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Endocrine Research Unit (S-W.J., N.L.E.) Thoracic Diseases Research Unit (K.W.) Department of Internal Medicine (S-W.J., K.W., N.L.E.) Department of Biochemistry & Molecular Biology (N.L.E.) Mayo Clinic Rochester, Minnesota 55905
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Our initial efforts to understand the mechanism of CSEn activation focused on TEF-1, the first cloned human GT-IIC binding factor that has been implicated in mediating SV40 enhancer function (10, 11, 12). We observed that TEF-1 is expressed in placental tissue and cultured human choriocarcinoma cell lines (BeWo and JEG-3) (8, 9). However, when TEF-1 function was tested by cotransfection with a TEF-1 expression vector and a reporter plasmid containing CSEn2, TEF-1 repressed, rather than stimulated, the reporter activity (8, 9). Also, down-regulation of endogenous TEF-1 levels with antisense oligonucleotides resulted in elevated CSEn2 as well as basal hCS promoter activity, suggesting that endogenous TEF-1 was acting as a repressor (9). We subsequently demonstrated that TEF-1 can interact in vitro with the TATA box-binding protein (TBP), inhibiting its ability to bind the TATA element (8). Thus TEF-1-TBP interactions may provide a possible mechanism by which TEF-1 exerts repressor activity on GT-IIC-containing enhancers. These data suggest that factor(s) other than TEF-1 may account for CSEn function in placental cells.
Recently, Jacquemin et al. (13) reported the cloning of an additional member of the hTEF-1 family, designated hTEF-5, that was shown to be related to the chicken DTEF-1 gene. They were able to show that hTEF-5 mRNA is most strongly expressed in placenta and choriocarcinoma cells (JEG-3), followed by skeletal and cardiac muscle. Jacquemin et al. (13) also demonstrated that hTEF-5 binds to several CSEn enhansons and defined a novel functional element comprised of repeated enhansons that bind TEF-5 in a cooperative manner, suggesting that hTEF-5 regulates CSEn activity. Nevertheless, attempts to express this gene product in a variety of cells, including JEG-3 cells, failed to demonstrate any CSEn activation (13). In the current study, we report the molecular cloning of an additional hTEF-5 clone from a human placental library that contains the identical open reading frame reported by Jacquemin et al. (13) but with an additional 300 bp of 5'-untranslated sequence. TEF-5 mRNA is highly expressed in placental, skeletal, and cardiac muscle tissues. The in vitro-generated TEF-5 is a polypeptide of approximately 53-kDa that specifically binds to GT-IIC and SphI/SphII enhansons. Overexpression of the complete, unmodified TEF-5 cDNA clone in the choriocarcinoma cell line BeWo resulted in activation of a variety of GT-IIC-containing enhancers. These data provide direct functional evidence that TEF-5 may play an important role in the placenta-specific expression of the hCS gene. In addition, its presence in muscle tissue indicates that it may be involved in the regulation of muscle gene expression and/or development.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and Fig. 1B). For the convenience of the
subcloning, BglII sites were fixed on each primer. After two
rounds of PCR, DNA fragments between 800 and 1200 bp were purified from
agarose gels. In subsequent BglII restriction digestions,
some of the DNA fragments were converted to 700- and 400-bp bands,
indicating the presence of an internal BglII site within
certain fragments. Two classes of TEA/ATTS domain genes were identified
from 18 positive clones in subsequent sequence analyses and database
comparisons. These included clones identical to TEF-1 (12) and clones
containing the BglII site that were related to the chicken
DTEF-1 gene (19) and corresponded to the hTEF-5 cDNA clone (13), which
were analyzed further.
