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• W2039103044 abstract "C1-tetrahydrofolate (THF) synthase is a trifunctional enzyme found in eukaryotes that contains the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase. The cytoplasmic isozyme of C1-THF synthase is well characterized in a number of mammals, including humans; but a mitochondrial isozyme has been previously identified only in the yeast Saccharomyces. Here, we report the identification and characterization of the human gene encoding a functional mitochondrial C1-THF synthase. The gene spans 236 kilobase pairs on chromosome 6 and consists of 28 exons plus one alternative exon. The gene encodes a protein of 978 amino acids, including an N-terminal mitochondrial targeting sequence. The mitochondrial isozyme is 61% identical to the human cytoplasmic isozyme. Expression of the gene was detected in most human tissues, but transcripts were highest in placenta, thymus, and brain. Two mRNAs were detected, a 3.6-kb transcript and a 1.1-kb transcript, and both transcripts were observed in varying ratios in each tissue. The shorter transcript results from an alternative splicing event, where exon 7 is spliced to exon 8a instead of exon 8. Exon 8a is derived from an exonized Alu sequence, sharing no homology with exon 8 of the long transcript, and encodes just 15 amino acids followed by a stop codon and a polyadenylation signal. This short transcript potentially encodes a bifunctional enzyme lacking 10-formyl-THF synthetase activity. Both transcripts initiate at the same 5′-site, 107 nucleotides up-stream of the ATG start codon. The full-length (2934 bp) cDNA fused to a C-terminal V5 epitope tag was expressed in Chinese hamster ovary cells. Immunoblots of subfractionated cells revealed a 107-kDa protein only in the mitochondrial fractions of these cells, confirming the mitochondrial localization of the protein. Yeast cells expressing the full-length human cDNA exhibited elevated 10-formyl-THF synthetase activity, confirming its identification as the human mitochondrial C1-THF synthase. C1-tetrahydrofolate (THF) synthase is a trifunctional enzyme found in eukaryotes that contains the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase. The cytoplasmic isozyme of C1-THF synthase is well characterized in a number of mammals, including humans; but a mitochondrial isozyme has been previously identified only in the yeast Saccharomyces. Here, we report the identification and characterization of the human gene encoding a functional mitochondrial C1-THF synthase. The gene spans 236 kilobase pairs on chromosome 6 and consists of 28 exons plus one alternative exon. The gene encodes a protein of 978 amino acids, including an N-terminal mitochondrial targeting sequence. The mitochondrial isozyme is 61% identical to the human cytoplasmic isozyme. Expression of the gene was detected in most human tissues, but transcripts were highest in placenta, thymus, and brain. Two mRNAs were detected, a 3.6-kb transcript and a 1.1-kb transcript, and both transcripts were observed in varying ratios in each tissue. The shorter transcript results from an alternative splicing event, where exon 7 is spliced to exon 8a instead of exon 8. Exon 8a is derived from an exonized Alu sequence, sharing no homology with exon 8 of the long transcript, and encodes just 15 amino acids followed by a stop codon and a polyadenylation signal. This short transcript potentially encodes a bifunctional enzyme lacking 10-formyl-THF synthetase activity. Both transcripts initiate at the same 5′-site, 107 nucleotides up-stream of the ATG start codon. The full-length (2934 bp) cDNA fused to a C-terminal V5 epitope tag was expressed in Chinese hamster ovary cells. Immunoblots of subfractionated cells revealed