From the Laboratory of Developmental Neurobiology, NICHD, National Institutes of Health, Bethesda, Maryland 20892
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thrombin has been shown to mediate neurite
retraction in neurons and synapse elimination at the neuromuscular
junction. The presence of prothrombin mRNA has been demonstrated in
brain and in muscle, but extra-hepatic regulation of the prothrombin
gene has not been investigated. To identify cis-acting DNA
elements involved in the expression of the prothrombin gene in muscle, we have isolated and analyzed a 1.3-kilobase pair promoter region of
the mouse prothrombin gene. Using a series of transiently transfected plasmid constructs in which gene segments of the prothrombin promoter were linked to the luciferase gene, we have identified a sequence, 302 to
210, essential for prothrombin promoter activity in
C2-myotubes. Fine analysis revealed that deletion of nucleotides
between
248 and
235 eliminated prothrombin promoter activity in
C2-myotubes. Furthermore, electrophoretic mobility shift assays
demonstrated that a nuclear factor present in C2-myotubes, but not in
C2-myoblasts or HepG2 hepatocytes, specifically binds to the sequence
241 to
225. Substitutional mutation of nucleotides
237 to
231
abolished myotube-specific promoter activity and inhibited the nuclear
factor binding. Quantitative reverse transcription polymerase chain
reaction demonstrated the expression of prothrombin mRNA in
myotubes, but not in myoblasts, of primary, C2, and G8 muscle cells.
This result correlates with the lack of prothrombin promoter activity
in C2-myoblasts. The data thus suggest that a myotube-specific nuclear
factor binds to a cis-acting sequence encompassing the core
nucleotides
237 to
231 and plays a critical role in
muscle-specific, differentiation-dependent expression of
the mouse prothrombin gene.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thrombin is synthesized and secreted from hepatocytes as a proteolytically incompetent zymogen form, prothrombin, the activation of which occurs at the sites of vascular injury. The function of thrombin has been extensively studied in the vascular system, and thrombin is known to play a key role in the maintenance of hemostasis (1). Recent reports have also identified extravascular cellular functions that are mediated by thrombin in the process of neural development. Thrombin has been shown to modulate the shape of astrocytes (2, 3) and to mediate neurite retraction in neuronal cells (4-7) and synapse elimination at the neuromuscular junction (8, 9). Most thrombin-mediated cellular effects are observed experimentally with the addition of exogenous thrombin or thrombin inhibitors to an in vitro culture system. The cellular localization of non-hepatic prothrombin mRNA is of interest because most thrombin-mediated cellular effects observed in vitro are thought to occur in the normal course of development, such as synapse reshaping. The recruitment of activated thrombin from blood in the absence of vascular injury during these events is unlikely. In support of this scenario, the expression of prothrombin mRNA has been demonstrated in brain and neural cell lines (10, 11), as well as in rodent skeletal muscle and primary skeletal muscle cultures (9, 12). Moreover, thrombin proteolytic activity was detected in the supernatants of mouse muscle cultures (12), and in extracts of mouse skeletal muscle (9). However, the transcriptional regulation of prothrombin gene expression in tissues other than the liver has not been studied.
We have investigated the expression of the mouse prothrombin gene in muscle, and in this report, we demonstrate that transcriptional regulation of the mouse prothrombin gene in skeletal muscle cells is distinct from that in liver cells. Furthermore, the prothrombin gene is not expressed in myoblasts but is activated upon differentiation of skeletal muscle in culture.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of the Mouse Prothrombin Promoter Region-- The 5'-flanking region of the mouse prothrombin gene was cloned using the Genome Walker Kit (CLONTECH). The kit provides five sets of mouse genomic libraries generated from mouse genomic DNA cut with different restriction enzymes and ligated to short adapter sequences. Primary and nested polymerase chain reactions (PCRs)1 were performed using forward primers complementary to the adapter sequence and reverse primers, 5'-TACCAGGCTAACCAGGGCAGCCAGAGC-3' and 5'-AGCCAGAGCCAAGCAGCCAGGGGAGGCC-3', which correspond to nucleotides 84-58 and nucleotides 66-37, respectively, of the mouse prothrombin cDNA sequence (13). A 1.3-kilobase pair (kbp) PCR product was cloned into the pCR 2.1 vector (Invitrogen), designated as pCR/1.3-kbp, and sequenced on both strands by a commercial service (Lark Technologies, Inc.).
