(Received for publication, June 5, 1995; and in revised form, August 9, 1995)
From the
The gene encoding the putative zinc finger protein tristetraprolin (TTP), Zfp-36, is rapidly induced by a variety of mitogens and growth factors. We show here that 77 base pairs 5` of the transcription start site are sufficient for full serum inducibility of the mouse Zfp-36 promoter. This region of the promoter includes consensus sequences for the binding of the transcription factors EGR-1, AP2, and Sp1. In addition, we have identified a previously undescribed element, TTP promoter element 1 (TPE1); this 10-base pair sequence includes a palindrome and is identical in the human, bovine, and mouse genes. Each of the three binding elements, EGR-1, AP2, and TPE1, contribute to the serum induction of Zfp-36 and can confer serum-inducible expression on a heterologous minimal promoter. Gel mobility shift assays demonstrated the formation of complexes consisting of this region of the promoter and cellular nuclear proteins and demonstrated that the extent of complex formation could be altered by treatment of the cells with serum or insulin. These results suggest that the response of Zfp-36 to serum and other mitogens is mediated by a series of cis-acting elements acting in concert to confer full inducible transcription of this gene.
An early response of many cells to serum and polypeptide growth
factors is the activated transcription of specific genes in the absence
of de novo protein synthesis. Many of these immediate-early
response genes encode regulatory proteins that mediate growth
responses, including transcription factors that modulate the expression
of other genes. These genes encode a number of well studied
transcription regulators such as the fos and jun families. One interesting member of the immediate-early response
class of genes encodes tristetraprolin (TTP), ()(1, 2) a zinc finger protein also known
as Nup475 (3) and TIS11(4) . This protein, which is
encoded by the gene Zfp-36(2) , is expressed at very
low levels in quiescent fibroblasts but is rapidly induced by serum,
polypeptide growth factors, and phorbol 12-myristate 13-acetate (PMA) (1, 3, 4) . This expression is transient,
with most mRNA disappearing after 2 h. In these cells, induction of Zfp-36 expression is independent of protein synthesis, and
treatment with cycloheximide results in superinduction of the gene (1, 3, 4, 5, 6) The
mechanisms that control the activation of Zfp-36 transcription
have not been defined.
To identify sequence elements involved in the transcriptional control of TTP synthesis, we have characterized the 5`-flanking region of Zfp-36 using site-directed mutagenesis and deletional analysis. Several potential binding sites for known transcription factors have been identified in this study as contributing to the serum-stimulated activity of this promoter. In addition, we have identified and characterized a previously unknown promoter element that appears to function as a transcriptional enhancer and that participates in the regulation of serum-induced Zfp-36 transcription.
A human genomic clone was obtained by screening a
human placental genomic library (Clontech) with the human TTP cDNA (2) (GenBank accession number for the human
genomic sequence: M19844). The bovine TTP cDNA was obtained from a
bovine aorta epithelial cell cDNA library (Stratagene) using the human
TTP cDNA as a probe(2) . The bovine cDNA was then used to
screen a bovine liver genomic library (Stratagene). The bovine genomic
sequence has been deposited in GenBank
(accession number
L42319).
Base pair numbers in this report refer to the transcription initiation site in the mouse genomic clone as determined by primer extension assays (data not shown). The position of the single intron was determined by sequence comparison of the genomic clones with the cDNAs and had the conventional sites for splice donors and acceptors(7) . Southern mapping, subcloning, and DNA sequencing were performed by standard techniques(8) .
In order to test potential promoter
elements, Glo48TTP was constructed as follows. The mouse TTP cDNA was
inserted into the EcoRI cloning site of pBS,
and a synthetic 48-mer double-stranded human
-globin promoter (9) with XbaI and BamHI coherent termini was
ligated 5` to the TTP cDNA at the XbaI-BamHI site of
the vector. Synthetic double-stranded serum response element (SRE,
5`-tcgacAGGATGTCCATATTAGG-3`; (10) ), EGR-1
(5`-tcgacGCGGGGGCG-3`; (11) ), AP2
(5`-tcgacTCTAGTGGCCACGCCCCCAGGC-3`; (12) ), and TTP promoter
element 1 (TPE1, 5`-tcgacCGTCCCGGAAGC-3`; this paper) DNA binding
sequences with SalI coherent termini were ligated individually
upstream of the minimal
-globin promoter at the SalI
cloning site to create reporter constructs containing these elements.
