INTRODUCTION

The intracellular accumulation of folic acid and reduced folates is essential for the survival of mammalian cells. Folates cannot be synthesized endogenously but are required as precursors for the biosynthesis of purines, pyrimidines, and amino acids.

There are two known systems for the internalization of folic acid and its reduced forms. One system consists of a membrane-bound 40-kDa glycoprotein folate receptor termed folate-binding protein. Folate-binding protein has a high affinity for folic acid and a low affinity for reduced folates including the chemotherapeutic agent methotrexate (Mtx)¹ (1, 2, 3, 4, 5, 6, 7, 8). The cDNA encoding folate-binding protein has been cloned from various sources (9, 10, 11, 12, 13, 14), and its genomic structure has been determined (15, 16, 17).

The second system consists of a 36-78-kDa glycoprotein termed the reduced folate carrier, which has a higher affinity for reduced folates including Mtx and a lower affinity for folic acid (18, 19, 20, 21, 22). Several cDNAs that putatively encode the reduced folate carrier have recently been cloned from human (23, 24, 25, 26), mouse (27), and hamster (28) cells.

The availability of cDNA clones from various sources provides a powerful tool for examining folate transport processes. However, at present very little is known about the molecular details of folate transport.

We have previously described the isolation of both a hamster genomic DNA cosmid clone (29) and a cDNA clone (pMtxT9) (28) that can, upon transfection, complement mutant Chinese hamster ovary (CHO) cell lines that are unable to take up folates. Mutant cells transfected with the cDNA are able to bind and take up Mtx and thus become sensitive to the cytotoxic action of the drug.

In our continuing studies to understand the molecular nature of the reduced folate carrier gene we have localized the gene for hamster rfc to chromosomes 1 and Z1 at position q2-q3 in CHO cells (30). The present study describes the organization of the hamster rfc gene, the identification of two alternatively spliced mRNA isoforms, and the mapping of the transcriptional start site.

EXPERIMENTAL PROCEDURES

The wild-type, Mtx-sensitive (Pro3) and Mtx-resistant (Pro3 MtxRII 5-3) cell lines and their maintenance have been previously described (31, 32). The cosmid clone, 100-2, containing CHO DNA sequences that complement a mutant cell line defective in Mtx transport has been previously described (29). The isolation of the cDNA clone, pMtxT9, and its properties have been described (28).

High molecular weight DNA was isolated from exponentially growing CHO cells by the procedure of Gross-Bellard et al. (33).

Cosmid and plasmid clones were propagated in Luria-Bertani medium supplemented with ampicillin or ampicillin plus tetracycline. DNA was isolated from overnight cultures using the Qiagen plasmid kit as described by the manufacturer.

High molecular weight DNA or cosmid DNA was digested with either BamHI or HindIII (Pharmacia Biotech Inc.) according to the conditions of the supplier. The digested DNA was separated on a 0.8% agarose gels and transferred onto Biotrans nylon paper (ICN Biomedicals) by the method of Southern (34). Blots were prehybridized for 24 h at 65 °C in 5 × SSC (1 × SSC is 0.15 M sodium chloride, 0.015 M sodium citrate), 5 × Denhardt's solution, 200 µg/ml salmon sperm DNA, and 0.1% SDS. Labeled DNA from the plasmid pMtxT9 was added to the hybridization mixture to a final concentration of 2-4 ng/ml.

After 48 h at 65 °C the blots were washed two times for 15 min with 2 × SSC, 0.1% SDS at room temperature and three times for 15 min with 0.1 × SSC, 0.1% SDS at 50 °C. Blots were exposed to x-ray film at 70 °C with intensifying screens.

The CHO rfc cDNA fragment from the plasmid pMtxT9 was used as probe and labeled with -[³²P]dCTP (ICN Biomedicals; 3000 Ci/mmol) by the random priming method described by Feinberg and Vogelstein (35). Labeled DNA was routinely obtained at a specific activity of 5-10 × 10⁸ cpm/µg.

Oligodeoxynucleotides used as probes were end-labeled with T4 polynucleotide kinase (Pharmacia) and -[³²P]ATP (ICN Biomedicals; 7000 Ci/mmol) to a specific activity of approximately 1 × 10⁸ cpm/µg.

The initial restriction endonuclease map of cosmid 100-2 was derived from partial endonuclease digestion and hybridization with either T3 or T7 end-labeled sequencing primers as described previously (29). Fine mapping of restriction endonuclease sites was done by hybridizing Southern blots with end-labeled oligodeoxynucleotide primers that span the cDNA sequence in pMtxT9.

