From the Graduate Institute of Molecular Biology,
College of Medicine, National Taiwan University, Taipei, Taiwan 100, Republic of China, § Institute of Biomedical Sciences,
Academia Sinica, Taipei, Taiwan 115, Republic of China,
Office
for Clinical Research, National Taiwan University Hospital,
** Department of Internal Medicine, School of Medicine, National Taiwan
University, Taipei, Taiwan 100, Republic of China, and the
§§ National Health Research Institute, Taipei,
Taiwan 115, Republic of China
Received for publication, September 8, 2000, and in revised form, September 25, 2000
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ABSTRACT |
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The p53 tumor suppressor protein functions as an
activator and also as a repressor of gene transcription. Currently, the
mechanism of transcriptional repression by p53 remains poorly
understood. To help clarify this mechanism, we carried out studies
designed to identify the minimal repression domain that inhibits p53
transcriptional activities. We found only eight amino acids (339)
of the COOH-terminal domain (termed P53MRD) that possess
activities of repression. The exact location of this minimal domain is
on the E6-binding region, and it lacks the ability of tetramerization.
P53MRD is able to repress the transcription of p53 while not affecting
VP16. The mutants (amino acids M340P and F341D) of native p53 also lost transcriptional repression of the thymidine kinase chloramphenicol acetyltransferase promoter. These results suggest that this
eight-amino acid element is required for the repression of p53.
The tumor suppressor p53 is a nuclear phosphoprotein and
transcriptional factor that plays a crucial role in the regulation of
cell growth, DNA repair, and apoptosis (1, 2). The wild-type p53
protein can be divided into four domains: an acidic transcriptional transactivation domain at the NH2 terminus (3-6); a
sequence-specific DNA-binding domain at the hydrophobic center portion
of the protein (7); a multifunctional COOH-terminal domain; and a
region near the COOH terminus containing a tetramerization domain that
may be required for the stabilization of DNA binding activity (8, 9).
Mutations in the p53 gene have been found in
approximately 50% of human cancers (10, 11). Most tumor-derived p53
mutants harbor single amino acid substitutions (missense mutations)
within the central region, which either prevent normal DNA contacting or alter the conformation of this domain (5). On the other hand, some
mutations in the NH2 terminus (codons 1-99) and in the
COOH terminus (codons 291-393) are non-missense mutations, such as
nonsense point mutations, small insertions or deletions, and splice
mutations (12-16). p53 is capable of both transactivating through
binding to specific DNA-binding sequences (17, 18) and repressing the
transcription of many cellular and viral promoters that do not contain
binding sequences. The promoters repressed are mainly those of which
initiation is dependent on the presence of a TATA box (19-23).
Depending on the physiological circumstances, p53 can mediate either
cell growth or apoptosis.
Three-dimensional nuclear magnetic resonance (7, 24) and x-ray
crystallography were used to examine the COOH terminus of p53, which
contains the tetramerization domain (25). The tetramerization region
was found to contain a To illuminate the mechanism of p53-mediated transcriptional repression,
we used a chimerical system to map the minimal repression domain of the
67 amino acids in the COOH terminus. In this report, we describe the
minimal domain containing eight amino acids (hereafter referred to as
P53MRD) that is the essential repression element. This minimal domain
exactly locates on the E6-binding region, which may possibly affect
transcriptional activity. Mutants of the eight amino acids exhibited
loss of their repression activity. Mutants (amino acids 340 and 341) of
native p53 also lost their transcriptional repression of TKCAT. We
demonstrate that P53MRD is indeed the element required for the
repression of p53.
Plasmid Construction--
To map the minimal repression domain
in p53, several vectors and their derivatives were constructed.
Plasmids pTKCAT, pG5E1bCAT, pSGVP, pSG424, pCEP4p53, pLEC, L6EG5C,
pSG424p53(1-318), and pLexA-VP16 have been previously described (27).