a 107-kDa protein only in the mitochondrial fractions of these cells, confirming the mitochondrial localization of the protein. Yeast cells expressing the full-length human cDNA exhibited elevated 10-formyl-THF synthetase activity, confirming its identification as the human mitochondrial C1-THF synthase. C1-tetrahydrofolate (THF) 1The abbreviations used are: THF, tetrahydrofolate; CHO, Chinese hamster ovary; nt, nucleotide(s); SOE, splice overlap extension; HMS, homogenization solution; TBS, Tris-buffered saline; RACE, rapid amplification of cDNA ends; EST, expressed sequence tag; GCS, glycine cleavage system. synthase is a trifunctional enzyme found in eukaryotes that contains the activities 10-formyl-THF synthetase (EC 6.3.4.3), 5,10-methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10-methylene-THF dehydrogenase (EC 1.5.1.5) (Fig. 1, reactions 1–3). These activities, along with serine hydroxymethyltransferase (Fig. 1, reaction 4), are central to the interconversion of the one-carbon units carried by the biologically active form of folic acid, THF. The activated one-carbon units are used in a variety of cellular processes, including de novo purine and thymidylate synthesis, serine and glycine interconversion, methionine biosynthesis, and protein synthesis in mitochondria and chloroplasts. In eukaryotic cells, the mitochondrial and cytosolic compartments each contain a parallel set of one-carbon unit-interconverting enzymes (1Appling D.R. FASEB J. 1991; 5: 2645-2651Crossref PubMed Scopus (304) Google Scholar). For example, in the yeast Saccharomyces cerevisiae, mitochondrial and cytoplasmic isozymes of C1-THF synthase (encoded by the nuclear genes MIS1 and ADE3, respectively) have been purified and characterized (2Paukert J.L. Williams G.R. Rabinowitz J.C. Biochem. Biophys. Res. Commun. 1977; 77: 147-154Crossref PubMed Scopus (48) Google Scholar, 3Shannon K.W. Rabinowitz J.C. J. Biol. Chem. 1986; 261: 12266-12271Abstract Full Text PDF PubMed Google Scholar). Both isozymes exist as homodimers of 100-kDa subunits. Each subunit consists of a C-terminal 10-formyl-THF synthetase domain of ∼70 kDa and an N-terminal bifunctional dehydrogenase/cyclohydrolase domain of ∼30 kDa linked via a proteolytically sensitive connector region. This subunit size and domain structure are shared by cytoplasmic isozymes from mammalian and avian sources (4Paukert J.L. Straus L.D.A. Rabinowitz J.C. J. Biol. Chem. 1976; 251: 5104-5111Abstract Full Text PDF PubMed Google Scholar, 5Tan L.U.L. MacKenzie R.E. Can. J. Biochem. 1979; 57: 806-812Crossref PubMed Scopus (12) Google Scholar, 6Smith G.K. Mueller W.T. Wasserman G.F. Taylor W.D. Benkovic S.J. Biochemistry. 1980; 19: 4313-4321Crossref PubMed Scopus (68) Google Scholar, 7Villar E. Schuster B. Peterson D. Schirch V. J. Biol. Chem. 1985; 260: 2245-2252Abstract Full Text PDF PubMed Google Scholar, 8Cheek W.D. Appling D.R. Arch. Biochem. Biophys. 1989; 270: 504-512Crossref PubMed Scopus (16) Google Scholar, 9Hum D.W. MacKenzie R.E. Protein Eng. 1991; 4: 493-500Crossref PubMed Scopus (35) Google Scholar). All three activities of C1-THF synthase are found in mammalian mitochondria as well (10Barlowe C.K. Appling D.R. Biofactors. 1988; 1: 171-176PubMed Google Scholar, 11Garcia-Martinez L.F. Appling D.R. Biochemistry. 1993; 32: 4671-4676Crossref PubMed Scopus (50) Google Scholar). Our studies with intact rat liver mitochondria and mitochondrial extracts demonstrated the ability of these organelles to oxidize carbon 3 of serine to formate by a folate-dependent pathway (Fig. 1, reactions 1–4) (11Garcia-Martinez L.F. Appling D.R. Biochemistry. 