Characterization of the Entire Transcription Unit of the Mouse Fibroblast Growth Factor 1 (FGF-1) Gene
TISSUE-SPECIFIC EXPRESSION OF THE FGF-1.A mRNA*

Francesca Madiai, Kevin V. Hackshaw, and Ing-Ming ChiuDagger

From the Department of Internal Medicine and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fibroblast growth factor 1 (FGF-1, also known as acidic FGF) is a mitogen for a variety of mesoderm- and neuroectoderm-derived cells, as well as an angiogenic factor in vivo. It has been implicated in angiogenic diseases including atherosclerosis, cancer and inflammatory diseases. In the present study, the entire transcriptional unit of the mouse FGF-1 gene, including four promoters, is characterized. By nucleotide sequence and RNase protection analyses, we have determined that its 3'-end resides 3.2 kilobase pairs downstream from the stop codon. We have previously cloned and characterized the mouse homologue of the human 1B promoter, as well as a novel upstream untranslated exon. In order to elucidate the regulatory mechanism of FGF-1 gene expression, the mouse promoter containing TATA and CAAT consensus sequences (FGF-1.A) was isolated from a P1 library and characterized. We further determined that the mouse heart is the most abundant source for the FGF-1.A mRNA. Finally, via both RNase protection analysis and 5'-rapid amplification of cDNA ends, we determined the transcription start site of the FGF-1.A mRNA.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fibroblast growth factor 1 (FGF-1, also known as acidic FGF)1 is the prototype, along with FGF-2, of a family of at least 17 related polypeptides that possess broad mitogenic and cell survival abilities (1-14). These peptides share 30-60% amino acid sequence identity, a similar exon/intron structure in the protein coding region of the gene, and an affinity for the glycosaminoglycan heparin.

FGF-1 exerts its effect on target cells through high affinity tyrosine kinase cell surface receptors (FGFR1, FGFR2, FGFR3, and FGFR4) (15) and low affinity heparan sulfate proteoglycans (16). Upon binding to these receptors, FGF-1 elicits a variety of cellular responses, such as differentiation and angiogenesis (17), as well as tissue repair (18), wound healing (19), and cell survival (20). Although the mechanism of FGF-1 transport and signaling has not been fully unraveled (21), the current data suggest that this growth factor is exported outside the cell, thereby interacting with cell surface receptors in an autocrine and/or paracrine manner. The lack of a classical signal peptide notwithstanding, endogenous FGF-1 is exported via unconventional secretion pathways, such as in response to oxidative stress (22), heat shock (23), and serum starvation (24).

FGF-1 is expressed in a variety of developing and adult tissues of both mesodermal and neuroectodermal origin, where it promotes their proliferation (25, 26). More specific to the cardiovascular system, FGF-1 has been shown to have a role in heart development, as it is expressed in heart (27), cardiac myocytes (28), smooth muscle cells of the vascular wall (29), and cultured vascular smooth muscle cells (30), where it stimulates cell growth by inducing angiotensin converting enzyme (31).

We have previously cloned and characterized the mouse FGF-1 coding region (32). It is composed of three protein coding exons of 203 (from the splice acceptor site), 104, and 192 bp (excluding the stop codon and the 3' untranslated sequence), similar to its human counterpart (33). The FGF-1 gene is characterized by both a long 3'-untranslated region (UTR) (34) and a complex 5'-UTR, which in the human gene is composed of at least four untranslated exons designated -1A, -1B, -1C, and -1D (35). The alternative splicing of these untranslated exons to the first protein coding exon generates mRNAs 1.A, 1.B, 1.C, and 1.D, respectively, which are expressed in a tissue-specific manner (35).

The 1.A cDNA, although initially isolated from human brain (36) and kidney (37), is predominantly expressed in heart and kidney (35, 38). The 1.B transcript is instead almost exclusively expressed in brain and several glioblastoma cell lines (35, 39). The remaining two mRNA species are present in various tissue culture cells, with 1.D mRNA being the major transcript (30, 40). Sufficient evidence now points to FGF-1.A and -1.B transcripts being involved in cell maintenance and survival, particularly in tissues of cardiac and neuronal origin, whereas FGF-1.C and -1.D possibly serve as markers for certain proliferating cells (38). So far, the most extensively characterized of these upstream exons is the brain-specific 1B promoter (41-43).

