Although the metabolic role of the NMDMC is not well understood, deletion of the gene in mice has been shown to be embryonic lethal beyond E13.5 (20Di Pietro E. Sirois J. Tremblay M.L. MacKenzie R.E. Mol. Cell. Biol. 2002; 22: 4158-4166Crossref PubMed Scopus (85) Google Scholar). At E12.5, the embryos appear smaller and pale when compared with their wild type littermates. They also demonstrate a failure of the liver to develop and to take over hematopoiesis from the yolk sac, despite the presence of hematopoietic precursor cells (20Di Pietro E. Sirois J. Tremblay M.L. MacKenzie R.E. Mol. Cell. Biol. 2002; 22: 4158-4166Crossref PubMed Scopus (85) Google Scholar). It was suggested earlier that the role of the enzyme was to provide formylTHF for the production of formylmethionyl-tRNA used in mitochondrial protein synthesis or to produce one-carbon units for purine synthesis (15Mejia N.R. MacKenzie R.E. Biochem. Biophys. Res. Comm. 1988; 155: 1-6Crossref PubMed Scopus (44) Google Scholar). Gene deletion studies indicated that the role of formylTHF in protein synthesis is not essential in yeast (21Li Y. Holmes W.B. Appling D.R. RajBhandary U.L. J. Bacteriol. 2000; 182: 2886-2892Crossref PubMed Scopus (60) Google Scholar), and examination of an NMDMC null mouse cell line indicated that mitochondrial protein synthesis is also not impaired when compared with a wild type control (20Di Pietro E. Sirois J. Tremblay M.L. MacKenzie R.E. Mol. Cell. Biol. 2002; 22: 4158-4166Crossref PubMed Scopus (85) Google Scholar). In this report, we establish and characterize immortalized cell lines and examine their nutritional requirements to further characterize the metabolic consequences caused by inactivation of the nmdmc gene. Embryonic Fibroblast Cell Lines—The targeted inactivation of NMDMC in ES cells and the generation of NMDMC knockout mice were described previously (20Di Pietro E. Sirois J. Tremblay M.L. MacKenzie R.E. Mol. Cell. Biol. 2002; 22: 4158-4166Crossref PubMed Scopus (85) Google Scholar). Heterozygous mice were generated from two independently targeted ES cells lines. To obtain primary embryonic fibroblast cell lines, E9.5 and E11.5 embryos were isolated from matings of heterozygous mice. Embryos were minced and trypsinized for 10–30 min. at 37 °C, and single cell suspensions were resuspended in Dulbecco's modified Eagle's medium containing 10–15% fetal bovine serum, supplemented with 1× non-essential amino acids, 1× glutamine, 1× penicillin/streptomycin (all from Invitrogen), as well as 50 μg/ml uridine (Sigma). This complete medium was supplemented with 100 μm sodium formate obtained from Sigma in all studies where formate was not a variable. Spontaneously immortalized cells (SF) were obtained by continuous passage in culture. Transformed cells (IF) were derived by infection of primary cultures with a recombinant retrovirus expressing the SV40 large T antigen for 2 h at 37 °C in serum-free medium (20Di Pietro E. Sirois J. Tremblay M.L. MacKenzie R.E. Mol. Cell. Biol. 2002; 22: 4158-4166Crossref PubMed Scopus (85) Google Scholar). Following infection, medium containing serum was added to the cells to obtain a final concentration of 15% fetal bovine serum. DNA was isolated from embryonic fibroblast cell lines using the Qiagen genomic DNA purification kit. Genotypes of embryos and cell lines were determined by as described previously (20Di Pietro E. Sirois J. Tremblay M.L. MacKenzie R.E. Mol. Cell. Biol. 2002; 22: 4158-4166Crossref PubMed Scopus (85) Google Scholar). The presence of the sequence SV40 large T antigen in transformed cells was by DNA from fibroblasts was used with and to a of the SV40 large T antigen were as each of and μm 1× Qiagen 2 and units of Qiagen in a final of 50 with at °C for at °C for °C for and °C for and at °C for min. were by Cell cell and the cell line from it was derived were from The cells were in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, 1× 1× glutamine, and 50 μg/ml with Mitotracker containing Mitotracker Red CMXRos in a of was