From the Departments of Neurochemistry and
** Pharmacology, and § Instituto de Neurociencias,
Universidad Miguel Hernández,
03550 San Juan, Alicante, Spain
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ABSTRACT |
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The 7 subunit is a component of
-bungarotoxin-sensitive nicotinic acetylcholine receptors expressed
in bovine adrenomedullary chromaffin cells. The proximal promoter of
the gene coding for this subunit contains several GC-boxes and one
E-box. Deletion analysis and transient transfections showed that a
120-base pair region (
77 to +43) including all of these elements gave
rise to ~70 and 95% of the maximal transcriptional activity
observed in chromaffin and SHSY-5Y neuroblastoma cells,
respectively. Site-directed mutagenesis of the different elements
indicated that both GC and E motifs contribute to the activity of the
7 gene in a very prominent way. Using electrophoretic mobility shift
assays, the upstream stimulatory factor (USF) was shown to be a
component of the complexes that interacted with the E-box when nuclear
extracts from chromaffin and SHSY-5Y cells were used. Binding of the
early growth response gene transcription factor (Egr-1) to three
different GC-boxes was also demonstrated by shift assays and DNase I
footprint analysis. Likewise,
7 promoter activity increased by up to
5-fold when
7 constructs and an Egr-1 expression vector were
cotransfected into chromaffin cell cultures. Mutagenesis of individual
GC-boxes had little effect on Egr-1 activation. By contrast, pairwise
suppression of GC-boxes abolished activation, especially when the most
promoter-proximal of the Egr-1 sites was removed. Taken together, these
studies indicate that the
7 gene is likely to be a target for
multiple signaling pathways, in which various regulatory elements are
involved.
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INTRODUCTION |
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Nicotinic acetylcholine receptors (nAChRs)1 are members of a supergene family of neurotransmitter-gated ion channels (1, 2). Neurons in the central and peripheral nervous systems express a diversity of nAChRs with different electrophysiological and pharmacological properties (3, 4). This variety arises, at least in part, from the different combinations of subunits that can form functional nAChRs (5). Since each of the subunits exhibits well defined and restricted expression patterns in vivo (1), it is of interest to elucidate the mechanisms controlling the expression of nAChR subunit genes. At present, both positive and negative transcriptional regulatory elements have been described in several nAChR subunits (6-17).
The chromaffin cells of the adrenal medulla constitute a good model
system in which to study the function and regulation of neuronal
nAChRs. These paraneurons express the two main types of nAChRs present
in the peripheral nervous system; the one sensitive to -bungarotoxin
is formed by
7 subunits (18), whereas the insensitive one is
probably composed of
3,
5, and
4 subunits (19, 20).
Acetylcholine triggers catecholamine secretion through a mechanism that
apparently involves the latter nAChR subtype (21).
The function of the -bungarotoxin-sensitive nAChRs in chromaffin
cells remains obscure. The high Ca2+ permeability of these
receptors (22) suggests their involvement in the control of
intracellular Ca2+ levels. Previously, we have shown that
they are differentially expressed in adrenergic cells (23), presumably
providing these cells with an additional way of epinephrine release.
Accordingly,
7 subunit expression is also restricted to adrenergic
cells by a mechanism that appears to be mediated by the immediate early gene transcription factor Egr-1. This protein was found to be expressed
exclusively in adrenergic cells and shown to bind to two sites within
the proximal promoter of the
7 gene (23).
The present study was undertaken to further characterize the elements acting at this promoter. We report here that, in addition to the two Egr-1 sites previously described, at least two other positive regulatory elements exist. One of these elements, a GC-box, was shown to interact with Egr-1 (and also Sp1), whereas the other, an E-box, is recognized by transcription factors USF1 and USF2.
