©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Upstream Organization of and Multiple Transcripts from the Human Folylpoly--glutamate Synthetase Gene (*)

Sarah J. Freemantle (1)(§), Shirley M. Taylor (2), Geoffrey Krystal (2), Richard G. Moran (1)(¶)

From the (1) Departments of Pharmacology and Toxicology and (2) Microbiology and the Massey Cancer Center, Medical College of Virginia, Richmond, Virginia 23298-0230

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Folylpoly--glutamate synthetase (FPGS) is essential for the survival of proliferating mammalian cells and central to the action of all ``classical'' folate antimetabolites. We report the isolation of cDNAs corresponding to the 5` ends of FPGS mRNA from both human and hamster cells which include a start codon upstream of and in-frame with the AUG in the previously reported FPGS open reading frame. The predicted hamster and human amino-terminal extension peptides have features consistent with a mitochondrial targeting sequence. Ribonuclease protection and 5`-rapid amplification of cDNA ends assays indicated multiple transcriptional start sites consistent with the sequence of the promoter region of this gene, which was highly GC-rich and did not contain TATA or CCAAT elements. These start sites would generate two classes of transcripts, one including the upstream AUG and one in which only the downstream AUG would be available for translation initiation. Transfection of the full length human cDNA into cells lacking FPGS restored their ability to grow in the absence of glycine, a product of mitochondrial folate metabolism, as well as of thymidine and purines. Therefore, we propose that the mitochondrial and cytosolic forms of FPGS are derived from the same gene, arising from the use of the two different translation initiation codons, and that the translation products differ by the presence of a 42-residue amino-terminal mitochondrial leader peptide.


INTRODUCTION

Folylpoly--glutamate synthetase (FPGS)() catalyzes the ATP-dependent formation of an amide bond between the -carboxyl group of the naturally occurring folates and the amino group of glutamic acid. The addition of glutamic acid moieties to folate compounds allows their intracellular retention and concentration for both the naturally occurring folate compounds (1, 2, 3) as well as for all of the ``classical'' folate antimetabolites studied to date. As a result of its role in the retention of folate cofactors in the cell, FPGS is essential for the survival of proliferating mammalian cells. This concept is most directly supported by the phenotype of a mutant CHO cell, AUXB1, which lacks measurable function of FPGS (3) and, as a result, is auxotrophic for thymidine, glycine, and purines (2) , i.e. it cannot provide 1-carbon units via folate metabolism.

The existence of mitochondrial and cytosolic folylpolyglutamate pools in mammalian cells and tissues has been known for some time (4, 5) . FPGS was previously thought to be located only in the cytoplasm, but subsequently it has also been found in mitochondria (6) . It is not clear whether the FPGS found in these two cellular compartments represents the same protein or how FPGS is targeted to the mitochondria. Recently, a cDNA that encoded a human FPGS was reported (7) . Upon transfection into the AUXB1 cells, this sequence complemented the auxotrophy for thymidine and purine, but not the requirement for glycine which is known to be synthesized in the mitochondria (8, 9) . This was unexpected, in view of the fact that AUXB1 cells transfected with high molecular weight human DNA regain prototrophy for thymidine and purines coincident with that for glycine and that such transfectants express FPGS in both cellular compartments (10) .

FPGS is abundant in tumors, and in normal gut and bone marrow stem cells, liver, and kidney but is, otherwise, not appreciably expressed in adult tissues (11, 12, 13, 14) . From a recent study in this laboratory (13) , it was concluded that FPGS levels are controlled by at least two mechanisms, one of which is linked to proliferation and the other acts during differentiation and is tissue-specific. Recent studies suggest that the sensitivity of human tumors to anti-folate chemotherapy is related to the level of expression of FPGS (13, 14, 15, 16) . In addition, initial evidence is supportive of the existence of isoforms of FPGS which accept different spectra of folate analogs as substrates (17) . Should appreciable differences exist in either the isoform or the control of expression of FPGS in normal stem cells and neoplastic cells, it would represent an opportunity for the design of selective cancer chemotherapy.

In this manuscript, we report the cDNA sequence corresponding to the 5` termini of mature FPGS transcripts and the organization and structure of the corresponding region of the human genomic locus. The sequences of the cDNA molecules studied and the transcriptional start site usage in human cell lines and in AUXB1 cells transfected with human genomic DNA indicated that two forms of FPGS mRNA were made from a single gene. One form corresponded to the published, apparently cytosolic form of the enzyme, and the other contained an additional 5` sequence compatible with a mitochondrial leader peptide which, when transfected into cells lacking FPGS, complemented the entire mutant phenotype.