We have previously cloned the mouse homologue of the -1B exon (44). Via in situ hybridization with RNA probes specific for the 1.B transcript, we determined that this message is expressed in the adult mouse sensory and motor nuclei in the midbrain, brain stem, and spinal cord, as well as in the granule cell layer and deep nuclei of the cerebellum (44). In addition, we have isolated a mouse genomic clone containing a novel 5' untranslated exon (45), which we designate -1G. This exon is the mouse homologue of the 5'-untranslated sequences present in a rat FGF-1 cDNA originally isolated from a rat prostate tumor cDNA library (46).

To gain a better insight into the expression of FGF-1, we isolated the mouse homologues of the exons -1A and -1C. We showed, via RNase protection analysis, that the mouse heart is the most abundant source for the FGF-1.A mRNA. DNA sequence analysis and mapping of the start site of this upstream untranslated exon (located 374 nt 5' of the -1A splice donor site) revealed great similarity with its human homologue (35). Additionally, we have determined the position of exon -1A, as well as that of exons -1B, -1C, and -1G, in the mouse FGF-1 locus. Furthermore, via DNA sequencing and RNase protection analysis, we have identified the 3'-end of the mouse FGF-1 gene, which resides 3.2 kb downstream of the stop codon. As such, the entire transcriptional unit of the mouse FGF-1 gene spans over 90 kb.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Library Screening-- P1 clones 7520, 7521, and 7522 were obtained (Genome Systems, Inc., St. Louis, MO) by polymerase chain reaction (PCR) screening of a P1 library using primers that amplify 109 bp of the mouse FGF-1.B exon. C129SVJ mouse genomic DNA had been partially digested with Sau3AI and cloned into the BamHI site of the P1 cloning vector, pAd10sacBII (47, 48).

Southern Blotting and Hybridization-- P1 DNA and plasmid DNA were prepared via standard methods (49) or using the BIGGERprep® and PERFECTprepTM plasmid isolation kits from 5 Prime right-arrow 3 Prime, Inc. (Boulder, CO). P1 DNA and plasmid DNA were digested with appropriate restriction enzymes, electrophoresed on agarose gels, and blotted onto HybondTM-N nitrocellulose filters according to standard procedures (49). Both prehybridization and hybridization were carried out in 6× SSC, 1× Denhardt's solution, 0.5% SDS, 100 µg ml-1 herring sperm DNA, and 1 × 106 cpm ml-1 of the appropriate 32P-labeled probes at 65 °C. The filters were washed in 1.0× SSC, 0.1% SDS and 2.0× SSC, 0.1% SDS either at 60-65 °C or at room temperature and exposed to x-ray film.

Polymerase Chain Reaction-- A 585-bp fragment containing 99 bp of exon -1A sequence present in the RT-PCR clones and 486 bp of upstream sequence, including CTAAT and TAATA, was amplified from pFM1A1 DNA using primers 3 and 4 (see Table I) with the eLONGaseTM enzyme mix. The reaction consisted of the following cycling temperatures and times: 94 °C for 30 s, 60 °C for 30 s, and 68 °C for 2 min. The resulting amplicon (nt 458-1042 in Fig. 3B) was subcloned into pBluescript KS(+) and designated pFM1A6.

To amplify a fragment spanning exons -1A and -1B, -1A-specific primer 2 and -1B-specific primer 7 (see Table I) were annealed to P1 7520 and 7521 DNA and then extended with the eLONGaseTM enzyme mix according to the following cycling temperatures and times: 94 °C for 30 s, 60 °C for 30 s, and 68 °C for 1 min/kb of product, for 35 cycles. The primers used to amplify DNA between exons -1G and 1 are primer 8 and primer 1 (see Table I), and the primers used to amplify DNA between exons -1C and -1G are primers 9 and 10 (see Table I). Both sets of primers were annealed to P1 980 DNA (see Fig. 8A) and extended with the eLONGaseTM enzyme mix, according to the same conditions described above.