To determine the transcription start site of the prothrombin gene, poly(A)+ mRNA from the leg muscles of embryonic day 16 mice was isolated using the Oligotex Direct mRNA purification kit (Qiagen). Muscle mRNA (100 ng) was subjected to the 5'-RACE, Version 2.0 (Life Technologies, Inc.). Reverse primers 5'-CACTGAATCACACACACTGTGT-3' and 5'-TCCTCCAGGAAGCCACTGTT-3', corresponding to nucleotides 300-281 and 170-151, respectively, of the mouse prothrombin cDNA sequence, were employed in primary and nested PCRs (13). Putative clones were identified by EcoRI and ApaI/SacII restriction enzyme digestion analysis, and four clones were sequenced using the T7 Sequenase kit (Amersham Pharmacia Biotech). All animals used for the study were handled in accordance with the National Institutes of Health Animal Care and Welfare protocol.Reporter Plasmids-- Prothrombin promoter gene segments containing various 5'-ends were generated by PCR using the pCR/1.3-kbp plasmid as a template. Forward primers used for PCR are shown in Table I. Promoter sequences containing substituted nucleotides, sequences M1 to M5, were also generated by PCR using forward primers with the indicated nucleotide substitutions (Table I). The numbering of nucleotides is relative to the transcription start site (+1) determined from the 5'-rapid amplification of cDNA ends. A common reverse primer, 5'-GTGAGATCTAGTGTGTCAGCTCCTG-3', contained a BglII site to facilitate cloning into the pGL3-basic vector (Promega) linearized with SmaI/BgllI. PCR was performed utilizing pfu polymerase (Stratagene). All the reporter plasmid constructs were sequenced on both strands to confirm their fidelity to the sequence of the 1.3-kbp mouse prothrombin gene. A series of pure clones was isolated by two consecutive platings on ampicillin-containing LB agar plates. Overnight cultures of 300 ml were subjected to the Maxi DNA preparation kit (Qiagen), and approximately 400 µg of plasmid was obtained. The stock reporter plasmids were diluted to 2 mg/ml in a buffer containing 10 mM Tris-Cl and 1 mM EDTA (TE), and kept at 4 °C.
|
Cell Culture, Transfection, and Luciferase Assay-- The mouse C2 skeletal muscle cell line was obtained from Dr. Christian Fuhrer (National Institutes of Health, Bethesda, MD) and maintained at 37 °C in Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum. The HepG2 liver cell line was purchased from ATCC (Rockville Pike, MD) and maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum. Transient transfection was performed by the method of calcium phosphate/DNA co-precipitation using a commercial kit (Life Technologies, Inc.). For the transfection of C2-myoblasts, mononucleated C2 cells at approximately 40% confluency were transfected, and the cells were harvested 48 h later. No signs of fusion were observed microscopically at the time of harvesting. For C2-myotubes, C2 cells 80-90% confluent were transfected with reporter constructs. After 24 h, the medium was replaced by Dulbecco's modified Eagle's medium supplemented with the 5% horse serum to induce differentiation into multinucleated myotubes. Within 48 h, fusion was complete, and C2-myotubes were harvested for the luciferase assay. pRL-CMV vectors were co-transfected with pGL3 reporter plasmids to normalize the pGL3 reporter luciferase activity according to the dual-luciferase reporter assay system (Promega). Measurements were made using a Lumat LB 9507 luminometer (EG & G Berthold, Wilbad, Germany).