All insertions were confirmed by dideoxy sequencing (U.S. Biochemical
Corp.).
HIR3.5 cells(15) , a generous gift from Dr. J. Whittaker (State University of New York, Stoney Brook, NY), were grown as described (16) .
Transient transfections were performed with plasmid
DNA in calcium-phosphate precipitates prepared in a transfection
solution containing 140 mM NaCl, 25 mM HEPES, 0.12
mM CaCl, and 0.75 mM sodium phosphate.
CEF cells were plated 1 day before the transfection in 10-cm tissue
culture dishes at a density of 3
10
cells/dish.
After removing the culture medium, 1 ml of transfection mixture
containing 15 µg of test plasmid and 5 µg of pXGH5 was added to
each dish for 30 min at room temperature. The cells were then incubated
at 37 °C following the addition of 9 ml of culture medium. Four h
after incubation with the plasmids, the cells were treated for 4 min
with 4 ml of 10% glycerol in HEPES-buffered saline and washed two times
with phosphate-buffered saline to remove the remaining precipitate. A
24-h incubation in complete culture medium followed to allow the
transfected DNA to be expressed. The cells were then incubated for 24 h
in medium containing 0.5% FBS to synchronize cells into quiescence and
were harvested to prepare total cellular RNA. Plasmid pXGH5 encoding
human growth hormone driven by the mouse metallothionein-1 promoter (17) was co-transfected as an internal control for transfection
efficiency. Growth hormone released was measured by the HGH transient
gene expression assay (Nichols Institute Diagnostics). HIR3.5 cells
were plated at the density of 1
10
cells/dish and
were transfected with 10 µg of test plasmid as described above.
The following synthetic oligonucleotides containing potential sequences for DNA binding factors were used in this study. For EGR1-AP2, two complementary synthetic oligonucleotides were annealed to form a double-stranded oligonucleotide corresponding to mouse Zfp-36 nucleotides -77 to -37. A 5-base single-stranded tail (SalI site) was included at the 5` end, and a 6-bp double-stranded EcoRV site was included at the other. For TPE1, two complementary synthetic oligonucleotides were annealed to form a 22-bp double-stranded oligonucleotide corresponding to bases -68 to -55 of mouse Zfp-36, with cloning sequences for HindIII and SalI site at either end.
To ensure
sequence fidelity, each oligonucleotide pair was cloned into Bluescribe
and sequenced. Double-stranded oligonucleotides were released by
digesting the plasmid with appropriate restriction enzymes, and the
ends were filled in with [-
P]dCTP (DuPont
NEN) and unlabeled dATP, dGTP, and dTTP (Life Technologies, Inc.).
Unlabeled dCTP was subsequently added to the reaction. The labeled DNA
was separated from unincorporated radioactivity and the cloning vector
by acrylamide gel purification.
Competitor fragments containing putative binding sequences for EGR-1 (tcgacGCGGGGGCG), AP2 (tcgaCCCCCAGGC), TPE1 (AGCTTCGTCCCGGAAGCTCG), and mutant TPE1(AGCTTCGTAACTTAAGCTCG) were made by annealing the complementary strands and filling in the ends with dNTPs using the Klenow fragment of DNA polymerase I.
Figure 1: Mouse TTP promoter-driven expression of human growth hormone. A, HIR3.5 cells were transfected with plasmids containing 5 kb (NcoI-NcoI), or 2.1 kb (XhoI-NcoI), of genomic sequence 5` of the TTP translation initiation site linked to the promoterless growth hormone gene (pØGH). Medium was sampled 48 h after transfection, and immunoreactive growth hormone was measured. The results shown are the means ± S.D. from three plates of cells in each assay; data from two independent experiments are shown. B, identical experiments were performed in which CEF cells were transfected with plasmids containing either 2.1 kb or 137 bp of genomic mouse TTP sequence 5` of the translation initiation site linked to the promoterless growth hormone gene (pØGH). Data shown are from three independent experiments. Individual experiments are indicated by the Roman numerals at the bottom of the graphs.