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EXPERIMENTAL PROCEDURES |
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Plasmid Constructions--
The isolation and characterization of
the 5'-flanking sequence of the bovine 7 subunit gene has been
previously described (23). All
7 promoter-LUC gene fusions were made
in the pGL2-Basic vector (Promega, Madison, WI), introducing in its
polylinker, upstream of the luciferase gene, the suitable
7 promoter
fragments. Deletion analysis was performed by partially digesting an
ApaI-HindIII fragment (from
339 to +43 in the
7 sequence) with BstUI, which leaves blunt ends at GCGC
sequences, and subcloning the partial digests into pBluescript
(Stratagene, Heidelberg, Germany) vector cut with
HincII-HindIII. Sequence analysis allowed
selection of the appropriate fragments, which were cloned into
pGL2-Basic and further transfected.
Cell Culture and Reporter Assays-- Chromaffin cells were isolated from bovine adrenal glands as described by Gandía et al. (24) and cultured in 90% Dulbecco's modified Eagle medium (Sigma, Madrid, Spain), 10% fetal calf serum, with 10 µM cytosine arabinoside and 10 µM 5-fluoro-2'-deoxyuridine (Sigma) added to prevent fibroblast proliferation. SH-SY5Y human neuroblastoma cells were grown in 90% Eagle's minimal essential medium with Glutamax-1 (Life Technologies, Inc., Barcelona, Spain), 10% fetal calf serum.
Plasmids were banded in two gradients of CsCl. Both cell types were transfected by the calcium phosphate procedure (25). Chromaffin cells on 48-well plates (5 × 105 cells/well) were incubated with 0.75 µg of pGL2 vector or an equivalent amount (in molar terms) of the different constructs derived from this vector and with 0.75 µg ofElectrophoretic Mobility Shift Assay--
Crude nuclear extracts
were prepared from chromaffin and SHSY-5Y cells as described by
Schreiber et al. (27). DNA fragments corresponding to the
region 77 to +43, mutated or not at the indicated elements, were
obtained by digesting pBluescript subclones with
EcoRI-HindIII and end-labeled by Klenow filling
with [
-32P]dATP. The DNA-protein binding reaction
volumes were 20 µl, containing 10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, 10% glycerol, 5 µg of bovine serum albumin, 2 µg
of poly(dA-dT)·(dA-dT) (Amersham Pharmacia Biotech), 2 µg of
nuclear extract protein, and 20,000 cpm of 32P-labeled
probe. Reactions were incubated for 10 min at room temperature, labeled
probe was added, and the incubation continued for an additional 20-min
period. For competition studies, the nuclear extract was incubated with
the competing probe prior to the labeled probe during 20 min.
Supershift assays were performed by preincubating nuclear extracts with
2 µl of antibodies against different transcription factors (Santa
Cruz Biotechnology, Santa Cruz, CA) or rabbit IgG (Sigma) for 3 h
on ice before the probe addition.
Western Blots-- 10 µg of nuclear proteins/lane were separated by 10% SDS-polyacrylamide gel electrophoresis. Western blots were carried out as described by Towbin et al. (29). After the transfer, nitrocellulose membranes were blocked overnight at 4 °C with 5% dry milk in phosphate-buffered saline, and incubated in the same way with the appropriate primary antibodies (1:500) in phosphate-buffered saline, 5% dry milk. After incubation with the secondary antibody at room temperature for 2 h, the bands were visualized by a chromogenic reaction (Sigma Fast, nitro blue tetrazolium; Sigma).
DNase I Footprinting--
The antisense strand corresponding to
region 77 to +43 of the
7 promoter was end-labeled by Klenow
filling with [
-32P]dATP. Assays were performed with
the Sure Track Footprinting kit from Amersham Pharmacia Biotech,
according to the manufacturer's instructions. Recombinant Egr-1 or Sp1
was incubated with the radiolabeled double-stranded fragment (~25,000
cpm) using the binding reaction conditions described above in the EMSA
experiments (except for the absence of EDTA and the presence of 2.5 mM MgCl2). Immediately following the 30-min
incubation at room temperature, 0.5 mM CaCl2
and 1 mM MgCl2 were added to the reactions.
This was followed by the addition of 1 unit of DNase I. The reactions were incubated at room temperature for 1 min and stopped with the
addition of stop solution (SDS/EDTA). The DNA was prepared for loading
onto an 8% polyacrylamide, 7 M urea sequencing gel (~15,000 cpm/lane), and the Maxam-Gilbert A/G chemical sequencing reaction was included as reference ladder.
