Characterization of the rat cytoplasmic C1-tetrahydrofolate synthase gene and analysis of its expression in liver regeneration and fetal development

Gene 319 (2003) 85 – 97 www.elsevier.com/locate/gene

Characterization of the rat cytoplasmic C1-tetrahydrofolate synthase gene and analysis of its expression in liver regeneration and fetal development Katherine M. Howard 1, Stephanie J. Muga 2, Liwen Zhang 3, Anice E. Thigpen, Dean R. Appling * Department of Chemistry and Biochemistry, The University of Texas at Austin, 1 University Station, Austin, TX 78712-0165, USA Received 12 March 2003; received in revised form 30 May 2003; accepted 24 June 2003 Received by W. Makalowski

Abstract The eukaryotic trifunctional enzyme, C1-tetrahydrofolate (THF) synthase, interconverts folic acid derivatives between various oxidation states and is critical for normal cellular function, growth, and differentiation. Using a rat C1-THF synthase cDNA and synthetic oligonucleotides, the rat C1-THF synthase gene was isolated and characterized. The gene consists of 28 exons and spans 67.5 kbp. Primer extension, RNase protection, and rapid amplification of cDNA ends (RACE) experiments indicate the presence of multiple transcription start points (tsp) within a 250-bp window located between 50 and 300 bp upstream from the start codon. The 5Vflanking region is devoid of a TATA consensus sequence motif, but putative regulatory elements, including NF-nh, HNF-4a1, RARa1, C/EBP, and PPAR are present in the promoter region. The 5Vflanking region also contains two sets of tetranucleotide repeats and two short interspersed nuclear elements (SINES). The initial 2500 bp of 5V flanking sequences of the rat and mouse cytoplasmic C1-THF synthase genes share 70% identity. However, comparison with the human gene from the Human Genome Data Bank revealed no significant homology in the 5Vflanking region. The gene structure characterization led to the identification of a pseudogene that is 94% identical to the C1-THF synthase gene and probably diverged 10 – 12 million years ago. In addition, the gene expression patterns of C1-THF synthase were investigated during liver regeneration and liver and kidney organogenesis, two highly regulated events. In both processes, C1-THF synthase expression correlated with increased nucleotide metabolism. This pattern suggests that the gene is regulated in response to changes in the demand for folate-dependent onecarbon units. D 2003 Elsevier B.V. All rights reserved. Keywords: Tetrahydrofolate; Transcription start site; Intron/exon organization; 5VUntranslated region; Promoter; Trifunctional enzyme

1. Introduction Folates serve as enzyme cofactors in one-carbon transfers and interconversions which link several major cellular proAbbreviations: 5VUTR, 5Vuntranslated region; aa, amino acids; FPGS, folylpolyglutamate synthetase; kbp, kilobase pair(s); nt, nucleotide(s); PHX, partial hepatectomy; RACE, rapid amplification of cDNA ends; RPA, ribonuclease protection assay; THF, tetrahydrofolate. * Corresponding author. Tel.: +1-512-471-5842; fax: +1-471-5849. E-mail address: [email protected] (D.R. Appling). 1 Present address: Department of Biochemistry, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA. 2 Present address: Department of Pathology and Microbiology, SC Cancer Center, University of South Carolina School of Medicine, Columbia, SC, USA. 3 Present address: Department of Atherosclerosis and Endocrinology, Merck Research Laboratories, Rahway, NJ 07065, USA. 0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-1119(03)00796-0

cesses, including nucleic acid biosynthesis, mitochondrial and chloroplast protein biosynthesis, amino acid metabolism, methyl group biogenesis, and vitamin metabolism. A central feature of folate-mediated one-carbon metabolism is interconversion of the one-carbon unit carried by the biologically active form of folic acid, tetrahydrofolate (THF). In eukaryotes, these interconversions are catalyzed by C1-THF synthase, a cytoplasmic trifunctional enzyme which contains the activities 10-formyl-THF synthetase (EC 6.3.4.3), 5,10methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10-methylene-THF dehydrogenase (EC 1.5.1.5). C1-THF synthase exists as a homodimer of 100-kDa subunits. Each subunit consists of a C-terminal 10-formyl-THF synthetase domain of about 70 kDa and an N-terminal bifunctional dehydrogenase/cyclohydrolase domain of about 30 kDa linked via a proteolytically sensitive connector region (Appling and Rabinowitz, 1985 and references therein).

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Our laboratory has used rat as a mammalian model system to investigate the expression and regulation of C1THF synthase. Previously, we observed that rat C1-THF synthase is differentially expressed among various adult rat tissues, with liver and kidney exhibiting the highest levels of the enzyme (Cheek and Appling, 1989). Using a rat C1-THF synthase cDNA as a probe, Thigpen et al. (1990) determined that transcript levels correlated with enzyme levels, suggesting that pretranslational events predominate in the tissuespecific expression. In order to further investigate the transcriptional regulation of C1-THF synthase, we have isolated and characterized rat genomic clones encoding the cytoplasmic C1-THF synthase. We have identified the promoter for this gene and determined the in vivo transcriptional start sites. Finally, we examined the expression profile of the rat C1-THF synthase gene during fetal development and liver regeneration, and identified several putative transcription factor binding sites which may be important in regulation of this multifunctional enzyme.

2. Materials and methods 2.1. Reagents and supplies All reagents were of the highest commercial grade available and were used in accordance with the manufacturer’s instructions. Restriction enzymes were purchased from Gibco-BRL, Promega, and United States Biochemical (USB). T4 DNA ligase was from New England Biolabs. Reagents for DNA sequencing and T4 polynucleotide kinase were from USB. T3 RNA polymerase, the RPA II Kit, RNase A/T1 and DNase I were from Ambion (Austin TX). The reagents used for RNA isolation were obtained from Biotecx (Friendswood, TX). M-MLV Superscript II reverse transcriptase was from Gibco-BRL, and Taq DNA polymerase was purchased from Perkin-Elmer Cetus. The E. coli strain XL1-B {endAl, hsdR17(rk , mK+), supE44, thi-l, recAl, gyrA96, relA7, (lac ), [FV, pro’AB, lac IqZDM15, Tn10, (tetr)]c} was used for transformations and plasmid DNA isolations. The cloning vector plasmid pBluescript II KS (Stratagene) was used for subcloning purposes. Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). Oligonucleotides are designated as sense (s) or antisense (as) and are given a number which corresponds to the position of the cDNA (GenBank accession no. J05519) where the 5V end of the oligonucleotide hybridizes. The numbering designates the A of the ATG start codon as + 1. 2.2. Isolation of RNA and genomic DNA All tissues were removed from male Sprague – Dawley rats (160 – 200 g) and flash frozen in liquid nitrogen. RNA isolation was based on a single-step RNA protocol (Chomc-