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DR2 phage cDNA library (CLONTECH Laboratories, Inc., Palo Alto, CA) was screened. Approximately
1.5 million plaques were screened and 22 positive clones were
identified. Sequence analysis indicate that all of these clones
contained partial sequences that were missing the extreme N-terminal
region. Using a probe from the 5'-end of the longest cDNA clone, we
screened an additional 1 million plaques and, from 14 positive clones,
isolated a 3.0-kb TEF-5 clone including more than 300 bp preceding the
TEA/ATTS domain (Fig. 1A
). Like other members of the TEA/ATTS family
(12, 18, 19, 20), no initiator methionine with perfect Kozak consensus
sequences was found in the 5'-region (Fig. 1A). Like TEF-1, TEF-5
contains an isoleucine codon (ATA) located 29 amino acids upstream of
the TEA domain (starting with the sequence DAEG) with a 9 of 13 match
with the Kozak consensus (GCCGCCRCCAUGG), including the three essential
positions at -3, +4, and +5. Xiao et al. (12) demonstrated
that the corresponding region in TEF-1 served as one of the two
translation initiation sites of TEF-1. Amino acid sequence analysis
indicates that TEF-5 contains a highly conserved TEA/ATTS domain and
typical proline-rich and STY-rich domains (Fig. 1A). Alignment of TEF-5
with the other cloned TEF-related factors revealed that TEF-5 is most
closely related to the chicken DTEF-1A transcription factor (Fig. 1B)(19).
TEF-5 Is Expressed Preferentially in Placenta and Muscle
Tissues
TEF-5 expression in different tissues was evaluated by Northern
analysis. A blot containing a panel of mRNAs from multiple human
tissues was hybridized with the TEF-5 cDNA probe. A 3.0-kb band is
readily detected from placenta, with somewhat lesser levels of mRNA
detectable in skeletal and cardiac muscle tissues (Fig. 2A). After longer exposure, a faint band
is also visible from liver tissue. In addition, a minor band at 6.5 kb
observed from placenta during longer exposure may indicate the presence
of partially spliced TEF-5 precursors. No hybridization signals were
observed in brain, lung, kidney, or pancreas. Also, TEF-5 expression
was characterized in several cell lines. Strong signals were found in
BeWo cells, a human choriocarcinoma cell line. In addition, TEF-5 is
also expressed in SV40 transformed human fibroblasts
(GM0637E) and osteosarcoma (MG63) cells (Fig. 2C). Low levels of TEF-5
mRNA are present in cervical carcinoma (HeLa), liver tumor (HepG2), and
human breast cancer (MCF-7) cells (Fig. 2C). GC and monkey kidney
(COS-1) cells contain very low or undetectable levels of TEF-5 mRNA
(Fig. 2C). These data indicate a restricted pattern of TEF-5
expression. Its high-level expression in placental tissue and BeWo
cells indicate TEF-5 could be involved in hCS gene
expression. However, the absence of a strong signal in COS-1 cells
indicates that TEF-5 is unlikely to represent the source of the
previously identified factor, PPf/CSEF-1, in these cells (9, 13), which
appears to be a proteolytic degradation product of multiple TEF factors
(13).
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). Xiao et al. (12) provided
evidence that in TEF-1, translation initiates from both the isoleucine
(AUA codon) and methionine (AUG codon), located 29 and 14 amino acids,
respectively, upstream of the TEA/ATTS domain, resulting in 53- and
51-kDa peptides. Examination of cDNA sequences reveals that DNA
sequences around the AUA codon were highly conserved (12 of 13 matches)
between hTEF-1 and TEF-5. However, there is no corresponding second
initiation site in TEF-5, since the methionine (ATG) is replaced by
arginine (CGG). The approximately 53-kDa single band of TEF-5 in
vitro translation products suggests that the isoleucine as
indicated in Fig. 1A, like the cDTEF-1, hTEF-3, and hTEF-1 homologs, is
the most likely translation initiation codon. Because TEF-1 and TEF-5
contain comparable numbers of methionine (12 for TEF-1 and 10 for
TEF-5), the difference in the density of in vitro
translation products observed in Fig. 3A may be due to different
translation efficiency, possibly caused by the single substitution in
their translation initiation site.