Double-stranded DNA sequencing was performed by the dideoxy chain termination method using the T7 sequencing kit supplied by Pharmacia. Sequences were obtained using either T7 or Sp6 sequencing primers or synthetic oligodeoxynucleotide primers spanning the cDNA sequence in pMtxT9.

Cosmid clone fragments chosen for sequence analysis were isolated and subcloned into the vectors pGEM3 or pGEM4 and propagated in Escherichia coli strains SURE®, XL1-Blue, or JM109.

Poly(A⁺) RNA was isolated from ~2 × 10⁸ exponentially growing Pro3 cells using the Fast Track mRNA isolation system (Invitrogen). Approximately 30 µg of poly(A⁺) RNA was obtained from this number of cells.

Five µg of poly(A⁺) RNA from Pro3 cells were resolved on a 1.2% agarose gel containing 10 mM sodium phosphate (pH 7.0) as described previously (28). After electrophoresis, the RNA was transferred to Biotrans nylon paper (ICN Biomedicals) as described previously (28). Hybridization, washing, and autoradiography were performed as described previously for Southern blotting (29).

First-strand cDNA was synthesized from 1 µg of poly(A⁺) RNA using Superscript II reverse transcriptase (Life Technologies, Inc.) according to the conditions recommended by the supplier. Sequences corresponding to pMtxT9 were amplified from the first-strand cDNA using a 20-mer forward primer (P1: 5-GTGTTGTAGTGCGCGTGGTG-3) that was designed at base pair 221 of the rfc mRNA1 sequence and a 20-mer reverse primer designed at either base pair 208 (P2: 5-GCACCGGAATGATCTCATTG-3) or base pair 2067 (P3: 5-TGTGCCTTGGCTGGTGTCTG-3) for 30 cycles of amplification using standard PCR conditions. The PCR products were separated by electrophoresis on 0.8% agarose gels or 5% acrylamide gels and visualized by ethidium bromide staining. The PCR products were subcloned using the TA cloning kit (Invitrogen) and were sequenced as described above.

The mRNA isoforms were amplified by RT-PCR from Pro3 cells using the 20-mer forward primer P1 and a 20-mer reverse primer designed at base pair 1733 (P4: 5-GGAGAGGTTGCTTAAGTCAG-3). The resulting PCR product spans full-length coding sequences. The Expand polymerase mixture (Boehringer-Mannheim) was used to minimize polymerase errors. The PCR products were directionally cloned into the pCR3 vector using the unidirectional TA cloning kit (Invitrogen). The PCR product clones were screened by restriction endonuclease digestion and DNA sequencing to identify clones of the larger DNA isoform.

Transfection of the PCR product clones was carried out by the polybrene procedure as described previously (36). Ten µg of DNA per 1-2 × 10⁵ cells were transfected into the mutant Pro3 MtxRII 5-3 cells. After transfection, cells were selected by growth in low levels of folinic acid (2 nM) (37). Cells able to grow in the selective medium were isolated and tested for sensitivity to Mtx as described previously (31).

Rapid Amplification of cDNA Ends (5-RACE)

Total RNA was isolated from 1 × 10⁷ Pro3 cells using a guanidinium-isothiocyanate-phenol-chloroform method (38). 5-RACE was performed using the 5-RACE System according to the conditions of the supplier (Life Technologies, Inc.). Reverse transcription was primed with a 20-mer reverse primer designed at base pair 500 (P5: 5-ACAGAGCTGGTGAAGACTCC-3) relative to the pMtxT9 sequence. Tailed products were amplified by PCR using the 5 anchor primer and a nested reverse primer (P2). These products were diluted 20- or 50-fold and reamplified using the nested universal amplification primer (Life Technologies, Inc.) and a specific reverse primer designed at base pair 149 (P6: 5-GGTGTGATGAAGCTCTCC-3). The RACE products were cloned using the TA cloning kit (Invitrogen) and sequenced as described above.

Antisense probes were prepared from a 400-bp subclone of the cosmid 100-2 in the vector pGEM4, which contained sequences extending 109 bp upstream of the first nucleotide of pMtxT9. Probes were synthesized using T3 polymerase (Pharmacia) incorporating -[³²P]CTP (DuPont NEN; 800 Ci/mmol). The probe was purified on a 5% denaturing acrylamide gel. Approximately 5 × 10⁵ cpm were combined with 2.5 µg of poly(A⁺) mRNA or 3 µg of yeast RNA and hybridized for 18 h at 45 °C. Ribonuclease digestion was performed using the RPA II kit according to the supplier's recommendations (Ambion Inc.). Protected fragments were separated on a 20% denaturing acrylamide gel. Sizes were approximated using ³²P end-labeled oligodeoxynucleotides.