added to cells on and at 37 °C for min. the medium containing the Mitotracker was with and the cells were by Cell Growth cell lines used in these experiments were in medium that Dulbecco's modified Eagle's medium that glycine, and were added that or of these amino be to the nutritional requirements of cell lines. medium also 1× glutamine, 1× 50 μg/ml 100 μm sodium formate, and 1× sodium and from was when of the dialyzed fetal bovine serum was further dialyzed 2 of for h with a of during the The cells were to the fetal bovine serum in a were at a of × in and h the medium was with medium glycine, or formate. The cells were by the at into were at a of × in and for h in medium but with glycine, or formate at of the The medium was with the of of medium with 2 × of or a control for DNA the cells were with In all experiments are compared where the in of thymidine was to of cell The cells were to for h in the presence of each precursor and and were with of They were in the of a 100 50 and μg/ml for h with at 37 The were to and with of The were with of by at °C for min. The DNA were resuspended in 100 of The DNA was to a membrane using a and with of was from the membrane with a and into a containing of This was in cell line and the for the of radioactivity into determined by were into were at a of × in and DNA isolated as described for the precursor incorporation into The cells were in the presence of 2 of medium containing × of the or The DNA were resuspended in of and to containing of of was added to each and the DNA was to at °C for min. The DNA were into their by using a of of the of DNA was in each to as The were in a and and each the was with a The were and in a containing of The of radioactivity into each was determined by This was in cell line and the were of with diet was that added glycine to the diet that glycine and heterozygous mice were on the diet the at was pregnant were Embryos were isolated from were and yolk were for a embryos from matings of heterozygous mice on the diet were also Genotypes of embryos were determined as described previously (20Di Pietro E. Sirois J. Tremblay M.L. MacKenzie R.E. Mol. Cell. Biol. 2002; 22: 4158-4166Crossref PubMed Scopus (85) Google Scholar). of with dams on the mouse diet were daily with 100 of of sodium formate in 100 of from with the to of from were embryos were were and yolk were for as described previously (20Di Pietro E. Sirois J. Tremblay M.L. MacKenzie R.E. Mol. Cell. Biol. 2002; 22: 4158-4166Crossref PubMed Scopus (85) Google Scholar). of NMDMC Cell Lines—The of the generation of NMDMC knockout mice have been described previously (20Di Pietro E. Sirois J. Tremblay M.L. MacKenzie R.E. Mol. Cell. Biol. 2002; 22: 4158-4166Crossref PubMed Scopus (85) Google Scholar). Primary embryonic fibroblasts derived from E9.5 and E11.5 embryos isolated from the matings of mice were spontaneously immortalized or transformed with the SV40 Large T and were determined by using the as described previously (20Di Pietro E. Sirois J. Tremblay M.L. MacKenzie R.E. Mol. Cell. Biol. 2002; 22: 4158-4166Crossref PubMed Scopus (85) Google Scholar). shown in the to a single DNA in of DNA from wild type embryonic fibroblasts and to an in from null mutant embryonic the of NMDMC in null mutant cells. mutant fibroblasts in earlier experiments were found to detectable NMDMC enzyme activity (20Di Pietro E. Sirois J. Tremblay M.L. MacKenzie R.E. Mol. Cell. Biol. 2002; 22: 4158-4166Crossref PubMed Scopus (85) Google Scholar). of previously that mitochondria of nmdmc(-/-) fibroblasts are intact by staining with the Mitotracker is by mitochondria of a membrane potential. a further of mitochondrial we fibroblasts with Mitotracker Red a mitochondrial membrane for are in the staining wild type and null mutant fibroblasts at the of used of the of the NMDMC enzyme in mitochondria and the that the nmdmc(-/-) cells a mitochondrial we to have a impaired cell line as a for nutritional studies serine and used cells that mitochondrial DNA G. PubMed Scopus Google