zynski and Sacchi, 1987). Tissues were pulverized in liquid nitrogen with a mortar and pestle, and RNA was isolated using either RNAzol B or Ultraspec RNA (Biotecx Laboratories). Briefly, 2 ml of RNAzol B or 1 ml of Ultraspec RNA was added per 10 –100 mg of tissue. For RT-PCR analyses, RNA samples were treated with RNase-free DNase I to remove any residual contaminating genomic DNA. The final preparation of total RNA had an A260/A280 ratio z 1.8 and an A260/A230 ratio z 2.0. For primer extension and ribonuclease protection (RPA) experiments, poly A+ RNA was isolated using oligo(dT) column chromatography (Ausubel et al., 2002). Rat genomic DNA was isolated from rat kidney tissue according to the procedure described in Ausubel et al. (2002). 2.3. Isolation and sequencing of the rat C1-THF synthase genomic clones Genomic clones were isolated from a rat genomic Charon 4A phage vector library (kindly provided by C. Glackin of Phytogen). Approximately 1  106 recombinants were screened by plaque-lift hybridizations using a nearly fulllength C1-THF synthase cDNA (clone Y in Thigpen et al., 1990), radiolabeled by random hexamers (Feinberg and Vogelstein, 1983). Prehybridizations, hybridizations, and high stringency washes were as described (Thigpen et al., 1990). Phage DNA was prepared by polyethylene glycol precipitation and DNA was purified using a Qiagen tip-500 column. Thirteen positively reacting, plaque purified phage were isolated and subsequently re-screened with four oligonucleotides corresponding to the 5V(as86), 5Vmiddle (s687), 3Vmiddle (as2250), and 3V(as2820) regions of the C1-THF synthase cDNA. Three clones, E7, E8, and E10, were identified as spanning the entire coding region of C1-THF synthase. To identify restriction fragments containing C1-THF synthase coding regions, genomic clone DNA was digested with restriction endonucleases, subjected to agarose gel electrophoresis, and analyzed by Southern blotting. Restriction fragments of interest were isolated from agarose gels by glass milk using a commercial kit (GeneClean, BIO 101). DNA fragments were ligated into the vector pBluescript II (cut with compatible restriction endonucleases) using T4 DNA ligase (Promega), and transformed into E. coli XL1B. A genomic clone containing exon 1 and the 5V flanking region of the C1-THF synthase gene was isolated from a rat genomic bacteriophage P1 artificial chromosome (PAC) library (Genome Systems, St. Louis, MO). C1-THF synthase PCR primers used to screen the library (s-288 and as-70) were designed from unique sequence obtained from the 5VRACE cDNA (see below) which preferentially amplified the 5V end of the C1-THF synthase gene but not the pseudogene. Two positive clones were obtained, P1-696 and P1-697. P1 plasmid DNA was isolated by a standard plasmid protocol (Birnboim and Doly, 1979). Restriction analysis revealed the two clones to be identical (data not

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shown). P1-696 contains approximately 60 kbp of DNA insert (less than the average insert size for P1 clones) with C1-THF synthase sequences located at the extreme 3V end (Fig. 1). The nucleotide sequence of 800 bp of 5V flanking sequence and the putative promoter region has been deposited in GenBank (accession no. U31032). 2.4. DNA sequence analyses A combination of methods was used to characterize the genomic clones and determine the intron/exon boundaries of the C1-THF synthase gene. In general, sequence information was obtained from subclones of restriction fragments or from PCR fragments generated from the recombinant phage DNA. Intron sizes were determined by a combination of restriction mapping, DNA sequencing, and PCR analyses and verified by comparison to newly released sequence reported by the Rat Genome Project (hgsc.bcm.tmc.edu/ projects/rat/). Sequence analyses were performed on doublestranded DNA templates using the dideoxy-chain termination method. Plasmid DNA used as a sequencing template was purified by the alkaline lysis method (Birnboim and Doly, 1979) and precipitated by PEG. Homology searches between the genomic sequences and the cDNA sequence were performed with MacVector software on a Macintosh computer. 2.5. Isolation of 5Vend of transcript by rapid amplification of cDNA ends (5VRACE) 5VRACE experiments were performed with 10 Ag of total kidney RNA and 2 pmol of the gene-specific primer as839 according to the manufacturer’s instructions (RACE Kit, Gibco-BRL). The first-strand cDNA synthesis was performed at 42 jC for 30 min in 20 mM Tris – HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl2, 0.1 mg/ml BSA, 10 mM DTT, 0.5 mM dNTPs, and 200 units SuperScript reverse transcriptase. The entire first-strand cDNA reaction was tailed with terminal deoxynucleotidyl transferase in the presence

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of dCTP. Five microliters of tailed cDNA in a total reaction volume of 50 Al was subjected to 35 cycles of amplification. Each cycle consisted of a 94 jC denaturation (45 s), 55 jC annealing (25 s), and 72 jC extension (2 min) step. The first round of PCR was performed with 0.4 AM each of the genespecific primer as460 and the anchor primer (Gibco-BRL). Ten percent of the PCR reactions were analyzed on 0.8% agarose –Tris acetate EDTA (TAE) gels. Southern analysis was performed to confirm C1-THF synthase-specific PCR products. Five microliters of a positively hybridizing sample was taken directly from the agarose gel and subjected to a second round of PCR using a nested gene-specific primer, as423, and the adapter primer (Gibco-BRL). Amplifications were performed for 30 cycles using standard PCR conditions. PCR products were visualized on a 1.2% agarose gel and TA cloned into pBS II KS (Marchuk et al., 1991). Five clones were isolated representing cDNAs extending 5V of the original cDNA. The longest clone, approximately 400 bp in length, was designated 5VRACE cDNA. 2.6. Transcript mapping by primer extension, ribonuclease protection, and 5VRACE analysis Synthetic oligomers (as19 and as86) were labeled with [g32P]ATP using T4 polynucleotide kinase to specific activities of 109 cpm/Ag, annealed with 50 Ag of total cellular RNA or 5 Ag poly A+ RNA at 42 jC for 2 h, and reversetranscribed in standard primer extension reaction conditions (Ausubel et al., 2002). The radiolabeled primer was extended for 30 min at 37 jC with either MMLV or AMV reverse transcriptase (Gibco-BRL). DNA sequencing reactions using genomic DNA and the same primer were used to size the primer extension products. For RPA analyses, total RNA samples from rat kidney and liver were hybridized with a C1-THF synthase antisense RNA probe according to established procedures (Krieg, 1991). The cRNA probe was synthesized using T3 RNA polymerase and [a32P]CTP from a 1.6-kbp BamHI fragment of clone P1-696 containing C1THF synthase 5V flanking sequence, the first exon, and 20

