From the Expression of the rat PRL-1 gene,
which encodes a unique nuclear protein tyrosine phosphatase, is
positively associated with cellular growth during liver development,
regeneration, and oncogenesis but with differentiation in intestine and
other tissues. Here, we analyzed the structure of the human
PRL-1 gene and localized it to chromosome 6 within band
q12. Human, rat, and mouse PRL-1 are 100% conserved at the amino acid
level and 55% identical to a newly identified Caenorhabditis
elegans PRL-1. The presence of two promoter activities, P1 and
P2, in the human PRL-1 gene were identified by primer
extension and RNase protection assays. A functional TATA box was
identified in promoter P1 upstream of the non-coding first exon. A
non-canonical internal promoter, P2, was found in the first intron that
results in PRL-1 transcripts beginning 8 base pairs
downstream of the 5'-end of exon 2 and causes no alteration in the
encoded protein. The first 200-base pair region of either promoter P1
or P2 conferred high basal transcriptional activity. An enhancer that
bound a developmentally regulated factor, PRL-1 intron
enhancer complex (PIEC), was localized to the first intron of the human
PRL-1 gene. The presence of PIEC correlated with the
ability of the intron enhancer to confer transcriptional activation in
HepG2 and F9 cells. The intron enhancer contributed significantly to
PRL-1 promoter activity in HepG2 cells which contain PIEC
but not to NIH 3T3 cells which do not.
Protein tyrosine kinases and phosphatases play important roles in
the regulation of cell growth, development, and differentiation. One of
the most interesting immediate early or primary growth response genes
first identified in regenerating liver encodes a novel 20-kDa nuclear
protein tyrosine phosphatase,
PTPase,1 PRL-1 (phosphatase
of regenerating liver). Other than the signature sequence for PTPases,
PRL-1 is not homologous to either the dual specificity PTPases (cdc25
and MKP-1) or monospecific PTPases (1, 2). A PRL-1-like protein, OV-1,
was isolated from human cells (3) and found to be 95% identical to rat
PRL-1 at the amino acid level but is not the human PRL-1 homologue.
Human PRL-1 (CAAX-1) and human OV-1
(CAAX-2) cDNAs were isolated as encoding proteins
containing isoprenylation sites (4). The finding of at least two
members of this PTPase family, PRL-1 and OV-1, with a high degree of
conservation suggests that PRL-1 and family members have important
cellular functions.
PRL-1 mRNA is elevated throughout the major
proliferative phase of liver regeneration when hepatocytes and
nonparenchymal cells in the liver are rapidly proliferating.
PRL-1 is expressed at high levels in other proliferating
cells including fetal liver and a number of tumor cell lines such as
hepatomas (1). PRL-1-transfected cells showed altered growth
characteristics, including a faster doubling time, growth to a greater
saturation density, altered morphology, and evidence of
anchorage-independent growth. Overexpression of human PRL-1 and OV-1 in
epithelial cells resulted in tumor formation in nude mice (4). Taken
together, these results suggest a positive role for PRL-1 in the growth
response.
However, in addition to regenerating liver, PRL-1 is
expressed at significant levels in brain and skeletal muscle, two
terminally differentiated tissues that are notable for their absence of
ongoing cellular proliferation. In intestinal epithelia which contain both terminally differentiated and proliferating cells, PRL-1 is
expressed in the terminally differentiated villus but not proliferating crypt enterocytes (5).
The correlation of PRL-1 expression with growth in some cellular
systems and differentiation in others suggests that PRL-1 may have
different roles depending on the cell type. Because of the interesting
pattern of regulation of this gene during cellular growth and
differentiation and its association with cellular transformation, we
cloned and characterized the human PRL-1 gene and determined its chromosomal localization. Moreover, we performed an analysis of the
regulatory regions of the human PRL-1 gene that are
necessary for its expression.
Isolation of Human PRL Genomic DNA--
A human lymphocyte
genomic library established in Chromosomal Localization--
To determine the chromosomal
localization of the human PRL-1, the 17.5-kb genomic DNA
clone hprl1 was used for fluorescence in situ hybridization.