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) interacted with both
GT-IICSV and GT-IICCS oligonucleotides, which
share the core sequence (GGAATGTG) but have different flanking
sequences (Fig. 3B). Mutation of the core sequences of
GT-IICSV effectively eliminated its ability to bind TEF-1
and TEF-5, indicating that TEF-5 can interact with the GT-IIC core
sequences. The binding specificity was further confirmed by a
competition experiment. The TEF-5·GT-IICCS interaction
was competed with increasing amounts of unlabeled GT-IICSV,
SphI, and SphII, but not the corresponding mutant
oligonucleotides (Fig. 3C). These probes were used because the binding
affinity of TEF-1 to each of these sequences has been well
characterized (10, 6) and the binding affinities occur in order:
GT-IIC > SphI 
SphII. In the
presence of a 300-fold excess of competitor, the TEF-5 complex was
completely competed by the GT-IIC oligonucleotide and greatly reduced
by the SphI oligonucleotide (Fig. 3C). Therefore, TEF-5 has
the same DNA binding specificity as the other TEA/ATTS domain factors
in interacting with both GT-IIC and SphI enhansons.
In previous studies, we and others have observed that GT-IIC probes
detected a two-band pattern from BeWo or JEG-3 cell extracts in gel
shift experiments (4, 9, 13, 23). Based on gel supershift experiments
with anti-TEF-1 antibodies, the slow migrating band contains TEF-1, and
the unknown fast migrating band was designated as PPf/CSEF-1 and
proposed to be a potential TEA/ATTS domain factor (9). Since TEF-1- and
TEF-5·GT-IIC complexes migrate identically in these experiments (Fig. 3B), it is unlikely that TEF-5 and PPf/CSEF-1 are the same factors.
Moreover, Jacquemin et al. (13) have shown that PPf/CSEF-1
appears to be a proteolytic breakdown product of TEF family members
that is immunologically related to the TEA/ATTS domain. However, the
exact precursor(s) for PPf/CSEF-1 are not known. Since PPf/CSEF-1 is
much more thermostable than TEF-1 (9), we performed thermolability
analyses of TEF-1 and TEF-5 to determine whether their relative
thermolabilities might provide some evidence about the origin of
PPf/CSEF-1. We reasoned that, if the proteolytic product were heat
stable, this was likely the result of a specific structural feature
that would be present in the precursor and might confer relative heat
stability to the precursor protein as well. Consistent with our
previous results, TEF-1 can withstand 60 C for 2 min but lost its
DNA-binding activity at 65 C (Fig. 3D). TEF-5 GT-IIC binding activity
is largely reduced at 50 C, indicating it is more sensitive to heat
exposure than TEF-1 (Fig. 3D). This suggests that it might be a less
likely precursor of the more thermostable PPf/CSEF-1.
TEF-5 Transactivates hCS and SV40 Enhancers
We next examined whether TEF-5 can transactivate
the hCS and SV40 enhancers when overexpressed in
BeWo cells. A cotransfection was performed with the pDR2
TEF-5 expression vector and a variety of hCSp.LUC
reporter plasmids (Fig. 4A). While TEF-5
overexpression did not affect hCS promoter activity, it
stimulated the hCSp.LUC gene carrying one copy of the
SV40 enhancer 2.9-fold (Fig. 4A). TEF-5 overexpression
increased luciferase expression 3.5-fold with constructs containing one
or two copies of CSEn2 (Fig. 4A). In addition, TEF-5 stimulated
luciferase activity 2.5-fold with a construct containing 12 copies of
the GT-IIC enhanson (Fig. 4A), providing direct evidence that TEF-5
transactivation is mediated by interaction with GT-IIC sequences.
