From the Departments of Neuroscience,
¶ Neurology, and
Biochemistry and Biophysics, University of
Pennsylvania Medical School, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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We have characterized a group of
cis-regulatory elements that control muscle-specific expression of the
rat skeletal muscle type 1 sodium channel (SkM1) gene. These elements
are located within a 3.1-kilobase fragment that encompasses the
5'-flanking region, first exon, and part of the first intron of SkM1.
We sequenced the region between 1062 and +311 and determined the
start sites of transcription; multiple sites were identified between +1
and +30. The basal promoter (
65/+11) lacks cell-type specificity, while an upstream repressor (
174/
65) confers muscle-specific expression. A positive element (+49/+254) increases muscle-specific expression. Within these broad elements, two E boxes play a pivotal role. One E box at
31/
26 within the promoter, acting in part through its ability to bind the myogenic basic helix-loop-helix proteins, recruits additional factor(s) that bind elsewhere within the
SkM1 sequence to control positive expression of the gene. A second E
box at
90/
85 within the repressor controls negative regulation of
the gene and acts through a different complex of proteins. Several of
these cis-regulatory elements share both sequence and functional
similarities with cis-regulatory elements of the acetylcholine receptor
-subunit; the different arrangement of these elements may contribute
to unique expression patterns for the two genes.
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INTRODUCTION |
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Sodium channels comprise a multigene family, the members of which are expressed in a tissue-specific and developmentally-regulated manner. The electrophysiological properties of the different channel isoforms differ subtly, and presumably reflect the underlying need for slight variations in electrical conduction in the different tissues. Several members of the sodium channel family have been implicated in various inherited disorders in muscle, heart, and neuronal tissues (1-3). Although significant progress has been made in understanding the structural defects of mutant channel proteins, the mechanisms that regulate transcriptional control of these genes have been examined in detail only for the rat brain II (RBII)1 and skeletal muscle type 2 (SkM2) isoforms (4-7). In this report, we characterize several of the cis-regulatory elements that control transcription of the skeletal muscle type 1 sodium channel (SkM1).
The two isoforms of skeletal muscle sodium channel, SkM1 and SkM2, are
regulated differentially during development. SkM2 is expressed in
embryonic muscle but is down-regulated postnatally; denervation of
adult muscle leads to the reactivation of SkM2 transcription (8, 9).
SkM1 comprises most of the sodium channel expressed in adult skeletal
muscle, with the mRNA levels up-regulating 10-fold postnatally
(8-10), and it is expressed in the same temporal manner as the
ancillary 1 subunit (11).
Since the SkM1 isoform is expressed in skeletal muscle, it seemed likely that it is regulated in part by muscle-specific transcription factors from the MyoD and MEF2 families (see Refs. 12-14, for reviews). Unlike most skeletal muscle genes, which are expressed at highest levels before birth, SkM1 reaches peak expression in adult muscle. Only a few other muscle genes, such as myoglobin, exhibit this late rise in expression levels (15), and novel factors may be involved in the regulation of such genes (16, 17). In addition, like the acetylcholine receptor (AChR), the sodium channel is expressed at higher levels beneath the neuromuscular junction than in the surrounding sarcolemma (18, 19); comparison of the SkM1 regulatory sequences to those for the AChR subunits may reveal common regulatory pathways.
We anticipate that the expression of the SkM1 gene is regulated by a
complex combination of tissue- and developmental-specific factors
working in conjunction with the general transcription apparatus. As a
first step toward elucidating the regulation of this gene, we have
carried out a detailed analysis of the cis-regulatory elements that
control its expression, elucidated similarities between some of these
elements and those of the AChR -subunit, and studied in more detail
the critical role played by two E boxes. One E box, located within the
promoter of the gene, plays a critical role in positive,
tissue-specific gene expression. We demonstrate that members of the
MyoD family of transcription factors contribute to this positive
regulation, although they are not wholly responsible for it. The second
E box plays a critical role in the repression of SkM1 expression in
non-muscle cells through binding a complex of proteins.