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RESULTS |
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Deletion Analysis of the Promoter for the Bovine nAChR 7
Subunit--
A series of constructs was generated to determine the
regions of the
7 subunit proximal promoter (Fig.
1A) that contributed to its
maximal activity. These constructs were introduced into SHSY-5Y and
chromaffin cells, a neuroblastoma cell line and a primary cell culture,
respectively, that express the
7 subunit endogenously (18, 30). In
SHSY-5Y cells, the construct containing 77 bp of
7 promoter sequence
plus 43 bp of 5'-noncoding region (p77
7LUC) showed the maximal
activity (Fig. 1B). No significant changes in relative
luciferase activity were observed when larger constructs were used
(p125, p161, and p199
7LUC). In chromaffin cells, these constructs,
particularly p199
7LUC, showed increased activity to yield ~145%
of p77
7LUC. The activity of p77
7LUC in SHSY-5Y was about 90%
reduced when 26 bp of the
7 promoter 5'-end were deleted further
(p51
7LUC). In chromaffin cells, this deletion caused a 75% decrease
in activity, with respect to p77
7LUC. Therefore, sequences in the
minimal promoter, within 77 bp upstream of the start site of
transcription, appear to be critical for basal transcription of the
7 subunit gene in transient transfection assays.
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Characterization of the Regulatory Elements Present at 77/
15 of
the
7 Promoter by Mutagenesis and Transient Transfections--
A
search for transcription factors that could interact with elements at
the proximal promoter region of the
7 subunit revealed the existence
of several GC-boxes (labeled 1, 2, and
4 in Fig. 2A) and
an E-box (labeled 3 in Fig. 2A). In fact, our
previous study on the bovine
7 promoter (23) characterized two of
the GC-boxes (1 and 2, Fig. 2A) as binding sites
for Egr-1 and demonstrated their involvement in activation of the
7
promoter by this transcription factor. However, construct p51
7LUC
displayed low promoter activity despite containing these elements,
which suggested the contribution of other elements between
51 and
77 to maximal promoter activity. Therefore, a systematic analysis of
these putative regulatory elements was carried out, by looking at the
functional effects produced by their mutagenesis in the context of
p77
7LUC (Fig. 2B).
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Characterization of the Regulatory Elements Present at 77/+43 of
the
7 Promoter by EMSA--
DNA fragments carrying the wild-type
77/+43 promoter region and the corresponding E-box mutant (Mut
3) were labeled and incubated with nuclear extracts from
chromaffin cells (Fig. 3) and SHSY-5Y (not shown). Several retarded bands, one of them very prominent (lane 2, large dot), were
observed when using the wild type fragment. By contrast, when the E-box
mutant was used as probe, the formation of several retarded complexes
was abolished (lane 5), as indicated by the lower
intensity of the main band. This suggests that some of these complexes
result from interactions with the E-box. Moreover, an excess of the
wild fragment could totally compete the formation of retarded complexes
with both wild-type and mutant probes (lanes 3 and 6, respectively), whereas the mutant one competed
totally the retarded complexes produced by itself (lane
7) but only partially the formation of the prominent band
observed with the wild-type probe (lane 4). Thus,
the formation of some complexes with the mutant probe (lane
5) suggested that other proteins, which were further
identified (see Fig. 4), were bound to
sites distinct from the E-box.
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Interactions of Egr-1 and Sp1 with the GC-boxes in the Proximal
7 Promoter--
Recombinant Egr-1 and Sp1 proteins were used in
EMSA and DNase I footprinting to document the preferences of these
transcription factors for the three GC-rich elements previously
characterized in transfection studies (Fig. 2). Sp1 was used despite
EMSA experiments that had shown the lack of complex formation by this
transcription factor in the conditions used in our assays. However, an
interaction with this transcription factor cannot be ruled out, since
it is expressed constitutively, and a very dynamic interplay between Egr-1 and Sp1 has been observed in a variety of promoters, depending on
specific physiological conditions (34-36). In order to detect multiple
interactions, two different amounts of protein were used in EMSA; the
larger one was chosen to generate higher order complexes in the event
that they could be formed. As shown in Fig.