Scholar) and produce the proteins of the but a mitochondrial membrane J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The mitochondria of these cells with Mitotracker Red CMXRos A. D.A. J. Cell Sci. Google Scholar), we also in this for NMDMC of experiments were on the NMDMC null mutant and wild type immortalized fibroblasts to their requirement for one-carbon and of folate-dependent metabolism. for in dialyzed serum and in that cell was by the of by This is not and immortalized mammalian cells a or ability to with in the medium In PubMed Scopus (60) Google Scholar, B. Res. 2000; Google Scholar). The nmdmc(-/-) cells are to well in the of serine but are glycine A and Growth in medium was not stimulated by the of the requirement for glycine used in synthesis nmdmc(-/-) cell lines obtained from mice derived from two independently targeted ES cells glycine for not be with added or formate in complete medium containing serum, formate and the of nmdmc(-/-) but not wild type is essential C and of thymidine on cell of the of established fibroblast cell cell incorporation of from formate relative to serine into total DNA of cell incorporation of from formate relative to serine into total DNA of cell lines in a whether cells that have mitochondrial are also glycine and the cell This cell as well as its cell are as and the of with not the of despite the fact that cells mitochondrial DNA and well on complete and into the DNA of a to the folate of these we a of experiments that the incorporation of glycine, and formate into the total DNA of nmdmc(-/-) using wild type cells for incorporation was in and was in null and wild type cells. The incorporation of and into DNA was for both wild type and null mutant cells A is seen in the incorporation of when compared with into total DNA of wild type and mutant cell lines. the used in these is from serine from formate in the wild type and the mutant cell line of from formate from serine This shown as a of radioactivity in is with all the wild type and mutant cell lines shown in the incorporation of from serine into and in a similar is for the wild type cell for the the incorporation of formate is in the wild type in the mutant cells The of cells the not this that its folate-dependent pathways are not of into the of DNA Following a of cells with the one-carbon serine and formate, radioactivity was in and of isolated The are expressed in and an of type and in a of with and nmdmc(-/-) fibroblasts are glycine we to the of null mutant embryos by the mouse diet of pregnant dams with that of a total of glycine not the of or the of embryos at or at we embryos from pregnant that the diet that with glycine not the phenotype of null of the pregnant dams with sodium formate in the embryonic to liver also not the phenotype of null at E13.5 and of on embryonic phenotype Embryos were isolated from dams a a glycine or a diet with daily injections of sodium of of in the number of in the number of embryos in a The of NMDMC suggested a role in embryogenesis (11Mejia N.R. MacKenzie R.E. J. Biol. Chem. 1985; 260: 14616-14620Abstract Full Text PDF PubMed Google Scholar, 12Smith G.K. Banks S.D. Monaco T.J. Rigual R. Duch D.S. Mullin R.J. Huber B.E. Arch. Biochem. Biophys. 1990; 283: 367-371Crossref PubMed Scopus (20) Google Scholar) that was by recent knockout of the gene demonstrating that at E13.5 to an of the liver to develop and take over hematopoiesis from the yolk (20Di Pietro E. Sirois J. Tremblay M.L. MacKenzie R.E. Mol. Cell. Biol. 2002; 22: 4158-4166Crossref PubMed Scopus (85) Google Scholar). were to demonstrate in the mitochondrial of nmdmc(-/-) cells and in mitochondrial protein synthesis (20Di Pietro E. Sirois J. Tremblay M.L. MacKenzie R.E. Mol. Cell. Biol. 2002; 22: 4158-4166Crossref PubMed Scopus (85) Google Scholar), and this is by the in this that the mitochondria a membrane potential. 