RESULTS

Previously we have isolated a genomic clone and the corresponding cDNA that are able to complement the phenotype of a mutant hamster cell line that is defective in Mtx uptake. The availability of both genomic and cDNA clones for this function allowed the determination of its genomic organization. To determine the intron/exon organization of the gene, synthetic oligodeoxynucleotides derived from regions spanning the cDNA were hybridized to DNA from the cosmid to identify complementary sequences. DNA sequencing was performed either directly on the cosmid DNA or on subcloned fragments to determine the intron/exon boundaries. These analyses revealed 7 exons and 6 introns that span approximately 15.3 kb (Fig. 1A). The donor and acceptor sequences (Table I) conform to consensus GT/AG splice site sequences with the exception of the donor splice site of intron 3. This site has a C replacing the consensus T at the +2 position of the intron. Although it is rare, this substitution has been observed in other donor splice sites (39).

Table I. Intron-exon boundary sequences of the hamster reduced folate carrier Exon sequences are shown in uppercase letters, and intron sequences are shown in lowercase letters. Exon Size Position in cDNA Donor Intron Size Acceptor Exon Exon Intron Intron Exon bp kb 1 53  221  to 169 GCTGAACCAG gtacaggcct 1 0.47 atccctctag AACCGAGTTC 2 2 121  168  to 48 TCCACGCCTG gtacgtggca 2 4.5 taccttccag GTGGAGTGTC 3 3 230  47  to 183 AATCGAGCAG gcatgttgtt 3 1.75 catcccacag GTGACCAATG 4 4 757 184  to 940 ACACTGCTGA gtatgcactg 4 0.5 ctacctccag GCGCCATCAC 5 5 211 941  to 1151 CCATTGCCAC gtgagtagga 5 1.7 acctccccag TTTTCAGATT 6 6 142 1152  to 1293 TGAAAAACAG gtaagctcca 6 3.6 tctctttcag TTTTGCATCT 7 7 1,233 1294  to 2526 AGACTCTGTT

The exon sequences were in agreement with the previously reported cDNA sequence (28) with one exception. The original cDNA was isolated from a CHO-K1 cDNA expression library. The cDNA contains the sequence AGA beginning at base pair 1459, which codes for arginine in the predicted amino acid sequence. The cosmid 100-2, which contains genomic DNA sequences from the Pro3 cell line, has the sequence GGA at this position, which codes for a glycine residue. This difference appears to be a polymorphism, because other cDNA clones from the CHO-K1 library contain the AGA sequence at this position, while cDNA clones and RT-PCR sequence data from the Pro3 cell line all contain the GGA sequence.² Although the significance of this change is not clear, both cosmid DNA from Pro3 and cDNA from CHO-K1 or Pro3 (see below) are able to complement the Mtx transport defect in the mutant cells, which indicates that this change may not be functionally important.

The cosmid 100-2 was isolated from a Pro3 genomic cosmid library by two rounds of genomic cloning (29). In order to ensure that the genomic organization of 100-2 reported above had not been altered during the selection and cloning process, these sequences were compared to genomic DNA isolated from the Pro3 cell line by Southern hybridization using the cDNA sequence as a probe (Fig. 2). The restriction endonuclease pattern of Pro3 genomic DNA as compared to cosmid 100-2 indicated a similar structure, with some exceptions. The largest fragments in both the BamHI and HindIII digests of cosmid 100-2 DNA are smaller than their counterparts in genomic DNA. This difference is due to truncation of the ends of the DNA in the cosmid 100-2 as a result of the cloning process (Fig. 1A). Fig. 1A also illustrates the change in the order of the restriction enzyme sites at the 3 end of the cosmid 100-2. This difference may also be due to the truncation of this end during the cloning process, which involved two rounds of genomic cloning in order to isolate the complementing genomic clone (29).

Another difference in the structure of the cosmid 100-2 is shown by the change in size of the smallest fragment in the BamHI digest. This fragment is approximately 500 bp smaller in the cosmid DNA as compared to the genomic DNA. This difference is due to a deletion in intron 2 of the cosmid 100-2 (Fig. 1A).

The other bands in the Southern blot analysis, including the 4.3-kb BamHI doublet and the 6.4-kb HindIII band, were comparable between the cosmid 100-2 and genomic sequences.