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear protein
extracts from C2-myoblasts, C2-myotubes, and HepG2 cells were prepared
as described (14) with the modifications described in Ref. 34. Nuclear
extracts were dialyzed in a buffer containing 20 mM HEPES
(pH 7.9), 20% glycerol, 100 mM KCl, 0.2 mM
EDTA, 2 mM phenylmethylsulfonyl fluoride, and 5 mM dithiothreitol at 4 °C. Any precipitation formed
during dialysis was removed by centrifugation. Protein concentrations
were determined using a commercially available Bradford assay
(Bio-Rad). For the EMSA probes, wild type (WT) and five mutant
constructs (M1-M5) containing nucleotides from 284 to
140 of the
prothrombin promoter sequence were generated by PCR amplification using
the pCR/1.3-kbp construct as a template. PCR was performed with WT or
mutant forward primers (Table I) and a common reverse primer,
5'-AGGTTCTCCTGGAACCAG-3' (
140 to
157). PCR products were purified
by low melting point agarose gel electrophoresis and end-labeled with
[
-32P]ATP (3000 Ci/mmol, Amersham Pharmacia Biotech)
using T4 polynucleotide kinase (Life Technologies, Inc.) and separated
from free nucleotides by Sephadex G-25 chromatography. Oligonucleotides
matching short segments of the WT fragment were synthesized by a
commercial service (Genosys) and purified by denaturing polyacrylamide
gel electrophoresis. For competitive EMSA, the double-stranded
oligomeric probes were prepared by annealing equal amounts of
complementary oligonucleotides. A 100-fold excess of unlabeled probe
was added in competitive binding reactions. DNA-protein binding
reactions were carried out in a total volume of 15 µl, which included
2 µg of nuclear proteins, 1 µg of poly(dI)·poly(dC), 50 fmol of
probes (~20,000 cpm), and a buffer consisting of 5% glycerol, 0.5 mM EDTA, 50 mM NaCl, 10 mM Tris-Cl,
pH 7.5. After incubation at room temperature for 30 min, DNA-protein
complexes were separated from the free oligonucleotides by
electrophoresis on a 4% polyacrylamide gel in buffer containing 50 mM Tris base, 380 mM glycine, and 2 mM EDTA. The gel was dried and autoradiographed.
Quantitative Reverse Transcription-PCR-- RNA samples from primary muscle and fibroblast cultures and skeletal muscle cell lines C2 and G8 were isolated using TRIzol (Life Technologies, Inc.). Primary mouse muscle cultures were prepared as described in Ref. 12, and primary fibroblasts were obtained from the preplating used to remove contaminating fibroblasts during muscle culture preparation. The G8 cell line was obtained from ATCC and maintained in Dulbecco's modified Eagle's medium with 10% horse serum and 10% fetal calf serum. G8 cells differentiated into multinucleated myotubes when at confluency without a reduction in serum content of the medium. cDNA was synthesized from 1 µg of total RNA by priming with oligo(dT)18 using the Advantage RT-for-PCR kit (CLONTECH).
Competitor mimic cDNAs flanked by the target gene primer sequences (5'-CAACATCAATGAGATACAGCCCAGCGTCC-3' and 5'-CATGCGTGTAGAAGCCGTATTTCCCCTTC-3' for prothrombin and 5'-GGCCAGATCCTGATGCCCAAC-3' and 5'-CAGCTGTGCTGCTCTTTCTAC-3' for rPL32) were generated using a PCR mimic construction kit (CLONTECH). The mimic cDNAs were initially diluted 10-fold ranging from 100 attomoles/µl to 10 ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Characterization of the Promoter Region of the Mouse Prothrombin
Gene--
The DNA sequence of the mouse prothrombin 1.3-kbp clone has
been submitted to GenBankTM. The sequence between 972 and
the translation initiation codon ATG had 69% identity with the
corresponding region of the human prothrombin gene, based on a GCG
sequence alignment program (Genetics Computer Group, Inc., Madison,
WI). The human prothrombin sequence used for the alignment was as
previously reported (16) with GenBankTM accession number
M65141. The sequence between
1211 and
1017 was 86% identical to
the mouse B1 repetitive sequence (17) found in numerous mouse genes
based on a National Center for Biotechnology Information BLAST search
(18). The transcription start site was determined to be five
nucleotides upstream from the translational initiation codon ATG based
on sequence analyses of the four clones resulting from the 5'-rapid
amplification of cDNA ends. The promoter region of the mouse
prothrombin gene does not contain a consensus CCAAT motif or a
canonical TATA box, as has been reported for the human prothrombin gene
(16). However, we have located a TATA-like sequence, TATTAA, at
46
with respect to the transcription start site.