Figure 2:
5`
sequences of the TTP promoter required for serum induction. A,
diagrammatic representation of the mouse TTP gene. B, CEF
cells were transfected with plasmids containing the full mouse mRNA
coding region, the single TTP intron, and various lengths of genomic
sequence 5` of the initiator codon: 1.7 kb (EcoRV), 0.9 kb (Sau3AI), and 137 bp (SstII) as indicated. 24 h after
transfection, the cells were serum-deprived for 24 h and then treated
with control conditions (C) or stimulated with 10% FBS (S) for 60 min. Each lane was loaded with 20 µg of total
cellular RNA, and the Northern blot was hybridized with the P-labeled TTP cDNA probe. Equivalent RNA loading in this
and subsequent figures was confirmed by acridine orange staining (data
reviewed but not shown). The positions of the 18 S and 28 S ribosomal
RNAs are indicated.
Computer analysis of the 137 bp 5` of the translational start site revealed a consensus H2A (+46 to +51) binding sequence for the eukaryotic core histone dimer H2A-H2B(21, 22) , and a consensus c-fos.5 binding site (+52 to +59)(23) . Neither the H2A site nor the c-fos.5 site were present in the human and bovine TTP sequences, and neither mutation of the putative H2A binding site (from CCATTC to GTCGAC) nor the putative c-fos.5 sequence (from GCGCCACC to GGTCGACC) affected either the basal or serum-stimulated expression of mouse TTP (data not shown).
The human, bovine, and mouse sequences were highly conserved in the proximal 5`-flanking region (Fig. 3A). Sequence analysis revealed consensus motifs for several transcription factors in the 5`-proximal region of all three TTP promoters, including sites for the TATA binding protein and Sp1 binding protein (Fig. 3A). Deletion of these sites from the mouse construct containing the 137-bp 5`-proximal sequence produced a predictable inhibition of TTP mRNA expression (Fig. 3B). Consensus sequences for the binding of transcription factors EGR-1 (11) and AP2 (12, 24) were also found in the proximal promoter region of the three animal species (Fig. 3A). To assess the contribution of the EGR-1 and AP2 elements to the induction of TTP by serum, deletion of each of these elements was made in the mouse TTP construct containing the 137-bp 5`-proximal sequence. The effects of these deletion mutations on serum induction of TTP mRNA expression by these plasmids were measured in starved CEF cells after transient transfection. Deletion of either of the two elements resulted in a reduced level of TTP mRNA accumulation induced by serum. Serum-stimulated TTP mRNA expression in cells expressing the deleted EGR-1 or AP2 constructs was decreased to 35 and 18%, respectively, of the wild-type level (Fig. 3C).
Figure 3:
Deletion analysis of the mouse TTP
promoter. Panel A, sequences of the first 100 bp of TTP
promoter from human, bovine, and mouse are compared. The consensus
sequences for the EGR-1, TPE1, AP2, and Sp1 elements and the TATA box
are indicated. The mouse TPE1 sequence shown was from the Balb/c clone.
The analogous sequence from the 129sv mouse genomic clone was identical
to the human and bovine sequences. Panel B, TTP plasmids
containing 77 bp of genomic sequence 5` of the TTP mRNA transcription
start site (TTP) deleted of the TTP intron (-Int), SP1, or TATA sequences, or deleted of the TTP intron (-Int), EGR-1, or AP2 sequences (panel C) were
transfected into CEF cells. 24 h after transfection, the cells were
deprived of serum for 24 h and then treated with control conditions (C) or 10% FBS (S) for 60 min. Northern blot analyses
were performed as described in the legend to Fig. 2.
TTP mRNA expression from constructs lacking the TTP intron was also greatly attenuated when compared with the corresponding constructs containing the same length of 5` sequence (Fig. 3, B and C). Full expression of human and bovine TTP constructs also required the presence of the intron (data not shown). The relationship between the promoter and the intron in the expression of Zfp-36 is not understood at present; these potential interactions are the subject of ongoing experimentation and will not be discussed further here.
Analysis of promoter sequences in this region also revealed a previously undescribed 10-bp sequence, TPE1, that was identical in the promoters of all three animal species (Fig. 3A). When this region was deleted from the mouse construct containing 137 bp of 5` DNA sequence, serum-induced TTP expression was decreased by 75% (Fig. 4A). In order to rule out the possibility that the diminished expression was due to nonspecific effects resulting from a shortened construct, we created a site-specific mutation at the palindromic sequence of TPE1 from TCCCGGA to TAACTTA. Serum-induced expression of the substitution mutant construct was decreased to a similar extent as that of the deletion mutant (Fig. 4A). Mutation of the same 4 bp also had profound effects on the expression of TTP from longer constructs containing 1.7 kb or 0.9 kb of promoter sequence (Fig. 4B, Table 1, Group 1), indicating that this palindrome is one of the key sites for the activation of the TTP gene during serum stimulation.