1993; 32: 4671-4676Crossref PubMed Scopus (50) Google Scholar). However, the existence, structure, and function of the folate-interconverting activities of C1-THF synthase in mammalian mitochondria have been controversial. MacKenzie and co-workers (12Mejia N.R. MacKenzie R.E. J. Biol. Chem. 1985; 260: 14616-14620Abstract Full Text PDF PubMed Google Scholar, 13Mejia N.R. Rios-Orlandi E.M. MacKenzie R.E. J. Biol. Chem. 1986; 261: 9509-9513Abstract Full Text PDF PubMed Google Scholar) characterized a bifunctional NAD-dependent 5,10-methylene-THF dehydrogenase/5,10-methenyl-THF cyclohydrolase, originally isolated from ascites tumor cells. This bifunctional enzyme lacks the large C-terminal domain catalyzing the 10-formyl-THF synthetase activity and thus is unable to produce formate. This enzyme was shown to be a nuclear encoded mitochondrial protein (14Mejia N.R. MacKenzie R.E. Biochem. Biophys. Res. Commun. 1988; 155: 1-6Crossref PubMed Scopus (41) Google Scholar, 15Belanger C. MacKenzie R.E. J. Biol. Chem. 1989; 264: 4837-4843Abstract Full Text PDF PubMed Google Scholar), detectable only in transformed mammalian cells and embryonic or non-differentiated tissues (12Mejia N.R. MacKenzie R.E. J. Biol. Chem. 1985; 260: 14616-14620Abstract Full Text PDF PubMed Google Scholar). Among adult differentiated tissues, NAD-dependent 5,10-methylene-THF dehydrogenase activity is detectable only in rat adrenal tissue (16Smith 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), although the mRNA encoding this enzyme is present at low levels in all tissues examined (17Peri K.G. MacKenzie R.E. Biochim. Biophys. Acta. 1993; 1171: 281-287Crossref PubMed Scopus (28) Google Scholar). MacKenzie and co-workers (18Yang X.-M. MacKenzie R.E. Biochemistry. 1993; 32: 11118-11123Crossref PubMed Scopus (37) Google Scholar, 19Di Pietro E. Sirois J. Tremblay M.L. MacKenzie R.E. Mol. Cell. Biol. 2002; 22: 4158-4166Crossref PubMed Scopus (83) Google Scholar) have argued that mammalian mitochondria lack a C1-THF synthase and that the bifunctional NAD-dependent dehydrogenase/cyclohydrolase is the mammalian homolog of the trifunctional mitochondrial enzyme. Here, we report the identification and characterization of the human gene encoding a functional mitochondrial C1-THF synthase. We show that it is expressed widely in adult human tissues and that the full-length cDNA encodes a protein that localizes to mitochondria when expressed in Chinese hamster ovary (CHO) cells. These data confirm the existence of C1-THF synthase in mammalian mitochondria, completing the folate-interconverting pathway shown in Fig. 1. Materials—All chemicals were of the highest available commercial quality. Difco media components were obtained from VWR (West Chester, PA). Restriction enzymes, shrimp alkaline phosphatase, calf intestinal alkaline phosphatase, and T4 DNA ligase were purchased from Invitrogen. Primers for PCR and sequencing were made by IDT (Coralville, IA). [α-32P]dATP (3000 Ci/mmol) was purchased from PerkinElmer Life Sciences. Construction of Full-length cDNA—A partial cDNA clone (DKFZp586G1517) constructed by the German Genome Project (RZPD German Research Center for Genome Research) (20Wiemann S. Weil B. Wellenreuther R. Gassenhuber J. Glassl S. Ansorge W. Bocher M. Blocker H. Bauersachs S. Blum H. Lauber J. Dusterhoft A. Beyer A. Kohrer K. Strack N. Mewes H.W. Ottenwalder B. Obermaier B. Tampe J. Heubner D. Wambutt R. Korn B. Klein M. Poustka A. Genome Res. 2001; 11: 422-435Crossref PubMed Scopus (160) Google Scholar) was identified in the GenBank™/EBI Data Bank (accession number AL117452) by a BLAST search using the cDNA sequence of the human cytoplasmic C1-THF synthase (21Hum D.W. Bell A.W. Rozen R. MacKenzie R.E. J. Biol. Chem. 1988; 263: 15946-15950Abstract Full Text PDF PubMed Google Scholar). This cDNA contains 390 nucleotides (nt) of 3′-noncoding sequence and a poly(A) tail, but lacks a start