From the Institute of Interdisciplinary Research,
School of Medicine, Université Libre de Bruxelles, 808 Route de
Lennik, 1070 Brussels, Belgium,
Sanofi-Synthelabo, 371 Rue du
professeur Joseph Blayac, 34084 Montpellier, France,
** Sanofi-Synthelabo, 195 Route d'Espagne, 31036 Toulouse, France, and
the
Department of Medical Chemistry, Erasme Hospital,
Université Libre de Bruxelles, 808 Route de Lennik,
1070 Brussels, Belgium
Received for publication, October 20, 2000, and in revised form, January 22, 2001
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ABSTRACT |
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The P2Y11 receptor is an
ATP receptor positively coupled to the cAMP and phosphoinositide
pathways. Ssf1 is a Saccharomyces cerevisiae nuclear
protein, which plays an important role in mating. The gene encoding the
human orthologue of SSF1 is adjacent to the
P2Y11 gene on chromosome 19. During the
screening of placenta cDNA libraries, we isolated a chimeric clone
resulting from the intergenic splicing between the
P2Y11 and SSF1 genes. The fusion protein was stably expressed in CHO-K1 cells where it generated a cAMP response to ATP qualitatively indistinguishable from that of the
P2Y11 receptor. According to both Western blotting and cAMP
response, the expression of the fusion protein in the transfected cells
was clearly lower than that of the P2Y11 receptor. Both P2Y11 and SSF1 probes detected a
5.6-kb messenger RNA with a similar pattern of intensity in each of 11 human tissues. The ubiquitous presence of chimeric transcripts and their up-regulation during granulocytic differentiation indicate that the transgenic splicing between the P2Y11 and the SSF1
genes is a common and regulated phenomenon. There are very few examples
of intergenic splicing in mammalian cells, and this is the first case
involving a G-protein-coupled receptor.
P2Y11 and SSF1 are adjacent
genes located on chromosome 19 (1). The P2Y11 receptor
belongs to the P2Y family of G-protein-coupled nucleotide receptors
(2); it is activated by ATP and positively coupled to the cAMP and the
phosphoinositide pathways. It has been cloned from a human cDNA
placenta library, but it is specifically expressed in the immune system
(3). In particular, P2Y11 messengers are present in HL-60
human promyelocytic leukemia cells and strongly up-regulated following
exposure to various agents inducing their differentiation into
neutrophil-like cells (4). Furthermore the induction of the
granulocytic differentiation of HL-60 cells by ATP is mediated through
the activation of P2Y11 receptors (5, 6). On the other
side, Ssf1 is a Saccharomyces cerevisiae nuclear protein, which plays an important role in mating (7, 8, 9).
Ssf1 and its close homologue Ssf2 have been
related to ppan, a gene involved in Drosophila
larval growth (10). The cloning of the human orthologue of yeast
Ssf1 was reported recently and the ubiquitous
expression of human Ssf1 mRNA is consistent with a
general role in cell growth (1). Ssf1 (a suppressor of swi
four) should not be confused with the homonymous
Ssf-1 (a second step splicing factor 1), an activity
involved in the second step of pre-mRNA splicing in S. cerevisiae (11).
The presence of chimeric messengers resulting from intergenic splicing
is not commonly observed in normal mammalian cells. It has been
reported that cotranscription and intergenic splicing of human
galactose-1-phosphate uridyltransferase and interleukin-11 receptor
During the screening of placenta cDNA libraries to isolate new P2Y
receptors, we have isolated a cDNA clone encoding a
SSF1-P2Y11 chimeric transcript. We have then investigated
the tissue expression of this fusion mRNA and the biological
activity of the corresponding fusion protein.
Materials--
Trypsin was from Flow Laboratories (Bioggio,
Switzerland). Culture media G418, fetal bovine serum, and
restriction enzymes Taq and Platinum® Pfx DNA
polymerases were purchased from Life Technologies, Inc. [ Cloning and Sequencing--
A human placenta cDNA library
was screened at moderate stringency with an
[ Reverse Transcription PCR Experiments--
RNA was extracted
from HL-60 cells with the Rneasy kit (Qiagen). The
reverse-transcription was performed with 2 µg of total RNA using the
Superscript kit (Life Technologies, Inc.). Specific primers located in
the genomic sequence located upstream of the second exon of the
P2Y11 gene and a specific reverse primer located in the third transmembrane region of the P2Y11 receptor
were synthesized and used in reverse-transcription PCR experiments. The
PCR amplification conditions with Taq DNA polymerase were
94 °C for 45 s, 50 °C for 30 s, and 72 °C for 1 min
30 s (35 cycles). The amplification products were subcloned in
pBluescript SK+ and sequenced using the BigDye Terminator
cycle sequencing kit (Applied Biosystems, Warrington, Great Britain).