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EXPERIMENTAL PROCEDURES |
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Isolation of Genomic Clones--
A rat spleen genomic library in
DASH was screened using probes derived from the 5'-untranslated
region of the SkM1 gene (20), and five overlapping clones were
obtained. Clones
14.2,
29.1, and
22.1A were positive in a
Southern blot to a PstI fragment encoding sequences
310 to
+119 relative to the published translation start site of the SkM1 gene
(20). Sequence analysis of
14.2, reported previously (21), encodes
the
401 to
226 segment but then deviates from that of the cDNA
clone (20), revealing the presence of an intron between
402 and
401. The library was screened using sequences
451 to
402 as a
probe, and two clones, designated
2633 and
2A.1.1, were obtained.
A restriction site map was derived using the enzymes
HindIII, EcoRI, BamHI, and
KpnI, indicating that the five clones encompass almost 40 kb
of DNA surrounding the transcription initiation site of the SkM1 gene.
A 3.1-kb HindIII fragment obtained from clone
2A.1.1 that
was positive in a Southern blot with the
451 to
402 probe was
cloned into pBluescript (KS) (SkM1-3.1) and used for all subsequent
analysis.
DNA Sequence Analysis--
DNA sequencing (Sequenase; U. S. Biochemical Corp.) was carried out on the SkM1-3.1 clone from the 3'
edge using T7 and a series of primers to the SkM1 sequence. Using the
resulting sequence, additional primers were synthesized and sequencing
performed on the complementary strand. Three clones containing sequence
between 1062 (BglII) and
438 (SphI),
438
(SphI) and +11 (SacI), and +11 (SacI),
and +254 (XbaI) were cloned into pAlter (Promega) and single
strand DNA generated. This DNA was both sequenced and used for
generation of site-directed mutations.
RNase Protection and 5'-RACE--
RNase protections were carried
out as described (4), using a [32P]UTP-labeled antisense
RNA probe containing sequences 640 to +127 hybridized to 50 µg of
total RNA obtained from rat skeletal muscle or liver.
Generation of Reporter Gene Constructs--
The SkM1-3.1 clone
was digested with HindIII (2.8) and XbaI (+49)
and the resulting fragment was ligated into pCAT-Basic (Promega)
digested with HindIII and XbaI using a linker
(XbaI-XhoI-XbaI). This clone was
designated SkM1CAT (
2800 to +49). 5' deletion mutants
1062 and +11
were created from SkM1CAT using naturally occurring restriction sites
at
1062 (BglII) and +11 (SacI) and linkers
(HindIII-BglII;
HindIII-SacI). The 5' deletion mutant
438
(SphI) was created by inserting the SphI to
XbaI fragment from SkM1CAT into pCAT-Basic cut with the same
enzymes. The 3' deletion extending from
2.8 (HindIII) to
438 (SphI) was created by inserting this fragment into
pCAT-Basic cut with HindIII and SphI and the 3'
deletion mutant ending at +11 (SacI) was created by
digesting SkM1CAT with SacI and XbaI and ligating
using a linker (SacI-XbaI). The 5' deletion
mutants
273,
174,
135,
95,
85, and
65 were created by PCR.
The 5' primers contained 20 bp of SkM1 sequence starting at the
designated point and a restriction site for enzyme indicated below for
cloning purposes; the 3' primer was complementary to +56 to +78. The
PCR products were digested with PstI and XbaI
(
273 and
174), SalI and XbaI (
65), or
HindIII and XbaI (
135,
95, and
85), and
cloned into pCAT-Basic digested with the same enzymes. All mutants
generated by PCR were sequenced. To generate the constructs XmaS and
XmaG, the XmaI fragment encoding sequences +50 to +254 was
cloned into the XmaI site of the SkM1CAT vector and clones
corresponding to the native (XmaS) and reverse (XmaG) orientation were
selected.
Generation of Linker-scanning Mutations--
Linker-scanning
mutations of the promoter were created by site-directed mutagenesis
using the Alter Sites II kit (Promega) according to the manufacturer's
directions. The entire region between 57 (HinfI) and +11
(SacI) was sequenced and introduced back into the SkM1
sequence of either the construct XmaS or the core promoter (
65 to
+49) at those restriction sites.