Fig. 1. Intron/exon structure of rat C1-THF synthase gene. Exons are shown as numbered black bars; introns as thin lines. Exon and intron sizes and positions are drawn roughly to scale, with the exception of introns 1, 2, and 8. The actual sizes of each exon and intron are listed in Table 1. P1-696, E7 and E10 are genomic clones used to characterize the gene. Restriction sites indicated as vertical lines are as follows: S, SstI; K, KpnI; R, EcoRI; H, HindIII.

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nucleotides of the first intron (sequences 1600 to + 41). After ribonuclease digestion, the protected fragments were analyzed on a sequencing gel and fragment sizes were determined from molecular weight markers or from sequencing ladders. RACE experiments to characterize the transcriptional start sites were performed as previously described (Section 2.5) except 1 Ag of poly A+ RNA was used as the starting template. The cDNA synthesis was primed with oligonucleotide as460. Two subsequent PCR reactions used the nested gene-specific primers as423 and as19. To insure contaminating genomic DNA was not amplified, a reaction lacking reverse transcriptase was conducted simultaneously. PCR products were visualized on a 1.2% agarose gel and TA cloned into pBS II. 32 white colonies were selected and screened by PCR using the universal primers 1201 and 1211 to determine insert size. Colonies were re-screened with the RACE adapter primer and an upstream gene-specific primer (as-70) to ascertain the different lengths of cDNA clones obtained. Several representative clones were selected and the DNA was isolated and sequenced. 2.7. RT-PCR assay for quantification of C1-THF synthase transcripts An RT-PCR assay using a competitor DNA as an internal control was used to quantitate C1-THF synthase transcripts in fetal tissues and regenerating liver. The competitor DNA was constructed by deleting 220 bp from a 605-bp StyI– ClaI fragment of the C1-THF synthase cDNA. This restriction fragment, from the 3Vend of the cDNA, was subcloned in pBluescript KS II. The competitor DNA was quantitated and serial dilutions made for use in the competitive PCR experiments. 2.7.1. First-strand cDNA synthesis Total RNA, isolated as described in Section 2.2, was reverse-transcribed using MMLV reverse transcriptase (RT) (Gibco-BRL). Approximately 1 Ag of total RNA and 2 pmol of gene-specific primer as3137, in a total reaction volume of 16 Al, were added to each of 12 replicate 0.5-ml Eppendorf tubes. The RNA and primer samples were denatured and quick cooled on ice. Two microliters of 10  buffer (0.67 M Tris – HC1, pH 8.8/166 mM (NH4)2SO4/67 mM MgC12/0.1 M 2-mercaptoethanol) (Davis et al., 1991) and 1 Al of 10 mM dNTPs were added to each tube. The tubes were preincubated at 37 jC for 2 min to allow primer annealing, and then 1 Al of MMLV RT (200 units) was added to each tube and the samples were incubated at 37 jC for 1 h. Control tubes minus RNA and minus RT were included in each experiment. Reactions were terminated by inactivation of the RT at 90– 100 jC for 5 min. The newly synthesized cDNA was concentrated by pooling 12 replicate RT reactions into a 1.5-ml Eppendorf tube and the cDNA precipitated with ethanol. The concentrated cDNA was stored at 20 jC until needed.

2.7.2. Competitive PCR reactions Serial dilutions of the competitor DNA ranging from 100 ng/Al to 3 fg/Al were made. Each PCR reaction contained 5 Al of cDNA (constant) and 1 Al of varying dilutions of competitor DNA. The total volume of each PCR reaction was 50 Al and consisted of the following components: 1  Voss buffer (26.8 mM Tris – HC1, pH 8.8/6.64 mM (NH4)2S04/1.6 mM MgC12/4 mM 2-mercaptoethanol), 50 mM KC1, 200 AM dNTPs, 0.1 AM oligonucleotides, 1 unit Taq DNA polymerase. The C1-THF synthase-specific oligonucleotides used were s2238 and as2718. Three negative controls were included: a minus DNA (5 Al H2O replaced the cDNA), a minus RT tube (5 Al of the minus RT reaction), and a minus RNA (5 Al of the minus RNA reaction). The positive control contained 5 Al of cDNA, but no competitor DNA. All PCR amplifications were performed in a Perkin Elmer DNA Thermal Cycler and consisted of 31 cycles of 1 min at 94 jC, 2 min at 55 jC, and 3 min at 72 jC. Ten microliters of each reaction was electrophoresed on a 0.8% agarose/TAE gel. Gels were photographed under UV illumination and the negatives scanned on a Hoefer Scientific Instruments GS300 densitometer. The resulting PCR products were quantitated using the Hoefer Scientific GS365W Electrophoresis Data System Software Package. The product generated from the target cDNA (C1-THF synthase mRNA) was 482 bp and the product generated from the competitor DNA was 259 bp. The log of ratio of target/competitor signal intensities was plotted as a function of the log of the initial input of molecules of competitor DNA. The equivalence point (log target/competitor = 0) was determined from the graph and the initial amount of cDNA extrapolated, reflecting the amount of C1-THF synthase mRNA in each sample. These equivalence points were multiplied by a constant to normalize for the amounts of cDNA (and total RNA) used in each experiment, such that final values are expressed as molecules C1-THF synthase/Ag total RNA. 2.8. Animal studies Adult male Sprague – Dawley rats (170 – 200 g) (Harlan) were used for the regeneration study. Animals used for the developmental experiments were obtained from the Animal Resource Center Breeding Facility at the University of Texas at Austin and were from Sprague – Dawley stock. All animal protocols conducted in these studies were performed in accordance with University of Texas Institutional Animal Care and Use Committee (IACUC) guidelines and were approved by the IACUC. Partial hepatectomies (PHX, removal of 2/3 of the liver) were performed according to Higgins and Anderson (1931). The excised liver tissue served as normal (control) liver for the regeneration studies. Following surgery, the animals were offered food and water ad libitum and maintained on a 12-h light/dark cycle. All surgeries were performed between 7 and 9 a.m. to control for any effects of circadian rhythms. The regenerating liver

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remnants were removed at 18, 24, 48, and 96 h post PHX, were frozen immediately in liquid nitrogen, and stored at 70 jC until needed. Timed pregnant female, neonatal, and young adult Sprague –Dawley rats were fed a standard laboratory chow ad libitum and maintained by the Animal Resource Center until needed. The time course for the developmental series included gestation, neonatal, young adult, and adult periods. The fetal rats were rapidly dissected from timed pregnant females at 17, 19, and 21 days of gestation. Neonatal animals were gathered on days 4, 8, 24, and 30 after birth. Young adults were designated as such 60 days after birth. All rats were sacrificed by cervical dislocation prior to harvesting of liver and kidney tissues.