The probe was labeled by nick translation with biotin-11-dUTP and
hybridized to human metaphase chromosomes. After fluorescence in
situ hybridization, the slide was destained and G-banded for
chromosomal identification (6).
RNA and RNA Analysis--
RNAs were prepared exactly as reported
using guanidinium isothiocyanate extraction (7, 8). Primer extension
was performed following the reported protocols (9) with minor
modifications. For P1 a 22-nucleotide antisense primer, corresponding
to bases +57-78 relative to the transcription start site (+1), and for P2 a 25-bp antisense primer from +3542-3567 were used. Primers were
end-labeled with T4 polynucleotide kinase and
[ Ribonuclease Protection Assay--
The antisense RNA probe was
generated from an EcoRI-linearized Bluescript KS plasmid
template (plasmid pP2-B) containing sequence 3269-3567 of human PRL
genomic DNA by transcription with T7 RNA polymerase in the presence of
[ Human PRL-1 Gene/Luciferase Fusion Plasmid
Construction--
Chimeric human promoter-luciferase plasmids were
constructed by cloning various lengths of 5'-flanking sequence and
intron sequence from the PRL-1 gene into the polylinker
region of the luciferase reporter plasmids pGL2-Basic or
pGL2-SV40-promoter (Promega). Unless indicated, the nomenclature used
for the constructs is based upon the succession of 5' restriction sites
within the fragments. The plasmid pP1-BglS was constructed by ligating
the 2219-bp (BglII-BglI blunted) fragment from
plasmid pPRL/SstI, which contains 6.1 kb of the 5'-flanking
region and 818 bp of the PRL-1 gene, into the
BglII and HindIII (blunted) sites of the vector
pGL2-Basic. A series of 5'-end deletion mutant plasmids were then
generated from the plasmid pP1-BglS (see Fig. 4A) by use of
various convenient restriction endonuclease sites. For construction of
the plasmid pP1-KpnS, pP1-Hind, pP1-Pst, and pP1-Sma, the plasmid
pP1-BglS was digested with KpnI,
KpnI/HindIII, KpnI/PstI, SmaI, respectively, and blunted by incubating with T4 DNA
polymerase and dNTP, and then the fragments were self-ligated to
generate corresponding plasmids. To construct the plasmids pP1-KpnL and pP1-BglL, the plasmid pPRL/SstI was cut by BglII
and KpnI respectively; the 3.8-kb KpnI fragment
and 1-kb BglII fragment were gel-purified and ligated to
KpnI and BglII sites of the plasmids pP1-KpnL and pP1-BglL, respectively. Orientation was determined by DNA sequence.
Department of Genetics and the
§ Department of Pediatrics,
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-FIX II (gift from E. Rand) was used
to isolate human PRL-1 gene clones. Approximately 1 × 106 plaques were screened with a 243-bp
EcoRI/SstI fragment derived from the 5'-end of
rat PRL-1 cDNA using high stringency conditions. Several
positive isolates proved to contain intronless PRL-1 genes and were believed to be pseudogenes. Two intron-containing positive phage clones were further characterized by restriction endonuclease mapping and Southern blotting. SstI fragments from the
positive phage clone, hprl1, were subcloned into the vector Bluescript KS(+) and denoted pPRL/SstI, pPRL/Sst2, etc. 12 kb of
continuous sequence was determined using ABI automated cycle sequencer
with Stretch upgrade (DNA Sequencing Core, Department of Genetics, University of Pennsylvania School of Medicine) and was analyzed with
Intelligenetics (Intelligenetics, Inc., Mountain View, CA) and GCG
sequence analysis software (Genetics Computer Group, Inc., Madison,
WI).
-32P]ATP and purified by Sephadex G-25 spin column.
50 µg of human HepG2 total cellular RNA was hybridized with the
radiolabeled primer (105 cpm) by heating at 80 °C for 10 min and then cooled to room temperature in a solution containing 50 mM Tris·HCl, pH 8.3, at 42 °C, 50 mM KCl,
10 mM MgCl2, 1 mM DTT, 1 mM dNTP mixture, and 0.5 mM spermidine. Then 2 units of avian myeloblastosis virus reverse transcriptase (Life
Technologies, Inc.) were added to the mixture, and the reaction was
incubated at 42 °C for 60 min. Following phenol/chloroform extraction, and ethanol precipitation with 50 µg/ml tRNA, the primer-extended products were analyzed on 6% polyacrylamide gel containing 8 M urea. Sequencing reactions were performed
using Sequenase 2.0 according to the manufacturer's protocol (U. S. Biochemical Corp.).