Northern Blot Analysis--
One blot containing 12 human
mRNAs (MTN 12:1 µg of poly(A)+ RNA/lane;
CLONTECH) was hybridized with specific probes
corresponding to the P2Y11 (2nd exon) and SSF1 (exons
1-11) coding sequences. The blot was prehybridized 8 h at
42 °C in a 50% formamide, 2% SDS solution and hybridized for
18 h in the same solution supplemented with the
Cell Culture and Transfection--
We have amplified 14 specific
PCR products encoding chimeric proteins starting at each of the ATG
codons present in the SSF1 part of the chimeric cDNA using the
Platinum® Pfx DNA polymerase (94 °C, 15 s;
50 °C, 30 s; 68 °C, 2 min for 30 cycles). The sequences
corresponding to P2Y11 or chimeric SSF1-P2Y11 receptors were subcloned between the HindIII and
XbaI sites of the bicistronic pEFIN3 expression vector and
checked for the absence of mutation. CHO-K1 cells were transfected with
the recombinant pEFIN3 plasmids or with the plasmid alone using the
FuGENETM 6 transfection reagent (Roche Molecular
Biochemicals). The CHO-K1-transfected cells were selected with
400 µg/ml G418 in complete medium (10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml
amphotericin B in Ham's F-12 medium) 2 days after transfection and
maintained in the same medium. HL-60 cells were cultured at 37 °C
with 5% CO2 in the following complete medium: 10% fetal
bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B, and 5 mM L-glutamine in
RPMI 1640 medium.
Cyclic AMP Assays--
Stably transfected CHO-K1 were spread on
Petri dishes (150,000 cells/dish) and cultured in Ham's F-12 medium
containing 10% fetal calf serum, antibiotics, amphotericin,
sodium pyruvate, and 400 µg/ml G418. Cells were preincubated for 30 min in Krebs-Ringer Hepes buffer with rolipram (25 µM) and incubated for different times in the presence of
the agonists (15 min in most experiments). The incubation was stopped
by the addition of 1 ml of 0.1 M HCl. The incubation
medium was dried up, resuspended in water, and diluted as required.
Cyclic AMP was quantified by radioimmunoassay after acetylation as
previously described (15).
Antibody Production--
An anti-P2Y11 polyclonal
antibody was generated in rabbits using a synthetic peptide located at
the extremity of the C-terminal part of the human
P2Y11 receptor (AAPKPSEPQSRELSQ). Rabbits were injected
subcutaneously with 2 mg of bovine serum albumin-peptide in 250 µl of
water and 250 µl of complete Freund's adjuvant. Animals were boosted
monthly under the same conditions. Blood was taken 10 days after the
second and subsequent injections. Sera were immunopurified on a
Affi-Gel tyrosine gel (Bio-Rad) modified with the P2Y11
C-terminal peptide according to the manufacturer's instructions. Briefly, 10 ml of immune serum was incubated overnight with 1 ml of
modified gel. After extensive washing, anti-P2Y11 was
eluted with 100 mM glycine/HCl (pH, 1.8) and neutralized
with 1 M Tris-NaOH. The pooled fractions were supplemented
with 10 mg/ml bovine serum albumin, concentrated, and dialyzed on a
filtron microsep 30-kDa membrane (Northborough, MA). Concentrated
antibody was stored in 50% glycerol at Western Blot Analysis--
At confluency, CHO-K1-transfected
cells were washed by PBS (pH, 7.3) (137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4·7H2O and 1.4 mM KH2PO4) and scraped, and the
pellets were solubilized in Laemmli buffer (10 (w/v) glycerol, 5%
(v/v) mercaptoethanol, 2.3% (w/v) SDS, 62.5 mM Tris-HCl
(pH 6.8). The protein concentration was determined using the method of
Minamide and Bamburg (16). The same amount of proteins for each
condition was electrophoresed in a 7.5% SDS-polyacrylamide gel
electrophoresis. Proteins were then transferred overnight at 60 V and
4 °C onto a nitrocellulose membrane using 20 mM Tris,
154 mM glycine, 20% (v/v) methanol as a transfer buffer.
