From the Institute for Molecular Virology, Saint
Louis University Health Sciences Center, St. Louis, Missouri 63110 and ¶ Apoptosis Technology, Inc.,
Cambridge, Massachusetts 02139
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The BCL-2 proto-oncogene contains unusually long untranslated 5' and 3' sequences. Deletion of the sequences flanking the BCL-2 open reading frame dramatically increases the level of protein expression. Transient high level BCL-2 protein expression mediated by plasmid transfection or by infection with recombinant adenovirus results in potent apoptosis of several cell lines. Detailed mutational (deletion and add-back) analysis reveals that both 5'- and 3'-flanking sequences contribute to the negative modulation of protein expression from the BCL-2 open reading frame. It appears that these sequences exert the negative regulatory effect in an orientation-dependent manner. Analysis of BCL-2 RNA levels indicate that elevated levels of mRNA may be the primary cause of elevated levels of protein expression. Apoptosis induced by adenovirus vectors expressing elevated levels of BCL-2 can be readily inhibited by the caspase inhibitor z-VAD-fmk, suggesting that high levels of BCL-2 expression induce apoptosis via the caspase cascade. Mutational analysis of BCL-2 indicates that its pro-apoptotic activity is separable from its anti-apoptosis activity. Our results raise the possibility that oncogenic conversion of BCL-2 may require somatic mutations in the pro-apoptotic activity, in addition to other activating mutations that result in enhanced expression. Consistent with this hypothesis, a somatic mutation of BCL-2 observed in multiple human tumors results in reduced apoptosis activity.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is well established that the BCL-2 proto-oncogene suppresses apoptosis induced by a multitude of stimuli in a variety of cell types (1, 2). In a seminal study by Vaux et al. (3), BCL-2 was shown to extend the survival of growth factor-dependent hematopoietic progenitor cells after factor withdrawal. In subsequent studies by Hockenbery et al. (4), it was shown that BCL-2 promotes survival of such factor-dependent cells by suppressing apoptosis. Most studies on the anti-apoptosis activity of BCL-2 have been based on stable cell lines (selected after transfection of BCL-2 expression vectors) that ectopically express elevated levels of BCL-2. In addition to the anti-apoptosis activity, recent studies have revealed that BCL-2 exhibits other novel activities. For example, BCL-2 has been shown to inhibit cell proliferation (5-9), which is a function separable from the anti-apoptosis activity (6, 9). BCL-2 has also been shown to promote cell proliferation in the central nervous system (10).
The human BCL-2 gene is characterized by unusually long 5'- and 3'-untranslated regions (11-13), including the entire first exon (14). The functions of the unusually long untranslated regions are not known. Interestingly, the 5'- untranslated region contains a sequence element that negatively regulates translation of BCL-2 (15). Here, we show that deletion of the 5'- and 3'-flanking sequences results in a large increase in BCL-2 protein expression, which results in cell death via the CED-3/ICE family caspase cascade.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmids--
pcDNA3Bcl2 was constructed by cloning an
EcoRI fragment from the plasmid pSFFV-BCL-2 (no. 58) (14)
into pcDNA3 cut with EcoRI. pcDNA3-Bcl2UTR (no.
58) was prepared by cloning a
PCR1-generated DNA fragment
(digested with HindIII and SalI) and cloned between the HindIII and XhoI sites of pcDNA3.
The PCR product was generated using oligonucleotides,
5'-GATCAAGCTTCCATGGCGCACGCTGGGAGA-3' (primer 1) and
5'-ACGCGTCGACTCACTTGTGGCCCAGATAG-3' (primer 2) as primers and the
BCL-2 cDNA clone no. 58 (14) as the template. pcDNA3-Bcl2
UTR (B4) was constructed similarly using pB4 (11) as
the template. pcDNA3-mBcl2 constructed by cloning the
EcoRI/XbaI fragment containing the mouse BCL-2
ORF from pUC19 (16) into pcDNA3. pcDNA3-Bcl2
UTR + 5'-UTR was
prepared by cloning a DNA fragment generated using the primer
5'-GTAGGGGCTGGGGCGAGAGGTGCCGTTGGC-3' (primer 3) and primer 2. The PCR
product was digested with SalI and cloned into the
EcoRV and XhoI sites of pcDNA3.