3. Results and discussion 3.1. Isolation of a full-length C1-THF synthase cDNA The original rat C1-THF synthase cDNA (clone Y) isolated by Thigpen et al. (1990) was truncated at its 5V end and extended only to within 30 nt downstream of the ATG codon. The isolation of a full-length C1-THF synthase cDNA would provide information concerning the length of the 5VUTR and provide sequence information to aid in the isolation and characterization of the C1-THF synthase promoter. The rapid amplification of cDNA ends (RACE) procedure was employed to isolate a 5V cDNA which extended upstream of the ATG. C1-THF synthase firststrand cDNA was primed with oligonucleotide as839 and a homopolymeric dC tail was incorporated using terminal deoxynucleotidyl transferase. After PCR amplification using the gene-specific primer as460 and the adapter primer, a smear of RACE products between 300 and 800 nucleotides in length was detected by Southern analysis using a labeled cDNA probe. No discrete product could be visualized by ethidium bromide staining (data not shown). Using the autoradiogram as a guide, a 10 Al agarose plug was removed from the most intensely hybridizing region of the gel and was subjected to a second round of PCR using the genespecific primer as423 and the adapter primer. PCR amplification resulted in a broad ethidium bromide stained DNA fragment approximately 800 nucleotides in length (data not shown). PCR products from the second amplification reaction were cloned into the vector pBluescript II KS . Five clones were isolated and represented C1-THF synthase cDNAs extending 5Vof the original cDNA. One of the five clones, designated 5VRACE, was approximately 200 nucleotides larger and was selected for sequence characterization. The 5Vend of the RACE clone contained 17 deoxycytosines and the sequence of the anchor and adapter primers verifying the clone was the result of a tailed and amplified RACE product. Counting the A of the start codon as + 1, this clone extended from 307 to the site of the antisense primer used in the RACE protocol ( + 423). Sequence analysis of the

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RACE clone revealed four nucleotides in the coding region that differed from the previously published cDNA sequence (Thigpen et al., 1990). The first two nucleotide differences, T to C at position + 24 and A to G, at + 27 (comparing clone Y to 5VRACE), did not alter the C1-THF synthase deduced amino acid sequence. The two other nucleotide differences were both T to G transitions. The first transition at + 56 changed codon 19 from leucine to arginine. The second transition at + 70 changed codon 24 from phenylalanine to valine. These two codon changes are downstream of the N-terminal amino acid sequence that was determined on purified C1-THF synthase protein (Thigpen et al., 1990). Thus, the amino acid discrepancies were not detected previously. Both amino acid differences detected in the 5VRACE clone are consistent with other C1-THF synthases and Arg19 and Val24 are also found in the human cytoplasmic C1-THF synthase (Hum et al., 1988). The cDNA sequence in the original submission (GenBank accession no. J05519) has been corrected to reflect both the changes in the length of the 5VUTR and the nucleotide differences revealed by the 5VRACE clone. 3.2. Isolation of rat C1-THF synthase genomic clones A lambda Charon 4A rat liver genomic library was screened with the truncated C1-THF synthase cDNA (clone Y). Three clones, E7, E8, and E10, were identified as spanning the entire coding region of C1-THF synthase (Fig. 1). Initially, E8 was identified as containing the ATG and 5V flanking sequences. Sequence from E8 (clone Z in Thigpen et al., 1990) was originally used to reconstruct a full-length cDNA. In E8, the first 45 nucleotides of C1-THF synthase coding sequence contains codons that exactly match the N-terminal amino acid sequence obtained from the purified protein. In addition, the E8 sequence overlapped the truncated cDNA sequence. Thigpen et al. (1990) noted a nucleotide transition from G to T at position 70, and attempted to resolve the sequence discrepancy by analyzing genomic DNA from two additional sources. Sequence analysis from both sources coded for phenylalanine. Subsequent sequence analyses revealed that E8 contained a large (over 830 nucleotides) intron-less open reading frame and several sequence mismatches to clone Y. In addition, the 5V flanking sequence of E8 was unable to function as a promoter in vitro (data not shown). Therefore, we conclude E8 is a processed pseudogene. It is now apparent that the PCR primers used by Thigpen et al. (1990) to investigate the sequence discrepancy amplified the pseudogene and, therefore, confirmed its own sequence. A comparison of the nucleotide sequences showed the 5VRACE clone and pseudogene were 94% identical. The analysis revealed numerous insertions, deletions, and transitions in the 5Vnoncoding region and four transitions in the coding region (see Section 3.1). Based on the 94% homology of the pseudogene to the 5VRACE sequence and using a neutral mutation rate of 5  10 9 nucleotide substitutions/

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site/year (Freytag et al., 1984), we estimate the pseudogene diverged 10 – 12 million years ago. The existence of a human C1-THF synthase pseudogene on the X chromosome has also been reported (Italiano et al., 1991). If one assumes the rat and human lineages diverged 75 million years ago (Waterston et al., 2002), then the rat C1-THF synthase pseudogene arose after rat and human divergence. Interestingly, although 94% identical in the region surrounding the major transcription start sites and extending to 268, all RACE clones sequenced to date represent authentic gene sequences. This further confirms that the pseudogene is not expressed and that important transcriptional regulatory sequences are located upstream of the region of homology. Genomic clone E7 contained 15 kbp of DNA insert and included exons 4 through 8 (Fig. 1). Southern analyses of E7 DNA with the C1-THF synthase cDNAs identified 5- and 2.5-kbp SstI fragments as containing exon sequences. These two fragments account for all coding region sequences in E7. Both of the SstI fragments were subcloned into pBluescript and the intron/exon boundaries were characterized. No additional coding sequences were found 5V of exon 4. Restriction analysis of genomic clone E10 indicated this clone contained at least 20 kbp of DNA insert. Sequence analyses revealed E10 contains exons 8 through 28 (Fig. 1).