6, both Egr-1 and Sp1 bound to the
wild-type
77/+43 fragment (lanes 2 and
4, respectively). The use of larger amounts of proteins
induced their binding to a second site (lanes 3 and 5, double dot). When a labeled
fragment mutated in site 4 was used as probe, the action of Egr-1 was
not significantly modified (lanes 7 and
8), except for a slight decrease in band intensity. By
contrast, Sp1 could just produce a small amount of retarded complexes
and only when used at high concentration (lane 10). Hence, it appears
that the main binding site for Sp1 is located within box 4. Moreover,
only if this site is intact, the binding of Sp1 to a second site seems to be facilitated (compare lanes 5 and
10). When the labeled probe had boxes 1 and 2 mutated, the
two factors could bind to a unique site (lanes 12 and 14), since higher order complexes were not observed
(lanes 13 and 15). This site is
presumably box 4, because a probe with the three GC-boxes mutated was
not retarded by any protein. Therefore, Egr-1 can bind to the three
boxes, whereas Sp1 binds preferentially to box 4, which may facilitate
the binding to a second site. These results were confirmed by DNase I
footprinting (Fig. 7), using the same
probe of the EMSA. Thus, Sp1 protected a domain that closely
corresponds to box 4 (lane 2), whereas Egr-1 showed several protected regions (lane 3), which
included the three GC-boxes, boxes 1, 2, and 4. Shown in Fig. 7 is the
antisense strand, which was yielding the best resolution. The sense
strand gave essentially the same footprint pattern.
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Relationship between the GC-boxes and the Activation of the 7
Promoter by Egr-1--
We have shown previously that an Egr-1
expression plasmid can activate the
7 promoter when cotransfected
with p38
7LUC (23), a construct that contains only two of the three
GC-boxes to which Egr-1 can bind (boxes 1 and 2). Given that the single
mutation of box 4, an element that is absent in p38
7LUC, or the
double mutation of boxes 1 and 2 produced a marked reduction on basal promoter activity (Fig. 2B), it was of interest to study the
functional effect of Egr-1 in the broader context of p77
7LUC and its
mutants with modified GC-boxes. As shown in Fig.
8, the activity of p77
7LUC in
chromaffin cells was increased by up to 5-fold when cotransfected with
an Egr-1 expression plasmid. Single mutations of boxes 2 and 4 (pmut
2
7LUC and pmut 4
7LUC,
respectively) did not modify this effect. A significant decrease in
Egr-1 activation did occur with the single mutation of box 1 (pmut
1
7LUC) or the simultaneous mutation of boxes 2 and 4 (pmut 4-2
7LUC). However, the maximal
reduction was observed when box 1 and any of the others were mutated at
once. Thus, activation was barely observed for pmut 4-1
7LUC, whereas pmut 2-1
7LUC and pmut
4-2-1
7LUC were insensible to the action of Egr-1. These
results suggested that GC-box number 1 is the main element for the
induction of the
7 promoter by Egr-1, although the presence of any
of the other GC-boxes is required for maximal activation.
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DISCUSSION |
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Neuronal nAChRs play a significant role in the central and
peripheral nervous systems, probably regulating transmitter release at
many synapses (reviewed in Ref. 4). Their functional importance in
these processes is evidenced just by taking into account the behavioral
and cognitive effects of nicotine abuse (37) and the involvement of
neuronal nAChRs in the pathogeny of many neurological disorders (38).
Several genes that encode nAChR subunits have been identified (1), and,
depending on the subunit composition, their heterologous expression has
shown clear differences in electrophysiological and pharmacological
properties (5). For this reason, it is important to understand how the
expression of the different subunits is regulated. The present study
was aimed to characterize the cis-elements and
trans-acting factors involved in the transcriptional regulation of the nAChR 7 subunit gene.