-32P]UTP using an in vitro transcription
kit (Stratagene, La Jolla, CA). The probe used contained 130 bp of exon
2 and 169 bp of intron 1 (see Fig. 3). 50-70 µg of total HepG2 RNA
was used for the assay. After overnight hybridization of 0.5-1 × 106 cpm of probe to the RNA, the hybrids were digested
according to the described protocol (9). The protected fragments were then analyzed on 6% polyacrylamide gel containing 8 M
urea.
Cell Culture, DNA Transfection--
Human hepatoma HepG2 cells,
mouse embryo testicular carcinoma cell line F9 cells, and mouse
fibroblast NIH 3T3 cells were grown in Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) supplemented with 10% fetal bovine
serum (Life Technologies, Inc.), 2 mM
L-glutamine (Flow Laboratories), 100 units of penicillin, and 50 units of streptomycin (Flow Laboratories) as described previously (1, 10). Human cervical carcinoma HeLa cells were grown in
Iscove's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, 100 units of penicillin, and 50 units of
streptomycin. Calcium phosphate-mediated transient transfections were
performed as described previously with minor modifications (10). Twenty
four hours before transfection, the cells were plated in 60-mm dishes
at 1-5 × 105 cells/cm per dish and then
cotransfected with various human PRL-luciferase chimeric plasmids (5 µg per dish) and 1 µg of the pSV--galactosidase reference
plasmid (Promega) as an internal standard for transfection efficiency.
After incubation with DNA-calcium phosphate coprecipitation buffer for
16 h, the cells were washed twice with serum-free culture medium;
complete medium was added, and then the cells were incubated for an
additional 24 h before harvesting for luciferase activity assays
performed with luciferase assay system (Promega) and the recommended
procedure by the supplier. Relative luciferase activity was reported
after normalization to
-galactosidase activity.
Preparation of Nuclear Protein Extracts--
All steps were
performed at 4 °C, and 2 µg/ml proteinase inhibitors antipain,
aprotinin, bestatin, and leupeptin were added into all buffers except
phosphate-buffered saline, pH 7.4. Crude nuclear extracts from
regenerating liver were prepared as reported (11). Crude nuclear
extracts from cell lines HepG2, H35, HeLa, F9, and NIH 3T3 were
prepared using the procedure of Seal et al. (12) with minor
modifications. Cells were washed twice and scraped into
phosphate-buffered saline solution. The cells were pelleted by
centrifugation at 500 × g for 10 min and lysed by
homogenization in approximately 9 volumes of Buffer A (10 mM HEPES, pH 7.6, 0.3 M sucrose, 60 mM KCl, 15 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 0.15 mM spermine, 0.5 mM
spermidine, 0.5% Triton X-100, 1 mM dithiothreitol (DTT),
0.5 mM phenylmethylsulfonyl fluoride). Aliquots of cell lysate were layered over 5 ml of Buffer B (same as Buffer A except that
the concentration of sucrose was 1 M) in 15-ml tubes and centrifuged at 10,000 × g for 10 min. The supernatant
was removed by aspiration, and the nuclei pellet was resuspended in
Buffer C (20 mM HEPES, pH 7.9, 0.2 mM EDTA, 0.1 mM EGTA, 1.5 mM MgCl2, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride,
20% glycerol). Nuclei were lysed by the dropwise addition of 1/9
volume of 4.2 M NaCl before being centrifuged at
50,000 × g for 50 min. The supernatant was dialyzed
against Buffer D (20 mM HEPES, pH 7.6, 0.2 mM
EDTA, 0.1 M KCl, 1 mM DTT, 0.5 mM
phenylmethylsulfonyl fluoride, 20% glycerol) for at least 6 h.