Cell Culture and Transient Expression Assays--
Culture of
primary muscle cells and NIH 3T3 cells was carried out as described (5,
11). Cells were transfected by CaPO4 precipitation as
described (22) using either 8.5 pmol of SkM1 reporter gene constructs
(25-41 µg of DNA), 5 µg of pCAT-Control as a positive control, or
24.4 µg (8.5 pmol) of pCAT-Basic as a negative control. Rous sarcoma
virus--galactosidase was co-transfected as a control for
transfection efficiency. Primary muscle cultures were transfected on
culture day 2 and harvested on culture days 6 or 7, following robust
formation of myotubes. NIH 3T3 cells were split to 40% confluence
prior to transfection and harvested 2 days following transfection.
Cells were harvested and assayed for CAT activity as described (18);
samples were also assayed for
-galactosidase activity (23).
Conversion of [14C]chloramphenicol was quantitated using
a PhosphorImager (Molecular Dynamics). The
-galactosidase assays
were used to normalize the samples within an individual set of
transfected cells; for comparison between transfections or cell types,
CAT activity was expressed relative to pCAT-Control or the RBII
promoter (5).
Gel Shift and DNase Footprint Assays--
Nuclear extracts were
prepared from muscle or 3T3 cells by the method of Dignam et
al. (24). The 32P-labeled 174/
65 and
135/
82
repressor probes and wild-type and mutant
34/
23 promoter probes
were purified from a nondenaturing acrylamide gel. Double-stranded
competitor DNAs were generated by annealing the wild-type or mutant
synthetic oligonucleotides. Gel shifts and DNase footprints were
carried out as described previously (19) with the following
modifications. The binding buffer for the repressor probes was 4 mM HEPES, pH 7.9, 1% glycerol, 1% Ficoll, 20 mM KCl, 50 µM EDTA, and 0.1 mM
dithiothreitol, and the gel was run in a low ionic strength buffer (6.4 mM Tris, pH 7.5, 3.3 mM sodium acetate, and 1 mM EDTA) at room temperature. The binding buffer and
conditions described by Simon and Burden (25) were used for gel shifts
involving myogenic factors, except that 10 µg of protein was used.
Supershift assays were carried out as described previously (25) using
commercially available antibodies to MyoD (Vector) and myogenin
(Dako).
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RESULTS |
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Isolation and Sequencing of Genomic Clones and Identification of
Transcriptional Start Sites--
Five SkM1 genomic clones were
obtained from a rat spleen genomic library, and the overlaps determined
by restriction mapping (Fig. 1). A 3.1-kb
HindIII fragment derived from 2A.1.1 was positive in a
Southern blot using a probe to sequences
451 to
402 relative to the
translational start site (20). This fragment was cloned into Bluescript
KSII, and used as the starting material for all subsequent analysis.
Sequence analysis between
1062 and +311 revealed that this fragment
contains 225 bp of the first intron, the first exon (87 bp), and
approximately 2.8 kb of 5'-flanking sequence (Fig.
2A).
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Deletion Analysis of the SkM1 Genomic Sequence Reveals Multiple
cis-Regulatory Elements That Control Expression of the Gene--
We
created a series of deletion mutants of the SkM1 genomic sequence
linked to the reporter gene for chloramphenicol acetyltransferase (CAT)
and transfected these mutants into either rat primary muscle cultures,
which express the SkM1 gene, or NIH 3T3 cells, which do not express the
gene. The series of 5' deletion mutants shown in Fig.
4A terminate at +49. The
longest construction of this group, beginning approximately 2800 bp
upstream of the transcription start sites, and 5' deletion mutants to
174, produced approximately 10-fold higher CAT levels in rat primary
muscle cells than in NIH 3T3 cells, demonstrating the ability of the
5'-flanking region to drive muscle-specific expression. Deletion of
sequences from
174 to
65 resulted in a 10-fold up-regulation in the
3T3 cell line, with only a 2-fold increase in primary muscle cells.