Myotube-specific Expression of Prothrombin mRNA-- mRNA expression of mouse prothrombin was investigated in primary mouse muscle cultures and in the mouse skeletal cell lines C2 and G8 (Fig. 1). A competitive PCR was used to quantitatively determine differences in prothrombin mRNA expression. The expression of prothrombin mRNA could be detected in myotubes of primary, C2, and G8 cells. Prothrombin mRNA in mononucleated myoblasts both from primary and from C2 and G8 was undetectable. The quantitation results of the competitive PCR on rPL32 demonstrated that housekeeping rPL32 mRNA levels were equivalent in myoblasts and myotubes (Fig. 1), thus confirming that prothrombin mRNA is present in differentiated multinucleated myotubes but not in undifferentiated myoblasts of primary, C2, and G8 cells. The expression of prothrombin mRNA was also not detected in the contaminating primary fibroblasts obtained during the muscle culture preparation (Fig. 1).
|
Differential Activity of the Mouse Prothrombin Promoter in
C2-Myotubes and HepG2 Cells--
To investigate the contributions of
specific promoter regions to prothrombin gene expression in muscle and
liver, varying lengths of the mouse prothrombin gene promoter
possessing the same 3'-end sequence were linked to the 5'-end of a
promoterless luciferase reporter gene in the pGL3-basic plasmid vector.
The reporter plasmid constructs were initially tested in human HepG2 hepatocytes to investigate the promoter activity of mouse prothrombin constructs in relation to that previously reported for the human prothrombin promoter in HepG2 cells (16). The overall pattern of
activity of the mouse reporter constructs in HepG2 cells (Fig. 2) was nearly identical to that of the
human prothrombin reporter constructs tested in HepG2 cells (16). In
the human gene, a liver-specific cis-acting sequence was
shown to be located between 940 and
860 (for numbering of the human
gene, see Ref. 16), and deletion of nucleotides in this region
virtually destroyed the activity of the human prothrombin gene promoter
in HepG2 cells (16). The GCG best-fit alignment indicated that residues
940 to
840 in human prothrombin align with
816 to
723 in the
mouse prothrombin sequence (data not shown), and deletion of this
region in mouse also drastically diminished promoter activity in HepG2 cells (Fig. 2). In addition, no significant differential activity was
observed among mouse promoter segments terminating between nucleotides
607 and
77 (Fig. 2), similar to the case of human gene (16). As
described for the corresponding sequence of human prothrombin, the
mouse sequence
816 and
723 was also flanked by Sp-1-like sequences.
Moreover, a putative liver-specific enhancer AATATTT (hepatic nuclear
factor 1) identified in the human promoter is also conserved in the
mouse prothrombin gene.
|
Localization of a cis-Acting Element in the Proximal Promoter
region of the Mouse Prothrombin Gene--
Five additional reporter
plasmids were constructed by progressive deletion of 5' sequence
spanning 302 to
210 and were transfected into C2-myotubes (Fig.
3). The luciferase activities of the
reporter constructs are shown relative to the activity of the construct
302/+5 as 100% activity. The
235/+5 construct was about half as
active as the longer constructs. Therefore, the proximal sequence between nucleotides
248 to
235 may contain an important
cis-acting regulatory sequence that is required for
prothrombin promoter activity in C2-myotubes.
|
Detection of trans-Acting Factors in C2-Myotube Nuclear
Extracts--
In pursuit of finding prothrombin gene-specific
trans-acting factors in C2 nuclear extract, EMSA was
performed with the probe 248/
140 and the nuclear extracts of
C2-myoblasts, C2-myotubes, and HepG2 cells (Fig.
4). The results demonstrated that complex C-I was present only in the nuclear extracts from C2-myotubes. Other
complexes (C-II, C-III, and C-IV) were shared between C2-myoblast and
C2-myotube nuclear extracts. The complexes CB-I and CB-II were observed
either exclusively or noticeably higher in C2-myoblasts nuclear
extracts. There was no apparent binding of nuclear factors from HepG2
cells specifically in the area of the C-I complex.
|
|
Mutational Analysis of the cis-Acting Sequence--
Because the
oligomeric probe 241/
225 successfully inhibited the binding of C-I
nuclear factor to the WT probe
248/
140 (Fig. 5), the region
241
to
227 was mutated by step-wise substitution of adenine nucleotides
(Fig. 6A). A nearly complete
inhibition of the binding of complex C-I was observed with the mutant
probes M2 and M3. The results were in agreement with transfection
analysis of the five mutant reporter plasmid constructs (Fig.