MATERIALS AND METHODS

Nitrocellulose was from Schleicher and Schuell. T4 DNA ligase, T4 polynucleotide kinase, restriction enzymes, and Taq polymerase were from Promega. MaeIII endonuclease was from Boehringer Mannheim. Total RNA was prepared by the method of Chomczynski and Sacchi (18) . Poly(A)-selected RNA was isolated using the FastTracksystem (Invitrogen). The human cell lines used were CEM (acute lymphoblastic leukemia, T cell), MCF7 (breast adenocarcinoma), H209 (small cell lung cancer), LAN-5 (neuroblastoma), COLO 205 (colon adenocarcinoma) and HT-29 (colon adenocarcinoma). Growth conditions for these cells followed ATCC recommendations.

5`-RACE and PCR

5`-RACE was carried out essentially as described by Frohman et al. (23) . First strand cDNA was synthesized using 1 µg of poly(A)-selected CCRF-CEM RNA and 2 pmol of antisense primer F8 (5`-CTTGGTGAAGAGCTCAGGACTG-3`). PCR was carried out for 35 cycles (95 °C, 61 °C, and 72 °C for 1 min each) with 2.5 µl of the poly(C)-tailed template in a 25-µl reaction with the anchored primer and primer F21 (5`-ACTCCGTGCCAGGTACAGTTCCATG-3`). The PCR products were ligated into the pCRII vector (Invitrogen) for restriction analysis and double-stranded sequencing. The 5` region of the hamster cDNA was PCR-amplified using the human FPGS primers Sta 1 (5`-ACTATGTCGCGGGCGCGGAGCCAC-3`) and FPE2 (5`-GGTACAGTTCCATGGCTTCCAACTGTGTCTGAGGG-3`) with cycling conditions as before. From this sequence, hamster-specific primers were designed for 5`-RACE which was performed as described above. First strand synthesis was from 1 µg of CHO poly(A)-selected RNA with primer HF1 (5`-TTTACCTGCTCCAGGTAGCTG-3`). PCR was then carried out with the anchored primer and the nested primer HF2 (5`-AGCTGGCATTGGTCTGCAGGGTG-3`).

Cloning and Nucleotide Sequencing of the Human FPGS Gene

A PCR-generated cDNA fragment representing the first 690 bp of the published human FPGS open reading frame (7) was used as a probe to screen a human male placenta genomic library in the FixII vector (Stratagene) (24) . Restriction analysis of positive clones demonstrated that the 5`-most sequence of the FPGS cDNA lay on a 6.0-kb HindIII- NotI fragment of BL. This fragment was subcloned into pBluescriptII SK, and a 2-kb region was sequenced in both directions.

Determination of the Transcriptional Start Sites Using Ribonuclease Protection Analysis

Ribonuclease protection assays were performed essentially as described (24) . The probe was synthesized from a genomic DNA template between two RsaI sites and covered the first 175 nt of exon I and 203 nt immediately upstream (see Fig. 2). 1-2.5 10cpm of labeled RNA was used per sample. Hybridization was performed overnight at 57 °C, and the mixture was digested in 350 µl of 100 µg/ml RNase A for 30 min at 30 °C. Protected fragments were resolved on a 6% polyacrylamide/urea gel.


Figure 2: Nucleotide sequence of the putative promoter and first three exons of the human FPGS gene. The thymidine which was the 5`-most transcription site defined by 5`-RACE analysis is numbered +1. The major transcription start sites defined by ribonuclease protection analysis are marked with vertical arrows. Putative SP1 binding sites are underlined, Y boxes are in bold type, and the two proposed start methionines are boxed. The position of the polymorphism is indicated at +106 as are the RsaI sites used to synthesize the ribonuclease protection probe.



Generation of AUXB1-Human Genomic DNA Transformants

AUXB1 cells (2) were transfected by calcium phosphate precipitation (19) , using high molecular weight DNA from human CEM cells (20 µg/10cells) mixed with the plasmid pY3 (5 µg/10cells), which encodes resistance to hygromycin B (20) . Initial selection with 400 µg/ml hygromycin B was applied 48 h after transfection, and double selection was applied 10-14 days later by the addition of media lacking deoxyribo- and ribonucleosides, supplemented with 10% dialyzed fetal bovine serum and with hygromycin B. The cloned cell lines used in this study, FB1/2C and FC2/2D, were independently derived secondary transfectants from DNA of primary transfectant cells generated in separate experiments (21) . FPGS expressed in both cell lines was judged as CEM-derived on the basis of a ratio of activity with ATP to that with dATP characteristic of human enzyme (22) .

