From the Departments of Human Genetics and Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618
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ABSTRACT |
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Type-1 plasminogen activator-inhibitor (PAI-1) is
a major physiologic inhibitor of plasminogen activation. Incubation of
HTC rat hepatoma cells with the cyclic nucleotide analogue,
8-bromo-cAMP, causes a dramatic increase in tissue-type plasminogen
activator activity secondary to a 90% decrease in PAI-1 mRNA.
Although 8-bromo-cAMP causes a modest decrease in PAI-1 transcription,
regulation is primarily the result of a 3-fold increase in the rate of
PAI-1 mRNA degradation. To determine the cis-acting
sequences required for cyclic nucleotide regulation, we have stably
transfected HTC cells with chimeric genes containing sequences from the
rat PAI-1 cDNA and the mouse -globin gene and examined the
effect of cyclic nucleotides on the decay rate of these transcripts.
The mRNA transcribed from the
-globin gene is stable and not
cyclic nucleotide-regulated, whereas the transcript from a construct
containing the
-globin coding region and the PAI-1 3'-untranslated
region (UTR) is destabilized in the presence of 8-bromo-cAMP,
suggesting that this response is mediated by sequences in the PAI-1
3'-UTR. Analyses by deletion of sequences from this chimeric construct
indicate that, whereas more than one region of the PAI-1 3'-UTR can
confer cyclic nucleotide responsiveness, the 3'-most 134-nucleotide
sequence alone is sufficient to do so. Insertion of PAI-1 sequences
within the
-globin 3'-UTR confirms that the 3'-most 134 nucleotides
of PAI-1 mRNA can confer cyclic nucleotide regulation of stability
on a heterologous transcript, suggesting that this sequence may play a
major role in hormonal regulation of PAI-1 mRNA stability.
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INTRODUCTION |
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Eukaryotic gene expression is determined not only by the rate of gene transcription, but also by the rate at which the transcript is degraded. The steady state concentration of a particular mRNA, as well as the rate at which a new transcriptionally induced steady state is attained, is directly related to the message half-life (1, 2). Numerous recent studies have been aimed at understanding the role of specific sequences within the mRNA in determining basal transcript stability and have led to the identification of consensus sequences that confer stability or instability to a transcript (2, 3). Although it is known that many stimuli alter mRNA stability, little is known about either the cis-acting sequences in the RNA or the cellular factors involved in regulation of message decay.
Plasminogen activators (PAs)1 are serine proteases that are critical for thrombolysis and also play a key role in other physiological functions involving tissue remodeling. Plasminogen activator-inhibitors (PAIs) are specific inhibitors of PAs and are members of the serine protease inhibitor (serpin) family of proteins. Type-1 PAI (PAI-1) is a 50 kDa glycoprotein found in plasma, platelets, and a variety of cell types; its expression is regulated by growth factors, cytokines, and hormones, including agents that alter cellular cAMP levels (4).
HTC rat hepatoma cells synthesize and secrete tissue-type plasminogen
activator (tPA) and PAI-1 (5). We have previously reported that
incubation with the synthetic glucocorticoid, dexamethasone, decreases
HTC cell tPA activity by increasing PAI-1 mRNA and protein 5-10-fold, an effect that is entirely explained by an increase in
PAI-1 gene transcription (5-7). In contrast, these cells respond to
cyclic nucleotides with a dramatic (50-fold) increase in tPA activity
that is primarily the result of a decrease in PAI-1 mRNA and
protein (8). This effect is time- and
concentration-dependent; a 90% decrease in PAI-1 mRNA
occurs after 12 h of incubation with 1 mM 8-bromo-cAMP
plus 1 mM isobutyl-1-methylxanthine (cA) (8, 9). Nuclear
run-on experiments have shown that cA causes about a 60% decrease in
PAI-1 transcription, not sufficient to account for the decrease in
message accumulation. We have determined the half-life of HTC cell
PAI-1 mRNA by following the decrease in message level either after
inhibition of transcription with actinomycin D or after washing out the
transcriptional inducer, dexamethasone. By either method, PAI-1
mRNA displays a half-life of 4.0-4.5 h. Incubation of cells with
cA accelerates PAI-1 mRNA decay, decreasing the half-life to
1.5 h. Thus, cyclic nucleotides regulate PAI-1 gene expression at
both a transcriptional and posttranscriptional level. Interestingly,
although actinomycin D does not alter the basal rate of PAI-1 mRNA
degradation, it prevents the cA-induced acceleration of decay;
incubation with cyclic nucleotides in the presence of actinomycin D
results in a decay rate identical to that seen in the presence of
actinomycin D alone (7).