Essentially identical results (3-fold stimulation) were obtained
with constructs containing one copy of CSEn2 with either one copy of
CSEn1 (enhancer copy downstream from the hCS-1 gene) or
CSEn5 (enhancer downstream of the hCS-5 gene). Since the CS
enhancer contains multiple enhansons and mutation of each of the
enhansons only partially removes enhancer activity (6), it is not
practical to generate a mutated CS enhancer. Instead, to directly test
the transactivation ability of TEF-5, we made two constructs,
(GT-IIC)5-CSp.LUC and
(OCT)5-CSp.LUC, containing either
tandemly repeated GT-IIC elements or tandemly repeated OCT elements to
serve as a control. The OCT elements are derived from a region of CSEn
within the footprint 2 region (3) and neither bind any of the
TEF factors nor exhibit any independent enhancer activity, but may
contribute to enhancer activity, since their mutation in the context of
an intact enhancer is associated with somewhat reduced enhancer
activity (3). Cotransfection of TEF-5 with this pair of reporter
plasmids shows that TEF-5 overexpression significantly stimulated the
GT-IIC artificial enhancer but not the control
(OCT)5-CSp.LUC or CSp.LUC
constructs (Fig. 4B). These experiments, together with transfection
studies using native hCS or SV40 enhancers, strongly support the
concept that TEF-5 is an effective transactivator and may account for
the constitutive CS and SV40 enhancer activities in placental
cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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To isolate additional TEA/ATTS family members we used a degenerate
oligonucleotide approach based on conserved domains within TEF-1 and
its family members (18). We isolated a clone that contained an open
reading frame identical to the TEF-5 homolog that was recently reported
by Jacquemin et al. (13). TEF-5 is related to the chicken
DTEF-1 family (19) (Fig. 1). In agreement with Jacquemin et
al. (13), high levels of TEF-5 mRNA were found in human placental
tissue and BeWo cells (Fig. 2), and its gene product was found to bind
specifically to GT-IIC sequences from both the hCS and
SV40 enhancers (Fig. 3). In contrast to Jacquemin et
al. (13), who were unable to demonstrate functional activity after
overexpressing TEF-5 in HeLa or JEG-3 cells, we demonstrate that it is
a transactivator of CSEn function in BeWo cells (Fig. 4). TEF-5
stimulates reporter constructs containing single copies of CSEn2 or
multiple enhancers, including CSEn5 and CSEn1, that occur downstream of
the hCS-5 and hCS-1 genes, respectively (Fig. 4).
Also, TEF-5 stimulated reporter constructs containing the
SV40 enhancer or an artificial enhancer comprised of GT-IIC
multimers. Taken together, these studies demonstrate that TEF-5 is a
transactivator in BeWo cells that operates through binding GT-IIC and
SphI/SphII enhansons and provide strong support
to the notion that TEF-5 plays a major role in mediating
hCS gene expression through the placental enhancer.
The disparity in our TEF-5 functional analyses vs. those of
Jacquemin et al. (13) are likely to be explained by the
different strategies that were used to express the protein and analyze
the protein. Thus, differences in the choriocarcinoma cell lines JEG-3
(13) and BeWo, which were used in the current studies, may well be a
factor contributing to the difference in behavior. In addition, in
constructing their TEF-5 expression vector, Jacquemin et al.
(13) modified the 5'-untranslated region and translation initiation
site of their TEF-5 cDNA clone, replacing the ATA isoleucine codon with
ATG, replacing the imperfect Kozak sequence (9 of 13 matches) with a
perfect Kozak sequence, and deleting any upstream 5'-untranslated
sequences. This was the identical strategy used for TEF-1 expression
(12). Our TEF-5 cDNA clone contained about 300 bp of 5'-untranslated
sequence and a much larger 3'-untranslated region, which was not
modified: the pDR2 TEF-5 expression vector was
obtained from the original DR2 bacteriophage clone via
loxP-dependent cre excision (15). These
differences in expression strategy suggest that expression efficiency
could be the result of engineering a more efficient translation
initiation site (ATG vs. AUA and perfect Kozak consensus
vs. 9 of 13 matched consensus) in the constructs. In
addition, the 5'- or 3'-untranslated regions could contain information
that either controls mRNA abundance or modifies translation efficiency.