For plasmid pSG424-p53(327-393), the polymerase chain
reaction-amplified fragment from COOH-terminal 327-393 of wild-type
p53 containing KpnI and XbaI sites was cloned into plasmid pSG424/KpnI/XbaI. For plasmid
pSG424-p53(1-125)-p53(327-393), the polymerase chain
reaction-amplified fragment from NH2-terminal 1-125 of
wild-type p53 containing KpnI and EcoRI sites was
cloned into plasmid
pSG424-p53(327-393)/KpnI/EcoRI. Plasmid
pSG424-VP16 was obtained by inserting VP16(413-489) into
pSG424-(BglII/del KpnI-XbaI).
The derivative vectors of pSG424-p53(1-125)-p53(327-393) were
obtained by replacing the () region of
pSG424-p53(1-125)(327-393) with (), (), (),
(339), and (), respectively. Three mutant clones derived
from pSG424p53(1-125)(339-346) mentioned above were constructed by the polymerase chain reaction mutagenesis method. The mutant clones
were pSG424p53(1-125)m(339-346)(M340P),
pSG424p53(1-125)m(339-346)(F341D), and
pSG424p53(1-125)m(339-346)(M340P,F341D), which were confirmed by sequencing.
The detailed procedures for these clones were described in a previous
report (28). Another mutant clone pCEP4p53(M340P,F341D) was obtained by
the same method.
Cell Culture, Transfection, and CAT
Assay--
p53-null human tumor cell lines Saos-2 and H1299
were maintained in Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum. Cells were seeded 12 h before
transfection at 1.2 × 106 cells/10-cm dish. Cells
were transfected by the calcium phosphate method. 5 µg of the CAT
reporter and activator plasmid was used, and 5 µg of a LacZ reporter
plasmid pCH110 (Amersham Pharmacia Biotech) was included to monitor the
transfection efficiency. Typically, the transfection lasted 12 h.
CAT activity was measured 48 h after transfection and quantitated
according to Carey et al. (29).
Protein Analysis--
For Western blotting, equal amounts of
lysates were boiled in a sample buffer (125 mM Tris-HCL, pH
6.8, 100 mM dithiothreitol, 2% SDS, 20% glycerol, 0.005%
bromphenol blue) for 5 min and then subjected to polyacrylamide gel
electrophoresis. After being transferred to an Immobilon membrane
(Millipore Asia Ltd.), p53 and the derivatives were detected
with antibodies (OD-1) against p53 using an ECL system (Amersham
Pharmacia Biotech).
Transactivation of Reporter Constructs pG5E1BCAT by Derivatives of
the GAL4-p53 Fusion Protein in Saos-2 Cells--
Fig.
1A shows that the deletion
mutants (lanes 3-7) compared with the positive control
(lane 2) had low relative CAT activity (RCA), indicating an
increased activity of transcriptional inhibition, but the RCA of
lane 8 (region 344-360) was recovered to 61%. A comparison of lanes 5, 7, and 8 clearly indicates that the COOH-terminal region (339) containing
eight amino acids was essential for the activity of transcriptional
inhibition. There was no apparent difference in the protein expression
level of p53 among all constructed clones (Fig. 1B). In
addition, a comparison of lanes 2, 7, and 8 in
Fig. 1B also indicates that the loss of activity of
transcriptional inhibition was not associated with the expression level
of p53. To validate this eight-amino acid minimal repression
domain (P53MRD), we constructed three point mutants as illustrated in
Fig. 2A, lanes
4-6. The corresponding constructs are listed below the CAT assay.
Lane 2 is the positive control in which no mutation was created. Lanes 3-6 showed a decreasing trend of repression
activity. This result pointed out the fact that this specific P53MRD
was very important for the activity of transcriptional inhibition and
that not any one single mutation, either a M340P or F341D mutant, could lead completely to its loss of transcriptional
repression function. At least 2-amino acid mutations at this region
were required to alter the activity of repression. Fig. 2B
shows there was no difference in the protein expression level of p53
among all constructed clones.