The aim of this study is to understand the mechanism by which cyclic
nucleotides regulate PAI-1 mRNA decay by determining the sequences
within the PAI-1 transcript that are required for 8-bromo-cAMP
regulation. We report here that the 3'-UTR of PAI-1 mRNA can confer
cyclic nucleotide regulation on the otherwise stable -globin
mRNA. Deletion analysis of the PAI-1 3'-UTR reveals the presence of
at least two cA-responsive regions; one of these is in the 3'-most 134 nt of PAI-1 mRNA. Furthermore, the 3'-most 134-nt sequence by
itself is able to confer cA responsiveness on the heterologous
-globin mRNA, suggesting that this sequence plays a major role
in the hormonal regulation of PAI-1 mRNA stability.
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EXPERIMENTAL PROCEDURES |
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Materials--
Eagle's minimal essential medium, glutamine,
fetal calf serum, calf serum, G418, restriction endonucleases (REs),
Taq DNA polymerase, T4 ligase, and TRIzol® were
purchased from Life Technologies, Inc. Bovine serum albumin was
purchased from Intergen Co. (Purchase, NY). Cycloheximide, 8-bromo-cAMP, and isobutyl-1-methylxanthine were purchased from Sigma.
T3 and T7 RNA polymerases, RNase inhibitor, RNase A, and RNase T1 were
purchased from Boehringer Mannheim. -32P-labeled UTP
(specific activity 800 Ci/mmol) was obtained from Amersham Pharmacia
Biotech. The vector pSVL was purchased from Amersham Pharmacia Biotech,
and pBluescriptKS(
) and Escherichia coli AG-1
competent bacteria were purchased from Stratagene (La Jolla, CA). Rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (10) was a
kind gift from Dr. Rodrigo Bravo (Bristol-Myers Squibb).
Oligonucleotide primers used in PCR were synthesized by the University
of Michigan Biomedical Research Core Facilities.
Constructs for Transfection Analysis-- The vector pSVL, which has an SV40 late promoter upstream and an SV40 polyadenylation signal downstream from the multicloning site (MCS), was used to drive expression of the DNA constructs used in these studies. Because the SV40 late promoter does not appear to be regulated by cyclic nucleotides, this vector allowed us to examine the effects of cA on message decay independent of transcriptional effects. All DNA vectors described below were amplified in E. coli AG-1 competent bacteria by established procedures (11).
MouseConstructs for Riboprobes--
A PCR-amplified fragment of
m-globin from bp 1256 to bp 1436 (which includes coding and intronic
sequence) was subcloned into the PstI and BamHI
sites of the pBluescriptKS(
) MCS. When linearized by
digestion with HindIII (site in the
pBluescriptKS(
)) and transcribed using T7 RNA polymerase,
this plasmid generates an antisense riboprobe complementary to 119 nt
of the m
-globin coding region mRNA (Fig. 1).
Preparation of Radiolabeled RNA-- Riboprobes were prepared by published methods (11). Plasmid DNA (approximately 500 ng) linearized with the appropriate RE was incubated with T7 or T3 RNA polymerase in transcription buffer (Boehringer Mannheim) containing RNase inhibitor, ATP, CTP, GTP, and [32P]UTP at 37 °C for 60 min. RNase-free DNase I was added, and, after 15 min at 37 °C, the reaction was extracted with phenol/chloroform (1:1) and the RNA was precipitated in the presence of ammonium acetate and ethanol. The labeled RNA was purified by electrophoresis through a 6% polyacrylamide, 8 M urea gel, eluted from the gel, and ethanol-precipitated.