Further deletion to +11, which falls within the cluster of
transcription start sites, reduced expression in both lines to levels
only 2-3-fold that of the pCAT-Basic vector itself. A 3' deletion
mutant that includes sequence between
2800 to +11 exhibited
expression levels in primary muscle cells similar to that of the
mutation that ends at +49, whereas further 3' deletion to
438
abolished expression in primary muscle cells. Thus, the rough
boundaries of the SkM1 "core" promoter fall between nucleotides
65 and +11, although for convenience, our core promoter constructs
extend from
65 to +49. This core promoter is not muscle-specific; the
repressor element located between
174 and
65 confers
muscle-specific expression on the SkM1 promoter.
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Analysis of the 174/
65 Repressor Element by Gel Shift and DNase
Footprint Assays--
Because the repressor located between
174 and
65 confers muscle-specific gene expression, we investigated this
element in greater detail. A 32P-labeled
174/
65 probe
was analyzed in a gel shift assay using nuclear extracts prepared
either from primary muscle cells at various stages of development or
from NIH 3T3 cells (Fig. 5A). As an independent control for the quality of the nuclear extracts, a
32P-labeled SP-1 recognition site synthetic oligonucleotide
was tested with the same extracts. A prominent shift, designated as the
upper band (UB), was detected at the same mobility in primary muscle
cells at all stages of development and in 3T3 cells. Although the
intensity of the upper band was greater in 3T3 cells than primary
muscle cells, the presence of a shift in the primary muscle cultures
once myotubes had fully formed (day 7) seemed at odds with the activity
profile in the two cell types shown in Fig. 4A. However, as
shown below, this factor is not only present in both cell types, but
also functional in both.
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An E Box at 90/
85 Plays a Crucial Role in the Function of the
Repressor--
Extending the results of the footprint analysis, we
constructed a series of mutations in which the
174/
65 repressor
element or various permutations of this sequence were transferred to a heterologous promoter. We chose a minimal rat brain type II sodium channel promoter containing sequences
130 to +177 (RBII) because it
is expressed strongly in many cell types and has structural similarities to the SkM1 promoter. As shown in Fig.
6A, the
174/
65 fragment, a
slightly longer
174/
50 fragment, and the footprinted region of
135/
82 all reduced expression of the rat brain promoter to
approximately 20% of its normal levels in both cell types, while a
fragment extending from
135 to
95 only reduced expression to 70%
of control. When a
93/
82 fragment, containing the E box at
90/
85, was transferred to the heterologous promoter, the level of
expression was also 20% of control levels. Thus, the sequence encoding
the E box conferred the same level of repression as observed for the
full
174/
65 repressor.
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Smaller Deletions between 174 and
65 on the Native Promoter
Reveal the Presence of a Muscle-specific cis-Regulatory
Element--
Deletions on the native promoter confirmed the importance
of the E box between
95 and
85 in the function of the SkM1
repressor (Fig. 6B). While deletions to
135 and
95 had only small
effects, deletions that included the E box produced a much higher
expression level in primary muscle than 3T3 cells. Further deletion to
65 greatly reduced this muscle-specific expression, indicating the presence of a positive element adjacent to the repressor. As noted above, there are two motifs within this region, AGATTG at
83/
78 and
GTTTC at
64/
60, that are conserved exactly between the SkM1 gene
and a region of the AChR
-subunit gene that acts as an enhancer (25,
27, 28).
An E Box at 31/
26 Coordinates a Positive Muscle-specific
Interaction of the SkM1 Promoter with Factors That Bind Elsewhere in
the Full-length SkM1 Regulatory Sequence--
We evaluated the SkM1
promoter by characterizing a series of linker-scanning mutations
through the region homologous to the
-subunit promoter. All
mutations were created within two different "backgrounds" to assess
their impact both on the promoter itself and on the larger, full-length
SkM1 regulatory sequence. Within the background of the core promoter,
all mutations reduced expression to 20-80% of wild-type levels in
both the positive and negative cell types, and mutations within the E
box itself did not have special significance (Fig.