pcDNA3-Bcl2
UTR + 3'-UTR was made by replacing a C-terminal
fragment (BamHI/XbaI) of pcDNA3-Bcl2
UTR
with a C-terminal fragment (BamHI/XbaI) from pcDNA3-Bcl2. pcDNA3-Bcl2
UTR + 3'- + 5'-UTR was constructed
by replacing a C-terminal fragment (SacII/XbaI)
of pcDNA3-Bcl2
UTR + 5'-UTR with that of pcDNA3-Bcl2
UTR + 3'-UTR. pcDNA3-Bcl2
5'-UTR was generated by digestion of
pcDNA3-Bcl2 with HindIII and EcoNI (partial)
followed by blunt-end ligation. pcDNA3-Bcl2
3'-UTR was made
by cloning the fragment generated by PstI (blunted) and
EcoRI from pSFFV-Bcl2 into pcDNA3 between
EcoRI and EcoRV sites. pcDNA3-Bcl2
5'-
3'-UTR was made by deletion of a KpnI and
EcoNI (partial) fragment from pcDNA3-Bcl2
3'-UTR. pcDNA3-Bcl2 + 3'-UTR-I was constructed by reversing the
orientation of the appropriate PstI-generated fragment of
pcDNA3-Bcl2 (no. 58). pcDNA3-Bcl2 + 3'-UTR-H by substituting a
1-kb fragment of pBR322 (NheI and blunted BsmI)
for a 1-kb fragment of pcDNA3-Bcl2 (no. 58) between PstI
(partial, blunt ended) and XbaI sites. All PCR reactions were carried out using Vent DNA polymerase (New England Biolabs) in 5%
Me2SO. The clones derived from PCR amplification were
sequenced by dideoxy or by automated sequencing.
Adenovirus Expression Vectors--
The BCL-2
expression cassettes, from plasmids pcDNA3-Bcl2 and
pcDNA3-Bcl2UTR, and the lacZ expression cassette,
from plasmid pCMV-
-galactosidase, were cloned into the adenovirus
transfer vector pAdLTR (17) between unique XhoI and
BglII sites. To construct the recombinant adenoviruses, 5 µg of each of the transfer plasmid was cotransfected with 5 µg of
the adenovirus genomic plasmid pBHGE3 (18) into 293 cells by the
calcium phosphate method. Seven days after transfection, plaques were
picked and screened for the presence of the transgene by restriction
analysis. Positives plaques were further purified through a second
plaque assay, amplified and titrated in 293 cells. In these
recombinants, the transgenes are expressed under the control of CMV-IE
promoter and interleukin-2 poly(A) site.
Cell Death Assays--
Transient cell death assays were carried
out using the human 293 cell line. Cells were transfected at 60%
confluency with 1.6 µg of various expression plasmids and 0.4 µg of
the reporter plasmid pCMV--galactosidase by the calcium phosphate
method in 12-well plates. Eight hours after transfection cells were
fixed and stained with X-gal (19). The number of blue cells was
counted, and the number of round (apoptotic) cells compared with the
total blue cell count was determined.
Analysis of DNA Fragmentation-- 293 cells (1 × 106 cells/60-mm dish) were infected at 100 plaque-forming units/cell. Twenty-four hours later, adherent and floating cells were collected and lysed, and small molecular weight DNA was prepared by Hirt extraction (21), treated with RNase, and analyzed on a 1% agarose gel.
Western Blot Analysis-- 3.4 × 104 MCF-7 cells were plated in 24-well plates in DMEM containing 10% fetal calf serum. Twenty-four hours after plating, cells were infected at 300 plaque-forming units/cell in 250 µl of serum-free DMEM for 4 h at 37 °C. Virus-containing media were aspirated and replaced with fresh DMEM containing 2.5% fetal calf serum. At various times post-infection, adherent cells from triplicate wells were harvested and pooled, and lysed in SDS-polyacrylamide gel electrophoresis sample buffer, and the equivalent of 7.5 × 104 adherent cells were loaded per lane of a 16% minigel (Novex). Following SDS-polyacrylamide gel electrophoresis, the proteins were electrotransferred onto a 0.2-µm nitrocellulose membrane and probed with a mouse anti-human BCL-2 primary antibody (Dako, MO887) at 1:300 dilution, followed by a sheep anti-mouse-horseradish peroxidase secondary antibody (Amersham NA931 used at 1:5000 dilution) and chemiluminescent detection (Amersham Corp.). For the analysis of transiently transfected cells, 293 cells were transfected at 60% confluency in 35-mm dishes with 4 µg of total DNA. Nine and a half hours later, cells were lysed in 100 µl of electrophoresis sample buffer, and 10-µl samples were loaded onto a 15% gel SDS-polyacrylamide gel electrophoresis, followed by transfer to a nitrocellulose membrane. A rabbit anti-glutathione S-transferase-BCL-2 polyclonal serum (1:1000) and donkey anti-rabbit-horseradish peroxidase (Amersham NA934 at 1:3000) were used to detect BCL-2.