The translational stop codon (TAA) resides 4 nucleotides from the 3Vend of exon 27; exon 28 contains 34 nucleotides of 3V UTR sequence at which point an EcoRI site is encountered. This Eco RI site is most likely the EcoRI linker site to the Charon 4A vector. A region encompassing exons 16 to 22, corresponding to nucleotides 1495– 2176, was not present in E10 DNA. Southern analysis of rat genomic DNA and of the lambda clone clearly showed the presence of a pseudogene and demonstrated a deletion in the E10 clone had occurred (data not shown). The bacteriophage P1 genomic clone (P1-696; see Section 3.3) contained exon 1 and part of the first intron, but did not overlap with E7. The sequence of the 5VRACE clone was identical to the genomic sequence obtained from P1-696. This indicates the absence of intronic sequences in the 5V flanking region. The missing sections of the rat gene (exons 2, 3, and 16 –22) were subsequently identified in the Rat Genome Database (NCBI). Fig. 1 illustrates the C1-THF synthase gene structure and the relationship between the different genomic clones isolated. The rat cytoplasmic C1-THF synthase gene is located on chromosome 6 and contains 28 exons spanning at least 67.5 kbp of DNA. Table 1 shows the locations and the sequence of the intron/exon boundaries. All the intron/exon

Table 1 Intron/exon splice junctions of the rat cytoplasmic C1-THF synthase gene Exon number

Exon length (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

348b 85 60 54 136 101 137 112 128 98 174 137 47 108 75 103 77 141 69 112 140 42 101 179 108 153 94 213 a b

Intron sequence

Exon sequence

5VJunction

tccctctag ttcttccag tttctgcag tacttttag ctgttttag ctcttccag tctccctag ctcaaccag tctgtccag tttttaaag tccttgaag gtcccctag cttttgtag ttctttaag tcttttcag ccactgtag tttccacag gtcttgcag tctccacag tccttgcag ttcttccag gttttacag cctgtacag gtacaccag cccacccag gcctcatag tctttccag

Intron sequence 3VJunction

TCAGAG—CTCCGC GCAAAT—CTGCAG GTTGGC—CAAGAG ATCGGG—TCGGAG GTGTTA—GGACGG GTTGAC—AGACAG GAGTGC—AAGGAG GTAAAT—TTCCAG ATGATA—ATGCAG AGCACA—ACCAAG TGACAT—GACTGG AATTAC—TAAAAG GTGGCG—GAAGAG TTTAAT—GACAAG GCTCTC—TTACGG AGGCTA—AGAGAG TGCTGG—CGCACG GCCCAG—GACCTG GGCGTG—TTAGAG GGCACG—TTGTAG TGACAG—CCCACG GTCACC—GAAGAG GACCTG—ATTCAA GACAGA—CTCAAG CTCTCA—AAGCAG GGCTTT—GGAACG ATGAGC—AAACAG ATCTTC—AAATGA

gtaagtact gtatgagag gtaaaggca gtgagcgaa gtgagtgct gtaagaatg gtaggaggt gtgagtggt gtatggcgt gtaggagtg gtgagtagg gtacctgtg gtaaagcag gtaggaagc gtaagcttt gtatgtacc gtaactgct gtaagtgac gtgagcagg gtaggttcc gtgaggacc gtaactacc gtaagtgca gtgggttgc gtaggtgtt gtaagtgga gtgagctat aataatttt

Introns are numbered starting with intron 1 between exons 1 and 2. The 5Vend of exon one, and therefore its length, is based on the longest 5VRACE clone isolated (see text).

Intron size (bp)

Intron numbera

14726 8815 670 164 114 2552 957 4867 396 197 1235 2283 677 162 3525 3096 770 1588 530 94 2951 4512 770 2316 447 4146 1692

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

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boundaries follow the GT/AG rule. Intron sizes range from 94 bp to greater than 14.7 kbp. Excluding the first and last exons, exon sizes range from 42 to 179 bp with the average exon size of 115 bp. This codes for an average of 38 amino acids, just slightly lower than the reported average of 44.6 amino acids (133.4 bp) (Traut, 1988). The structures of the mouse and human cytoplasmic C1THF synthase genes were recently deduced (Patel et al., 2002). Comparison of the three mammalian genes reveals that their intron/exon organizations are remarkably conserved. All three possess 28 exons, and all except the first and last exons are nearly identical in size (Table 2). Furthermore, the corresponding introns are similarly sized and in nearly identical positions. Clearly, this gene arose before the divergence of the human and rodent lineages, more than 75 million years ago (Waterston et al., 2002). C 1 -THF synthase is composed of two functional domains, an N-terminal dehydrogenase/cyclohydrolase domain (aa 1 to f 300) and a C-terminal synthetase domain (aa f 301 to 935). As reported for the mouse homolog (Patel et al., 2002), the rat dehydrogenase/cyclohydrolase domain is encoded by exons 1 – 10, and the synthetase domain is encoded by exons 10 – 27. The domain junction sequences are encoded in exon 10. A bifunctional NAD+dependent methylenetetrahydrofolate dehydrogenase –methenyltetrahydrofolate cyclohydrolase is expressed in mammalian mitochondria during embryonic development (Mejia Table 2 Exon sizes of cytoplasmic C1-THF synthase genes from rat, human, and mouse Exon

Rat

Human

Mouse

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

348 85 60 54 136 101 137 112 128 98 174 137 47 108 75 103 77 141 69 112 140 42 101 179 108 153 94 ?