Schematic Representation of the 67 Amino Acids of the COOH Terminus
of GAL4-p53 Derivatives Required for Transcriptional
Repression--
Fig. 3 shows a summary
of the relationship between the activities of transcriptional
repression and the regions on the COOH terminus of p53. A comparison of
the second and third clones shows that the loss in repression activity
was increased from 39-98%, demonstrating that the six-amino acid
(region 339-344) deletion resulted in an additional 60% loss of
repression activity. A comparison of the fifth and eighth clones
was surprising in that the activity was down to 2%, i.e.
more than 95% of repression activity was lost. Of any single mutation
(sixth or seventh clone) compared with the fifth clone, only about 10%
of repression activity was lost.
Transactivation of the Reporter pG5E1BCAT by GAL4-VP16
Derivatives--
To understand whether this P53MRD was specific to
p53, we used VP16 that has an acidic activation domain (1, 30, 31) like
p53 to examine the activity of transcriptional repression. Fig.
4A shows that both the wild
type (lane 3) and mutants (lanes 4-6) of this
eight-amino acid element did not exhibit any repression activity. This
indicated that the repression activity of the P53MRD in transcription
was specific for p53. As shown in Fig. 4B, the difference in
the protein expression levels of VP16 among all constructed clones was
normalized to eliminate the effect of the loading amount. The
RCA value was recalculated as 1, 100, 153, 184, 125, and 118, respectively. The normalized RCA value reveals no significant effect in
the repression activity.
Effect of the Eight-Amino Acid Minimal Repression Domain on the DNA
Binding Activity of the Gal4 Chimera--
To further verify that the
repressive function of the identified minimal domain was directly on
the transcriptional activation domain and was not affected by
the influence of the binding action of GAL4(1-147), a molecular
interference assay was employed (27, 32). The construct
L6EG5C (Fig. 5)
contains five repeats of the GAL4-binding element (5 × GAL4)
placed behind the EIbTATA box (2). The GAL4 derivative protein binds to
this element and blocks the transcriptional initiation complex.
Therefore, the CAT activity is reduced. pSG424 (previously referred to
as GAL4(1-147) and its derivatives pSG424p53(1-318),
pSG424p53(1-125), and pSG424p53(1-125)m(339-346)(M340P,F341D)
blocked the CAT activity with different repressive levels (Fig. 5,
upper left). A comparison of lanes 3-6 with
lane 2 shows that there was a 5-15-fold difference in
transcriptional repression using various RCA ratios. In contrast, the
construct L6EC containing no GAL4-binding element did not influence transcriptional activity when co-transfected with the activator and blockers (Fig. 5, upper right). A comparison
of lanes 9-12 with lane 8 showed that
the RCA of these four constructs had little or almost no
difference.
Effect of Wild-type p53 and p53(M340P,F341D) Double Mutant on TKCAT
Promoter Activity--
To understand whether this region in native p53
also regulates the activity of transcriptional repression, we assayed
its repression activity using the reporter pTKCAT (33). The constructs used in the assay of TKCAT promoter activity are listed in Fig. 6A. The control of protein
levels is illustrated in Fig. 6B. The double mutation, which
is the same as described in Fig. 2A, was performed on
wild-type p53. Fig. 6A (lane 1) shows that there was no p53 and thus, no repression activity. In lane 2,
wild-type p53 existed, and repression was present. In lane
3, the double mutation of the eight-amino acid region of p53
decreased the repression activity by 8-fold.
Assay of the Ability of Different Mutants Mediation of
Transcriptional Repression of pTKCAT in H1299 Cells--
As shown in
Fig. 7, the mutants on the hot spot
(V143A, V173L, N247I, R248W, and R273H) of the DNA-binding domain
retained repression activity (compare the lanes with
decreasing RCA with lane 1). The relative ratio of
repression activity was greater than 5-fold among all mutants compared
with the control pCEP4 and showed a significant difference. A
comparison among lanes 8 and 9 and lanes
2 and 10 on the transcriptional activity domain also
retained significant repression activity, indicating that the
NH2-terminal transcriptional domain was not necessary for the repression of TKCAT in p53.