codon, indicating that it is truncated at the 5′-end. The truncated cDNA clone was obtained from RZPD, and its sequence was confirmed by the DNA Analysis Facility of the University of Texas (Austin, TX). The Human Genome Database contains the entire gene corresponding to this cDNA and predicts an additional 5′-exon that encodes 60 additional N-terminal amino acids. The missing 5′-exon (exon 1) (see Fig. 4) was PCR-amplified from a genomic P1 artificial chromosome clone (dJ44A20) obtained from the Sanger Centre (Cambridge, UK). The PCR-amplified product was gel-purified using a QIAGEN gel extraction kit and subcloned into the pGEM-T Easy vector (Promega, Madison WI), and its sequence was verified. It was necessary to use MasterAmp Tfl DNA polymerase (Epicentre Technologies Corp., Madison, WI) in the PCR due to the high GC content of exon 1 (see “Results”). The partial cDNA clone and the exon 1 clone were then used as templates in a splice overlap extension (SOE)-PCR (22Horton R.M. Ho S.N. Pullen J.K. Hunt H.D. Cai Z. Pease L.R. Methods Enzymol. 1993; 217: 270-279Crossref PubMed Scopus (431) Google Scholar) to produce the full-length cDNA. The exon 1 fragment (230 bp) was amplified using Tfl polymerase and primers TOPO5′ (5′-CACCATGGGCACGCGTCTGCCGCTC-3′, with the ATG start codon underlined) and humitoSOE3′ (5′-CTTCTCTGACGATGGAGTCCCG-3′). The 2719-bp cDNA fragment was PCR-amplified using Pfu polymerase and primers GS5′SOE (5′-GGGACTCCATCGTCAGAGAAG-3′) and TOPO3′ (5′-GAACAAGCCTTTAACTTGTTCTGTTTC-3′). Primer TOPO3′ is complementary to the last nine codons of the open reading frame before the stop codon. Both products were gel-purified using the QIAGEN gel extraction kit. The 230- and 2719-bp PCR products served as templates in the SOE-PCR using primers TOPO5′ and TOPO3′ and Tfl polymerase. The full-length cDNA product (2934 bp) was gel-purified and cloned into the mammalian expression vector pcDNA3.1D/V5-His-TOPO (Invitrogen) using directional TOPO cloning according to the manufacturer's instructions. The TOPO cloning reaction was transformed into One-Shot chemically competent Escherichia coli (Invitrogen) by chemical transformation, and positive colonies were selected on YT (0.5% yeast extract, 0.8% Tryptone, and 0.5% NaCl) plates containing 50 μg/ml ampicillin. The colonies were screened by PCR with a vector primer and a gene-specific primer, and positive plasmids were prepared using a QIAGEN miniplasmid preparation kit. Sequence analysis revealed a base substitution in the full-length clone compared with the original cDNA and genomic sequences, presumably incorporated during the PCRs. (Tfl polymerase, which was chosen due to the high GC content of exon 1, lacks a 3′ → 5′ proofreading activity.) This substitution was repaired using the QuikChange site-directed mutagenesis kit (Stratagene). The repaired full-length cDNA clone, pcDNA3.1-humito, was sequenced completely, and the correct sequence was confirmed (GenBank™/EBI accession number AY374130). CHO Cell Transfection—CHO cells (1.5 × 105) were plated on 35-mm diameter dishes and cultured in α-minimal Eagle's medium supplemented with 10% (v/v) fetal bovine serum. Duplicate plates were then transfected with 2 μg of pcDNA3.1-humito/plate using the LipofectAMINE 2000 reagent method (Invitrogen). After transfection, cells were cultured for an additional 48 h in regular medium before a G418-containing selective medium (0.8 mg/ml) was applied. The selective medium was applied for ∼1 week until antibiotic-resistant colonies developed. Resistant colonies were picked, replated, cultured, and collected. Preparation of Cell Homogenates and Subcellular Fractions—Transfected cells were cultured in two 150-cm2 T-flasks to yield 1–2 × 108 cells. The monolayer was rinsed with phosphate-buffered