Cell Culture-- HTC cells were maintained in monolayer cultures as described previously (5). In preparation for experiments, cells were plated in 60-mm tissue culture dishes and grown to confluence. Cultures were washed in sterile phosphate-buffered saline (PBS) and incubated in serum-free medium containing 0.1% bovine serum albumin with or without 0.1 mM cycloheximide. After 10-16 h, the medium was removed, the monolayers washed twice with PBS to remove cycloheximide, and fresh medium with or without cA was added. Cells were harvested at the times indicated for each experiment. For experiments in which the RNA from the transfected genes was to be analyzed, incubation with cycloheximide provides two major advantages. First, cycloheximide significantly enhances expression from the SV40 late promoter (5-50-fold), resulting in easier detection of transcript from the transfected gene (13). Second, cycloheximide can be readily removed and its effects completely reversed by washing the monolayers with PBS and adding fresh medium (15, 16), resulting in a dramatic decrease in SV40 promoter activity. This protocol allowed us to measure mRNA degradation without using actinomycin D, which not only inhibits transcription but also blocks the cA effect on PAI-1 mRNA (7). Cycloheximide is known to alter the decay rates of some transcripts (2); however, the degradation rate of endogenous PAI-1 mRNA (determined using actinomycin D to block transcription) in cells from which cycloheximide has been removed after a 12-h incubation with the inhibitor, is nearly identical to that in cells not treated with cycloheximide. Furthermore, in cells transiently treated with cycloheximide, as in untreated cells, endogenous PAI-1 mRNA decay is accelerated after incubation of the cells with cA.
Transfections-- HTC cells were transfected using the Ca2PO4 DNA precipitation technique, and stably transfected cells were selected in media containing 1 mg/ml G418 as described previously (17). Colonies of stably transfected cells were pooled to minimize the possible effects of different integration sites.
Ribonuclease Protection Analysis-- Total cellular RNA was isolated either by the method of Chomczynski and Sacchi (7, 18) or by the TRIzol® reagent method as described by Life Technologies, Inc. Ribonuclease protection analysis was carried out essentially as described (11). Total cellular RNA (20-40 µg/sample) was precipitated and resuspended in 30 µl of hybridization buffer (40 mM Pipes, pH 6.4, 400 mM NaCl, 1 mM EDTA, 80% formamide) containing 1-5 × 105 cpm of 32P-labeled riboprobe. After 5 min at 85 °C, the samples were placed at 50 °C for 16-20 h. RNase digestion buffer (350 µl of 10 mM Tris-Cl, pH 7.4, 300 mM NaCl, 5 mM EDTA, 20 µg/ml RNase A, and 100 units/ml RNase T1) was added to each reaction. After 30 min at 30 °C, SDS (0.5% final concentration) and proteinase K (125 µg/ml) were added and incubation continued at 37 °C for 30 min. The samples were then extracted with phenol/chloroform and ethanol-precipitated. The RNA pellets were washed with 80% ethanol, allowed to air-dry, and resuspended in an 80% formamide gel-loading buffer. Samples were subjected to electrophoresis through 6% polyacrylamide, 8 M urea gels and the gels were dried under vacuum. The amount of radioactivity in each protected fragment was determined by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA). For each sample the signal in the specific band of interest was normalized to the signal in the 80-nt protected fragment of GAPDH.
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RESULTS |
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The 3'-UTR of PAI-1 mRNA Confers Cyclic Nucleotide
Responsiveness onto the -Globin Transcript--
To determine the
sequences in the PAI-1 mRNA involved in the cA regulation of
message stability, we stably transfected HTC cells with DNA constructs
carrying portions of the PAI-1 sequence and examined the degradation
rates of the transcribed mRNAs. The pSVL vector was chosen because
the SV40-late promoter, unlike some other viral promoters, does not
appear to be regulated by 8-bromo-cAMP.2 To confirm
that the cA regulation can be faithfully reproduced in a transfection
system, we first prepared a PAI-1 mini-gene using the SstI
recognition site to delete 530 bp of the coding region, and subcloned
it into the vector pSVLneo. The mRNA transcribed from
the PAI mini-gene can be distinguished from the endogenous PAI-1
mRNA in ribonuclease protection analysis using a PAI-1 riboprobe that overlaps the deleted sequence. The half-life of the transcript from the PAI-1 mini-gene is 4.0 h and is decreased to 2.0 h
in cells treated with cA (data not shown). These results are nearly identical to those observed for endogenous PAI-1 mRNA (7) and confirm that the cyclic nucleotide regulation of the transfected mini-gene faithfully reflects the regulation of the endogenous gene.