Northern Blot Analysis--
Total cytoplasmic RNA was isolated
using RNAzol B solution (TEL-TEST Inc.) and purified by CsCl density
gradient centrifugation. 12 µg of RNA were loaded in each lane,
subjected to electrophoresis in a formaldehyde-agarose gel, transferred
to a Duralon nylon membrane (Stratagene), and hybridized with the
BCL-2 probe. The probe was prepared by random primed
extension (DuPont) and the HindIII/XbaI fragment
from pcDNA3-Bcl2UTR as the template.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transient Expression of BCL-2--
The human BCL-2
cDNA contains 1.4 kb of 5'- and 3.9 kb of 3'-untranslated sequences
(Fig. 1A). A widely used
cDNA clone (clone no. 58) (14) contains 58 bp of 5'- and 900 bp of
3'-untranslated sequences. We constructed a derivative of this cDNA
clone by PCR that lacks the untranslated sequences
(pcDNA3-Bcl2UTR). We observed that, after transfection of
pcDNA3-Bcl2
UTR, the number of G418-resistant colonies induced
was substantially lower than that induced either by the pcDNA3
empty vector or the vector expressing the parental cDNA
(pcDNA3-Bcl2). This finding prompted us to examine whether the
clone lacking the untranslated regions (pcDNA3-Bcl2
UTR) caused any cell death in transfected cells. For this purpose, 293 cells were
transfected with a reporter plasmid expressing the Escherichia coli lacZ gene along with the pDNA3 vector or pcDNA3-Bcl2 or
pcDNA3-Bcl2
UTR. Cells expressing
-galactosidase were detected
by X-gal staining, and the percent of apoptotic cells was calculated
from the number of round and fragmenting blue cells out of the total
number of blue cells (19). Transfection of the pcDNA3 vector or the
vector expressing the parental cDNA (pcDNA3-Bcl2) did not
induce significant cell death (Fig. 1B). In contrast, about
80% of the cells transfected with pcDNA3-Bcl2
UTR were round.
The observed cell death activity of BCL-2 was confirmed with two
different independently derived clones that lack the flanking sequences
(not shown). None of the pcDNA3-Bcl2
UTR clones contained any
mutations compared with parental cDNA. Similar results were also
obtained with a different human BCL-2 cDNA (11) and a
mouse BCL-2 cDNA (22).
|
Adenovirus Vector-mediated Expression of BCL-2--
To further
substantiate the above results, we constructed recombinant adenoviruses
that express the two versions of the BCL-2 cDNA. The
effects of the recombinant adenoviruses on cell viability of three
different human cell lines were determined by the MTT assay. Infection
of cell lines MRC-5, MCF-7, and DU145 with an adenovirus vector
expressing the E. coli lacZ gene (AdE1-lacZ) did not result in significant cell death compared with uninfected control cells (Fig. 2). Similarly,
infection with the vector that expresses the parental cDNA clone
(Ad
E1-Bcl2) also did not result in any significant cell death. In
contrast, infection with the adenovirus vector expressing the
BCL-2 cDNA lacking the flanking sequences
(Ad
E1-Bcl2
UTR) resulted in severe cell death in MRC-5 (Fig.