saline (4 × 5 ml) at 4 °C and then incubated with phosphate-buffered saline containing 10 mm EDTA (10 ml) at room temperature until the cells detached (5–10 min). The flasks were tapped gently to dislodge the cells, and the cells were transferred to a 50-ml plastic conical tube. Cells were pelleted by centrifugation at 300 × g for 5 min at room temperature, and the cell pellet was washed with 15 ml of homogenization solution (HMS; 250 mm sucrose and 1 mm EDTA (pH 6.9)) at 4 °C. The cell pellet was resuspended in HMS (2 ml) at 4 °C, transferred to a Kontes nitrogen cavitation device, and exposed to a pressure of 36 p.s.i. for 30 min at 4 °C. The suspension of disrupted cells was collected into a 3-ml conical ground-glass Duall tissue grinder and further disrupted with four strokes of the homogenizer (23Lin B.-F. Huang R.-F.S. Shane B. J. Biol. Chem. 1993; 268: 21674-21679Abstract Full Text PDF PubMed Google Scholar). Nuclei and unbroken cells were sedimented by centrifugation at 900 × g for 6 min. The supernatant was removed carefully, transferred to another centrifuge tube, and stored on ice. The pellet was resuspended in HMS (1 ml) and further dispersed by four strokes in the grinder. After centrifugation at 900 × g for 6 min, the supernatant was combined with the first supernatant and stored on ice. The pellet was washed with HMS (3 × 1 ml), and the final viscous pellet (nuclear fraction) was resuspended in HMS (1 ml). The combined supernatants were centrifuged at 900 × g for 5 min, and any pellet was discarded. The volume of the supernatant (total post-nuclear supernatant fraction) was increased to 5 ml by the addition of HMS. The post-nuclear supernatant was centrifuged at 10,000 × g for 15 min, and the pellet was stored on ice. The supernatant was recentrifuged at 10,000 × g for 15 min to give a final supernatant (cytosolic fraction). The second pellet was combined with the first, washed with HMS (2 ml), and resuspended in HMS (1 ml) to give the mitochondrial fraction. Glutamate dehydrogenase activity (24Schmidt E. Bergmeyer H.U. Methods of Enzymatic Analysis. 2nd Ed. Vol. 2. Academic Press, Inc., New York1974: 650-656Crossref Google Scholar) was used as a mitochondrial marker, and lactate dehydrogenase activity (25Kornberg A. Methods Enzymol. 1955; 1: 441-443Crossref Scopus (619) Google Scholar) was used as a cytoplasmic marker. Immunoblotting—The protein concentration of the cytosolic and mitochondrial fractions was determined using the Bradford assay (26Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar) with bovine serum albumin as a standard. Eighty μg of cytosolic and mitochondrial protein from transfected and untransfected CHO cells were fractionated on a 7.5% SDS-polyacrylamide gel for 50 min at 180 V. One-half of the gel was stained, and the proteins on the other half were transferred onto a nitrocellulose membrane (Midwest Scientific, Valley Park, MO) by electroblotting for 90 min at 250 mA. The membrane was then washed with distilled water (3 × 5 min each) and blocked in 2% dry milk in Tris-buffered saline (TBS; 10 mm Tris base and 0.15 m NaCl (pH 8.0)) for 1 h at room temperature. The blocked membrane was incubated with mouse anti-V5 primary antibody (1:1000 dilution; Invitrogen) diluted in TBS and 1% dry milk for 1 h at room temperature. The membrane was then washed with TBS containing 0.0025% Tween 20 (TBST; 3 × 5 min each) and incubated with goat anti-mouse secondary antibody (1:2000 dilution; Zymed Laboratories Inc., San Francisco, CA) for 1 h at room temperature. The membrane was finally washed with TBST and TBS (2 × 5 min each) and rinsed with water before visualizing the bands. Reacting bands were visualized by enhanced chemiluminescence detection (ECL, Amersham Bioscience). Expression in Yeast and Enzyme Assays—The full-length human cDNA was subcloned from pcDNA3.1-humito into the BamHI and XhoI sites of the yeast expression vector pVT103U (27Vernet T. Dignard D. Thomas D.Y. Gene (Amst.). 