In this regard it is interesting that the 5'-untranslated region of
TEF-5 contains several polypyrimidine tracts (see Fig. 1A) that are
similar to those that have been shown to negatively regulate
translation intitiation in a number of genes, including the androgren
receptor (20) and ribosomal protein rpL32 gene (21). Since most
proteins that are derived from mRNAs with non-AUG initiation
codons contain methionine as the initiating amino acid (30), the
question of whether a unique NH2-terminal structure may
contribute to TEF-5 structure/function is considered less likely. While
the translation initiation site for TEF-5 has not been directly
measured, based on the size of the expressed protein product, its size
relative to TEF-1, its amino acid sequence homology with TEF-1, and the
relative absence of other likely translation initiation sites, the
isoleucine initiation codon (AUA, nucleotide 306, Fig. 1A, GenBank Accession No. AF142482) is the most probable. Consistent
with this interpretation is the observation that Jacquemin et
al. (13) did observe squelching with their pXJ41 TEF-5 expression
construct in JEG-3 cells. These observations are consistent with the
concept that, like TEF-1, TEF-5 may operate through the obligate
requirement of a cofactor that is present in limited abundance.
Nevertheless, when higher concentrations of TEF-5 expression plasmid
were used in otherwise identical experiments, several reporter
constructs containing CSEn2 were stimulated to even higher levels of
activity (Fig. 4C), indicating that TEF-5 stimulates over widely
varying concentrations. Thus, if expression efficiency explains the
difference in functional behavior between these two otherwise identical
clones, either large differences in the translational efficiency or
negative regulatory sequences in the 5'- and/or 3'-untranslated regions
or both are very likely to be involved.
DTEF-1 was cloned originally from chicken skeletal muscle by Azakie
et al. (19) and was found to be expressed at high levels in
cardiac muscle, but low levels in skeletal muscle. Moderate expression
of this gene was also found in gizzard and lung, and lower levels were
present in kidney. The transcriptional potential of this gene has not
been examined. The alignment of TEA/ATTS domain genes (Fig. 1)
indicates that DTEF-1 and TEF-5 are most closely related, sharing 87%
identity and 91% similarity at the amino acid level. This suggests
that TEF-5 is the human homolog of chicken DTEF-1. However, the
tissue-specific distribution between the two species appears to be
different. In humans, TEF-5 is expressed at comparable levels in
cardiac and skeletal muscle, and no expression was observed in lung and
kidney even after long exposure. The relatively high level expression
of TEF-5 in muscle tissues suggest that TEF-5 may participate in muscle
gene expression and development.
Currently the TEA/ATTS gene family is comprised of four members, including TEF-1, TEF-3 (RTEF-1), TEF-4, and TEF-5 (DTEF-1). These factors are thought to play important, but partially redundant roles in myogenesis, cardiogenesis, and central nervous system development. Stewart et al. (22) cloned RTEF-1 from human heart. RTEF-1 was shown to be expressed at high levels in skeletal muscle and pancreas and at lower levels in placenta. Interestingly, RTEF-1 shares 98% identity with human TEF-3, a gene whose mouse homolog was found to be expressed mainly in embryonic mouse skeletal muscles (18). From the human placental library, we isolated a clone that is identical to hTEF-3 (data not shown), confirming the expression of this homolog in placenta reported by Stewart et al. (22). Thus at least three of the TEA/ATTS homologs, TEF-1, RTEF-1/TEF-3, and TEF-5, are expressed in the placenta and could all play varying roles in mediating CSEn function and placental development.