2A). Similarly, infection of MCF-7 and DU145 cells with Ad
E1-Bcl2
UTR also resulted in significant cell death (Fig. 2, B and C), although the effect on DU145 was less
pronounced (Fig. 2C). These results indicate that the
BCL-2 cDNA lacking the 5'- and 3'-flanking sequences
induce cell death during transient expression. It should be noted that
the DU145 cell line is resistant to apoptosis induced by Fas and tumor
necrosis factor-
(23).
|
BCL-2 mRNA and Protein Expression--
To determine the
possible mechanism by which the flanking sequences dictate the opposing
activities of BCL-2, we determined the levels of expression of
BCL-2 mRNA and protein. These levels were determined
from MCF-7 cells infected with AdE1-Bcl2
UTR or Ad
E1-Bcl2.
Total cytoplasmic RNA was extracted from infected cells (adherent and
floating) and analyzed by Northern blot analysis. In cells infected
with Ad
E1-Bcl2, an mRNA band of 2.6 kb (that corresponds to the
BCL-2 ORF and flanking vector sequences) was observed (Fig.
3A). In cells infected with
Ad
E1-Bcl2
UTR, significantly higher levels of a 1.2-kb mRNA
were observed. These results suggest that deletion of the flanking
sequences of the BCL-2 cDNA significantly increases the
level of BCL-2 mRNA expression.
|
Mutational Analysis of the Flanking Sequences--
To determine
the role of the 5'- and 3'-flanking sequences separately, we
constructed a series of add-back mutants by adding either the 5'- or
the 3'-flanking sequences (by PCR) to pcDNA3-Bcl2UTR (Fig.
4A). Similarly, a series of
5'- or 3'-deletion mutants of pcDNA3-Bcl2 were also constucted by
exploiting conveniently located restriction sites (Fig. 4B).
The cell death activities of the various mutants were then determined
by transient transfection (Fig. 4D) as described in the
legend to Fig. 1B. Addition of the 5'-flanking sequences to
pcDNA3-Bcl2
UTR (+5' in Fig. 4D) reduced the
cell death activity of pcDNA3-Bcl2
UTR. Similarly, addition of
the 3'-flanking sequences also reduced cell death activity (+3' in Fig. 4D). Addition of both 5'- and
3'-flanking sequences significantly reduced the cell death activity,
close to the level of pcDNA3-Bcl2. Comparable results were obtained
by analysis of deletion mutants (Fig. 4B). Deletion of the
5'-untranslated region (
7; EcoNI) increased the apoptosis
activity of pcDNA3-Bcl2. Similarly, deletion of the 3'-flanking
sequences (+752; PstI) also increased the apoptotic
activity. Deletion of both 5'- and 3'-flanking sequences increased the
apoptotic activity in an additive manner. These results provide
evidence that both 5'- and 3'-flanking sequences have a combinatorial
effect in modulating the cell death activity of BCL-2.
|
Effect of BCL-2 Mutations-- To determine if the apoptotic activity of BCL-2 is linked to its anti-apoptotic activity, we examined the effect of two previously characterized mutants, G145E (BH1) and W188A (BH2) which were found to be severely defective in the anti-apoptotic activity (24). The BH2 mutant (W188A) did not significantly affect the apoptotic activity (Fig. 5). In contrast, the BH1 mutant (G145E) exhibited significantly reduced apoptotic activity (Fig. 5). These results suggest that the pro- and anti-apoptotic activities of BCL-2 are separable and that a protein region located around the BH1 domain may be important for the apoptotic activity.
|
Effect of Caspase Inhibitor--
Since CED-3/ICE family caspases
are activated during apoptosis induced by several different stimuli
(reviewed in Refs. 25 and 26), we determined if the cell death induced
by higher levels of BCL-2 expression is also dependent on caspases
(Fig. 6, A and B).
293 cells infected with AdE1-BCL-2
UTR induced extensive internucleosomal DNA fragmentation (Fig. 6A, left
panel). In contrast, there was no significant DNA fragmentation in
mock-infected cells or in cells infected either with
Ad
E1-lacZ or Ad
E1-BCL-2. We then examined if the
caspase inhibitor z-VAD-fmk can inhibit DNA fragmentation induced by
Ad
E1-BCL-2
UTR. Addition of 25 or 50 µM z-VAD-fmk
significantly inhibited the extent of DNA fragmentation induced by
Ad
E1-BCL-2
UTR. Similarly, addition of z-VAD-fmk also inhibited
the extent of cell death (determined by vital dye exclusion) induced by
Ad
E1-BCL-2
UTR (Fig. 6B). These results suggest that a
distal step in the cell death pathway activated by infection with
Ad
E1-Bcl2
UTR may involve caspases.