Nucleotide Sequencing and DNA Sequence Comparison-- All DNA fragments to be sequenced were subcloned into pBluescript KS(+) vector (Stratagene, La Jolla, CA) using T4 DNA ligase (Roche Molecular Biochemicals and Life Technologies). Double stranded sequencing was carried out via the dideoxy chain termination method (50) using the Sequenase version 2.0 kit (Amersham Pharmacia Biotech) and T3 and T7 oligonucleotide primers (Roche Molecular Biochemicals) as well as synthetic primers derived from newly obtained sequences. The ALIGN and DOTPLOT computer programs of DNA Star (DNA Star, Madison, WI) were used to compare mouse and human DNA sequences in the 1A promoter region of Fgf1.

RNase Protection Analyses-- Mouse tissues were harvested and either frozen away at -80 °C or immediately homogenized in 2 ml of RNAzolTM (Tel-Test, Inc., Friendswood, TX)/100 mg of tissue with a glass-Teflon homogenizer. Total RNA was isolated according to the manufacturer's instructions, and its quality was determined on a 1% agarose formamide gel following ethidium bromide staining. RNase protection analysis was carried out as outlined by Ausubel et al. (51) with minor modifications. Briefly, 50 µg of total RNA were first mixed with either the antisense riboprobe (5 × 105 cpm) alone, or with 2 × 104 cpm of mouse glyceraldehyde-3-phosphate dehydrogenase, in a solution of 40 mM PIPES, pH 6.4, 0.4 M NaCl, 1 mM EDTA, pH 8.0, and 80% formamide. The samples were heated at 85-90 °C for 5 min, and then immediately transferred to a 42 °C water bath and incubated overnight. RNase A (7 µg/ml) and T1 (0.35 µg/ml) were used to treat the hybridization reaction products, followed by the addition of SDS-proteinase K (50 µg). The protected fragments were recovered by ethanol precipitation and analyzed on a 6% denaturing polyacrylamide gel.

To determine the 3'-end of the mouse FGF-1 gene, a 219-bp PstI/PvuII fragment (nt 3608-3826 in Fig. 1) was subcloned into the PstI/EcoRV site of pBluescript KS(+) in such an orientation that T3 RNA polymerase could be used for in vitro transcription. The resulting plasmid was designated pFM3E. For the analysis of FGF-1.A versus non- 1.A expression, a 445-bp EcoRI/BamHI containing 295 nt of -1A exon and 150 nt of exon 1 (nt 1-295 and 296-446, respectively, in Fig. 5C) was cloned in pBluescript KS(+). The resulting plasmid, designated pFM1A5, was linearized with EcoRI and in vitro transcribed using T7 RNA polymerase. The x-ray films were scanned and analyzed on a LKB 2400 GelScan XL scanner (LKB, Bromma, Sweden). In order to determine the start site of the 1A promoter, pFM1A6 was in vitro transcribed with T7 RNA polymerase.

Reverse Transcription-PCR-- Total RNA was prepared from mouse tissues, and its quality was tested as described above. First strand cDNA synthesis was carried out using 1-5 µg of RNA by SUPERSCRIPT IITM RNase H- reverse transcriptase provided with the SUPERSCRIPTTM preamplification system (Life Technologies) using primer 1 (see Table I) as an exon 1-specific antisense primer. The subsequent amplification of the 1.A-exon 1 product was carried out with the eLONGaseTM enzyme according to the manufacturer's instructions (Life Technologies) with primers 1 and 2 (see Table I). Following a 30-s denaturation step at 94 °C, the samples were subjected to 35 cycles of amplification at the following temperatures: 94 °C for 30 s, 60 °C for 30 s, and 68 °C for 2 min. The amplified fragment was cloned into EcoRV-digested T-tailed pBluescript KS(+) and designated pFM1A4.

5'-Rapid Amplification of cDNA Ends (RACE)-- Antisense primer 4 or 5 (see Table I) was annealed to 1 µg of total mouse heart RNA and extended according to the manufacturer's instructions of the 5'-RACE system, version 2.0 (Life Technologies, Inc.). The dC-tailed cDNA product was subsequently amplified using 400 nM of both abridged anchor primer and primer 6 (Table I). The eLONGaseTM enzyme mix (Life Technologies) was used in this reaction, which involved the following cycling temperatures and times: 94 °C for 30 s, 60 °C for 30 s, and 68 °C for 60 s for 35 cycles. The resulting amplified products were subcloned into EcoRV-digested T-tailed pBluescript KS(+) and sequenced.