From 5'-end deletion analysis (Fig. 1), we determined that the region
located between nucleotides 77 and +43 was necessary for the basal
promoter activity observed in SHSY-5Y cells. Additional positive
elements, outside this region, may be operative in chromaffin cells, as
demonstrated by the slight increase in the activity of larger
constructs (see Fig. 1 and also Ref. 23 for a comparison of larger
fragments of the
7 promoter). However, the most striking fact was
the large stepwise loss in promoter activity of p51
7LUC with respect
to a larger construct (p77
7LUC). Since the region between
51 and
77 contains an E-box and a GC-rich sequence (labeled 3 and
4 in Fig. 1A, respectively), these elements
appeared to be essential for promoter activity. Consequently, when any
of these elements was mutated in the context of p77
7LUC, promoter activity was reduced to ~50% (Fig. 2). Nevertheless, these elements seem necessary but not sufficient for maximal promoter activity, since
simultaneous mutation of other two downstream GC-boxes (labeled 1 and 2 in Fig. 1A), while keeping
intact elements 3 and 4, produced a large decrease in activity (Fig.
2). This is also the case for other double mutant in which elements 1 and 3 had been removed. Therefore, a primary conclusion of this study
is that the basal activity of the
7 subunit promoter is not defined
by a predominant unique element; it rather seems to reflect the
cumulative and concerted action of several transcription factors.
Element 3 corresponds to an E-box (Fig. 1). Several class B E-box
binding proteins have been identified and shown to bind the CACGTG
consensus site (39). These include USF, which preferentially binds to
this region when a thymidine residue precedes the consensus region
(TCACGTG; see Ref. 39). Element 3 in the 7 promoter region has
exactly this sequence. Our band shift assays identified USF1 and USF2
as the transcription factors that are binding to element 3. Although
some minor complexes were only displaced by USF1 antibodies, the major
band was supershifted by both anti-USF1 and USF2 antibodies, indicating
that a heterodimer of both proteins constitutes the main E-box binding
factor. Interestingly, nuclear extracts of human SHSY-5Y and bovine
chromaffin cells yielded essentially the same band shift pattern,
suggesting that regulation of the
7 promoter may follow similar
pathways in both species. This assumption, however, will have to be
confirmed once information about the human
7 promoter is available.
The avian
7 promoter, by contrast, does not contain such an
unambiguous USF binding site (6), although several CANNTG sequences are
present in the proximal promoter region. Their functionality, if any,
has not been explored.
The other main elements in the core region of the 7 promoter are
composed of GC-boxes. Our previous study indicated that the immediate
early gene transcription factor Egr-1 was able to bind to elements 1 and 2 in the context of p38
7LUC (23). Here we have confirmed these
results in a broader context (p77
7LUC) and shown that Egr-1 can also
bind to another site (element 4). Moreover, the functionality of these
elements is demonstrated when they are eliminated from p77
7LUC by
site-directed mutagenesis; the three elements are needed for maximal
basal activity, although they do not appear equivalent (Fig. 2). Thus,
it is interesting that the individual mutation of site 4 produced a
marked decrease in promoter activity, whereas its counterparts at sites
1 and 2 did not produce this effect, unless the mutations take place simultaneously. Therefore, a certain hierarchical order is suggested, in which site 4 appears critical for basal promoter activity, whereas
at least one of the other two needs also to be operative. The
co-transfection experiments with the Egr-1 expression plasmid (Fig. 8)
confirmed that the three GC-boxes are involved in Egr-1 activation, but
again in a nonequivalent manner. Thus, sites 2 and 4 can be
individually removed without affecting Egr-1 activation, but site 1 needs to remain intact for maximal activation. In addition, when this
site and any of the other two are simultaneously mutated, Egr-1
activation is abolished. Taken together, these results indicate that
Egr-1 can bind to the three sites and perform its action by binding at
least to two of them (preferentially the most promoter-proximal and
-distant ones), probably through a synergistic mechanism.