To determine the nature of the messenger RNA produced in Pro3 cells, a Northern blot of poly(A⁺) selected mRNA was probed with the cDNA sequence (Fig. 3A). The Northern analysis indicated that there were two mRNA species approximately 2.5 kb and 2.6 kb in size. The two mRNAs were present in similar amounts in Pro3 cells, since the band intensities are comparable (Fig. 3A). Amplification of the hamster rfc gene using first-strand cDNA synthesized from Pro3 cells confirmed the presence of two mRNA species that differed in size by 121 bp (Fig. 3B). Using PCR primers specific for the 5 end of pMtxT9, the 121-bp difference was localized to this portion of the pMtxT9 sequence (Fig. 3C). The PCR products shown in Fig. 3, B and C, were cloned and sequenced. The sequence of the lower molecular weight species was identical to the sequence of pMtxT9. The sequence of the higher molecular weight species contained an additional 121 bp inserted at position 48, which was known to be a splice junction (Fig. 4). An oligodeoxynucleotide specific for the 121-bp sequence was used to map the new exon relative to the genomic sequence in the cosmid 100-2. This exon was labeled exon 2 and the numbering of the 5-untranslated region of the previously reported pMtxT9 sequence (28) was changed to include the new exon (Fig. 4). Therefore the hamster rfc gene codes for two mRNA species (Fig. 1B), rfc mRNA1, which contains exons 1 through 7, and rfc mRNA2, in which exon 2 is absent.

DNA sequences at the 5 end of the

The addition of this exon occurs 5 to the putative translational start site. Thus it was anticipated that there should be no functional difference between the protein products encoded by the two messages. To confirm this, the two mRNA species were amplified by RT-PCR and cloned into the expression vector pCR3 (Invitrogen). The resulting RT-PCR clones were sequenced, and three clones of the large mRNA isoform, which contain the 121-bp second exon, were transfected into the mutant Pro3 MtxRII 5-3 cell line and selected for the ability to complement the mutant phenotype. All three clones were able to rescue the phenotype at a frequency comparable to that of the cDNA clone pMtxT9, which corresponds to the mRNA2 sequence.² This indicates that there is no apparent functional difference between the mRNA isoforms with respect to their ability to complement the mutant phenotype.

To ensure that DNA fidelity was maintained during the amplification process, the sequence of the RT-PCR clones was compared to the cDNA sequence. Three sequence alterations that were presumably due to polymerase error were noted in the three RT-PCR clones. One of the three clones had no sequence alterations, while another clone had a silent mutation at base pair 780. The third clone had two point mutations, a silent mutation at base pair 1648 and a mutation at base pair 853 that resulted in the substitution of methionine for leucine at amino acid 285 in the predicted amino acid sequence. This change is relatively conservative and did not appear to affect the ability of the clone to rescue the mutant phenotype.

The 5-region of the Hamster Mtx Transport Gene

The transcriptional start site of the hamster rfc gene was mapped using 5-RACE and RNase protection analysis. The results of the 5-RACE analysis are indicated in Fig. 4. Thirty-three clones were obtained and sequenced. The majority of the clones mapped to six sites near the beginning of the pMtxT9 sequence with two clones mapping to a site at base pair 53 of the putative open reading frame.

RNase protection was used to confirm the location of the transcription start sites. Six RNase protection products were detected (Fig. 5) whose positions corresponded well with the 5-RACE products near the beginning of the pMtxT9 sequence (Fig. 4). The predominant RNase protection product appears to correspond to the first base pair of the pMtxT9 clone (Fig. 4). Two minor bands were seen in the RNase protection approximately 50 bp upstream of the major RNase protection product. These bands were not very intense but may indicate that there is a low level usage of an upstream transcription start site. No 5-RACE products corresponding to an mRNA of this size were detected.

Two hundred sixty-one base pairs of sequence upstream of the major transcriptional start site from the cosmid 100-2 are also shown in Fig. 4. This sequence is consistent with a ``housekeeping'' gene promoter sequence in that it is fairly GC-rich, does not contain a TATA box element, and contains a consensus binding site for the Sp1 transcription factor at 346 (40).

DISCUSSION

This report describes the organization of the hamster rfc gene, the characterization of two alternatively spliced transcripts, and the identification of the transcriptional start sites. The gene is organized into seven exons and six introns. The sequences at the splice site junctions conform to the consensus GT/AG with the exception of the donor splice site of intron 3, which contains a C instead of a T at the +2 position of the intron. Although this splice site does not conform to the consensus, it appears to function efficiently in CHO cells. We have not been able to detect a splicing error at this intron/exon junction in mRNA from Pro3 cells using RT-PCR analysis.