Generation of AUXB1-Human cDNA Transformants

The published FPGS cDNA was generated by reverse transcriptase PCR amplification of mRNA from CEM lymphoblasts and a fragment stretching from the proposed (7) downstream start methionine to 200 bp into the 3`-untranslated region was inserted into the eukaryotic expression vector pcDNA3 (Invitrogen) (pC-FPGS). To construct the vector which would express the putative mitochondrial isoform (pM-FPGS), the coding sequence identified in Fig. 1 a was inserted into pC-FPGS so that the two vectors differed only by the presence of the 42-codon putative leader sequence. Transfection was carried out essentially as described above (5 µg of plasmid/5 10cells), and selection was applied 48 h after transfection using G418 (800 µg/ml) in either complete -minimal essential medium or nucleoside-deficient medium. Three independent clones were selected by two subcultures of individual colonies from the pC-FPGS and pM-FPGS transfected AUXB1 cells surviving double selection. In the colony-forming assays, combinations of glycine (133 µ M), adenosine (36 µ M), and thymidine (36 µ M) were added to -minimal essential medium formulated without these components.


Figure 1: a, transcriptional start sites for human FPGS mRNA as determined by 5`-RACE analysis and comparison of amino acid sequence with hamster FPGS. Arrows represent the positions of the tsps; the numbers over the top represent site frequency (when >1) from 2 separate experiments. The consensus sequences for translation initiation are underlined with the codons for the proposed start methionines in bold type. Dots indicate identity between human and hamster sequences. b, helical wheel analysis of the amino-terminal extension peptide. Hydrophobic residues are boxed, and positive charges are indicated.




RESULTS

The 5` Sequence of FPGS Transcripts

We used the PCR-based 5`-RACE technique (23) to determine whether there was additional sequence upstream from the published GC-rich FPGS cDNA (7) represented in poly(A)-selected mRNA from CEM human leukemic cells. PCR with an anchor primer and the internally nested primer, located 202 bp from the 5` end of the published sequence, resulted in the amplification of a set of cDNAs ranging in size from approximately 150-300 nt. These PCR products were ligated into the pCR II plasmid; 25 clones were selected and sequenced with some bias toward longer clones. All of the inserts examined contained the published human FPGS sequence (7) , but 20/25 cloned inserts extended further upstream, with as much as 99 additional base pairs corresponding to the most 5` region of the CEM FPGS transcripts. Three transcriptional initiation starts were repetitively identified by this analysis (Fig. 1 a). A methionine codon was found in this upstream sequence which fit the consensus for efficient eukaryotic ribosome binding (25) and was in-frame with the previously defined (7) downstream translational initiation codon. The 42-amino acid sequence (Fig. 1 a) encoded by the nucleotides between the two ATGs had a noticeable absence of acidic residues, and a preponderance of arginine (6/42), serine (4/42), and leucine (4/42) residues relative to what would be expected for the amino terminus of cytosolic proteins. Helical wheel analysis of the first 36 residues from the amino terminus revealed the potential to form an amphipathic -helix with positively charged and hydrophobic faces (Fig. 1 b). These are all features thought to be essential in a leader peptide needed for the passage of proteins through the mitochondrial membranes (26, 27, 28) . Reported vertebrate leader peptides, which are proteolytically cleaved after entry into the mitochondria, range in size from 20 to 60 amino acids (26, 27) .

A Comparison of the 5` Termini of the FPGS Transcripts Found in Human and Hamster Cells

We were able to amplify a 250-bp region of the hamster FPGS cDNA using primers chosen from the upstream human cDNA sequence. The sequence of this region was homologous to the human FPGS sequence (Fig. 1 a). When the hamster sequence was extended using 5`-RACE, an upstream ATG was found at an identical location as in the human sequence and, again, this codon was in-frame with the downstream ATG and obeyed Kozak's consensus sequence (25) . The entire putative mitochondrial leader peptide was predicted to be 78% identical in hamster and human sequences. Downstream of the 3`-methionine, the amino acid homology was 93% over the sequence studied.