A previous report described the region of COOH-terminal amino
acids () on p53 required for repression activity (2). Subler et al. (19) also showed that the COOH-terminal 67 amino
acids were crucial for transcriptional inhibition by deleting this
region for transcriptional activity. However, the exact location and mechanism of p53-mediated repression remained unclear.
In this study, we investigated the activity of transcriptional
inhibition by minimizing the transcriptional domain. A chimeric assay method was employed to detect the transcriptional activity and
was performed by fusing arbitrary fragments of the p53 COOH-terminal to
an activator Gal4-p53N(1-125). We have found that the COOH-terminal region (339) containing eight amino acids was essential for the activity of transcriptional inhibition, and the loss of activity of
transcriptional inhibition was not associated with the expression level
of p53. Among these eight amino acids, it was reasonable to suspect
that the loss of six amino acids (region 339-344) would create an
unstable binding site for certain factors. Our findings clearly
demonstrated that the substitution of at least two amino acids at this
region was required to alter the activity of repression. Amino acids
340 and 341, which were hydrophobic, were the most crucial in
determining the activity of inhibition. Further studies are needed to
clarify the exact mechanism of how these two amino acid substitutions
affect p53 transcriptional activity.
Overall, our results demonstrated that only eight amino acids are
required to determine the transcriptional activity of p53, and the
minimal repression domain only influenced the activation domain. In
addition, the chimera protein still maintained its activity for DNA
binding as illustrated by the unchanged binding activity of GAL4. The
use of powerful chimera protein assays also validated that amino acids
340 and 341 play a key role in transcriptional inhibition.
To clarify whether P53MRD in native p53 could regulate the activity of
transcriptional repression, we assayed its repression activity using
the reporter pTKCAT. We chose the thymidine kinase promoter as a
reporter because this promoter, unlike the E1bTATA promoter, possesses
a detectable basal transcriptional activity that is necessary for the
assay of repression. Our results confirmed that the region in native
p53 still functioned in regulating transcriptional repression.
The eight-amino acid region identified is indeed the minimal and
specific domain for the transcriptional repression of p53. The
transcriptional activity domain in both VP16 and p53 has the same
effect on transcriptional regulation, but only the P53MRD could repress
the transcription of p53 and not affect that of VP16. This fact
indicated that the transcriptional inhibition or transcriptional
repression was p53-dependent and p53-specific.
Pellegata et al. (34) showed that mutant p53 There are two possible mechanisms for the P53MRD repression. First, the
P53MRD may directly hinder the transcriptional activity domain of the
NH2 terminus and interfere with the transcriptional complex
to achieve repression. A previous report (26) demonstrated this
mechanism whereby the COOH-terminal repression domain (amino acids
327-393) achieved repression activity by mediating the binding of
TATA-binding protein. Second, the transcriptional repression may be
accomplished by a putative factor, such as the effect of E6 on p53. The
E6 protein of human papillomaviruses could target p53 in two
ways. The first, which may be a more immediate response, is the
abrogation of p53 transcription activity by binding to the cofactor
CREB-binding protein/p300 (37, 38). The second is the removal of
the p53 protein through E6-associated protein-dependent degradation (39). In the present study, we found that the P53MRD was located on the E6-binding region of p53. Therefore, the P53MRD might mediate certain cellular factors like the E6 protein to achieve repression.
On the other hand, recent work by several groups has established an
evolutionary conserved role for histone deacetylases in the mechanism
of repression by transcription factors, such as Mad, Max, and the
nuclear hormone receptors (40-42). Transcriptional repression by
wild-type p53 also employs histone deacetylases mediated by interaction
with mSin3a, and the domain necessary for the p53-mSin3a interaction to
two regions of the p53 protein was mapped from 40-160 and 320-360
(43). We suspect that the P53MRD (amino acids 339-346) might mediate
the same pathway to carry out p53-specific repression.