7A). The basal promoter
activity arose from a distributed sequence of nucleotides, such that
all mutations diminished promoter activity while none abolished it.
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MyoD and Myogenin Form Part of the Complex of Proteins That Bind to
the SkM1 Promoter E Box--
A 32P-labeled probe
encompassing the E box (34/
23) gave rise to a prominent group of
shifted bands in primary muscle cells, while fewer bands were seen in
3T3 cells (Figs. 7B and 8). In primary muscle cells, this pattern consisted of an upper band (UB), two
closely spaced middle bands (UMB and LMB), and a lower band (LB).
Competition with both the wild-type sequence and the mutants revealed a
close correlation between the functional assay and the degree to which
each mutant disrupted the gel shift (Fig. 7B). In 3T3 cells,
there was a band of similar size and intensity to the UMB and two lower
bands of different size from those in primary muscle cells.
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DISCUSSION |
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We have identified multiple cis-regulatory elements that control
expression of the SkM1 gene in a primary muscle culture system. The
combination of 5' and 3' deletion analysis suggests that the core
promoter of the SkM1 gene is located between nucleotides 65 and +11.
Within this sequence lie consensus binding sites for two families of
transcription factors. The CG-rich sequence (
20 to
9) is
characteristic of housekeeping genes and includes a binding site for
the AP-2 factor, while the E box at
31/
26 is a binding site for the
basic helix-loop-helix (bHLH) proteins that include members of the
muscle-specific MyoD family. Linker-scanning mutations of nucleotides
40 to
11 within the background of a basal promoter sequence of
65
to +49 indicates that both families of factors are involved in basal
expression of the gene, since no single mutation eliminates promoter
function, although all reduce function. This core promoter drives
transcription in both primary muscle cells and NIH 3T3 cells,
indicating that it lacks muscle specificity.
The linker-scanning analysis of the nucleotides 40 to
11 within the
larger background of the full-length SkM1 regulatory sequence reveals
that the
31/
26 promoter E box plays a role in coordination of
positive muscle-specific expression of the SkM1 gene. Because this
property is seen only in the background of the full-length SkM1
regulatory sequence, factors that bind to this E box must work in
concert with factors that bind elsewhere in the full-length SkM1
sequence. The gel shift competition and supershift assays implicate the
myogenic bHLH factors in this function.
An element located between +50 and +254 exerts a powerful positive effect on the overall expression of the SkM1 gene. This region encodes the 3' end of the first exon and the first 167 nucleotides of the first intron. Because this element exerts at least 50% of its effect in the reverse orientation, it likely acts by binding a transcription factor rather than by stabilizing the RNA structure. Since this element is included in the full-length SkM1 regulatory sequence, it is a candidate for interacting with the promoter E box.
A repressor element located between 174 and
65 confers
muscle-specific expression on the native SkM1 promoter, although this
specificity is not seen with a heterologous promoter. Interactions between the native promoter and the repressor must produce the muscle
specificity observed on the native promoter, although we have yet to
discover the nature of this interaction. The footprint of the
174/
65 probe localizes the binding of the repressor complex of
proteins to
135/
82, and this fragment confers the same degree of
repressor activity as the larger fragment. Although the E box at
90/
85 exhibits only low-affinity binding restricted to the upper
band, it is required for repressor activity, implicating the factor
which gives rise to this gel shift in the activity of the repressor.
This factor is present in both muscle and non-muscle cell types, which
lack the MyoD family of proteins.