                              
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Table I
List of primers
The name of each primer, along with its sequence (in the 5' to 3' direction) is listed. The position of each primer is also indicated, along with the figure showing the sequence from which it is derived.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characterization of the 3'-End of the Mouse FGF-1 Gene-- We have previously isolated overlapping genomic clones that span the mouse FGF-1 coding region (32), which is composed of three exons. To further characterize the mouse FGF-1 gene and to identify the polyadenylation signal sequences, we sequenced 4005 bp in the 3'-end (Fig. 1). Analysis of 3512 bp of 3' untranslated sequence revealed the presence of six putative polyadenylation signals (Fig. 1) located at nt 2408, 2411, 2573, 2654, 3275, and 3725. The human FGF-1 gene contains two polyadenylation sites located at 3089 and 3101 bp downstream of the termination codon (34), and the length of the mouse FGF-1 3'-UTR was expected to be similar to that of human. Because the size of the major mouse FGF-1 mRNA transcript is 4 kb (52), we expected the AATAAA spanning nt 3725-3730 to be the polyadenylation site of the mouse FGF-1 gene. To demonstrate this point, RNase protection analysis using RNA from kidney, brain, heart, and liver C3H/HeJ mice was carried out (Fig. 2). BamHI-digested pFM3E DNA, the 219-bp PstI/EcoRV insert of which contains the last putative polyadenylation sequence (AATAAA), was in vitro transcribed with T3 RNA polymerase and used as the riboprobe (Fig. 2). Only one protected fragment of the expected size (150 nt) was detected, confirming that AATAAA spanning nt 3725-3730 is the polyadenylation signal sequence. This result demonstrated that the mouse Fgf1 3' untranslated sequence is 3.2 kb.


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Fig. 1.   Nucleotide sequence of the 3'-end of the mouse FGF-1 gene. The arrow indicates the splice acceptor site. Selected restriction enzyme sites in the sequence are indicated for reference. Putative polyadenylation sequences are underlined. The polyadenylation signal sequence, verified by RNase protection experiments, is shown in boldface and underlined. GenBankTM accession number, AF067190.


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Fig. 2.   Mapping of the 3'-end of the mouse FGF-1 gene. An RNA riboprobe was transcribed in vitro with T3 RNA polymerase using BamHI-digested plasmid pFM3E DNA as a template. The striped box represents the sequence derived from the mouse FGF-1 gene, including 96 bp beyond the major polyadenylation signal AATAAA. The protected fragment is indicated by an arrow in the top panel. MspI-digested pBR322 DNA was used as a marker.

Isolation and Characterization of Mouse FGF-1.A Genomic Clones-- The FGF-1A exon was isolated by screening of a mouse P1 library using primers designed to amplify a 109-bp fragment from the -1B exon (43). Three positive clones (P1 7520, 7521, and 7522) were analyzed by restriction enzyme digestion followed by hybridization to a 0.8-kb EcoRI/HindIII fragment containing the rat -1A exon (data not shown). A 5.6-kb BglII hybridizing fragment from P1 7521 was subcloned into the BamHI site of pBluescript KS(+) and designated pFM1A. Subsequently, it was characterized by restriction enzyme digestion followed by hybridization to the 0.8-kb EcoRI/HindIII fragment containing the rat -1A exon (Fig. 3A). In order to confirm the presence of the mouse -1A sequence, a 1375-bp HindIII/EcoRI fragment hybridizing to the rat -1A probe was subcloned into pBluescript KS(+) and named pFM1A1. Its DNA sequence was determined (Fig. 3B) and was aligned with that of the human -1A exon (data not shown), thus enabling us to identify the promoter region. Furthermore, putative CAAT and TATA consensus sequences were also identified at the appropriate position expected, on the basis of their position in the human FGF-1 A exon.