93 84 59 53 136 100 136 111 127 97 173 136 46 107 74 102 76 140 68 111 139 41 100 177 107 152 93 245

100 85 59 54 137 101 137 112 128 98 174 137 47 108 75 103 77 141 69 112 140 42 101 178 108 153 94 215

91

and MacKenzie, 1985). This enzyme is f 40% identical at the amino acid level to the dehydrogenase/cyclohydrolase domain of the trifunctional C1-THF synthase. The mouse gene encoding the bifunctional enzyme contains eight exons varying in size from 92 to 1104 bp (Belanger and MacKenzie, 1991). Comparison of the exon organization of this gene to exons 1– 10 of C1-THF synthase fails to reveal any apparent similarities. Recently, however, Patel et al. (2002) reported homology between sequences in the long 3V UTR of the bifunctional gene and sequences in the trifunctional genes. They speculate that the gene encoding the bifunctional mitochondrial protein arose from a trifunctional precursor through inactivation of the C-terminal synthetase domain. 3.3. Isolation of C1-THF synthase promoter The presence of a pseudogene in the rat genome and two large introns near the 5V end of the C1-THF synthase gene created severe difficulties in the isolation of the 5Vflanking region. To obtain the promoter sequences, it was necessary to first isolate a 5V extended cDNA. RACE experiments produced this full-length cDNA (see Section 3.1) and allowed a direct comparison of the 5V UTR and the pseudogene sequence. The 5VRACE clone and the pseudogene were >94% identical from + 93 until 268, where the two sequences diverged completely. This divergence allowed the design of a primer (s-288) specific for the authentic gene. PCR reactions using s-288 and another oligonucleotide, as70, amplified a 220-bp fragment from rat genomic DNA. Subsequent cloning and sequencing of this PCR product revealed that the authentic gene, rather than the pseudogene, had been amplified. This PCR assay, specific for the extreme 5V end of the authentic gene, was used to isolate C1-THF synthase genomic clones from a rat P1 bacteriophage library (Genome Systems). Two positive P1 clones were obtained and restriction analyses revealed the two clones to be identical (data not shown). P1-696 contains approximately 60 kbp of DNA insert (less than the average insert size for P1 clones) with C1-THF synthase sequences located at the extreme 3V end. The C1-THF synthase sequence is located in the middle of a 4-kbp SstI/KpnI fragment and represents the first exon of C1-THF synthase: from 307 to + 41 including the translational start codon. There is an additional 3.5 kbp of DNA between the KpnI site and end of the P1 vector indicating the first intron is at least 5 kbp. The 1.6-kbp BamHI subclone of P1-696 was used to obtain sequence information, to create riboprobes for transcript mapping, and as a template for in vitro transcription reactions to investigate promoter activity. 3.4. Determination of transcriptional start sites To determine the in vivo transcription initiation sites, primer extension and RPA analyses were conducted using both kidney and liver RNA. In addition, 5VRACE products

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were cloned and sequenced to characterize the transcriptional start sites and to confirm that no unusual splicing events were occurring in the 5VUTR. Transcription initiation sites were first investigated by primer extension analyses. Because of the low abundance of C1-THF synthase transcripts and previous indications of extensive secondary structure near the 5V end, primer extension reactions were performed with two different reverse transcriptases. A synthetic oligonucleotide (as19), end-labeled with T4 polynucleotide kinase and [g-32P]ATP, was annealed to total RNA at 42 jC for 2 h. The radiolabeled primer was extended at 37 jC with either MMLV or AMV (not shown) reverse transcriptase (Fig. 2). Both enzymes produced similar primer extension results; however, the MMLV

Fig. 2. Primer extension analysis of rat liver and kidney RNA. Total RNA (50 Ag) from rat kidney or liver was used as template in primer extension reactions using MMLV RT and radiolabeled oligonucleotide as19 primer. The same primer was used in sequencing reactions on a genomic DNA clone (lanes G, A, T, C). Samples were separated on an 8% polyacrylamide sequencing gel and autoradiographed. Numbering is relative to the A of the ATG start codon as + 1.

reverse transcriptase appeared to be more efficient. The results revealed three major primer extended products at 55, 75, and 87 relative to the ATG. Minor start sites ranging from 90 to 308 were also detected. To exclude the possibility that the extension products were a result of nonspecific primer extension due to the low annealing temperature of 42 jC, primer extensions were repeated with kidney total RNA and with tRNA (data not shown) at both 50 and 55 jC with identical results. Brain, heart, lung, and skeletal muscle were also examined. The 55 extension product was detected in brain RNA; however, extension products for heart, lung, and skeletal muscle were not detected (data not shown). A second oligonucleotide (as86) produced an identical pattern of transcription start sites in primer extension experiments (data not shown). Reverse transcriptase can pause and prematurely terminate in regions of high GC content or extensive secondary structure in the target mRNA. Ribonuclease protection assays (RPA) were conducted to verify that the numerous start sites identified by primer extension represent true heterogeneity in the 5Vends. A 1.6-kb antisense RNA probe generated with T3 RNA polymerase was hybridized in solution to 50 Ag of total liver and kidney RNA, then digested with RNase A and RNase T1. Three major fragments were protected from RNase digestion, which correspond to start sites at 55, 87, and 110 (Fig. 3). Minor protected fragments mapping to start sites as far as 300 were also detected. Both kidney and liver RNA produced nearly identical results in ribonuclease protection experiments. The presence of multiple transcriptional start sites spanning a 250-nucleotide region was demonstrated by both primer extensions and ribonuclease protections. There was good agreement in both major and minor start sites with the exception of the 75 band detected by primer extensions that was not observed in protection experiments. The 75 primer extended product may be a result of premature termination of reverse transcriptase due to secondary structure. RACE experiments were conducted to verify that the longer transcripts detected in primer extension and RPA experiments were authentic products and to confirm the lack of introns in the 5V UTR. RACE experiments were essentially performed as before; however, the first-strand cDNA was synthesized from 1 Ag poly A+ RNA. The cDNA synthesis was primed with oligonucleotide as460. Subsequent PCR reactions used the gene-specific primers as423 and then as19. To insure contaminating genomic DNA was not being amplified, a reaction lacking reverse transcriptase was conducted simultaneously. No specific RACE products were detected after the first round of PCR. Five Al of each first round PCR reaction was subjected to a second round of PCR using nested primers. The final PCR products were cloned into pBluescript II. Of 32 colonies screened by PCR, 9 corresponded to the amplification of a non-insert containing plasmid while the remaining 23 colonies contained C1-