In addition to the human TEA/ATTS domain factors described above, a mouse gene ETRF-1, closely related to RTEF-1, has been isolated (7). However, its ubiquitous expression pattern in all tested tissues, including lung, kidney, muscle, heart, liver, brain, thymus, spleen, and testis, argues against its assignment as the mouse homolog of hRTEF-1. Similarly, another mouse gene, ETRF-2, showed high level expression in lung, a situation unique to all the other TEA/ATTS domain factors (7). Two more mouse TEA/ATTS factors, FR19 (26) and TEFR-1 (27), were isolated from NIH3T3 cells and heart libraries, respectively. Both genes were shown to be expressed in lung and muscle tissues. More recently, Kaneko and associates (28, 29) have demonstrated that mouse mTEAD-2, the hTEF-4 homolog, is expressed throughout early mammalian development before the onset of zygotic gene expression, followed by expression of all the other TEF family members, suggesting a selective role for this homolog in development. With the exception of TEFR-1, which was shown to have M-CAT (chloramphenicol acetyltransferase) binding ability and function as transactivator in HeLa cells, the transcriptional potential of these gene products has not been characterized.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). BglII sites were incorporated into each oligonucleotide
at the 3'-end to facilitate efficient subcloning. In the first round
PCR, the outside pair of oligonucleotides, TIC11 and TIC12, with a
total sequence complexity of 256, were used to amplify two human
placental libraries subcloned in gt11
(Stratagene, La Jolla, CA) and DR2
(CLONTECH Laboratories, Inc., Palo Alto, CA). The
reactions contained 10 µl 10x PCR extender buffer
(Stratagene), 2.5 µg each primer, 10 mM
deoxynucleoside triphosphates, 2.0 µl library DNA template in
a total volume of 100 µl. After 5 min of initial denaturing at 94 C,
10 U of Taq DNA polymerase and 10 µl of Taq
Extender (Stratagene) were added, and 25 cycles of PCR
were performed at the following conditions: denaturation, 94 C for 1
min; annealing, 50 C for 1.5 min; and elongation, 72 C for 2 min. PCR
products were extracted with phenol-chloroform, precipitated with
ethanol, and resolved on 1.5% agarose gels. Faint DNA bands
corresponding to about 800-1200 bp were excised from the gel, purified
by GeneClean (BIO 101, Vista, CA), and dissolved in 10 µl 10
mM Tris·HCl (pH 7.5), 1 mM EDTA.
For second-round PCR reactions, 1.0 µl templates from the first-round
PCR reactions were reamplified using 2.5 µg primers TIC11 and TIC12
(Table 1) with a combined complexity of 1024. PCR was performed for 35
cycles under the same conditions as first-round PCR. After agarose gel
electrophoresis, 1100-bp products were cut out and purified by
GeneClean. Restriction digestion with BglII, the site used
for subcloning, rendered some of the 1100-bp DNA to 700- and 400-bp
fragments, indicating the presence of an internal BglII
site. These DNA fragments were purified and subcloned individually into
BglII-digested pA3LUC vector. Eighteen positive
clones were purified and sequenced by automatic dye termination
sequencing (Molecular Biology Core Facility, Mayo Clinic) using M13 and
pA3 P12 primers (Table 1). Sequencing results were analyzed using the
FASTA program (GCG, Madison, WI).
Screening of cDNA Libraries
The 700-bp and 400-bp DNA fragments of TEF-5 were purified from
agarose gels and used as probes to screen a DR2 cDNA
library. The 700-bp and 400-bp DNA fragments (20 ng) were combined and
labeled by [32P]dCTP (8000 Ci/mmol,
Amersham, Arlington Heights, IL) using a random primed DNA
labeling kit (Boehringer Mannheim, Indianapolis, IN) and
purified on BioGel P-60 columns. Plaques (50,000 per 15-cm plate)
were transferred onto nitrocellulose membranes (Protron,
Schleicher & Schuell, Inc., Keene, NH) as described (14).
The membranes were hybridized with 1.5 x 106 cpm/ml
of denatured probe dissolved in ExpressHyb solution (CLONTECH Laboratories, Inc.) and washed according to the manufacturer’s
recommendations. The membranes were exposed to Kodak x-ray film
(Eastman Kodak Co., Rochester, NY) for 18–36 h. Positive
plaques were purified by two additional rounds of selection and
subjected to lox-mediated automatic excision for releasing
of pDR2 plasmid (15). DNA sequences from several clones were
organized by contig analysis, and the longest sequence was
identified. A more complete TEF-5 clone was obtained by using the most
5'-sequences as probes to screen the library for additional clones.