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Fig. 3.   Restriction map of a 5.6-kb BglII fragment from P1 7521 containing the mouse FGF-1.A exon (A) and nucleotide sequence of a 1375-bp EcoRI/HindIII fragment containing the mouse FGF-1.A exon (B). A 5.6-kb BglII fragment from P1 7521, hybridizing to the rat -1A sequence, was subcloned into the BamHI site of pBluescript KS(+) and analyzed by restriction enzyme mapping. E, EcoRI; H, HindIII; X, XbaI. The box represents the -1A exon. The letters above the diagonal lines indicate vector sites; XbaI sites in the insert were not mapped. The area marked by the dashed line contains more than one EcoRI site. The sequence of the 1375-bp EcoRI/HindIII fragment from pFM1A was determined. The putative CAAT and TATA boxes are underlined. The closed diamond represents the transcription start site as determined by RNase protection analysis. The sequence present in the cDNA clones obtained by RT-PCR is double underlined, except for the internal c-Myb and AP-1 binding sites, which are marked by a single underline. GenBankTM accession number, AF067191.

When comparing 629 bp of mouse (nt 747-1375 in Fig. 3B) with 668 bp of human -1A sequence (nt 1-668, as determined by Myers et al. (35)), 66% sequence similarity was observed. In addition, DOTPLOT analysis between mouse and human revealed the presence of six regions with greater than 70% sequence similarity (Fig. 4), including sequences immediately upstream from the start site.


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Fig. 4.   DNA sequence homology between the mouse and human FGF-1 A exon. The DNA sequences compared are 668 nt of human FGF-1 A (nt 1-668 as determined by Myers et al. (35)) and 629 nt of the corresponding mouse sequence (nt 747-1375 in Fig. 3B). The hatched box corresponds to the sequence that begins with the transcription start site (nt 135 for human and nt 865 for mouse) and ends with the splice donor site (nt 532 for human and nt 1238 in mouse). The parameters used for the DOTPLOT program were 70% sequence similarity with a 30-nt window size.

Characterization of the FGF-1.A Promoter and Expression of its mRNA-- In order to obtain an FGF-1.A cDNA, we used RT-PCR. Primer 1, specific to the first protein coding exon, was annealed to RNA derived from mouse kidney, brain, heart, and liver and extended. The resulting cDNAs were amplified using an exon -1A-specific sense primer (primer 2) as well as primer 1, and visualized on a 1.2% agarose gel (Fig. 5A), and then hybridized to the pFM1A1 1375-bp EcoRI/HindIII fragment (Fig. 5B). The 498-bp product amplified from heart RNA was subcloned into EcoRV, T-tailed pBluescript KS(+) and named pFM1A4; sequence analysis confirmed the presence of both the -1A exon and exon 1, spliced together at the expected site, 34 nucleotides upstream from the ATG (Fig. 5C).


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Fig. 5.   RT-PCR of the mouse FGF-1.A mRNA. Total RNA was isolated from mouse C3H/HeJ kidney (lane 1), brain (lane 2), heart (lane 3), and liver (lane 4). Three µg of RNA were reverse-transcribed using primer 1 (see Table I). The cDNA was then amplified by PCR with primers 1 and 2 from Table I (underlined in C). The resulting products were first electrophoresed on a 1.2% agarose gel (A) and then hybridized to the 1375-bp EcoRI/HindIII genomic fragment containing the mouse -1A exon (B). The marker is bacteriophage lambda  DNA digested with HindIII and phi X174 DNA digested with HaeIII. The 498-bp PCR product in A, lane 3, was cloned into pBluescript KS(+) and designated pFM1A4. Its sequence is shown in C. The arrow (C) indicates the splicing of -1A to exon 1. The initiation codon is underlined.

Based on the study of the human tissues, we know that mononuclear cells, placenta, ovary, prostate, and breast epithelial cells do not express any FGF-1 transcripts (27, 30, 33, 34, 39) and that stomach, duodenum, appendix, and testes are negative for FGF-1 (53). We further showed that macrophages, spleen and lymph nodes did not express FGF-1 mRNA (data not shown). Because human heart and kidney are the most abundant sources of FGF-1.A mRNA, we chose these same mouse tissues, as well as brain, liver, and lung, to assess the expression pattern of the FGF-1.A mRNA species, via RNase protection analysis. The riboprobe used was derived from pFM1A5, which was generated by subcloning a 445-bp EcoRI/BamHI fragment (which lacks 53 bp of the 3'-end of exon 1) from pFM1A4 into pBluescript KS(+) (Fig. 6). The results, summarized in Table II, confirmed the RT-PCR data by showing the FGF-1.A transcript being more abundant in heart than in any other tissues assayed (Fig. 6). The 445-nt protected fragment, corresponding to the 1.A mRNA, represents 26.2% of the total FGF-1 mRNA in the heart, whereas in kidney and lung, the 1.A mRNA transcript only comprises 0.4 and 0.3%, respectively, of the total FGF-1 mRNA. Brain and liver displayed only the 150-nt protected fragment, indicating the presence of FGF-1 transcripts other than 1.A in these tissues.