Finally, the extract was centrifuged at 12,000 × g for
10 min, and aliquots were quick-frozen in dry ice, 95% ethanol mixture
and stored at 70 °C. Protein concentrations were determined using
the Bio-Rad protein determination method per manufacturer's
instructions.
Electrophoretic Gel Mobility Shift Assays (EMSA)--
The
synthesized single polynucleotides were annealed in 10 mM
Tris·HCl, pH 8.0, 1 mM EDTA, 0.3 M NaCl at
90-95 °C for 10 min, and then cooled to room temperature. After
being kept at room temperature for 24 h, the annealed
oligonucleotide was purified by 15% native polyacrylamide. End
labeling of oligonucleotides was carried with T4 polynucleotide kinase
and [-32P]ATP and purified by Sephadex G-25 spin
column. Binding reactions were performed essentially as reported (11,
13). Nuclear protein extracts (3-6 µg) were incubated with 2 µg of
poly(dI-dC) in 10 µl of binding buffer containing 20 mM
HEPES, pH 7.9, 1 mM EDTA, 60 mM NaCl, 1 mM DTT, 1.5 mM NaCl, 10% glycerol for 15 min
at room temperature. 32P-Labeled probe (0.5-1 × 104 cpm) was then added to the reaction, and incubation was
continued for another 20 min. DNA-protein binding complexes were
analyzed on 6% native polyacrylamide gel at 300 V using 0.5× TBE as
electrophoresis buffer. Gels were dried and exposed to Kodak X-Omat AR
films overnight. The sequence of the oligonucleotide used in the
EMSA was as follows, 5'GCCGCCTCCGCCCTTGGCCCTTTGGTCTG3'.
DNase I Footprinting-- DNase I footprinting was carried out as described in Ohlsson and Edlund (13). Briefly, 0-150 µg of nuclear protein extract was preincubated for 20 min at room temperature with 4 µg of poly(dI-dC) in 50 µl of a mixture containing 10 mM HEPES, pH 7.9, 10% glycerol, 50 mM KCl, 0.5 mM EDTA, 1 mM dithiothreitol, and 0.25 mM fluoride. 1-2 × 104 cpm of 32P-end-labeled DNA probe was then added. After the binding reaction was incubated for 40 min at room temperature, MgCl2 and CaCl2 were added to a final concentration of 5 and 2 mM, respectively. An empirically adjusted amount of freshly diluted DNase I was added, and the reaction was allowed to proceed for exact 45 s at room temperature. The reaction was stopped by the addition of 100 µl of buffer containing 100 mM Tris·HCl, pH 8.0, 100 mM NaCl, 1% SDS, 20 mM EDTA, 50 mg/ml yeast tRNA, and 100 mg/ml proteinase K and was incubated for 15 min at 37 °C. Nucleic acids were extracted twice with phenol/chloroform, precipitated by ethanol/sodium acetate, and fractionated on 6% polyacrylamide, 8 M urea gel, alongside sequencing ladder G + A that was produced by chemical cleavage of aliquots of the probe DNA (14).
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RESULTS |
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Isolation and Characterization of Human PRL-1 Gene--
Two
overlapping genomic clones, designated as hprl1 (17.5 kB) and hprl2
(14.5 kB), containing the human PRL-1 gene were isolated (Fig. 1A). Results from
detailed restriction mapping and Southern hybridization demonstrated
that both clones spanned the entire human PRL-1 gene but had
different 5' and 3' boundaries (Fig. 1). The clone hprl1, with a
17.5-kb insert, was subcloned, and 12 kb were sequenced from the
upstream BglII site (2179) to 0.8 kb past the 3'-end of
the second polyadenylation signal. The human PRL-1 gene is
composed of 6 exons and 5 introns. The boundaries of each exon
display the canonical splice donor (GT) and splice acceptor (AG)
consensus sequences (Fig. 1A). The first exon contains no
coding sequence and is separated from the second exon by the largest
intron (3.2 kb) in the gene. An ATG initiation codon is located in the
middle of the second exon. Based on GenBankTM analyses,
rat, human, and mouse PRL-1 are identical at the amino acid level. We
found that human PRL-1 had 55% amino acid identity with a
Caenorhabditis elegans "similar to the protein tyrosine phosphatase (U42846)" sequence identified by C. elegans
genomic sequencing (15) (Fig. 1B). Interesting features of
this conservation include virtual identity within the enzymatic domain
(around amino acid 120), size similarity, and absolute conservation of
the C-terminal CAAX (CCIQ) sequence.