These results also confirm that under these conditions cA regulation of
message stability can be reproduced in cells that have been transiently
treated with cycloheximide.
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Deletion Analysis of the PAI-1 3'-UTR-- In an effort to locate the region within the PAI-1 3'-UTR responsible for cyclic nucleotide responsiveness, we used convenient restriction endonuclease sites to delete selected portions of the PAI-1 cDNA in the context of the G/P construct. The 3'-UTR of PAI-1 mRNA has several sequences of potential interest (14) illustrated in the top portion of Fig. 3. AU-rich elements (AREs) that include AUUUA motifs flanked with U-rich stretches have been implicated in the instability of mRNAs of cytokines and oncogenes (3). The PAI-1 3'-UTR has three AUUUA motifs (ATTTA in the cDNA), two in close proximity at nt 2053 and 2117, and a third farther downstream at position 2728, close to the two 70-nt U-rich stretches (T-rich in the cDNA) (nt 2821-3023). PAI-1 has a highly conserved region in its 3'-UTR; the 127-nt sequence in rat PAI-1 from nt 2506 to nt 2632 is 80% identical in mink, murine, bovine, porcine, and human PAI-1 and shares 85-97% identity when compared pairwise with each of these species (14, 22-26). Finally, PAI-1 has an unusual stretch of 39 GpA dinucleotides between nt 2363 and 2442. The location of these regions within the 3'-UTR, the RE sites that flank them and the designations given them are shown in the top of Fig. 3. Regions "A" and "C" are so named for lack of putative regulatory sequences.
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Insertion Analysis: The 3'-most 134 nt of PAI-1 Is Sufficient to
Confer Cyclic Nucleotide Regulation--
To more clearly define the
cis-acting regions involved in the cA regulation, we
prepared constructs in which selected portions of the PAI-1 3'-UTR
sequence were inserted into the 3'-UTR of the -globin gene (Fig.
5). These constructs were then stably transfected into HTC cells and experiments performed as described above
for the deletion constructs. Results from experiments with four of
these constructs are shown in Fig. 6. The
mRNA transcribed from pSVL G/G, like the
-globin transcript, is
stable and not regulated by cyclic nucleotides (Fig. 6A).
However, when the 3'-most 130 nt of PAI-1 sequence (lacking the final 6 A nucleotides; see Fig. 8) was inserted into the globin 3'-UTR (pSVL
G/G + U II), the resultant construct was regulated by cA. Fig.
6B shows a composite of three such experiments; the mean
half-life of 9 h was decreased to 4 h upon incubation with
cA, a 2.2-fold change. The construct pSVL G/G + 1400 nt has an
insertion of 1378 nt of the 5' end of the PAI-1 3'-UTR. The transcript
from this construct displayed a half-life of 4.7 h, which was
decreased to 2.4 h upon incubation with cA (Fig. 6D).
In contrast, the message transcribed from pSVL G/G+U I, which has the
upstream U-rich region, is not cA-regulated (Fig. 6C). These
results confirm that the 3'-most 134-nt region of PAI-1 3'-UTR is
sufficient to confer cyclic nucleotide regulation and that there is at
least one other regulatory element in the upstream region of the
3'-UTR. The PAI-1 sequence between bp 2770 and bp 2923, containing the
upstream U-rich stretch (U I), does not appear to confer cyclic
nucleotide regulation.