Cloning of the Human FPGS Genomic Locus

Forty positive plaques were isolated from a screen of 1.5 10recombinants; three classes of overlapping inserts were found which covered the entire human FPGS genomic locus. Mapping studies indicated that one clone (BL) contained the 5` end of the FPGS open reading frame and at least 10 kb of sequence upstream. A 6.0-kb NotI- HindIII fragment of BL which hybridized with the most upstream probes from the FPGS cDNA was subcloned into pBluescript II SKfor fine mapping and partial sequencing.

The first 363 nt of the FPGS cDNA (Fig. 2) were located within the 6.0-kb fragment, distributed onto three exons that were 180, 129, and 54 bp in length. The sequences of these exons, the positions of introns I and II which were 1039 and 85 nt long, respectively, and 361 bp immediately upstream from exon I are shown in Fig. 2 . Both upstream and downstream translational start sites were located on exon I, an observation that rules out alternative exon usage of this gene as the mechanism for the formation of cytosolic and mitochondrial forms of FPGS. The 5`-flanking region of the FPGS gene did not contain a canonical TATA sequence nor a CCAAT motif. However, two inverted CCAAT boxes, i.e. Y boxes, were present at positions -57 and -20 nt (Fig. 2). The 5`-flanking sequence and exon I were very GC-rich (74% from nt -221 to nt +180) and contained eight forward (GGGCGG) and one reverse (CCGCCC) SP1 binding sites.

Ribonuclease Protection Analysis of the Human FPGS Gene

Initial experiments using total RNA from CEM cells (Fig. 3 b, lane 9) indicated multiple transcriptional start points ( tsps) with major bands representing start sites at positions -2, +6, +46, +78, and +107 (with respect to the 5`-most transcription start site detected by RACE). The abundance of the small protected fragment at 69 bp (indicated by the hatched arrow on Fig. 3) had not been predicted by the RACE experiments (Fig. 1 a). Analysis of the sequence of the 5`-RACE-derived human cDNA clones demonstrated a single base pair polymorphism 69 bp from the 3` end of the riboprobe in CEM cell RNA (see Fig. 2): 63% of the clones analyzed had an A at this position and 37% had a G residue ( n = 19) (the residue present in the riboprobe). Because a single base mismatch is often sufficient for ribonuclease cleavage, this polymorphism introduced an uncertainty in the interpretation of these ribonuclease protection experiments and suggested that the 69-bp band was artifactual.


Figure 3: Definition of the transcriptional start sites of the human FPGS gene by ribonuclease protection. The RNA probe used was a 378-nt fragment positioned between the RsaI sites indicated in Fig. 2. a, lane 1 contains 1000 cpm of undigested riboprobe. Protection analysis was performed on 30 µg of yeast tRNA and on 2 µg of poly(A)RNA from AUXB1/CEM DNA transfectants FC2/2D RNA and FB1/2C RNA ( lanes 2-4, respectively). b, analysis was also performed on 30 µg of yeast tRNA and 30 µg of L1210 murine total RNA as controls ( lanes 5 and 6) and on 30 µg of total RNA from COLO 205, HT-29, CEM, LAN-5, MCF7, and H209 human cell lines ( lanes 7-12, respectively). Filled arrows represent major transcription start sites, the bracket represents a group of minor start sites, the hatched arrow indicates the position of the major band caused by the polymorphism, and the hollow arrows represent other cleavage products of the polymorphism, most clearly visible in lane 3 but also evident in lanes 7 and 8. The marker set used was HinfI-digested X174 DNA.



We used two complementary approaches to determine which bands represented bona fide tsps and which resulted from this polymorphism. In the first, we used cell lines in which the fpgsphenotype of AUXB1 cells was complemented by calcium phosphate-mediated transfection with high molecular weight DNA from CEM cells (21) . These transfectants would be expected to be haploid for the human fpgs locus due to the low frequency of complementation and the low amount of human DNA stably integrated per transfectant (19) . Two of these transfected cell lines were screened for the polymorphism using the fact that one of the allelic variants represented the recognition sequence (5`-GTNAC-3`) for the restriction enzyme MaeIII. Cell line FB1/2C contained only the human fpgs allele with a G at position 106, whereas FC2/2D contained only the other allele. In the second approach, several human cell lines were screened for the polymorphism using the MaeIII site and a selection of cell lines found to be either homozygotic and heterozygotic by this criterion were analyzed further.

