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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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
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.
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RESULTS |
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.
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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.
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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.
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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 DNA
digested with HindIII and 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.
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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.
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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.
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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.
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DISCUSSION |
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-
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.