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DISCUSSION |
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In this study, we have examined the mechanism by which cyclic
nucleotides accelerate the rate of rat HTC cell PAI-1 mRNA
degradation. We have shown that sequences in the 3'-UTR of PAI-1 can
confer both instability and cA regulation of degradation on the
otherwise stable and non-regulated mouse -globin mRNA. The PAI-1
3'-UTR has at least two cA-responsive elements; one of these elements is in the 3'-most 134 nt, which by itself is able to confer cA regulation on the globin message.
Studies from several laboratories have defined specific elements in the 3'-UTR of transcripts that are responsible for their relative stability. With a few exceptions (2, 27), these have all been instability determinants. The most extensively studied instability determinants are the AU-rich elements, or AREs, found in the 3'-UTR of many highly labile transcripts including those of proto-oncogenes (28, 29), growth factors (30), and cytokines (31, 32). AREs may have either multiple AUUUA motifs resulting in the nonomer UUAUUUA(U/A)(U/A) (28), AUUUA motifs scattered in a generally U-rich sequence, or an AU-rich region without an AUUUA motif (3). In addition, two U-rich domains in the yeast MFA2 3'-UTR appear to be involved in regulation of poly(A) nuclease activity (33) and a 29-nt U-rich sequence in human amyloid precursor protein mRNA has been implicated in regulation of its degradation (34).
Rat PAI-1 has two AUUUAs in close proximity at nt positions 2053 and
2117, but these are not in an otherwise AU-rich region. A third AUUUA
sequence is near the two 70-nt U-rich stretches (nt 2821-2892 is 57%
U and nt 2952-3023 is 51% U, separated by a 60-nt sequence that is
only 20% U), making this region a candidate as an instability
determinant. Interestingly, one of these U-rich regions is included in
the 3'-most 134-nt fragment that is able to confer both an increase in
basal degradation rate and cA regulation of decay on the -globin
transcript. Although the U-rich region may be involved in the cyclic
nucleotide responsiveness, it is not by itself sufficient as seen by
the results shown in Fig. 7. Decay rates of transcripts from G/G
constructs carrying either one or both of the U-rich sequences, but
lacking the last 30 nt, are not regulated by cA. In addition, the
upstream functional element(s), is not simply the upstream U-rich
sequence, as evidenced by the fact that constructs lacking both U I and
U II (G/P
U I + U II (Fig. 3) and G/G + 1400 nt (Figs. 6 and 7))
display cA regulation of mRNA decay.
The growing number of reports of hormone, growth factor, and cytokine
regulation of mRNA stability reflects the growing realization of
the importance of this process in overall regulation of gene expression. Rapid changes in the abundance of a short-lived transcript can be effected by a small change in the half-life (2). There are now
several studies that have identified cis-acting sequences and/or trans-acting factors potentially involved in hormonal
regulation of degradation. The dramatic stabilization of the frog liver
vitellogenin mRNA is mediated by binding of an estrogen-inducible
protein, recently identified as the KH domain protein, vigilin, to a
27-nt sequence within the 3'-UTR (35, 36). Tumor necrosis factor- induces a 4-fold stabilization of GLUT1 mRNA in 3T3-L1
pre-adipocytes, which appears to be mediated by a 105-nt GC-rich
portion of the 3'-UTR and is correlated with increased protein binding
to the 3'-UTR (37). Stabilization of amyloid precursor protein mRNA in activated peripheral blood mononuclear cells is accompanied by an
increase in binding of a protein to a 29-nt U-rich element in the
3'-UTR (38). Finally, in rat pituitary cultures, thyroid hormone
accelerates degradation of thyrotropin
message and causes an
increased binding of a cellular factor to a 41-nt portion of the 3'-UTR
of the transcript (39). This 41 nt includes a 12-nt consensus sequence
found in several unstable mRNAs, including the 3'-most 134 nt of
PAI-1 mRNA (11 of the 12-nt consensus at nt 3010-3020).
Agents that elevate cellular cAMP levels also have been shown to
regulate mRNA degradation, in some cases stabilizing message (40-42), but more often causing down-regulation of mRNAs.