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Although the cell lines used in this and into that cell line glycine this be in that the in the nmdmc(-/-) cell lines provide derived one-carbon are on the cytoplasmic folate wild type of the nmdmc(-/-) cell lines is stimulated by added formate that be by with but not by results demonstrate that the nmdmc(-/-) cells provide one-carbon units cytoplasmic pathways to for optimal purine synthesis. This conclusion is also supported by studies with that a of formate over serine incorporation by the nmdmc(-/-) cells relative to the wild type cells. the mitochondria of nmdmc(-/-) cells produce formate, then the formate added to the medium is into The glycine that the immortalized or transformed cells not a mitochondrial DC or DCS a enzyme were expressed to THF, we not the glycine of evidence have on the of the NMDMC kinetic demonstrated a ability to use and suggested that NMDMC from an NADP+-dependent DC by using inorganic and Mg2+ to the (17Yang X-M. MacKenzie R.E. Biochemistry. 1993; 32: 11118-11123Crossref PubMed Scopus (37) Google Scholar, 18Pawelek P.D. MacKenzie R.E. Biochim. Biophys. Acta. 1996; 1296: 47-54Crossref PubMed Scopus (7) Google Scholar). recent that the 3′-untranslated region of the NMDMC cDNA 100 each with to the synthetase domain in both the mouse and DCS enzymes and two in the (19Patel H. Christensen K.E. Mejia N.R. MacKenzie R.E. Arch. Biochem. Biophys. 2002; 403: 145-148Crossref PubMed Scopus (18) Google Scholar) the for from a trifunctional precursor of the S two NADP+-dependent trifunctional DCS located in mitochondria and the in the cytoplasm (6Shannon K.W. Rabinowitz J.C. J. Biol. Chem. 1986; 261: 12266-12271Abstract Full Text PDF PubMed Google Scholar). It that the NMDMC protein from a mammalian mitochondrial DCS a of requirement and the of the synthetase The of from to has been to the methyleneTHF and formylTHF in mitochondria the MacKenzie R.E. Biochemistry. PubMed Scopus Google Scholar) and the production of formate from NMDMC is the only DC expressed in the mitochondria of these cell lines, then is synthetase domain to the formate from formylTHF as is proposed in the model of the yeast (7Pasternack L.B. Laude D.A. Appling D.R. Biochemistry. 1994; 33: 74-82Crossref PubMed Scopus (50) Google Scholar, 8West M.G. Horne D.W. Appling D.R. Biochemistry. 1996; 35: 3122-3132Crossref PubMed Scopus (49) Google Scholar, 9Kastanos E.K. Woldman Y.Y. Appling D.R. Biochemistry. 1997; 36: 14956-14964Crossref PubMed Scopus (85) Google Scholar, 10Piper M.D. Hong S.P. Ball G.E. Dawes I.W. J. Biol. Chem. 2000; 275: 30987-30995Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). It is that is a mitochondrial synthetase enzyme in mammalian but it is not as to this be over a synthetase domain to the DC the synthetase in the in the presence of of is a to formate for example, the of a putative formylTHF mitochondrial protein synthesis not to a activity be the of formylTHF of the mitochondria for use in the cytoplasm the and provide formylTHF to cytoplasmic but this is to be a to metabolic activity Biochemistry. PubMed Scopus Google Scholar, Acta. PubMed Scopus Google Scholar, D.W. R.J. Arch. Biochem. Biophys. PubMed Scopus Google Scholar, Biochem. 1991; Biochem. PubMed Scopus Google Scholar). metabolic studies have used immortalized mammalian cell lines to of and all the of formate by all immortalized mammalian cell lines only this single mitochondrial DC, then the mechanism of this formate production further with the nmdmc(-/-) cell lines that a metabolic role for NMDMC is to provide one-carbon units for purine synthesis. Despite recognition of the metabolic consequences of the gene deletion at a cellular we the embryonic The ability of heterozygous dams to glycine, the of glycine in the and the failure of glycine to the phenotype of embryos make it that embryonic is to the of Although formate the of null mutant of sodium formate into pregnant also not the phenotype of embryos at the nmdmc(-/-) in mitochondrial folate the to establish hematopoiesis in the liver is not for the cell lines and and Mejia from this for

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