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Fig. 6.   FGF-1.A mRNA expression in various mouse tissues. Plasmid DNA pFM1A5 was linearized with EcoRI and used as a DNA template for in vitro transcription with the use of T7 RNA polymerase. Fifty µg of total RNA from C3H/HeJ kidney, brain, heart, liver and lung were hybridized to the riboprobe. The sizes of protected fragments are 150 nt for non-1.A mRNA and 445 nt for FGF-1.A mRNA. pBR322 digested with MspI was used as size marker. In vitro transcribed mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is included for normalization of the amounts of RNA used.

                              
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Table II
Expression of FGF-1.A versus non-1.A mRNA
The abundance of FGF-1.A and non-1.A transcripts relative to total FGF-1 mRNA, as determined by densitometric analysis, is expressed in percentage values.

Identification of the Transcription Start Site for FGF-1.A mRNA by RNase Protection Analysis and 5'-RACE-- In human, the FGF-1.A transcription start site is located 25 nt downstream of the TATA box (35). Due to the high sequence similarity between the two species, we expected the mouse -1A start site to reside at a comparable position. To obtain a suitable template to carry out the RNase protection experiment, primers 3 and 4 were used to amplify a 585-bp product from pFM1A1. The amplicon contains 99 bp of -1A sequence present in the cDNA clones and 486 bp of upstream sequence containing putative CAAT and TATA boxes. Following subcloning of this fragment into pBluescript KS(+), the resulting DNA, designated pFM1A6, was linearized with HindIII and transcribed in vitro with T7 RNA polymerase (Fig. 7). The obtained riboprobe was hybridized to 50 µg of total mouse kidney and heart RNA. A protected fragment of 176 nt was obtained in the heart lane (Fig. 7), thus placing the -1A start site 374 nt upstream of the splice donor site of exon -1A.


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Fig. 7.   Mapping of the FGF-1.A transcription start site via RNase protection analysis. HindIII-digested pFM1A6 was transcribed in vitro with T7 RNA polymerase to generate an antisense riboprobe. The striped box represents 585 nt of -1A sequence (nt 458-1042 in Fig. 3B). Total RNA from mouse C3H/HeJ kidney (lane 1) and heart (lane 2), and yeast tRNA (lane 3) were hybridized to the riboprobe. A protected fragment of 176 nt was observed in lane 2. MspI-digested pBR322 DNA and a sequencing ladder were used as size markers.

To confirm this finding, 5'-RACE was carried out. Heart RNA was used as a template to extend -1A cDNA using antisense primer 4 and/or 5. Following PCR amplification using primer 6 as an antisense nested primer and an abridged anchor primer as a sense primer, the samples were run on a 1.2% agarose gel and transferred to a nylon filter. Hybridization to the pFM1A6 EcoRI/HindIII insert containing the 5'-end of the 1A promoter revealed a band of approximately 150 bp (data not shown). This band was observed only in the heart lanes, consistent with our RT-PCR and RNase protection results. Upon nucleotide sequence analysis of five independent clones, obtained from the cloning of this ~150-bp fragment into T-tailed pBluescript KS(+), we established that two of the clones had their 5'-end at nt 872 and three at nt 873 (Fig. 3B), which is seven and eight nucleotides downstream of the start site, respectively, as determined by RNase protection analysis.