Immunodetection was achieved using the enhanced chemiluminescence
Western blotting detection system (ECL, Amersham Pharmacia Biotech)
using a biotinylated-secondary rabbit antibody (1/50000). The
anti-P2Y11 polyclonal antibody was used at a
1/200-dilution.
A human placenta cDNA library was screened at moderate
stringency with a P2Y4 probe (spanning transmembrane
domains 3-7) to isolate novel P2Y receptors. Some clones encoded a
novel P2Y receptor that has been characterized and named
P2Y11 receptor (3). One of the positive clones was clearly
longer than the others (2500 base pairs (bp)). A 2385-bp open reading
frame was identified in this clone (GenBankTM/EBI accession
number: AJ300588). The first half of the corresponding protein
displayed 40% amino acid identity with a S. cerevisiae protein involved in mating called Ssf1 (1), whereas the second half exactly matched the P2Y11 sequence (3).
We have then obtained the complete cDNA and genomic sequences
of human SSF1, which is split into 12 exons, and have shown that its
mRNA is expressed in all human tissues tested (1). We have also
shown that the P2Y11 and SSF1 genes
are contiguous on chromosome 19p31 (Fig.
1). The genomic organization of the SSF1 gene has been previously discussed (1). Existence of
the fusion cDNA is due to a transgenic splicing removing the
genomic sequence included between the first third of the last exon of the SSF1 gene and the second exon of the
P2Y11 gene (Fig. 1A). This splicing
occurs in the absence of a consensus splicing donor site (residue 426 of the SSF1 protein) (Fig. 1B). The last 47 amino acids of
the SSF1 protein (residues 427 to 473) are truncated in the fusion
SSF1-P2Y11 protein. From these observations, it was clear
that in fact the first three amino acids, MDR, of the P2Y11
sequence, which we have previously published (3), were coming from the
SSF1 sequence. In the placenta cDNA library, we had first obtained
a partial clone of the SSF1-P2Y11 fusion protein in which
these three amino acids appeared to be the beginning of the
P2Y11 cDNA sequence (3). After we obtained the complete SSF1-P2Y11 fusion protein sequence and the genomic
organization of the SSF1 gene, we performed PCR experiments
to identify the true first exon of the P2Y11
gene to clarify its genomic organization. We have identified the first
exon of the P2Y11 receptor by reverse-transcription PCR
experiments using primers located upstream of potential ATG starting
codons in the genomic sequence included between the last exon of the
SSF1 gene and the second exon of the
P2Y11 gene. The reverse primer was a specific
primer located in the third transmembrane domain of the human
P2Y11 receptor and contained a BamHI restriction site (in italic) (5'-TCGCGGATCCATGCCCAGGTAGCGGTTGAG-3').