A comparison among p53/p53C The fact that the COOH-terminal domain of p53 has transcriptional
repression function has been known for some time. p53 might activate
the transcription of death genes or repress the transcription of
survival genes to promote apoptosis. Although several studies have
indicated a role for transcriptional repression in
p53-dependent apoptosis (2), the molecular basis of this
activity remains poorly understood. This P53MRD may serve to regulate
repression and even perhaps regulate apoptosis. More studies are needed
to further investigate these possibilities and are currently in
progress in our laboratory.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet-turn-
-helix motif (). The
COOH-terminal segment of p53 mediates repression when bound to DNA as a
GAL4-p53 fusion protein (26). A COOH-terminal deletion of up to amino
acid 327 (del 327-393) eliminates the repression of
cytomegalovirus-CAT1
(17). However, very little is known about the transcriptional repression ability of p53 deletion mutants. This repression activity may potentially affect the biological function of p53 and is important for further analysis. In the present study, we further clarified the
importance of the COOH-terminal domain of human p53 in relation to its
ability to repress transcriptional activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Transactivation of reporter constructs
pG5E1BCAT by derivatives of the GAL4-p53 fusion protein in Saos-2
cells. A, plasmid pCH110 (Amersham Pharmacia Biotech),
which contains a functional lacZ gene, was used as
an internal control to monitor transfection efficiency. An
autoradiogram of a typical experiment is shown. A list of the construct
of the activators are shown below the autoradiogram. The
activator and RCA are indicated above each track of the
autoradiogram. B, the expression of GAL4-p53 derivatives in
transfected Saos-2 cells. Approximately 50 µg of proteins of Saos-2
cells transiently transfected with the vector (lane 1) or
GAL4-p53 derivatives (lanes 2-8) were fractionated on a
10% SDS-polyacrylamide gel electrophoresis gel. GAL4-p53 derivatives
were detected by immunoblotting as described under "Experimental
Procedures." The GAL4-p53 derivatives used as activators in
A are indicated above each track of the
immunoblot. All experiments were performed in triplicate.
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Fig. 2.
Transactivation of reporter constructs
pG5E1BCAT by derivatives of the GAL4-p53 fusion protein in Saos-2
cells. A, experiments were performed as described in
Fig. 1A except that the activators were either single or
double point mutations of the eight-amino acid element of p53
as shown below the autoradiogram. B, the expression of
GAL4-p53 derivatives in transfected Saos-2 cells. Experiments were
performed as described in Fig. 1B except that single
or double point mutations of GAL4-p53 were used.
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Fig. 3.
Schematic representation of the 67 amino
acids of the COOH terminus of GAL4-p53 derivatives required for
transcriptional repression. Deletion peptides of the amino acids
327-393 were generated from the COOH-terminal direction (clones
3-8) and from both directions (clone 1). The
peptide deletion of clones 6-8 was the same as in clone
5, but additional mutations were created: clone 6 (M340P), clone 7 (F341D), and clone 8 (M340P,F341D). Eight clones were tested for transcriptional repression
after transfection in the chimera assay. The percentage of
transcriptional repression is derived from RCA in Figs. 1 and 2. The
transcriptional repression for each clone is shown at the
upper right side.
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Fig. 4.
Transactivation of the reporter
pG5E1BCAT by GAL4-VP16 derivatives. A, procedures were
performed as described in Fig. 1A except that the activators
are GAL4VP16-p53 derivatives. A list of their structures are shown
below the autoradiogram. The RCA value was obtained by
normalization with -galactosidase activity. Both the wild type
(lane 3) and mutants (lanes 4-6) of this
eight-amino acid element do not reveal any repression activity.
B, approximately 50 µg of protein from transfected Saos-2
cells were fractionated on a 12.5% SDS-polyacrylamide electrophoresis
gel. GAL4-VP derivatives that were expressed were detected with an
anti-GAL4(1-147) antibody. The RCA values were recalculated by
VP16-derived protein of all constructed clones. The values were 1, 100, 153, 184, 125, and 118, respectively. The RCA values normalized by the
expressed protein levels still showed no significant effect on the
repression activity.
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Fig. 5.
Effect of the eight-amino acid minimal
repression domain on the DNA binding activity of the Gal4 chimera.