Characterization of the Mouse FGF-1 Gene-- The position of exon -1A relative to -1B was determined via long range PCR using a set of primers specific to each exon (primers 2 and 7, respectively). A PCR product of approximately 10 kb was detected. Following digestion with EcoRI and BglII, both alone and together, this fragment was hybridized to the following 32P-labeled fragments: a 1375-bp EcoRI/HindIII fragment containing the -1A exon, a 2.5-kb EcoRI fragment of the mouse FGF-1 cDNA containing 112 bp of -1B exon, and the PCR product itself. Based on the hybridization results (data not shown) and on the basis of the available restriction map around these two exons, we determined the size of the distance between -1A and -1B to be 10.7 kb (Fig. 8B). To determine the distance between exons -1C and -1G and between exons -1G and 1, we used long range PCR as described under "Experimental Procedures." The sizes of the amplicons are 16 and 8.2 kb, respectively (data not shown). Therefore, the position of these two upstream untranslated exons are 23.6 and 7.8 kb upstream of exon 1, respectively (Fig. 8A). Based on the size of the P1 980 DNA (78.5 kb), the position of -1B relative to the 5'-end of P1 980, and the overlapping region between clones 980 and 981, we determined the size of the intron separating -1B and the first protein-coding exon to be 59 kb (Fig. 8A). Therefore, the mouse FGF-1 gene spans over 90 kb.


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Fig. 8.   Map of the mouse Fgf1 transcription unit. A, overlapping mouse genomic DNA clones 980, 981, and 7521 were obtained by screening of a P1 library. Subclones pFM1A, pKA7, pFMP4, pFMP2, and pFMP3, containing exons -1A (374 bp), -1B (109 bp), 1 (203 bp), 2 (104 bp), and 3 (3.2 kb), respectively, were characterized by restriction mapping and DNA sequence analysis. PCR 1 designates the 11.1-kb PCR fragment spanning exons -1A and -1B (detailed in B); PCR 2 designates the 11.7-kb PCR product spanning the first and second protein coding exons; fran1 is a phage clone containing a 12.9-kb insert that spans exons 2 and 3. The positions of exons -1C and -1G were determined by long range PCR amplification of DNA encompassing exons -1C and -1G and exons -1G and 1, respectively. The dashed lines represent the splicing of -1A and -1B to the first protein coding exon. The DNA in fran1 not derived from the Fgf1 locus is indicated with a dotted line. The double dashed line denotes that the boundary of P1 7521 DNA has not been determined. B, primers 2 and 7 (see Table I) were used to amplify an 11.1-kb fragment from P1 7521 DNA. E, EcoRI; G, BglII; N, NotI. The boxes represent the amplified regions of exons -1A (left) and -1B (right), which are 295 and 151 bp, respectively. pFM1A was subcloned from P1 7521 DNA, whereas pKA7 and pKA8 were subcloned from P1 980 DNA. The insert sizes are shown below each clone. The double dashed line represents vector DNA derived from P1 clone 980. The NotI site shown in pKA8 is in the multiple cloning site of the P1 vector.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mapping of the end of the mouse FGF-1 gene revealed the presence of a long 3'-untranslated region, consistent with the human FGF-1 gene structure. We have identified the major polyadenylation site, residing 3231 bp downstream from the stop codon, by RNase protection analysis (Fig. 2). Furthermore, no additional AATAAA sequences were detected 275 bp beyond this polyadenylation site. In comparison, the two major polyadenylation signals in human reside 3090 and 3102 bp 3' of the stop codon (34). Minor differences between the two species are to be expected, although the crucial features of this gene are conserved. Indeed, the 3'-UTR of the mouse and human FGF-1 genes share 60.4% sequence similarity, with stretches sharing more than 80% similarity. The region immediately upstream from the major polyadenylation signal (nt 3696-3734 in Fig. 1) shares 87% sequence similarity with the corresponding human sequence. In contrast, the sequence similarity of the region downstream from the polyadenylation signal (nt 3735-3794) dropped to 45%.

The conservation observed in the Fgf1 3'-UTR among different species, along with the presence of long 3'-UTRs in genes involved in modulation of cellular proliferation (54, 55), implies a functional significance of this area. Indeed, the expression of the hst/K-FGF proto-oncogene is controlled by regulatory elements present in the 3'-end (56). Both human (34) and mouse Fgf1 3'-UTRs contain AU-rich elements (57), which may be partly responsible for the transient expression of certain messages, particularly FGF-1.C (30, 38). In fact, the levels of this particular mRNA are dramatically increased upon phorbol 12-myristate 13-acetate, serum, and transforming growth factor-beta stimulation. These observations point to a more complex mechanism of Fgf1 regulation, as it cannot be ruled out that the 5' and 3' untranslated regions work in concert to dictate the fate of a particular FGF-1 message in response to external stimuli in a given cellular milieu.