Figure 4:
Effect of deletion or mutation of the TPE1
sequence on the expression of mouse TTP. A, CEF cells were
transfected with the mouse TTP plasmid containing the wild-type 137 bp
5`-proximal sequence or the same sequence with the TPE1 site deleted (delTPE1) or mutated from TCCCGGA to TAACTTA (TPE1). B, CEF cells were transfected with mouse
TTP plasmids containing varying lengths of promoter with (
TPE1) or without (Wt) the analogous mutation of
the TPE1 site described above. Cell treatments, abbreviations, and
other details are as described in the legend to Fig. 2.
The above results suggest that the EGR-1,
TPE1, and AP2 elements within the first 77 bp upstream of the mouse TTP
transcription start site all contribute to transcriptional activation
of Zfp-36 by serum in CEF cells (Table 1, Group 2).
Since deletion or mutation of each single element reduced, but did not
abolish, serum-induced expression, we examined whether any of these
elements was able to impart serum-responsiveness to a minimal promoter.
We therefore constructed plasmids containing a single copy of EGR-1,
AP2, or TPE1 5` of a hybrid insert, Glo48TTP. This hybrid insert was
constructed of a 48-mer 5`-flanking sequence from the human
-globin gene and the mouse TTP cDNA. Function of this minimal
human
-globin promoter has only been observed in vectors that
include an enhancer element(9, 25) . The 5` 48-mer
only contained a TATA box and no other promoter elements. We did not
include the TTP intron in these plasmids because this intron may also
contain enhancer activities (Fig. 3, B and C).
A plasmid with the c-fos SRE sequence inserted 5` to Glo48TTP served as
a positive control, since the SRE has been demonstrated to be
sufficient for the induction by serum in heterologous
constructs(10) . Using transient transfection in CEF cells, we
tested the ability of these putative elements, EGR-1, TPE1 and AP2, to
confer serum responsiveness on the silent human
-globin promoter.
The expression of Glo48TTP plasmids containing each of the test
sequences was induced by serum as compared with the plasmid lacking
these sequences (Fig. 5), both in the presence and absence of
cycloheximide. In addition, the presence of all three putative enhancer
elements together resulted in a 1.5-fold increase in expression
compared with the sum of expression from each element individually (Fig. 5). Finally, the reverse orientation of the TPE1 element
resulted in a similar increase in serum-induced expression to that of
the forward construct (data not shown).
Figure 5: Effect of TTP promoter elements on the activity of a silent promoter. CEF cells were transfected with Glo48TTP alone(-), or with Glo48TTP constructs containing a single copy of the indicated TTP promoter elements or the c-fos SRE. Cells were deprived of serum for 24 h after 24 h of recovery from transfection; FBS (10%) was then added to the treatment group (S) for 60 min at 37 °C, compared with control conditions (C). The position of the TTP mRNA is indicated. Other details describing the Northern blotting are contained in the legend to Fig. 2.
Figure 6:
Gel mobility shift assay for nuclear
proteins binding to the TTP promoter. Panel A, nuclear
extracts (12 µg of protein) from HIR3.5 cells treated for 10 min
with 70 nM insulin (I) or control conditions (C) were then allowed to bind to the EGR1-AP2 probe containing
-35 to -77 of the mouse TTP promoter. These assays were
performed with or without specific oligonucleotide competitors
comprising either the AP2 or TPE1 sites, as indicated at the top of the gel. C1-C4 represent DNA-protein complexes 1-4
as described in the text. Panel B, nuclear extracts (5 µg
of protein) from CEF cells treated for 10 min with 10% FBS (S)
or control conditions (C) were allowed to bind to the TPE1
probe. Panel C, nuclear extracts (5 µg of protein) from
HIR3.5 cells treated with control conditions (C) were allowed
to bind to the wild-type or the mutant (TPE1) TPE1 probes. All
lanes contained 20
10
cpm of
P-labeled
double-stranded oligonucleotide, in the presence of poly(dI-dC) at a
final concentration of 50 µg/ml. The unlabeled competitors were
present at 1 µg/reaction when indicated. Assay conditions are
described under ``Experimental Procedures.'' The sequences of
the probes used were: EGR1-AP2,
ctagaGCGGGGGCGCGTCCCGGAAGCTCTAGTGGCCACGCCCCCAGGCgatatc; TPE1,
tcgacGTCCGGGAAGCGtcga;
TPE1, tcgacGTAAGTTAAGCGtcga, where the underlined bases indicate the core sequences of the consensus
protein binding sites, and the lowercase bases indicate
portions of the restriction sites used for
subcloning.