1 µg of the reporter DNA, 1 µg of the activator DNA, and 13 µg of
the specified blocker DNA were used for calcium phosphate-mediated DNA
transfection. A Gal4 derivative GAL4(1-147), which contains residues
1-147 of the yeast GAL4 and possesses DNA binding activity, and
pSG424p53(1-318) were used as positive controls. Cells were harvested
48 h after transfection. The absence ( ) or presence (+) of the
activator is indicated above each track of the autoradiogram
as are the blockers and RCA. The reporter was
pL6EG5C (lanes 1-6) or
pL6EC (lanes 7-12). A list of the structure of
the activator, reporters, and blockers are shown below the
autoradiogram.
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Fig. 6.
Effect of wild-type p53 and p53(M340P,F341D)
double mutant on TKCAT promoter activity. A, pTKCAT (5 µg)
was co-transfected into H1299 cells with 5 µg of either the pCEP4
expression vector, the wild-type p53 expression plasmid, or the p53
double mutant and the carrier DNA for a total of 20 µg of
DNA/transfection. A list of the structure of the activators are shown
below the autoradiogram. Cells were harvested and CAT was
assayed after 48 h. The figure represents an autoradiogram of the
CAT assay. B, approximately 50 µg of protein from
transfected H1299 cells were fractionated on a 12.5%
SDS-polyacrylamide electrophoresis gel. Wild-type p53 and mutant
p53(p53M340P,F341D) were detected with an anti-p53(DO-1)
antibody.
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Fig. 7.
Assay of the ability of different mutant
mediations of transcriptional repression of pTKCAT in H1299 cells.
H1299 cells were transfected with 5 µg of pTKCAT and 5 µg of either
the pCEP4 expression vector, the wild-type p53 expression plasmid, or
the mutant p53 and the carrier DNA for a total of 20 µg of
DNA/transfection. Cells were harvested 48 h after transfection.
CAT assays were performed as described under "Experimental
Procedures." The figure represents an autoradiogram of the CAT
assay.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
363 was the
only truncated p53 protein that retained the ability to repress transcription from the TATA element. The monomeric forms of p53 (p53
333 and p53
303) did not display any repression of
transcription. This finding was consistent with our results that the
eight amino acids of the P53MRD were essential for repression. On the
other hand, minimal tetramerization was located on amino acids 325-355 (24, 35). Thus, the P53MRD containing amino acids 339-346 (part of
-helix structure) do not constitute the tetramerization. We
suspect that repression is not associated with tetramerization but is
similar to the binding of E6 on p53, which does not require oligomerization (36).
30 and mutants (p53Q22S23 and
p53N
100C
30) on the transcriptional activity domain retained
significant repression activity, indicating that the
NH2-terminal transcriptional domain was not necessary for
the repression of TKCAT in p53. This observation ruled out any
squelching effect (44). Only the double mutant on hydrophobic amino
acids 340 and 341 lost its repression activity. This result was the
same as that obtained using the GAL4-fusion chimeric protein system as
described above (mutated on amino acids 340 and 341). We concluded that
the eight-amino acid region identified has a crucial role in
transcriptional repression.
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ACKNOWLEDGEMENTS |
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We thank Drs. Young-Sun Lin and Jeou-Yuan Chen for providing some plasmid constructs used in this work and Douglas Platt for providing helpful comments and English editing on this manuscript prior to submission.
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FOOTNOTES |
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* This work was supported by grants from Academia Sinica.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.
¶ Both authors contributed equally to this work.
To whom correspondence should be addressed: Dept. of Internal
Medicine, National Taiwan University Hospital, 7, Chung-Shan South
Road, Taipei 100, Taiwan. Tel.: 886-2-23562116; Fax: 886-2-23582867 or
886-2-23934176; E-mail: pcyang@ha.mc.ntu.edu.tw.
Published, JBC Papers in Press, September 27, 2000, DOI 10.1074/jbc.M008231200
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ABBREVIATIONS |
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The abbreviations used are: CAT, chloramphenicol acetyltransferase; TKCAT, thymidine kinase chloramphenicol acetyltransferase; RCA, relative CAT activity.
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