Screening of a mouse P1 library using primers specific to the mouse -1B exon allowed for the isolation of exon -1A. In addition, having obtained a clone spanning both exons allowed us to determine the size of the intron separating them, which is very similar to that of human (10.7 versus 11.7 kb) (Fig. 8B). In contrast, when DNA sequence comparison was carried out in order to find the -1A chicken homologue among 5 upstream untranslated exons recently isolated (58), no significant sequence similarity was observed with any of the 5'-UTRs tested. These differences between the mouse and chicken upstream untranslated exons are possibly due to the greater evolutionary distance that exists between avian and mammalian species. Nonetheless, the presence of 5'-UTRs, as diverse as they may be, in species that are evolutionary divergent, further confirms the complexity of this growth factor gene, and the need for a stringent regulation of its expression.

Finally, further characterization of the mouse -1A revealed that, like that of human, this promoter also contains TATA and CAAT sequence variants, located at comparable distance from the transcriptional start site of the exon. Indeed, as established by RNase protection analysis (Fig. 7), the mouse -1A exon is 374 bp in size, which differs from its human homologue by only 24 bp.

While attempting to obtain an RT-PCR product containing the -1A exon spliced to exon 1, we determined that the mouse heart is the most abundant source of FGF-1.A mRNA (Fig. 5). These data were further confirmed by RNase protection (Fig. 6), although this analysis also demonstrated that other FGF-1 mRNA species are present in this particular tissue. Upon searching for putative transcription factor binding sites in the FGF-1.A promoter region (59), we discovered the presence of several AP-1 binding sites (Fig. 3B), and a single putative binding site for c-Myb. It is noteworthy that both c-Myb and FGF-1 are involved in vascular smooth muscle cell proliferation, and tissue repair of the neointima following angioplasty (60). Although there is no evidence that these two genes are involved in the same signaling pathway, it is not to be excluded that c-Myb is activated first, and in turn this nuclear protein activates FGF-1 transcription. Antisense nucleotides against c-Myb inhibit cell proliferation and neointima formation (61), as well as reducing the steady state levels of the corresponding mRNA and protein in a dose-dependent manner in vitro. Thus, it is likely that c-Myb expression in response to vascular injury serves as an activator for FGF-1 and other growth factors that promote angiogenesis and tissue repair. Ultimately, however, DNase I footprinting and electromobility shift assay would demonstrate this interaction, the functionality of which would in turn be proven by the appropriate promoter studies.

Characterization of the entire transcription unit of the mouse FGF-1 gene (Fig. 8A) has enabled us to compare it with its human homologue. This information will be valuable for the development of knockout constructs used in homologous recombination for generating transgenic mice in an effort to determine the functional significance of the 1A promoter in heart. Furthermore, it will be important to examine the expression of FGF-1.A in the developing mouse, in order to assess any changes during the life span of the animal, from the embryonic stage to adulthood. Second, because FGF-1 expression has been correlated with cell survival in vascular endothelial cells, such function could be attributable specifically to the FGF-1.A mRNA. Finally, having determined the position of the mouse -1G exon in the Fgf1 locus will be useful in isolating the human homologue of this recently identified exon.

    FOOTNOTES

* This work was supported by grants R01CA45611 (to I.-M. C.) and K11AI01048 (to K. V. H.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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 GenBankTM/EMBL Data Bank with accession number(s) AF067190 and AF067191.

Dagger To whom correspondence should be addressed: Dept. of Internal Medicine, The Ohio State University, 480 W. 9th Ave., Columbus, OH 43210. Tel.: 614-293-5814; Fax: 614-293-5631; E-mail: chiu.1{at}osu.edu.

    ABBREVIATIONS

The abbreviations used are: FGF, fibroblast growth factor; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; RACE, rapid amplification of cDNA ends; UTR, untranslated region; nt, nucleotide(s); bp, base pair(s); kb, kilobase pair(s); PIPES, 1,4-piperazinediethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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