These experiments were performed with RNA extracted from HL-60
cells in which it is known that the P2Y11 receptor is
expressed (4). One of the PCR products sequenced has allowed us to
identify the first exon of the P2Y11 gene, which
is located 1.9 kb upstream of the second one. This 445-bp product was
obtained using the reverse primer and the following 5'-primer
containing an EcoRI restriction site (in italic)
5'-TCCGGAATTCTAGCAGACACAGGCTGAGGA-3'. The exon encodes the six first amino acids of the P2Y11
receptor, MAANVS (Fig. 1B). An in-phase stop codon (in bold
and underlined in the 5'-primer) is located 33 bp upstream of the
starting codon, which is in a Kozak consensus. In conclusion, the
sequence MAANVSGAK is the true beginning of the non-chimeric
P2Y11 receptor, whereas MDRGAK represents the
junction between the SSF1 and P2Y11 gene products (Fig. 1B). The correct non-chimeric
P2Y11 sequence has been submitted to
GenBankTM/EMBL (accession number: AJ298334).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain genes generates a fusion transcript in normal cells (12). The
intergenic splicing between the MDS1 and EVI1 genes has also been described (13). EVI1 is a protooncogene encoding a nuclear protein with several zinc finger domains, whereas MDS1 has been cloned as one of the partner genes of
AML1 in the t(3;21)(q26;q22) translocation associated with
myeloid leukemia. A third case reported in the literature is the
intergenic splicing involving the murine Prnd and
Prnp genes encoding the prion protein PrP and the PrP-like
protein Doppel, respectively (14). It has been speculated that
intergenic splicing would be a mechanism for generating new multidomain
proteins and could therefore have major evolutionary implications.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (800 Ci/mmol) was from Amersham Pharmacia
Biotech. ATP, ATP
S (adenosine
5'-O-(3-thiotriphosphate)), benzoyl ATP (2'- and
3'-O-(4-benzoyl-benzoyl)adenosine 5'-triphosphate), ADP,
UTP, UDP and all-trans-retinoic acid were obtained from
Sigma. Rolipram was a gift from the Laboratoires Jacques Logeais
(Trappes, France). The human placenta cDNA library was kindly given
by Prof. P. Chambon (Strasbourg, France). pEFIN3 is an expression
vector developed by Euroscreen (Brussels, Belgium). The human Multiple
Tissue Northern (MTN) blots was from CLONTECH (Palo
Alto, CA). HL-60 cells were obtained from American Type Culture
Collection (Manassas, VA.). P2Y11 C-terminal peptide
(AAPKPSEPQSRELSQ) and bovine serum albumin-conjugated peptides
(conjugation through an additional tyrosine) were from Neosystem
(Strasbourg, France).
-32P]dATP-labeled P2Y4 receptor probe
corresponding to a partial sequence covering the third to the seventh
transmembrane domains. The hybridization conditions for screening were
6× SSC (1× SSC: 0.15 M NaCl, 0.015 M sodium
citrate) and 40% formamide at 42 °C for 14 h, and the final
washing conditions were 0.5× SSC, 0.1% SDS at 60 °C. One of the
purified clones displayed an insert of 2.5-kb1 length and was sequenced
on both strands after subcloning of overlapping restriction fragments
in M13mp18 and M13mp19 using the Sanger dideoxynucleotide chain
termination method.
-32P-labeled probe. The final washing conditions were
0.2× SSC and 0.1% SDS at 55 °C. The blot was exposed during 6 days
and visualized as an autoradiograph or by using the PhosphorImager SI
(Molecular Dynamics). A HL-60 blot hybridized previously with a
P2Y11 probe (4) was hybridized with a SSF1 probe. To
realize this blot, total RNA was extracted using the Rneasy kit
(Quiagen) from HL-60 cells undifferentiated or differentiated for
various times with 1 µM retinoic acid.
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (34K):
[in a new window]
Fig. 1.
Schematic representation of the intergenic
splicing of the SSF1 and
P2Y11 genes. A, the boxes represent
the exons of the two genes (1-12 for SSF1; 1' and 2' for
P2Y11). Gray, coding sequence;
white, non-coding sequence. The RNA obtained after
cotranscription of the SSF1 and P2Y11
genes leads to the formation of a chimeric SSF1-P2Y11
messenger RNA. B, representation of the junction sites
observed in non-chimeric and chimeric P2Y11 messengers. The
residues 424-426 of the SSF1 protein are represented to the
left. They are located at the end of the first third of exon
12, which encodes residues 401-473 of the SSF1 protein. Residues
427-473 of the SSF1 protein and the six residues (MAANVS,
potential N-glycosylation site in bold) encoded by exon 1'
of the P2Y11 gene are truncated in the fusion
protein.
We have then investigated whether the fusion transcript could be translated into a functionally active chimeric receptor. Because there are 14 potential starting codons in the first half of the clone corresponding to the SSF1 part and potential extracellular region of this chimeric receptor, we have amplified 14 specific PCR products encoding chimeric proteins starting at each of these ATG codons. The sequences of these products have been checked after insertion in the pEFIN3 expression vector and transfected into CHO-K1 cell lines.