In Fig. 9, we postulate a hypothesis on
the basal transcriptional regulation of the 7 subunit gene, which
takes into account our results and have been suggested by a recent
model applied to the promoter of the nAChR
4 subunit gene (16). As
happens with several nAChR subunits, the promoter of the
7 subunit
is in a class of promoters that lack a canonical TATA box and, like many of them, it contains several GC-boxes (boxes 1, 2, and 4; Fig. 9)
to which Egr-1 can bind. Upon binding to at least two elements, Egr-1
may be involved in interactions with other transcriptional co-activators and the basal transcription machinery to activate
7
subunit expression. The Egr-1 sites that have been detected are
probably interrelated, perhaps by direct Egr-1 contacts (elements 1 and
2) or through other proteins (elements 1 and 2 with element 4). In
addition, USF proteins are also critical in transcriptional activation
upon interacting with the E-box of element 3. It is believed that USF1
affects transcription by interacting with the TFIID complex (40, 41)
and, specifically, with the TFIID subunit TAFII55 (42). In
the case of the
7 subunit promoter, the USF binding element is not
closely adjacent to the transcription initiation site; hence, USF
binding proteins may carry out their action through an additional
protein, as it happens with PC5, a co-factor that has been demonstrated
to mediate transcriptional activation by USF1 (43). Finally, a
cooperative effect of Egr-1 (at site 1) and USF (at site 3) on
transcriptional initiation appears plausible, if we consider that the
double mutant of these elements (pmut 3-1
7LUC) showed
a decrease in promoter activity larger than the mere addition of the
effects produced by the respective single mutants (pmut 1
7LUC and pmut 3
7LUC). For this reason, we have
depicted a complex of co-activators whose action may depend on their
simultaneous interactions with USF and Egr-1 proteins.
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Although a number of neuronal genes appear to be regulated in a cell
type-specific manner by silencer elements (44, 45), we have not
detected them in the 7 subunit promoter. If they exist, they might
be acting at sites not included in our constructs. Another possibility
of tissue- or cell-specific regulation could be through the
antagonistic effect that other factors can exert by binding at the E-
and GC-boxes. For instance, members of the Sp transcription factor
family could bind to G/C-rich elements and block the activating effect
of Egr-1. Although neither Sp1 nor Sp3 was shown to interact with the
GC-boxes in the context of our gel retardation assays with nuclear
extracts from chromaffin and SHSY-5Y cells, we have demonstrated that
recombinant Sp1 can bind to element 4 in the basal promoter of the
7
subunit. Therefore, depending on the concentration, cell
cycle-regulated expression, or phosphorylation state of Egr-1 and Sp
factors, an interplay between them could be established, as has been
demonstrated for other promoters (34, 35, 36, 46-48). A similar effect
could take place upon binding of Myc family members to the E-box
element 3. According to a previous study (39), the surrounding sequence of element 3 in the
7 subunit promoter is optimal for the binding of
USF proteins, but certain physiological conditions could alter binding
specificity or protein availability. Finally, if certain co-activators
are needed for coupling to the RNA polymerase II complex, as we propose
in Fig. 9, their concentration, functionality, etc. in a determined
cellular type would also be significant in regulating
7 subunit
expression.
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ACKNOWLEDGEMENTS |
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We thank Y. Wang, V. P. Sukhatme, and X. Cao for Egr-1 plasmids. The excellent technical assistance of Eva Martínez is also appreciated.
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FOOTNOTES |
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* This work was supported by grants from the Ministry of Education (Dirección General de Investigación Científica y Técnica, PB95-0690 and PM95-0110) of Spain, the Comission of the European Economic Community (SC1*CT91-0666), and the Generalitat Valenciana (GV-D-VS-20-158-96).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.
¶ A predoctoral fellow from Generalitat Valenciana.
Recipient of a Consejo Superior de Investigaciones
Científicas-Bancaja postdoctoral fellowship.
To whom correspondence should be addressed. Tel.: 34 965919479;
Fax: 34 965919484; E-mail: Manuel.Criado{at}umh.es.
The abbreviations used are: nAChR, nicotinic acetylcholine receptor; USF, upstream stimulatory factor; EMSA, electrophoretic mobility shift assays; bp, base pair(s).
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REFERENCES |
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