This clone, which represents the most complete human TEF homolog cDNA
cloned to date, was sequenced at least three times over its entire
length on both strands.
Northern Blot Analysis
Cells grown to 50% confluence were washed three times
with PBS (Gibco BRL, Gaithersburg, MD), scraped off, and
collected by centrifugation. Total RNA was purified from cell cultures
using a RNA STAT 60 kit (Tel-Test, "B",
Friendswood, TX) according to the manufacturer’s instructions. RNA (20
µg) was resolved in a 1% denaturing agarose gel and stained with
ethidium bromide for evaluation of RNA loading and integrity. The RNA
was transferred onto a nylon membrane (Boehringer Mannheim) by capillary elution and immobilized by UV
cross-linking. The TEF-5 cDNA-containing pDR2 plasmid was
digested with BamHI and XbaI restriction enzymes,
and TEF-5 cDNA was purified by GeneClean from agarose gel. TEF-5 cDNA
probes were labeled, purified, and hybridized to the cellular RNA blot
or a human multiple tissue Northern blot (MTN, CLONTECH Laboratories, Inc.) as described above. Blots were washed two
times each sequentially with 2x SSC and 1x SSC at 42 C, a
nonstringent condition which, in the case of the ß-actin probe,
detects the 2.1-kb ß-actin mRNA and the muscle-specific 1.6- to
1.7-kb 
-actin mRNA in muscle tissues only (31, 32). Autoradiography
was performed at -70 C for 1–3 days with two intensifying screens. To
assess RNA loading, the multiple tissue blot was also probed with a
ß-actin cDNA probe under identical conditions.
Cell Culture, Transfection, and Luciferase Assays
BeWo cells purchased from American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 (Gibco BRL). The medium was supplemented with 10% FBS (BioWhittaker,
Walkersville, MD), 100 U/ml penicillin (Gibco BRL),
100 g/ml (Gibco BRL), and 2 mM
L-glutamine (Gibco BRL). Cells were maintained
at 37 C in an atmosphere containing 5% CO2 and 100%
humidity. For transfection studies, BeWo cells grown to confluence in
T175 flasks were rinsed with 10 ml PBS and harvested by treatment with
5 ml 1x trypsin (Gibco BRL). Cells were transfected by
electroporation at 960 µFarads and 250 V using a GenePulser
(Bio-Rad Laboratories, Inc.). After 18–20 h of
incubation, cells were harvested for luciferase and protein assays,
which have been described in detail previously (3). Transfection data
expressed as relative light units/µg protein were analyzed by
Student’s t test. Luciferase reporter plasmids used in
transfection were constructed in pA3LUC (16) as described
previously (3, 8, 9). All plasmid DNAs were grown in Escherichia
coli HB101 and purified by double CsCl2 gradient
ultracentrifugation.
Csp.LUC is the control plasmid and contains 500 bp of the
hCS proximal promoter and 5'-flanking DNA, but no enhancer (7);
SVEn CSp.LUC and
CMV CSp.LUC contain SV40 and CMV enhancers,
respectively (7); CSEn2 CSp.LUC and
(CSEn2)2_CSp.LUC contain
one and two copies of hCS2 gene enhancers, respectively (7);
(GT-IIC)12 CSp.LUC
contains 12 tandemly repeated GT-IIC enhansons (7);
CSEn1 CSEn2 CSp.LUC and
CSEn5 CSEn2 CSp.LUC are plasmids
containing consecutive homologous enhancers associated with the
hCS1, hCS2, and hCS5 genes, either
CSEn1 and CSEn2 or CSEn5 and CSEn2, respectively (7). The
(GT-IIC)5 CSp.LUC and
(OCT)5 CSp.LUC genes
were constructed using a ligation-coupled PCR protocol with the
oligonucleotides GT5 and OCT (Table 1) that places each of the tandemly
arrayed sequences in the same orientation as described (17). The
amplified inserts were subsequently cloned into the BglII
site of the hCSp.LUC, upstream of the polyadenylation
repeats that prevent upstream transcription initiation (16).