In the transfected cells, the construction inducing the greatest
functional response corresponded to the P2Y11 receptor
alone, previously characterized following stable expression in CHO-K1 cells (3, 6). However each transfected chimeric construction led to a
significant but much lower cAMP response to ATP (100 µM),
even the construction corresponding to the full-length
SSF1-P2Y11 fusion protein (Fig.
2). The basal level was considerably
lower in the cell lines transfected with the chimeric transcript as compared with the P2Y11-transfected cells, suggesting a
constitutive activity of the P2Y11 receptor. No ATP
response was observed with CHO-K1 cells transfected with the pEFIN3
empty vector, whereas the forskolin-induced cAMP accumulation was
comparable in all the transfected cell lines (Fig. 2). The cAMP data
obtained for the intermediate constructions were similar to those
obtained for the full chimeric receptor (data not shown). Northern
blotting revealed comparable amounts of messengers for the different
constructions in the various transfected CHO-K1 cell lines (data not
shown).
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Other adenine nucleotides, known to produce a strong activation of the
P2Y11 receptor, were tested on cells transfected with the
chimeric SSF1-P2Y11 construction. ATPS and benzoyl ATP
behaved as full agonists of the chimeric receptor and were apparently more potent than ATP (Fig. 2) as observed previously for the
recombinant P2Y11 receptor (6). No effect of ADP, UTP, or
UDP was observed (data not shown).
We have used a polyclonal antibody generated in the rabbit against a
peptide located at the extremity of the C terminus of the
P2Y11 receptor (AAPKPSEPQSRELSQ). This antibody was used on CHO-K1 cells transfected with P2Y11,
full-length-SSF1-P2Y11, and with the empty vector (Fig.
3). In P2Y11-transfected
cells, three strong bands were clearly detected around 45 kDa. A weak
single band was detected in SSF1-P2Y11-transfected cells
around 90 kDa (Fig. 3). These bands were not detected in the
presence of 2 µg/ml of the corresponding peptide (data not shown). No
band was detected in CHO-K1 cells transfected with pEFIN3 vector alone
in the absence (Fig. 3) or the presence of the corresponding peptide
(data not shown).
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Northern blotting experiments were performed with specific probes of
the SSF1 and P2Y11 genes (Fig.
4, A and B) on a
blot containing mRNA from 11 human tissues and blood leukocytes.
With an SSF1 probe, two prominent messengers (1.7 and 5.6 kb) were revealed in each tissue. Additional weaker bands were also detected (2.6 and 3.5 kb) (Fig. 4A). As shown previously, a
P2Y11 probe hybridized to a 2-kb mRNA in human spleen
(3) and liver (Fig. 4B). However, a second band was present
in each tissue (Fig. 4B) and had a size indistinguishable
from that revealed by the SSF1 probe at 5.6 kb. It seems that 1.7-, 2.6-, and 3.5-kb messengers were only detected with a SSF1 probe and
correspond to SSF1 messengers (Fig. 4A), whereas 2-kb
messengers shown in panel B correspond to P2Y11
messengers. The 5.6-kb band detected with the two probes corresponds
apparently to a chimeric SSF1-P2Y11 messenger. This 5.6-kb
unique band was also detected with a probe corresponding to the
chimeric transcript and not with other unrelated probes (data not
shown). A 5.6-kb messenger detected with a P2Y11 probe in
the HL-60 cells (4) was also revealed with an SSF1 probe with a similar
pattern (Fig. 4C) and corresponded apparently to the
chimeric SSF1-P2Y11 messenger. This messenger was
up-regulated during granulocytic differentiation of HL-60 cells by
retinoic acid (Fig. 4C).
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DISCUSSION |
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Intergenic splicing is extremely uncommon in mammalian cells with
only three cases reported in the literature: MDS1 and
EVI1 (13), galactose-1-phosphate uridyltransferase and
interleukin-11 receptor -chain (12), and Prnd and
Prnp (14). In this paper we have reported a new case of
fusion mRNA resulting from in-frame intergenic splicing between the
human SSF1 and P2Y11 genes; this is
the first case involving a G-protein-coupled receptor. It is interesting to note that the transgenic splicing between these two
unrelated genes leads to the addition of a potential ATP binding site
present in SSF1 (GVGEGK, residues 289 to 294) to the sequence of a
purinergic receptor. However, apparently this has no major effect on the responsiveness to nucleotides.