The two patterns expected for opposite single alleles were seen in the reaction using RNA from the AUXB1 cells transfected with CEM DNA: RNA from the FC2/2D cell line did not protect the probe at position 106 permitting RNA cleavage at this site resulting in the presence of the 69-bp fragment, whereas RNA from the FB1/2C transfectant showed a simple pattern of multiple protected bands without the 69-bp fragment (Fig. 3 a). The fragments protected by RNA from FC2/2D cells ( lane 3, Fig. 3, hollow arrows) were exactly 69 nt less than the three longest major products protected by RNA from FB1/2C and appeared to be generated by cleavage at the tsp and at the polymorphic mismatch. The protection of RNA from six cell lines differing in homozygosity at position 106 of the fpgs locus is shown in Fig. 3 b. RNA from homozygous cell lines with sequence matching the probe (MCF7 and H209) did not result in a 69-bp fragment, and, hence, the protected bands from lanes 11 and 12 in Fig. 3 b directly indicate transcriptional start site usage. The reactions run with RNA isolated from the homozygous cell lines bearing the opposite allelic variant at position 106 resulted in the appearance of the 69-bp fragment ( lanes 7 and 8, Fig. 3 b). RNA from the heterozygous cell lines CEM and LAN-5 ( lanes 9 and 10) showed protection of the longer bands and allowed the cleavage of probe to the 69-bp fragment. The probe fragments protected from ribonuclease by RNA from the MCF7 and H209 cells and from the FB1/2C cells indicated the same set of multiple transcriptional start sites in all of these cell lines; the major sites are mapped to the upstream genomic structure of the FPGS gene in Fig. 2at positions -2, +6, +46, and +78.

The 5`-RACE and the ribonuclease protection experiments both indicate at least three major and multiple minor transcriptional start sites. The positions of the sites are consistent between the experiments within the constraints of these techniques.

Transfection of FPGS cDNAs into AUXB1 Cells

The ability of FPGS cDNAs carrying the additional upstream ATG to complement the adenosine, thymidine, and glycine auxotrophy in AUXB1 cells was evaluated using stable AUXB1 transfectants. The transfection efficiency of AUXB1 cells with the pC-FPGS and pM-FPGS constructs ranged between 1 in 400 to 1 in 800. Of the AUXB1 colonies which had acquired G418 resistance from the FPGS constructs, approximately 80% were viable on media lacking nucleosides.

The ability of several clonally isolated transfectant cell lines to form colonies with and without glycine, adenosine, and thymidine is shown in . As expected, the parental AUXB1 cells can form colonies only in the presence of glycine, adenosine, and thymidine, whereas cells transfected with pC-FPGS form colonies in the absence of nucleosides but still require glycine. Cells transfected with pM-FPGS did not require glycine or nucleosides, mimicking the wild type CHO and cells transfected with CEM genomic DNA (FC2/2D cells).


DISCUSSION

We have shown that the human FPGS promoter drives transcription from multiple tsps spread over approximately 80 bp. These mRNAs contain up to 99 bp of previously unreported 5` sequence including an AUG codon which is in-frame with the previously reported open reading frame and satisfies the requirements for efficient translation defined by Kozak (25) . Transfection of the published FPGS cDNA complemented only the thymidine and purine auxotrophy resultant from FPGS deficiency (7 and ). However, transfection of a cDNA corresponding to the extended message complemented the entire FPGSphenotype including auxotrophy for glycine, which is synthesized in the mitochondria. We propose therefore that differential usage of the two translational start codons determines the cytosolic or mitochondrial location of FPGS.

In yeast there are several enzymes present in both the cytoplasm and the mitochondria which originate from a single genetic locus. These include histidine-tRNA synthetase (29) , isopropylmalate synthetase (30) , fumarase (31) , valyl-tRNA synthetase (32) , and -isopentenyl pyrophosphate:tRNA isopentanyltransferase I (33) . Each of these genes contain two in-frame ATG codons, both of which can be used to initiate translation resulting in the production of proteins differing only by the presence or absence of an amino-terminal extension peptide. Although less frequent, this phenomenon has also been documented in mammals, examples of which include rat liver mitochondrial and peroxisomal serine:pyruvate aminotransferase (34) , and rat cytosolic and mitochondrial fuma-rases (35) . In both of these cases, the two potential translation initiation codons are found within exon I as in the FPGS gene. The mRNA which codes for rat mitochondrial serine:pyruvate aminotransferase is transcribed from a start site 70 bp upstream of that for the cytosolic form and encodes an amino-terminal 22-residue peptide essential for the translocation of the protein into the mitochondria. For the rat fumarases, it has been proposed that both mitochondrial and cytosolic forms are translated from one species of mRNA and that it is the secondary structure of the 5`-noncoding region which determines initiation codon usage (35) . The ratio of cytosolic to mitochondrial FPGS levels may therefore be determined simply by the relative levels of start site usage or there may be a component of translational regulation.