1987; 52: 225-233Crossref PubMed Scopus (465) Google Scholar). In the resulting construct, pVT-humito, the entire human mitochondrial C1-THF synthase open reading frame, including the mitochondrial presequence, is expressed from the ADH promoter of the vector. Yeast strain DAY3 (ser1 ura3-52 trp1 leu2 ade3-130) (28West M.G. Horne D.W. Appling D.R. Biochemistry. 1996; 35: 3122-3132Crossref PubMed Scopus (48) Google Scholar) was transformed with pVT-humito or empty pVT103U vector using a lithium acetate method (29Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar) modified as described. 2Details are available upon request from the corresponding author. Cells were grown in synthetic minimal medium, and extracts were prepared and assayed for NAD+- and NADP+-dependent methylene-THF dehydrogenase activity as described (30West M.G. Barlowe C.K. Appling D.R. J. Biol. Chem. 1993; 268: 153-160Abstract Full Text PDF PubMed Google Scholar). 10-Formyl-THF synthetase activity was determined according to Kirksey and Appling (31Kirksey T.J. Appling D.R. Arch. Biochem. Biophys. 1996; 333: 251-259Crossref PubMed Scopus (6) Google Scholar). Northern Analysis—A FirstChoice Northern human blot I kit was obtained from Ambion Inc. (Austin, TX), with poly(A)+ mRNA from the following adult human tissues: brain, placenta, skeletal muscle, heart, kidney, pancreas, liver, lung, spleen, and thymus. Probes were synthesized by asymmetric PCR using reagents supplied in the kit and [α-32P]dATP according to the kit manufacturer's instructions. The two probes represented the 5′- and 3′-ends of the putative mitochondrial C1-THF synthase cDNA. The 5′-end probe was synthesized using primer GS5′SOE for the sense strand and primer GSI (5′-CCGCTCGAGCAAGGCATTGAGGACTTTGTTGCT-3′) for the antisense strand. This 304-bp probe covered nt +215 to +518. (The A of the ATG start codon is designated +1.) The 3′-end probe was synthesized using primer DRA3 (5′-GATGCAGTCCCCTGCTATCA-3′) for the sense strand and primer TOPO3′ for the antisense strand. This 465-bp probe covered nt +2469 to +2933, ending just before the stop codon. A probe was also synthesized for detection of the human cytoplasmic C1-THF synthase. The plasmid pUC13/HS230 (obtained from Dr. R. E. MacKenzie, McGill University), which contains a 230-bp fragment near the 3′-end of the human cytoplasmic C1-THF synthase cDNA (21Hum D.W. Bell A.W. Rozen R. MacKenzie R.E. J. Biol. Chem. 1988; 263: 15946-15950Abstract Full Text PDF PubMed Google Scholar), was linearized by digestion with SacI. A linear PCR amplification method (following the kit manufacturer's instructions) was used to synthesize the probe. The antisense primer used was 5′-GTAAAACGACGGCCAGT-3′, which is complementary to the vector sequences flanking the insert. The membrane was subjected to a 1-h prehybridization at 42 °C with Ultrahyb ultrasensitive hybridization buffer (Ambion Inc.). The probe was added at 106 cpm/ml of hybridization buffer and allowed to hybridize at 42 °C overnight in a roller bottle. The membrane was then washed twice with NorthernMax low stringency wash solution (equivalent to 2× SSC; Ambion Inc.) for 10 min at 42 °C and twice with NorthernMax high stringency wash solution (equivalent to 0.1 × SSC) for 30 min at 42 °C. The membrane was exposed to a storage phosphor screen (Amersham Biosciences) for 48 h and imaged using an Amersham Biosciences 445 SI PhosphorImager. The same blot was stripped and reconstituted for hybridization with each probe according to the kit manufacturer's instructions. Transcript Mapping—The 