We have clarified the genomic organization of the P2Y11 gene. An exon encoding the first six residues of the non-chimeric P2Y11 receptor has been identified 1.9 kb upstream of the second exon which encodes the seven transmembrane regions of the receptor. This first exon contains the starting codon and encodes a potential N-glycosylation site (MAANVS). This first exon is not present in the chimeric messenger. The first three residues of the previously published P2Y11 sequence, MDR, (3) were thus a consequence of the intergenic splicing and belong in fact to the SSF1 sequence.
Theoretically the fusion transcript encodes a receptor with a very large extracellular domain displaying no peptide signal sequence. Because binding assays using radiolabeled nucleotides are not a valid method to quantitate P2Y receptors (17-19), we have performed functional assays to determine whether the transfection of the chimeric cDNA could lead to a biochemical response to ATP in the transfected cells. Indeed cAMP assays showed that CHO-K1 cells transfected with a SSF1-P2Y11 construction exhibited a cAMP response to nucleotides qualitatively similar to that observed in cells expressing the P2Y11 receptor alone (6). However, both the basal cAMP level (reflecting possible constitutive activity) and the maximum accumulation of cAMP in response to ATP were much lower in cells expressing the fusion protein than in P2Y11-expressing cells. The pharmacological data could be correlated with the level of expression of the fusion protein, which seems clearly lower than that of the P2Y11 receptor. Whereas three strong bands, probably corresponding to different degrees of glycosylation, were observed in cells expressing the P2Y11 receptor, a weak 90-kDa band corresponding to the expected molecular mass of the fusion protein was detected in cells transfected with the SSF1-P2Y11 construction. Although the level of mRNA detected was comparable between cells transfected with the chimeric receptor and the P2Y11 receptor, we can speculate that the fusion protein is less translated or less stable than the P2Y11 receptor. It appears that the ATP response observed in cells expressing the fusion protein is not due to its cleavage at the fusion site because no lower band corresponding to the P2Y11 receptor was detected.
Both P2Y11 and SSF1 probes detected the same 5.6-kb
messenger with a similar pattern of intensity in each tissue. The
detection of the chimeric transcript in all the tested tissues was
surprising as was its up-regulation in HL-60 cells in response to an
agent inducing granulocytic differentiation. It indicates that the
cotranscription and transgenic splicing between the
P2Y11 and the SSF1 genes is a
frequent, ubiquitous, and regulated phenomenon. However its functional
significance remains unclear. It is important to emphasize that we have
obtained cAMP data for the chimeric receptor in a system of
overexpression of the recombinant fusion protein, but it is unclear
whether the chimeric transcript observed in all the tested tissues is
translated into fusion protein in vivo. Indeed in most cells
ATP is unable to increase cAMP. Alternatively, the production of
SSF1-P2Y11 fusion mRNAs could be a way to down-regulate the expression of active P2Y11 receptors by misleading
transcription, or the fusion protein might have another function which
remains to be determined.
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ACKNOWLEDGEMENTS |
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We thank Profs. J. E. Dumont, G. Vassart, and J. M. Herbert for helpful advice and discussions. We thank Prof. P. Chambon for the generous gift of the cDNA placenta library.
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FOOTNOTES |
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* This work was supported by an Action de Recherche Concertée of the Communauté Française de Belgique, by the Belgian Program on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Federal Service for Science, Technology, and Culture, by grants of the Fonds de la Recherche Scientifique Médicale, the Bekales Foundation, the Fonds Médical Reine Elisabeth and Boehringer Ingelheim, and Fonds Emile DEFAY.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) AJ298334 and AJ300588.
§ Chargé de recherches of the FNRS. To whom correspondence should be addressed: Inst. of Interdisciplinary Research, Campus Erasme, Bld. C, 5th Floor (local C5-145), 808 Route de Lennik, 1070 Brussels, Belgium. Tel.: 32-2-555-41-59; Fax: 32-2-555-46-55; E-mail: communid@ulb.ac.be.
¶ Supported by Euroscreen.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M009609200
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ABBREVIATIONS |
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The abbreviations used are: kb, kilobase(s), PCR, polymerase chain reaction; CHO, Chinese hamster ovary; kb, kilobase(s); bp, base pair(s).
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REFERENCES |
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