In CHO cells, 50-65% of total cellular FPGS activity was reported to be associated with the mitochondrial fraction (6) . We quantitated the intensity of RNase-protected bands in the Ghomozygous cell lines using a phosphorimaging system. From four evaluable cell lines, 66-81% of transcripts contain sufficient upstream sequence for translation of the mitochondrial FPGS precursor. In a recent study, Escherichia coli FPGS cDNA was transfected into AUXB1 cells (36) . E. coli FPGS was expressed only in the cytosol, and this expression reversed the auxotrophy for purines and thymidine of these cells, but not that for glycine, the synthesis of which is thought to occur mainly in the mitochondria (8, 9) . E. coli FPGS directed to the mitochondria only, using a synthetic leader sequence, complemented all three growth requirements. We now report the occurrence, sequence, and function of the endogenous mitochondrial leader sequence for FPGS. It would appear that folylpolyglutamates cannot enter the mitochondria, but their function is required in that organelle for glycine synthesis.

Analysis of the putative FPGS promoter revealed a GC-rich region with no canonical TATA or CCAAT sequences, a structure usually associated with ``housekeeping'' genes and proto-oncogenes. TATA-less promoters were previously believed to drive unregulated, constitutive expression but have since been shown to respond to a variety of stimuli with a range of transcriptional regulatory responses. This group includes genes that are expressed differentially during embryogenesis, are tissue-specific, and respond to viral and pharmacological stimuli (reviewed in Ref. 37). Many of these genes are growth-regulated with low levels in nongrowing cells which become elevated as cells are stimulated to proliferate. This is consistent with the distribution of FPGS activity in different cell types and various growth and differentiation states (12, 13) . Numerous transcription factor binding sites were found in the promoter region and in intron 1 (not shown), but their relative importance to FPGS gene regulation remains to be determined. Of particular interest will be the significance of the two Y boxes in the immediate promoter area. In MHC II genes, the Y box consensus sequence is required for constitutive and cytokine-mediated gene expression (38) . Y box consensus sequences in the proximal promoter region are also required for the basal expression of the human MDR1 gene (39) and the thymidine kinase gene (40, 41) .

This study supports the hypothesis that the mitochondrial and cytosolic forms of FPGS are derived from a single gene. The control of expression of this gene is of practical significance: four recent reports of pediatric acute lymphoblastic leukemia (13, 14, 15, 16) have suggested a correlation between levels of FPGS activity in leukemias and the outcome of antifolate chemotherapy. Clearly, a basic understanding of the regulation of this enzyme in normal stem cells and neoplastic cells is now needed.

  
Table: Viability of CHO cell transfectants in media with and without exogenous glycine

Colony-forming assays were carried out in 100-mm tissue culture dishes using 10 ml of media per plate. 150 cells were plated per dish in GAT media and left overnight. The next day, cells were washed once in PBS, and the selective media were applied. After 8 days, the colonies were stained and counted.



FOOTNOTES

*
The work was supported by Grant CA 27605 from the DHHS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U14938 and U14939 (hamster and human sequences, respectively).

§
Fellow of the Leukemia Society of America.

To whom correspondence and reprint requests should be addressed: Massey Cancer Center, Medical College of Virginia, MCV Station Box 980230, Richmond, VA 23298. Tel.: 804-828-9645; Fax: 804-828-5782; E-mail: rmoran@gems.vcu.edu.

The abbreviations used are: FPGS, folylpoly--glutamate synthetase; 5`-RACE, 5`-rapid amplification of cDNA ends; tsp, transcription start point; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s); nt, nucleotide(s).


ACKNOWLEDGEMENTS

We thank Drs. Julie L.-C. Kan and Eric Westin for their useful discussions throughout this work. We also appreciate the guidance and encouragement of Dr. Norman Davidson during the early phases of related work which led to this study.


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