Wild-type and mutant TPE1
double-stranded oligonucleotides were next radiolabeled for use as
probes in gel shift assays. Nuclear extracts from both serum-treated
and control cells produced a single DNA-protein complex with the
wild-type TPE1 probe, which corresponded in eletrophoretic mobility to
complex C4 seen with the EGR1-AP2 probe. As with the EGR1-AP2 probe,
formation of this complex was decreased in extracts from serum-treated
cells. A mutant TPE1 oligonucleotide competitor containing base
changes from TCCCGGA to TAACTTA had no effect on formation of the TPE1
complex (Fig. 6B). The mutant
TPE1 oligonucleotide
probe was also directly radiolabeled and shown to be unable to form
this complex (Fig. 6C). These results suggested that
the TPE1 element was recognized by one or more specific nuclear
proteins. The small but consistent decrease in the intensity of the
TPE1 complex following serum treatment, seen with both the EGR1-AP2
(C4) and the TPE1-specific probe, suggests that serum treatment might
modify these binding proteins in such a way as to decrease their
binding to the TPE1 element. Similar decreases in intensity of complex
C4 were seen when HIR3.5 cells were treated with either insulin (70
nM) (Fig. 6A) or PMA (1.6 µM)
(not shown) for 10 min.
These studies establish that the first 77 bp 5` upstream of the transcriptional start site are sufficient for maximal serum induction of the mouse TTP gene (Zfp-36) when expressed in CEF cells; deletions 3` of this point dramatically decrease serum inducibility of this gene. Within this minimal effective promoter, we have also identified several putative transcription factor binding sites in the mouse TTP promoter, all of which are present in the human and bovine genes. The presence of each is necessary for the full, serum-inducible expression of the gene. Finally, we have identified binding proteins in cell nuclear extracts that bind specifically to some of these DNA motifs and whose binding is altered by prior treatment of the cells with mitogens. These studies have begun to evaluate the mechanisms by which serum and other mitogens rapidly and dramatically stimulate the transcription of this immediate-early response gene.
One conserved transcription factor binding site
identified in the present study is the Sp1 site, located at -35
to -30 5` of the transcription start site in the mouse promoter.
Sp1 is a well characterized zinc finger-containing transcription
factor, which enhances transcription by RNA polymerase II from
promoters that contain at least one properly positioned GGGCGG
hexanucleotide (for review, see (26) ). Sp1 has been shown to
regulate transcription of certain proto-oncogenes (27, 28) and growth factor genes(29) . When
the consensus binding sequence for Sp1 was deleted from the
TTP plasmid, an 80% decrease in TTP expression
resulted, indicating that the TTP promoter is Sp1-responsive (Fig. 4B); however, we have not demonstrated directly
that Sp1 binds to its hexanucleotide binding site in the TTP promoter.
The TTP promoter construct with the Sp1 site deleted remained
serum-responsive but to a lesser extent than the wild-type construct,
implicating other promoter elements in the serum-induced expression of
TTP mRNA.
EGR-1 is another zinc finger-containing transcription factor, also known as NGF1-A(30) , KROX24(31) , TIS-8(4) , and Zif268(32) . It binds to a GC-rich consensus sequence, GCGGGGGCG, that is found in the 5`-flanking regions of many genes involved in cell growth such as proto-oncogenes and genes encoding mitogens and mitogen receptors. Several immediate-early response genes also have EGR-1 binding sites in their promoters(33) . Deletion of the EGR-1 binding sequence from the TTP promoter decreased its serum-stimulated expression by 65%. Although we found no direct evidence of EGR-1 binding to the TTP promoter in our gel shift assays, our deletional analysis indicates that EGR-1 may contribute to the regulation of TTP expression.