Immediately downstream of the repressor E box, there is an additional
muscle-specific positive element at 85/
60, within which occur two
sequence motifs, AGATTG at
83/
78 and GTTTC at
64/
60, also found
in an AChR
-subunit enhancer (25). In our primary muscle culture
model system, the
90/
85 E box masks the activity of this element.
The overall effect of the repressor is therefore both to counteract the
activity of the
85/
60 element and to confer muscle specificity on
the native SkM1 promoter. The possibility that the
85/
60 positive
element mediates the muscle-specific aspect of the interaction between
the repressor and promoter was tested by using a construction extending
from
174 to
50 on the heterologous promoter, but inclusion of the
85/
60 element fails to supply muscle specificity. Clearly, the interaction between the repressor, promoter, and
85/
60 positive element is complex, and it is unclear that their activity in
vitro reflects their activity in vivo. In the
-subunit, the region of the second motif (GTTTC) was shown to be
important in the subsynaptic expression of that gene (28). Since sodium
channels, like AChR, are clustered preferentially at the neuromuscular
junction, it will be important to determine if these elements play a
role in localized gene expression in vivo.
One candidate for the factor that binds the repressor E box is the transrepressor ZEB, which binds to a subset of E boxes that encode CACCTG (29-31). Like our factor, ZEB is a direct transrepressor, and is found in both non-muscle cells and in adult skeletal muscle (29, 31). Others have shown that there is competition between the bHLH proteins and ZEB for binding to E boxes and that this competition reduces ZEB's activity in muscle (29, 31).
Based on this competitive mechanism, we suggest a model that could
explain the difference in expression patterns of SkM1 and AChR
-subunit. Following denervation, SkM1 mRNA levels remain relatively constant (9), while the
-subunit mRNA levels increase 30-fold (32). Although there is considerable similarity between the
5'-flanking regions of the two genes, the layout of the cis-regulatory elements is different. The
-subunit contains one E box that controls both positive and negative regulation (25), while the SkM1 contains two
E boxes that control these functions separately. For the SkM1 gene, we
propose that the repressor E box is always bound by a repressor factor,
and the promoter E box is always bound by a bHLH protein. The 40-fold
increase in myogenin levels and 15-fold increase in MyoD levels
following denervation (33) have minimal impact since both E boxes are
already occupied. In contrast, the bHLH proteins could displace
repressor factor(s) bound at the sole E box of the
-subunit,
resulting in the dramatic increase in mRNA levels
post-denervation.
In the course of this work, we have identified several cis-regulatory
elements that control expression of the SkM1 gene and have begun to
address the question of how they work together. These elements include:
1) a core promoter, which lies between 65 and +11 and lacks muscle
specificity; 2) a repressor, which lies between
135 and
82 and
confers muscle specificity on the promoter; 3) a muscle-specific
positive element at
85 to
60, which partially overlaps the
repressor and the action of which is masked by the repressor; and 4) a
muscle-specific positive element that lies between +50 and +254 and
confers a 10-fold up-regulation of expression. The organization of
these four elements, and others yet to be identified, contribute to
aspects of SkM1 gene regulation shared with other muscle genes, as well
as those unique to itself.
Within the these larger regions, two E boxes play a pivotal role in the
regulation of the gene, with an E box at 90/
85 controlling negative
regulation and an E box at
31/
26 controlling positive regulation.
Although some of the transcription factors binding these elements are
the same factors that control expression of other genes, the
arrangement of the cis-regulatory elements in SkM1 may lead to
interactions between those factors that give rise to the unique
regulation of this gene.
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ACKNOWLEDGEMENTS |
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We thank Martha Sholl, Huanying Zhou, and Virginia Harris for superb technical assistance, Hui Zhang and Jim O'Malley for helpful discussions, and Robert Maue and Gail Mandel for reviewing previous drafts of this manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants NS-34954 (to R. L. B.) and NS-01852 (to M. R.).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) AF042092.
§ To whom correspondence should be addressed: 223 Stemmler Hall, Dept. of Neuroscience, University of Pennsylvania Medical Center, Philadelphia, PA 19104-6074. Tel.: 215-898-0521; Fax: 215-573-2015; E-mail: kraner{at}mail.med.upenn.edu.
1 The abbreviations used are: RBII, rat brain II; SkM1 or -2, skeletal muscle type 1 or 2; AChR, acetylcholine receptor; kb, kilobase(s); bp, base pair(s); RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; UB, upper band; LB, lower band; bHLH, basis helix-loop-helix.
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REFERENCES |
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