5′- and 3′-ends of the transcripts were mapped by RNA ligase-mediated rapid amplification of cDNA ends using the FirstChoice RLM-RACE kit from Ambion Inc. Human placental total RNA (Ambion Inc.) was used to map the 5′-end of the transcript. Nested antisense primers specific to the cDNA were designed for use with the two nested 5′-RACE primers provided in the kit (see Fig. 6). The cDNA-specific inner primer (GSI2, 5′-CGCCTCGAGACGGCTGGTTCTCAGGGGACAC-3′, with the XhoI site underlined) was complementary to nt –9 to –30 in the 5′-untranslated region. The cDNA-specific outer primer (GSO2, 5′-AGCGCGACAGGGCACACGGAG-3′) was complementary to nt +93 to +73. The 5′-RACE inner primer and the cDNA-specific inner primer had BamHI and XhoI sites, respectively, at their 5′-ends to facilitate cloning. For mapping the 3′-end of the 1.1-kb transcript, first-strand cDNA was synthesized from human placental total RNA using the supplied 3′-RACE adapter. Nested sense primers specific to the cDNA were designed for use with the two nested 3′-RACE primers provided in the kit. The cDNA-specific inner primer (3′-RACE GSI, 5′-CGCCTCGAGGAACTTGTTTAGCAACAAAGTCCT-3′, with the XhoI site underlined) was equivalent to nt +485 to +508. The cDNA-specific outer primer (3′-RACE GSO, 5′-CGCCTCGAGCTCCCTCCAGATAGCAGTGAA-3′) was equivalent to nt +390 to +410. The 3′-RACE inner primer and the cDNA-specific inner primer had BamHI and XhoI sites, respectively, at their 5′-ends to facilitate cloning. PCR fragments generated in the “inner” PCRs of both 5′- and 3′-RACE were gel-purified, digested with BamHI and XhoI, and ligated separately into BamHI/XhoI-digested pBluescript II KS(+) vector (Stratagene, La Jolla, CA). The ligation reactions were transformed into chemically competent XL1-Blue cells (Stratagene), and positive colonies were selected on YT/ampicillin plates. Colonies were screened by PCR using T7 reverse (5′-GTAATACGACTCACTATAGGGC-3′) and T3 forward (5′-AATTAACCCTCACTAAAGGG-3′) vector primers, and plasmids were prepared for sequence analysis. This 1.1-kb cDNA has been submitted to the GenBank™/EBI Data Bank under accession number AY374131. cDNA Identification and Cloning—A cDNA encoding an open reading frame with high similarity to human cytoplasmic C1-THF synthase was cloned from human uterine RNA by the German Genome Project (RZPD; GenBank™/EBI accession number AL117452). The homology extends the length of the proteins, suggesting that the cDNA encodes another trifunctional C1-THF synthase (Fig. 2). This cDNA encodes 917 amino acids plus 390 nt of 3′-noncoding sequence and a poly(A) tail, but lacks a start codon, suggesting that it is truncated at the 5′-end. Blasting this sequence against the Human Genome Database (NCBI Protein Database) revealed the corresponding gene on chromosome 6 at 6q25.2. This gene spans 236 kilobase pairs and encodes the entire cDNA sequence in 27 exons plus an additional 5′-exon that encodes 60 additional N-terminal amino acids. The predicted initiator codon sits within a near-perfect expanded Kozak consensus sequence (32Kozak M. J. Mol. Biol. 1987; 196: 947-950Crossref PubMed Scopus (997) Google Scholar). The first half of this N-terminal extension has the characteristics of a mitochondrial leader sequence, including the potential to form a positively charged amphipathic α-helix. Truncation of the original cDNA clone was due to the presence of a NotI site near the 3′-end of the first exon; NotI was used in the cDNA cloning procedure (20Wiemann S. Weil B. Wellenreuther R. Gassenhuber J. Glassl S. Ansorge W. Bocher M. Blocker H. Bauersachs S. Blum H. Lauber J. Dusterhoft A. Beyer A. Kohrer K. Strack N. Mewes H.W. Ottenwalder B. Obermaier B. Tampe J. Heubner D. Wambutt R. Korn B. Klein M. Poustka A. Genome Res. 2001; 11: 422-435Crossref PubMed Scopus (160) Google Scholar). Subsequently, the RIKEN Mouse Gene Encyclopedia Project (33Kawai J. Shinagawa A. Shibata K. Yoshino M. Itoh M. Ishii Y. Arakawa T. Hara A. Fukunishi Y. Konno H. Adachi J. Fukuda S. Aizawa K. Izawa M. Nishi K. et al.Nature. 