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insert detected were sequenced. The 5V ends of the three RACE clones sequenced were 148, 200, and 308 relative to the ATG, and correspond to minor transcription start sites detected in both primer extension and ribonuclease protection experiments (summarized in Fig. 4). Although none of the RACE clones sequences corresponded to the major transcripts detected in primer extension and ribonuclease protection experiments, the lack of shorter transcripts likely resulted from experimental technique in that longer cloned PCR products were preferentially selected for sequencing. 3.5. Analysis of the 5Vflanking sequence A 1.6-kbp BamHI fragment from the genomic clone P1696 provided the C1-THF synthase 5V flanking sequence. The sequence upstream of the translation start site has been characterized (Fig. 4). Major transcription start sites, as determined from primer extension and ribonuclease protection experiments, are indicated with bold arrows at positions 55, 87, and 110 relative to the ATG. Several of the more prominent minor start sites at positions 148, 163, 199, and 307 are indicated with smaller arrows. The asterisks denote the 5V ends of the four RACE clones that were isolated and sequenced. The 5Vflanking sequence contains no obvious TATA box. However, like many TATAless promoters, several other potential transcription factor binding sites are present, including NF-nB, PPAR, HFH-8, RARa and h, Sp1, HNF4-a1, and C/EBPa. There are 12 repeats of the tetranucleotide (AAAT) and 15 repeats of the tetranucleotide (AAAG). The two sets of tetranucleotide repeats are separated by a 54 bp AG repeat sequence. In addition, two short interspersed nuclear elements (SINEs) (Smit, 1996) are found at 1860 and 1210 in the 5Vflanking region of the rat gene. Smith –Waterman alignment of the 5V flanking regions (2300 bp) of the rat and mouse genes reveals that they share 70% identity (Fig. 4). This homology extends at least 2500 bp upstream of the ATG start codon of each gene. However, comparison with the human gene from the Human Genome Data Bank revealed no significant homology in the 5Vflanking region. Fig. 3. Ribonuclease protection assay of rat liver and kidney RNA. Total RNA (10 Ag) from rat kidney or liver was hybridized in solution with 5  104 cpm of labeled RNA probe. tRNA (10 Ag) was used as a negative control. The 1.6-kb probe RNA contained 5Vflanking sequences, exon 1, and the first 20 nucleotides of intron 1. Undigested probe remains substantially intact (1.6 kb) during the hybridization. The samples were subjected to digestion by RNase A and T1 and the protected fragments were separated on an 8% polyacrylamide sequencing gel and autoradiographed. A sequencing ladder is included for size determination (lanes G, A, T, C). Numbering is relative to the A of the ATG start codon as + 1.

THF synthase sequences. With the exception of two clones that contained a small insert of approximately 50 –75 bp, all of the clones contained inserts between 100 and 300 bp in size. Three clones representing the three major sizes of

3.6. C1-THF synthase gene expression during fetal development and liver regeneration The RT-competitive PCR assay used here was validated with several preliminary experiments. The relative efficiencies of amplification of target and competitor DNAs was determined by evaluating the signal intensity of product vs. cycle number in a PCR reaction containing equal amounts of the two templates. The slopes of the linear portion of the graphs were nearly identical (data not shown). In a second experiment, total rat liver RNA was reverse-transcribed on three separate occasions and the cDNA from each reaction was subjected to competitive PCR. The three assays yielded values ranging from 6.8  104 to 9.1  104 molecules C1-

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Fig. 4. Comparison of the 5Vflanking regions of the rat (top) and mouse (bottom) cytoplasmic C1-THF synthase genes. The ATG start codons are shown in bold, and the numbering is relative to the A of the ATG. The arrows indicate transcriptional start sites, with the size of the arrow roughly proportional to the abundance of transcripts from each position. The asterisks indicate the 5Vends of sequenced RACE clones. Putative transcription factor binding sites [predicted by the TESS web server (http://www.cbil.upenn.edu/tess) using the TRANSFAC v4.0 database] are shown in brackets, and include retinoic acid receptor-a1 (RAR-a1), NF-nB, peroxisome proliferator activated receptor (PPAR), HNF-3/Fork-head homolog 8 (HFH-8), GAGA, RAR-b, Sp1, C/EBP, HNF-4a1, C/EBPa .

THF synthase cDNA/Ag RNA, with an average of 8.1  104 molecules C1-THF synthase cDNA/Ag RNA and a standard error of the mean (S.E.M.) of 6.9  103 (8.5%). 3.6.1. C1-THF synthase expression during liver regeneration RT-competitive PCR was performed on total RNA from normal liver and from 18-, 24-, 48-, and 96-h regenerating rat liver. Twenty percent of each PCR reaction was electrophoresed on an agarose gel, photographed, and quantified by densitometry. Fig. 5A shows data from 18-h regenerating liver. The log of the ratio of the signal intensities of the target/competitor products was plotted as a function of the log of the initial input of competitor DNA in Fig. 5B. The equivalence point was determined from the graph and the initial amount of C1-THF synthase cDNA was calculated as described in Materials and methods. In this example, the C1THF synthase message is about 2.5-fold more abundant in RNA from the control or normal liver sample than in RNA

from 18-h regenerating liver (1.6  106 vs. 6.5  105 molecules/Ag RNA). Similar analyses were carried out on the 24-, 48-, and 96-h regenerating liver samples. The data are summarized in Fig. 6. C1-THF synthase transcript levels in regenerating liver are represented as a percentage of control (normal) liver F S.E.M. The transcript level at 18 h is only 40% of normal, but returns to normal levels by 24 h and remains there through the remainder of the regeneration time course. However, due to the small sample size, and animal-to-animal variation, these differences did not reach statistical significance ( p < 0.05). In liver regeneration, the compensatory growth that ensues in the remaining lobes after PHX is highly controlled and regulated while maintaining the histological architecture of the organ. Initial responses to PHX include increases of both RNA and DNA synthesis. In the rodent model, DNA synthesis in periportal hepatocytes starts about 12– 16 h after PHX with a peak at 22– 24 h (Grisham, 1962). A second peak of DNA synthesis in the centrilobular hepatocytes is

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Fig. 5. RT-competitive PCR analysis of C1-THF synthase transcript levels in 18-h regenerating liver. (A) RT-PCR reaction products were electrophoresed on 0.8% agarose/TAE gels and stained with propidium iodide. Competitor DNA concentration in the reactions decreased from left to right, as indicated. The product generated from the target cDNA is 482 bp and the product generated from the internal standard DNA is 259 bp. These bands were scanned with a densitometer and the area underneath the peaks was integrated and plotted. (B) The log of the ratio of the target/competitor signal intensities is plotted as a function of the log of the initial input of molecules of competitor DNA. The equivalence point (log target/ competitor = 0) was determined from the graph and the initial amount of cDNA extrapolated, reflecting the amount of C1-THF synthase mRNA in each sample. These equivalence points were multiplied by a constant to normalize for the amounts of cDNA (and total RNA) used in each experiment, such that final values are expressed as molecules C1-THF synthase/Ag total RNA. In this experiment, the calculated values for normal liver and 18-h regenerating liver were 1.6  106 and 6.5  105 molecules/Ag RNA, respectively.