In Vitro Translation
TEF-1 expression plasmid pXJ-40-TEF1A was kindly
provided by Drs. Pierre Chambon and Irwine Davidson (Institut de Chemie
Biologique, Strasbourg CEDEX, France). The 3.3-kb TEF-5
BamHI/XbaI fragment was resolved by agarose gel
electrophoresis and purified by GeneClean treatment. The fragment was
ligated into BamHI/SpeI- digested pBluescript KS
(Stratagene) to yield pKS TEF-5.
The construct was confirmed by DNA sequencing and used for in
vitro expression of TEF-5.
Standard in vitro transcription-translation reactions were performed with TNT-coupled rabbit reticulocyte lysate (Promega Corp., Madison, WI). Reactions were carried out at 30 C for 2 h with 25 µl TNT, 2 µl reaction buffer, 1 µl T7 polymerase (for TEF-1) or T3 (for TEF-5) RNA polymerase, 1.5 µl ribonuclease (RNase) inhibitor (RNasin, 40 U/µl, Boehringer Mannheim), 1 µl amino acid mixture lacking methionine, 1 µg of pXJ-40-TEF1A, or pKS TEF-5 template, and 4.0 µl [35S]methionine (1000 Ci/mmol, Amersham) in a total volume of 50 µl. To characterize the reaction products, 5 µl from each reaction were mixed with the same volume of 2x SDS loading buffer, heated to 95 C for 2 min, and resolved on a 10% SDS polyacrylamide gel. Prestained protein standards (10 µl, Bio-Rad Laboratories, Inc.) were loaded alongside to estimate molecular size. After electrophoresis, the gels were dried and autoradiography performed at -80 C for 18 h. For gel shift experiments, in vitro-generated TEF-1 and TEF-5 were prepared as described above except that labeled methionine was omitted and the amino acid mixture was supplemented with unlabeled methionine.
Gel Shift Assays
Double-stranded oligonucleotides containing wild-type or mutated
GT-IIC sequences were labeled as described previously (8, 9). In
vitro-generated TEF-1 and TEF-5 (5 µl) were mixed with 28 µl
binding buffer containing 15 µl 20 mM HEPES (pH 7.9), 100
mM KCl, 20% glycerol, 0.2 mM EDTA, 0.5
mM PMSF, 0.5 mM dithiothreitol, 10
mM MgCl2, and 100 µg/ml poly dI·dC. Binding
reactions were initiated by addition of 30,000 cpm DNA probes in 2 µl
10 mM Tris·HCl (pH 7.5), 1 mM EDTA. After
incubation on ice for 30 min, the samples were loaded immediately onto
a 4.0% polyacrylamide gel that had been prerun for 1 h.
Electrophoresis was performed in 0.85x TBE buffer at room temperature
for 2 h at 220 V (constant voltage), with cold water circulation.
After electrophoresis, gels were dried in vacuo and exposed
to Kodak x-ray film (Eastman Kodak Co.) at -80 C with
intensifying screens for 16 h. To characterize their heat
stability, in vitro-generated TEF-1 and TEF-5 were heated in
a PCR cycler at various temperatures for 2 min. Treated samples were
cleared by centrifugation at 13,000 rpm x 5 min, and supernatants
were used in gel shift experiments.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by NIH Grants DK-41206 and DK-51492 (N.L.E.).
Received for publication June 11, 1998. Revision received February 9, 1999. Accepted for publication March 16, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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DR2 episomal vector
system. Methods: A Companion to Methods in Enzymology 4:111–131
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