2001; 409: 685-690Crossref PubMed Scopus (573) Google Scholar) identified a full-length mouse cDNA (ID22289) that predicts a protein with 88% identity to the human protein, including the N-terminal extension (Fig. 2). The mouse cDNA lacks the NotI site that caused truncation of the human cDNA. These data suggest that the gene on human chromosome 6 encodes a mitochondrial C1-THF synthase. Attempts to construct a full-length cDNA by RACE using human uterine RNA were unsuccessful, probably due to the extremely high GC content (>80%) of the first exon. Instead, a genomic P1 artificial chromosome clone (dJ44A20, Sanger Centre) was used to PCR-amplify the 5′-exon. This was then spliced to the remaining cDNA by SOE-PCR to construct a full-length cDNA encoding the human protein (GenBank™/EBI accession number AY374130). CHO Cell Expression and Subcellular Localization—To determine whether the protein encoded by this cDNA is, in fact, mitochondrial, we expressed the cDNA in CHO cells. The full-length cDNA was cloned into the mammalian expression vector pcDNA3.1D/V5-His-TOPO. This construct fused the 14-amino acid V5 epitope and a His6 tag to the C terminus of the 2934-bp coding region. Expression of the insert in mammalian cells is driven by the cytomegalovirus promoter. The resulting plasmid, pcDNA3.1-humito, was transfected into CHO cells, and G418-resistant colonies were selected and grown. The cytosolic and mitochondrial fractions from transfected and untransfected (control) CHO cells were isolated as described under “Experimental Procedures.” Each fraction was assayed for the mitochondrial marker enzyme glutamate dehydrogenase and the cytoplasmic marker enzyme lactate dehydrogenase. Glutamate dehydrogenase activity ranged from 68 to 95 μmol/min/mg of protein in the mitochondrial fractions, compared with 2.4–4 μmol/min/mg of protein in the cytoplasmic fractions. The lactate dehydrogenase activity of the mitochondrial fraction was only one-seventh that of the cytoplasmic fraction. These subcellular fractions were then subjected to SDS-PAGE and immunoblotting using antibodies against the V5 epitope (Fig. 3). A clear signal at ∼107 kDa was detected in the mitochondrial fraction of the transfected CHO cell line (lane 2), but not in the cytoplasmic fraction (lane 1). This mobility is consistent with the expected size of the epitope-tagged construct (∼1000 amino acids). No signal was seen in either fraction of the untransfected CHO cell line (lanes 3 and 4). These results confirm that this cDNA encodes a protein that localizes exclusively to mitochondria in a mammalian cell line. Expression in Yeast—The full-length human mitochondrial C1-THF synthase cDNA, including the 62-codon N-terminal extension, was subcloned into a yeast expression vector (pVT103U) and transformed into an ade3 deletion strain (DAY3). Disruption of the ADE3 gene, which encodes the cytoplasmic C1-THF synthase, results in yeast cells with very low 10-formyl-THF synthetase and 5,10-methylene-THF dehydrogenas" @default.
• W2039103044 created "2016-06-24" @default.
• W2039103044 creator A5003143584 @default.
• W2039103044 creator A5011519595 @default.
• W2039103044 creator A5016223938 @default.
• W2039103044 creator A5028185029 @default.
• W2039103044 creator A5088297113 @default.
• W2039103044 date "2003-10-01" @default.
• W2039103044 modified "2023-09-26" @default.
• W2039103044 title "Human Mitochondrial C1-Tetrahydrofolate Synthase" @default.
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