detected at 48 h after surgery. Other cells (endothelial cells, Kupffer cells, Ito cells, and bile duct cells) also participate in the growth process and show elevated DNA synthesis approximately one day later than hepatocytes. Hypertrophy dominates the period from 72 to 96 h after PHX (Grisham, 1962). The decrease in expression observed at 18 h PHX suggests that the basal levels of C1-THF synthase are able to supply enough one-carbon units for purine synthesis required for this period of rapid growth. This is followed by an increase in C 1-THF synthase message levels corresponding to the onset of the first wave of DNA synthesis (24 h after surgery). This suggests that as one-carbon units become depleted, C1-THF synthase expression increases to support higher one-carbon fluxes. The C1-THF synthase response is consistent with other findings. Weber et al. (1976) addressed the enzymatic activity of some of the anabolic and catabolic enzymes in the production of inosine monophosphate, a precursor for purine synthesis, during liver regeneration. The first observation was that IMP dehydrogenase activity in regenerating liver increased 5.8-fold over normal liver 18 h PHX. In

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addition, PRPP amidotransferase, the first enzyme committed to de novo purine biosynthesis, increased 1.5- to 1.7-fold at 24– 48 h PHX. The increased activity of both enzymes was followed by a return to normal activity levels 96 h after surgery. The second observation noted that the other purine synthesizing enzymes did not show significant changes during liver regeneration. Finally, Weber et al. (1976) observed that the purine catabolic enzymes, xanthine oxidase and uricase, remained unaltered during the process. More recent studies have found that the rate of de novo purine synthesis in regenerating rat liver does increase in response to the surgery (Itakura et al., 1986a,b,c). The rate of de novo purine synthesis was shown to increase 2.4-fold over sham-operated animals at 6 –18 h after surgery with the largest peak occurring 12 h PHX. However, they did not find statistically significant changes in the net purine concentrations in regenerating and normal livers (Itakura et al., 1986b). Information on the role that folate-dependent pathways may play in liver regeneration is somewhat limited. Barbiroli et al. (1975) examined several aspects of folate metabolism in rats subjected to partial hepatectomy. They observed a decrease in total liver folates, and especially the 5-CH3-THF pool, during the first 12 h of regeneration. Two of the activities of C1-THF synthase, 5,10-methyleneTHF dehydrogenase and 10-formyl-THF synthetase, also decreased during this time period. At 12 h PHX, these activities began to increase, reaching normal levels by 48– 72 h. This exactly parallels the response we observed in C1THF synthase transcript levels (Fig. 6). Barbiroli et al. (1975) speculate that the initial 5-CH3-THF pool is large enough to meet the increased requirements for folate coenzymes during the early phase of liver regeneration. When these pools begin to decline, enzyme activity increases to replenish the folate pool in the later phases of regeneration.

Fig. 6. C1-THF synthase transcript levels during liver regeneration. Transcript levels were determined by RT-PCR as described in Fig. 5, and are expressed as a percentage of the level in normal liver. Three animals were used for each time point, and samples from each animal were measured in at least three independent RT-PCR assays. Data represent means F S.E.M. A t-test was used to compare each time point to the normal liver; none of the differences were statistically significant ( p < 0.05).

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Thymidylate synthase (Labow et al., 1969) and folylpolyglutamate synthetase (FPGS) (Tolomelli et al., 1987) activities also increase during liver regeneration. Whereas thymidylate synthase activity peaked at 36 h, and then declined back to normal levels (Labow et al., 1969), FPGS reached its highest level at 36 h, and remained high relative to control liver (Tolomelli et al., 1987). Thus, it appears that the folate enzymes required to support nucleotide and amino acid biosynthesis (C1-THF synthase, thymidylate synthase, FPGS) are all induced in the early stages of liver regeneration in order to support the increased demands of RNA and DNA synthesis. 3.6.2. C1-THF synthase expression during development RT-competitive PCR was performed on total RNA from liver and kidney during the gestation, neonatal, young adult, and adult stages of development. Fig. 7 shows the C1-THF synthase transcript levels expressed as a percentage of adult liver or kidney levels. Significant changes in transcript levels were seen in both organs during the development time course. At gestation day 17, liver C1-THF synthase message levels were less than 20% of adult levels, but then gradually increased throughout the prenatal stages up through day 8 of the neonatal period. The transcript level decreased at day 24, but returned to adult levels by day 30 (Fig. 7). The expression pattern observed in developing kidney was very different (Fig. 7). C1-THF synthase transcript levels were already at adult levels in the prenatal period. There was then a dramatic increase in transcript levels (4-fold) during the early phase of neonatal growth. C 1 -THF synthase transcript levels remained elevated throughout the neonatal period, but trended down towards adult levels by day 60. It is interesting to note that at 24 days after birth, we observed a decrease in C1-THF synthase transcript levels in both kidney and liver. Our results for transcript levels are consistent with two earlier reports that followed C1-THF synthase enzyme activities during fetal development. Mejia and MacKenzie (1985) reported that fetal rat liver (day 17 of gestation) contained half the 5,10-methylene-THF dehydrogenase activity of adult liver. The 10-formyl-THF synthetase and 5,10-methyleneTHF dehydrogenase activities fluctuate in the developing chick embryo by approximately 2-fold (Silber and Mansour, 1971). Studies with human infants showed that preterm infants had lower levels of liver 5,10-methylene-THF dehydrogenase activity than one year old infants (Kalnitsky et al., 1982). Other folate-dependent enzymes also exhibit dynamic expression patterns during development. Thompson et al. (2001) studied the expression of enzymes involved in serine and one-carbon unit metabolism during development in rabbits. The patterns they observed for 5,10-methyleneTHF reductase, methionine synthase, 5,10-methenyltetrahydrofolate synthetase, and serine hydroxymethyltransferase suggested a metabolic response to ensure adequate 5,10methylene-THF to support the rapid growth characteristic of the late prenatal and postnatal periods (Thompson et al.,

Fig. 7. C1-THF synthase transcript levels during liver and kidney development. Transcript levels were determined by RT-PCR as described in Fig. 5, and are expressed as a percentage of the level in the adult tissue. Tissues from each period of development were measured in at least three independent RT-PCR assays. Data represent means F S.E.M.

2001). The increased expression of C1-THF synthase during this same period in both liver and kidney is entirely consistent with this proposal, as the trifunctional enzyme would allow oxidation of 5,10-methylene-THF to 10-formyl-THF to support de novo purine synthesis.

Acknowledgements This study was supported by Grant DK36913 from the National Institutes of Health.

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