Splicing errors at other splice site junctions do seem to occur in the hamster rfc gene. In screening the mRNA from Pro3 cells by RT-PCR, we were able to detect a potential splice error at the splice acceptor site of exon 6. Two RT-PCR clones have been isolated that contain either a 7- or 29-bp deletion at the beginning of exon 6.² The two deletions both begin at the first base pair of exon 6 and end after the next two downstream AG sequences, indicating that they may be cryptic splice acceptor sites. The deletions in these RT-PCR products correspond to the 29-bp deletion previously reported in Williams et al. (28) for clones pMtxT5 and pMtxT7. Since the 29-bp deletion is found in both a cDNA library and by RT-PCR analysis, there must be a proportion of mRNAs in the cell that contain the deletion. Although our PCR conditions were not quantitative, the majority of PCR products isolated did not contain the 29- or the 7-bp deletion.

Another possible splicing error that involves the splice site junctions that flank exon 4 has been reported in the literature. The hamster rfc cDNA clone pMtxT5 (28) contains a 757-bp deletion which is the result of the loss of exon 4. The 3 end of this deletion corresponds to the 3 end of the 119-bp deletion reported by Brigle et al. (41) for the mouse rfc message. Although the intron/exon structure of the mouse rfc gene is not known, it is likely that this deletion also involves a splice site junction. In the present study, we have not been able to detect any sequence alterations that would correspond to either of the above deletions using RT-PCR analysis on mRNA from Pro3 cells.

These deletions may represent aberrantly spliced messenger RNAs that are present at very low levels and are detected due to the sensitivity of the polymerase chain reaction. We have not been able to detect mRNAs containing these deletions by Northern blot analysis, although we can detect the alternatively spliced messages that only differ in size by 121 bp. All of these deletions occur at splice junctions, but it is not clear whether they are simply splice errors or if they represent another level of regulation of this gene. It should be noted that a construct containing the full-length cDNA sequence with the 29-bp deletion at the beginning of exon 6 is not able to complement our mutant cells upon transfection.² This indicates that the protein produced from a transcript containing the 29-bp deletion is not functional, at least in our assay system.

The hamster rfc gene encodes two alternatively spliced transcripts. The transcripts differ with respect to the presence or absence of a 121-bp exon in the putative 5-untranslated region. Upon transfection, the larger mRNA isoform is able to complement the mutant phenotype at a frequency similar to the original cDNA clone pMtxT9, indicating that there is no significant functional difference between them in this assay system. Although this is the first report of an alternative splice in the rfc gene, we also see a doublet band on a Northern blot of mouse RNA.² It will be interesting to see if the two mRNA isoforms show any differences in spatial/temporal expression or translational regulation in an intact organism.

We have mapped the transcriptional start sites of the rfc gene to several positions approximately 200 bp upstream of the translational start site. The RACE data and RNase protection data taken together indicate that there are approximately six transcription start sites close to the beginning of the pMtxT9 cDNA clone. The size of transcripts generated from these start sites agree with the observed size of the rfc mRNA on a Northern blot. The use of imprecise transcriptional start sites is common in TATA-less ``housekeeping'' genes (40).

Two additional bands in the RNase protection assay indicate that there may be a low level usage of an upstream transcriptional start site. We have not been able to detect any 5-RACE products that would correspond to these bands, nor do we see a larger mRNA on a Northern blot. The possible use of an upstream transcriptional start site is currently being investigated using RT-PCR.

Brigle et al. (41) have shown an alignment of the translated nucleotide sequence upstream of the predicted ATG initiation codon from the CHO and L1210 rfc cDNAs. The authors point out that the degree of similarity and lack of an upstream in-frame termination codon may indicate that these sequences encode additional protein information. However, an analysis of the putative 5-untranslated region of the human cDNA sequence, which does not contain an in-frame stop codon, revealed very little homology with the hamster or mouse sequences. This is in contrast with the amino acid sequence downstream of the putative ATG initiation codon, which is highly conserved among hamster, mouse, and human (23). These observations combined with the transcriptional start site mapping data presented in this paper and the size of the messenger RNA on a Northern blot imply that the predicted ATG initiation codon is the major translational start site.

The information obtained in these studies should prove useful for determining the state of the rfc gene in both normal and transport-deficient cell systems.