The AP2 consensus
binding sequence GCCNNNGGC (34) is present in the minimal
effective promoter of TTP from all three animal species tested. This
sequence, when bound by AP2 homodimers, has been identified as a
control element for several viral and cellular
genes(24, 34) . AP2 mediates regulation of gene
expression in response to a number of different signal transduction
pathways(35) . The activity of AP2 is increased in response to
treatment of cells with phorbol esters and agents that elevate cAMP
levels(34, 35, 36) . When the AP2 consensus
sequence in the mouse TTP promoter was deleted, induction of TTP
expression by serum was decreased by 72%. We have previously shown that
PMA could induce TTP expression(1) , making it possible that
the AP2 binding site was involved in PMA-induced TTP expression.
However, when the AP2 binding sequence was deleted from the
TTP construct, PMA still induced TTP expression to a
similar extent as the serum-induced response (data not shown). These
results indicate that the PMA-stimulated TTP expression does not depend
solely upon the consensus AP2 binding sequence in the TTP promoter.
We also identified a previously undescribed promoter element at -66 to -60 5` of the cap site in the mouse gene that we have called TPE1. It contains a palindromic element with dyad symmetry, TCC(C/G)GGA. It is perfectly conserved in the TTP promoter from all three animal species we have studied. Deletion or mutation of this element led to a 75% decrease in serum-induced TTP expression. Mutation of the TPE1 palindrome also severely impaired serum responsiveness of the TTP promoter when introduced into longer promoter constructs. Both orientations of the TPE1 element could also confer serum responsiveness to the silent promoter Glo48, indicating that this element behaves as a transcriptional enhancer.
The TPE1 palindrome also appears to represent a binding site for a nuclear protein, as demonstrated by gel shift analysis. Mutations within the palindrome that impaired serum responsiveness of the promoter also abolished binding of this nuclear protein. In addition, nuclear extracts from cells stimulated with serum or other mitogens for 10 min showed a small but consistent decrease in band intensity in the gel shift assays. This suggests, but does not prove, that the TPE1 element binding protein is a target of the signaling cascade responsible for serum induction of the TTP promoter.
We have located consensus TPE1 binding sites in the promoters of a number of genes, including c-ha-ras1(27) , mdm2(37) , and fosb(38) . fosb is induced by serum with early response gene characteristics. It will be interesting to investigate the possibility that this motif is involved in the regulated expression of these and other genes.
Because the serum-responsive region in the TTP promoter contains multiple potential promoter elements, it is likely that a number of nuclear proteins interact in its regulation. The DNA mobility shift assay using the EGR1-AP2 probe provides evidence to suggest that several proteins interact with the -77 to -35 sequence to mediate the serum response. We observed small but consistent changes in the intensity of a number of protein-DNA complexes using nuclear extracts from serum-treated and control cells. Serum-induced transcriptional activation of TTP is likely to result from post-transcriptional modification of pre-existing nuclear protein complexes, since TTP gene transcription is very rapidly stimulated by insulin or serum, even when cells have been pretreated with the protein synthesis inhibitor cycloheximide(1) . Post-transcriptional modification of such factors following serum treatment could result in changes in the affinity of these factors for their DNA binding sites. Isolation and characterization of the TPE1 protein should lead to a better understanding of the transcription factor interactions that are involved in the regulation of TTP expression.
Our results suggest
cooperative interactions among nuclear proteins binding to closely
positioned cis-acting elements in the TTP promoter. For
example, the EGR-1, TPE1, and AP2 elements from the TTP promoter each
conferred only low levels of serum-inducible expression of
-globin-TTP hybrid constructs, but together they produced greater
than additive expression. The gel mobility shift data also suggest the
possibility of cooperative interactions among several transcription
factors; for example, there was a reciprocal increase in the binding of
nuclear proteins to the AP2 binding site and decreased protein binding
to the TPE1 site. Finally, the presence of the single intron in the TTP
gene markedly enhanced serum induction of the gene; we suspect this is
due to enhanced transcription, although we cannot exclude effects of
the intron on processing or stability of the mRNA(39) . Further
experimentation will be necessary to determine whether the intron
and/or proteins that bind to it interact in some way with the other
serum-responsive elements of the TTP promoter.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) M19844[GenBank], L42319[GenBank], and L42317[GenBank].