6B). The luciferase activity of the constructs was evaluated
relative to the activity of the WT construct. Constructs containing
mutations M2 (
237/
235) and M3 (
233/
231) had diminished
activity, comparable to the activity loss observed with the
235/+5
construct. The M1 mutation resulted in a moderate inhibition of the C-I
binding but did not result in a significant decrease in reporter
activity. Taken together, these results indicate that C-I interacts
with the cis-region encompassing the core nucleotides
237
to
231 and plays a critical role in prothrombin reporter activity in C2-myotubes.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Until the early 1990s, prothrombin was considered to be a model gene for studying liver-specific gene regulation because of its abundant and exclusive expression in the liver (16). Recent reports, however, strongly indicate that the regulation of prothrombin gene expression in the skeletal muscle might be independent of that in the liver. In particular, the expression of prothrombin mRNA in muscle appears to be innervation-dependent, being high at birth and drastically diminishing after the period of stable synapse formation (9). Moreover, we have recently found that the denervation of adult limb muscle significantly up-regulates the expression of prothrombin.2 Nevertheless, the localization of prothrombin mRNA in muscle has been problematic due to extremely low mRNA levels. In our experience, the expression of prothrombin mRNA in neonatal mouse skeletal muscle was up to 3 orders of magnitude lower than in neonatal and adult mouse liver. Although a sensitive reverse transcription-PCR analysis detected the expression of prothrombin mRNA in the leg muscle at mouse embryonic days 12 and 16 (9), the cellular localization of prothrombin using in situ hybridization revealed the expression of prothrombin exclusively in the liver of the corresponding days of embryos (19). It has been reported recently that application of exogenous thrombin to C2-myoblasts resulted in a delay in the formation of myotubes without being mitogenic (20). However, in that study, the authors did not investigate endogenous prothrombin expression in C2 cells. Earlier, exogenous thrombin had been shown to be mitogenic in rat primary myoblasts (21). Using a quantitative reverse transcription-PCR, we have demonstrated here that prothrombin mRNA is exclusively expressed in differentiated myotubes but not in myoblasts of both primary muscle culture and skeletal muscle cells C2 and G8. The prothrombin message appears upon fusion of muscle cells, and thus, it is intriguing to speculate about the in vivo function of endogenous prothrombin in the muscle fibers. The proteolytic activity of thrombin has already seen associated with the process of synapse elimination at the neuromuscular junction (8, 9).
Our experiments revealed that the activity of the prothrombin gene in
C2-myotubes was significantly dependent on a C2-myotube nuclear factor
C-I binding to the sequence between 241 and
225 encompassing the
nucleotides TGATTCA (
236/
230), a sequence previously reported to
bind to AP-1 (22). The substitution of either nucleotides
237/
235
or
233/
231 abolished both the binding of C-I and reporter activity
in C2-myotubes. A major component of the AP-1 binding factor, c-Jun, is
known to form homodimers as well as heterodimers with Jun B, Jun D,
c-Fos, Fra-1, Fra-2, and Fos B. A putative AP-1 binding site is located
in a corresponding region of the human prothrombin sequence based on a
search using Transcription Element Search Software on the World Wide
Web.3 We therefore performed
both supershift EMSA and EMSA coupled with Western blot analysis (data
not shown) using antibodies (purchased from Santa Cruz Biotechnology)
specific to c-Jun (cross-reactivity with c-Jun, Jun B, and Jun D) and
c-Fos (cross-reactivity with c-Fos, Fos B, Fra-1, and Fra-2). In
addition, we have performed competitive EMSA with the 26-mer DNA probe
containing a consensus AP-1 binding motif TGACTCA (Santa Cruz
Biotechnology). None of the antibodies alone or combined showed a
reactivity with the C2-myotube nuclear complex C-I. The 26-mer AP-1
sequence failed to compete for the binding of C-I to the
248/
140
probe (data not shown), whereas the 16-mer oligomeric
241/
225
probe, encompassing the sequence TGATTCA, competitively abolished the
binding of C-I to
248/
140. It has been previously reported that the
expression of c-jun mRNA is down-regulated during differentiation
of C2 cells and that the constitutive expression of c-Jun in
C2-myoblasts inhibits the myogenic differentiation of C2 cells (23).
Thus, we have concluded that the possibility of the C-I complex
containing AP-1 is remote. We are currently engaged in purification of
the C-I factor, and further analysis will determine its identity.