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Chromosomal Localization-- The human hprl1 genomic probe specifically hybridized to the centromeric region of the long arm of a C group chromosome in 20 out of 30 metaphases that were scored. Sequential G-banding localized the probe to chromosome 6, within band q12 (Fig. 2).
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Identification of PRL-1 Promoters P1 and P2-- Examination of the nucleotide sequence of the human PRL-1 gene 5'-flanking region revealed that the promoter region was highly GC-rich and contained a canonical TATA box. The transcription start site of the gene was identified from total RNA of human hepatoma cell line HepG2 using primer extension analysis with a primer contained within exon 1 (Fig. 3A). A single major primer extension product was identified at a position 25 bp 3' to the TATA box. We designated it as promoter P1.
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Functional Analysis of Promoters P1 and P2 of the Human PRL-1
Gene--
To establish that P1 and P2 are functional and begin to
delineate the sequences essential for transcription of the human
PRL-1 gene in cell culture systems, we first constructed a
series of the human PRL-1 P1 luciferase chimeric plasmids in
which various lengths of the 5'-flanking region of the human
PRL-1 gene including the P1 RNA initiation site were fused
with the coding region of the bacterial luciferase gene (Fig.
4A). A plasmid containing the
SV40 promoter and enhancer (pGL-C) was used as a positive control for
transfection. Sequences necessary for high level basal transcription of
the PRL-1 P1 promoter in both HepG2 and mouse NIH 3T3 cells
were found within the first 200 bp of the P1 promoter, and a negative
element may exist in the region between 3200 and
2179 bp.
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Identification of a Transcriptional Enhancer in Intron 1--
The
fact that the first exon of the PRL-1 gene is non-coding and
the gene contains two transcription start sites in exons 1 and 2 suggested that transcriptional regulatory sequences may reside in
intron 1. The sequence of the first intron shows a high content of GC
bases in the first 900 bp downstream of exon 1 and contains several
consensus sites for transcription factor binding, including SP-1, AP-2,
Myo D, NF-B, HNF5, and C/EBP elements. To test for the presence of
an enhancer sequence within the first intron, constructs were made that
contained promoters P1, various lengths of the first intron, and the
first 130 bp of exon 2 (including P2) inserted upstream of the
luciferase gene. Analyses suggested that removal of the
BglI/SstI fragment (+40 to +862) greatly
diminished transcriptional activity in HepG2 cells (Fig.
5A). This could be explained
by the fact that this deletion removed the splice donor for exon 1 and
destabilized P1-derived transcripts, and/or the region contained a
transcriptional enhancer. This region was subdivided into three
fragments to test for enhancer activity. Only the
SmaI/SmaI fragment significantly enhanced
transcription from the PRL-1 P1 or SV40 promoter luciferase
constructs (Fig. 5B). As shown, this fragment conferred
orientation independent activity. Sequence analysis of the region
showed a high percent of GC composition (76.8%). Detailed deletion
with exonuclease Bal 31 narrowed the enhancer activity to a 90-bp
region (Fig. 5C).
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DISCUSSION |
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The rat PRL-1 gene is highly expressed in proliferating liver tissues in development, regeneration, and oncogenesis and is associated with differentiation in other cell types. As an initial step in understanding the regulation of the PRL-1 gene, we isolated the human PRL-1 genomic clone. We showed that the PRL-1 gene is highly conserved in mammals and contains a C. elegans homologue. We identified its chromosomal position as 6q12. We demonstrated the presence of two functional promoters near exons 1 and 2 of the PRL-1 gene and identified a developmentally regulated enhancer site within the first intron of the PRL-1 gene.