Treatment of rat Sertoli cells with follicle-stimulating hormone causes destabilization of androgen receptor, follicle-stimulating hormone receptor, and G-protein -subunit mRNAs (43-45). In both rat
ovary and human endometrial stromal cells, luteinizing hormone/human chorionic gonadotropin receptor mRNA is down-regulated by human chorionic gonadotropin, apparently through accelerated degradation (46,
47). Cyclic nucleotide analogues also destabilize asialoglycoprotein receptor mRNA in HepG2 cells (48), tyrosine aminotransferase in H4
rat hepatoma cells (49), angiotensin type I and type II receptor
mRNA (50, 51) and the kidney-specific transcription factor, LFB3,
mRNA in porcine kidney cells in culture (52). In two cases, cA
regulation of message stability has been associated with binding of
proteins to the 3'-UTR of the transcript. Chlorophenylthio-cAMP both
stabilizes rat hepatoma cell phosphoenolpyruvate carboxykinase mRNA
10-fold and decreases the binding of a 100-kDa protein to the 3'-UTR of
this transcript (53). In hamster smooth muscle cells, isoproterenol
destabilizes
2-adrenergic receptor mRNA and
increases the binding of both an AUF1 related protein and a more
specific
ARB protein to the 3'-UTR of
2-adrenergic
receptor mRNA (54, 55). Sequences in human and hamster
2-adrenergic receptor mRNAs that are A+U-rich have
been implicated in both protein binding and regulation of message
stability (56, 57). The 134-nt region that we find to confer cA
regulation of PAI-1 mRNA stability has both a U-rich and an A-rich
region (Fig. 8), similar but not identical to those in
2-adrenergic receptor mRNA.
The cyclic nucleotide regulation of PAI-1 mRNA accumulation, first observed in HTC cells (8), has also been demonstrated in rat testicular peritubular cells (58), astrocytes (59) and osteoblasts (60), mink lung epithelial cells (61), human fibrosarcoma cells (62), umbilical vein endothelial cells (63), and synovial cells (64). Our previous experiments have shown that in rat HTC cells, whereas transcriptional regulation plays a role in the cA-induced decrease in PAI-1, the major effect is on message stability. The studies reported here demonstrate that at least two elements in the 3'-UTR of PAI-1 mRNA are involved in regulation of PAI-1 message decay. One of these regulatory elements is the 3'-most 134 nt. We have found that a number of cellular proteins interact with the same PAI-1 sequence that can confer cA regulation,3 suggesting that one or more of these factors may play a role in cyclic nucleotide regulation. Our current studies are aimed at delineating the exact sequences responsible for the cyclic nucleotide regulation of mRNA degradation and defining the binding proteins involved in this regulation, as well as identifying the other cis-acting element(s) in the PAI-1 3'-UTR.
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ACKNOWLEDGEMENT |
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We thank Erin Janssen for technical assistance during her tenure on a Summer Student Research Program Fellowship from the Michigan Diabetes Research and Training Center.
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FOOTNOTES |
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* This work was supported by Public Health Service Grant CA22729 from the National Cancer Institute (to T. D. G.) and National Research Service Award DK09437 from the National Institutes of Health (to M. T.-B.).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.
To whom correspondence should be addressed: Dept. of Human
Genetics, M4708 MSII/0618, University of Michigan Medical School, 1301 E. Catherine St., Ann Arbor, MI 48109-0618. Tel.: 734-763-3460 or
734-764-5491; Fax: 734-763-5831; E-mail: heatonj{at}umich.edu.
1 The abbreviations used are: PA, plasminogen activator; PAI, plasminogen activator-inhibitor; PAI-1, type-1 PAI; UTR, untranslated region; tPA, tissue-type plasminogen activator; PCR, polymerase chain reaction; nt, nucleotide(s); bp, base pair(s); Pipes, 1,4-piperazinediethanesulfonic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; MCS, multicloning site; cA, 8-bromo-cAMP plus isobutyl-1-methylxanthine; ARE, AU-rich element; RE, restriction endonuclease.
2 J. H. Heaton and T. D. Gelehrter, unpublished observation.
3 M. Tillmann-Bogush, J. H. Heaton, and T. D. Gelehrter, manuscript in preparation.
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REFERENCES |
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