A family of myogenic basic helix-loop-helix DNA-binding proteins,
mainly MyoD, Myf5, myogenin, and MEF4, play a pivotal role in the
determination of muscle lineage and the differentiation of skeletal
muscle fibers (reviewed in Refs. 24 and 25). These myogenic factors
bind E-box motifs (CANNTG) found in control regions of muscle-specific
genes, and the binding of the myogenic factors to the E-box has been
shown to play a critical role in the expression of muscle-specific
genes (reviewed in Refs. 24 and 25). We have identified four E-box
motifs in the 1.3-kbp promoter region of the mouse prothrombin gene.
However, the three E-boxes located between 1218 and
802 did not
play a positive role in reporter activity in transient transfection
because the highest luciferase activity was obtained with the construct
802/+5. Also, the construct
151/+5, possessing a proximal E-box,
did not exhibit any appreciable level of luciferase activity in
C2-myotubes (Fig. 2). Although maximal expression of many
muscle-specific genes is controlled by the presence of more than one
E-box, it has been reported that certain muscle-specific genes can be
controlled by a single E-box accompanied by other regulatory sites
(26-28). There have also been examples of muscle-specific genes that
do not contain E-boxes within their regulatory regions (29-31). Such
regulatory regions were shown to contain motifs, such as CArG (29),
M-CAT (30), and MEF-2 (32, 33), that were also shown to participate in the activation of muscle-specific genes. However, the C-I binding site,
CTTATTGATTC (
241/
231), does not resemble CArG, M-CAT, or MEF-2
sequences. Whether myogenic factors interact in cis with the
C-I factor or C-I is indirectly activated by myogenic factors during
muscle differentiation remains to be determined.
The myotube-specific complex C-I readily interacts with the probe
248/
140; however, C-I did not bind to the oligomeric probes
241/
210 and
241/
225. Instead, a ubiquitous nuclear factor (not
AP-1) present in both C2-myoblasts and C2-myotubes bound to these two
probes, as did nuclear factors in HepG2 cells (data not shown). Most
importantly, there was no evidence of differential binding activity of
C2-myotube nuclear extracts to either
241/
210 or
241/
225 probes
in comparison with nuclear extracts from C2-myoblasts and HepG2 cells.
Nevertheless, when these probes were used in excess in the competitive
EMSA, they could compete the binding of C-I to the probe
280/
140.
This might indicate a cooperative interaction between the C-I complex
and nuclear complexes bound to the
209 to
148 that resulted in an
increased binding affinity of C-I to its site.
In this report, we have demonstrated that regulation of the prothrombin
gene in muscle is quite distinct from that in liver and that its
activation is dependent upon the terminal differentiation of muscle
cells. Detection of the endogenous prothrombin mRNA in C2-myotubes
but not in myoblasts is in agreement with the observation that the
prothrombin promoter is active in C2-myotubes but not in C2-myoblasts.
It has been shown that exogenously applied thrombin inhibitors can
completely block activity-dependent synapse elimination in
a nerve-muscle co-culture system (8) and that cholinergic stimulation
of aneural myotubes results in the augmentation of prothrombin mRNA
expression (12). It will thus be interesting to investigate whether the
cis-acting sequence TTGATTC (237 to
231), identified
here to be critical for the transcription in differentiated muscle, can
also confer activity-dependent regulation of the
prothrombin gene in myotubes.
![]() |
ACKNOWLEDGEMENTS |
---|
We express our appreciation to Drs. Gretchen Gibney and James Brady for their critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* 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) AF049131.
To whom correspondence should be addressed: Bldg. 49, Rm. 5A38,
Laboratory of Developmental Neurobiology, NICHD, National Institutes of
Health, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-402-1484;
Fax: 301-496-9939; E-mail: shkim{at}helix.nih.gov.
1 The abbreviations used are: PCR, polymerase chain reaction; kbp, kilobase pair(s); EMSA, electrophoretic mobility shift assay; WT, wild type.
2 S. Kim, unpublished data.
3 J. Schug and G. C. Overton (1997) Technical report CBIL-TR-1997-1001-v0.0, Computational Biology and Informatics Laboratory, School of Medicine, University of Pennsylvania, (available at agave.humgen.upenn.edu/tessl).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|