The 100% conservation of the mammalian PRL-1 protein, strong similarity of a family member OV-1, and conservation within simple eucaryotes (C. elegans) suggest that the PRL-1 protein has a critical function in cellular regulation. Of particular interest is the near identity of the active site region between mammalian and C. elegans PRL-1 and the 100% conservation of the unusual isoprenylation sequence, CCIQ. This suggests that prenylation indeed plays an important role in posttranslational regulation of PRL-1 activity.
The localization of the PRL-1 gene to chromosomal segment 6q12 by in situ hybridization correlates with the recent mapping of the PRL-1 ESTs to between the D6S1695 and D6S21 markers in this region (NCBI, the Human Gene Map). This region of chromosome 6 does not have any known closely linked markers to disease genes. Although chromosome 6 is frequently abnormal in solid tumor malignancies, these abnormalities usually involve segment q15 to the terminus (17).
Both P1 and P2 promoters of the PRL-1 gene contribute to the
activity of the gene. In HepG2 cells P1 contributes more to the steady
state transcript (Fig. 3B), but both promoters show high activity in transfection studies. In HepG2 and NIH 3T3 cells, major
basal regulation is still retained within 200 bp of the RNA initiation
site. We have identified several transcription factor binding sites
including Egr-1 and SP1 elements within this sequence. Because Egr-1 is
strongly up-regulated during liver regeneration, we have explored the
contribution of Egr-1 to regulation of the PRL-1 gene in
liver regeneration.2 A major regulatory site of the
TATA-less P2 promoter has been identified within a 39-bp segment
upstream of P2. This segment contains a C/EBP-binding site, and
C/EBP like Egr-1 is also increased during liver regeneration
(18).2
PRL-1 is also expressed at a high level in terminally differentiated intestinal cells (5). The Cdx class of homeodomain proteins has been shown to be of critical importance in the regulation of genes that are expressed in differentiated intestinal cells (16). Of interest is the significant number of consensus Cdx elements found upstream of PRL-1 promoter P1 including several within a few hundred bases of the TATA element. Subsequent studies examining the ability of Cdx to regulate the PRL-1 promoter will be of interest.
Promoter P1, upstream of untranslated exon 1, and P2, upstream of exon 2, are separated by the large intron 1. We assessed the possible contribution of sequences within intron 1 to promoter regulation. We localized a novel enhancer sequence within intron 1. This sequence fulfilled the requirement for an enhancer because it was position-independent and acted upstream of various promoters. A specific complex (PIEC) bound to the strongly footprinted region within the enhancer sequence and allowed us to derive a consensus binding sequence. In different cells, different specific protein complexes were observed that bound this sequence, and thus far PIEC has only been detected in cells of a fetal lineage including HepG2 and F9. In addition, fetal but not adult liver contained PIEC, and PIEC was not induced in regenerating liver. There was a correlation between the presence of PIEC in four cell lines tested and the ability of this sequence to function as an enhancer. Moreover, the intron enhancer contributed significantly to total PRL-1 promoter activity in HepG2 which contain PIEC but not NIH 3T3 cells which do not. However, the presence of PIEC does not necessarily correlate with the level of steady state PRL-1 mRNA in every tissue or cell line. HeLa cells and regenerating liver express high levels of PRL-1 mRNA but do not contain PIEC. Other regions of the PRL-1 promoter are likely to contribute to regulation in these cells; post-transcriptional controls could play a role as well.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants DK49629 (to R. T.) and DK44237 (to R. T. and R. H. D.).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) AF051160 and U42840.
To whom correspondence should be addressed: Dept. of Genetics,
University of Pennsylvania School of Medicine, 705a Stellar-Chance, 422 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-898-9131; Fax: 215-573-2195 or 215-573-9411; E-mail: taubra{at}mail.med.upenn.edu.
1 The abbreviations used are: PTPase, protein tyrosine phosphatase; PRL-1, phosphatase of regenerating liver; PIEC, PRL-1 intron enhancer complex; EMSA, electrophoretic gel mobility shift assay; kb, kilobase pairs; bp, base pair(s); DTT, dithiothreitol; PCR, polymerase chain reaction.
2 Y. Peng, K. Du, S. Ramirez, and R. Taub, submitted for publication.
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REFERENCES |
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