|
Effect of Oncogenic Mutant--
Expression of BCL-2 is activated
in a number of human tumors. Our results raise the possibility that
oncogenic conversion of BCL-2 may require somatic mutations in the
pro-apoptotic activity, in addition to other activating mutations that
result in enhanced expression. To test this hypothesis, we examined the
apoptotic activity of a BCL-2 mutant, P59S, located within the
nonconserved region of BCL-2. This mutant was chosen since mutations
involving the Pro59 residue (Pro59 Ser or
Pro59
Leu) have been detected in three different human
non-Hodgkin's lymphomas (27, 28). The P59S mutant has previously been
shown to efficiently protect against apoptosis
(27).2 Transient transfection
of the plasmid containing this mutant (pcDNA3-Bcl2
UTR/P59S; lacking
the flanking sequences) or pcDNA3 Bcl2
UTR expressed elevated
levels of BCL-2 compared with cells transfected with pcDNA3-Bcl2
(wild type) (Fig. 7A), as
expected. Transfection of pcDNA3-Bcl2
UTR/P59S consistently
exhibited less pronounced cell death compared with pcDNA3-Bcl2
UTR
(Fig. 7B). These results suggest that at least one of the
somatic mutations observed in human tumors exhibit a reduced apoptosis
activity.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this communication, we have identified a surprising cell death
activity of BCL-2 manifested during high levels of transient protein
expression. We believe that a similar activity has not thus far been
identified since most assays used to determine the activity of BCL-2
have employed cell lines that have been selected to grow in tissue
culture. It should be noted that cell lines established by transfection
of pcDNA3-Bcl2UTR also protect against cell death similar to
cell lines established by transfection of pcDNA3-Bcl2 (not shown),
suggesting that cells transfected with pcDNA3-Bcl2
UTR get
selected for survival during the selection procedure. We believe that
our results may, at least partially, explain the effect of BCL-2
reported by Pietenpol et al. (5). These investigators have
observed that transfection of a BCL-2 expression vector in
certain tumor cell lines strongly inhibited G418-resistant colony
formation. Employing one of the tumor cell lines, SW480, used by these
investigators, we have observed that transfection of
pcDNA3-Bcl2
UTR strongly reduced the G418-resistant colony number
compared with cells transfected with pcDNA3 or pcDNA3-Bcl2 (not
shown).
Our study does not address the physiological relevance of the cell death activity of BCL-2. However, the observation that CED-9, the Caenorhabditis elegans anti-cell death protein also contains a minor cell death activity (29) raises the possibility that the cell death activity of BCL-2 may also be physiologically relevant under certain yet unidentified settings. It could be speculated that the unusually long flanking sequences of the BCL-2 gene might have evolved to regulate the opposing activities of BCL-2 protein. Under certain physiological or pathological conditions, shorter transcripts may be generated, and such transcripts may be preferentially translated leading to cell death rather than survival. There is some basis for such a speculation, because the BCL-2 gene has also been shown to be transcribed from a minor promoter (30), resulting in a transcript that would have a 5' end similar to the 5' ends of the mutants described here. The presence of a separate cis-acting translational inhibitory sequence in the 5'-untranslated sequences (15) also lends support for negative regulation of BCL-2 protein expression by flanking sequences. The modulation of BCL-2 expression by the flanking sequences would be similar to the role of the 3'- flanking sequences in regulation of p53 protein expression in response to DNA damage (31).
In the present study, we have observed that deletion of the UTRs enhanced BCL-2 mRNA accumulation and protein expression. It is possible that transcriptional and post-transcriptional regulation such as mRNA stability may contribute to increased accumulation of BCL-2 mRNA. Although we have observed both elevated levels of mRNA and protein expression, the role of flanking sequences in negative modulation of translational efficiency cannot be ruled out. Interestingly, we have observed that both 5'- and 3'-flanking sequences of the BCL-2 mRNA could potentially form extensive secondary structures (not shown). It is possible that such secondary structures may also suppress translational efficiency of the BCL-2 mRNA.
The molecular basis by which elevated levels of BCL-2 induce cell death
is not known. It is possible that, during high level expression of
BCL-2, N-terminal-deleted versions are expressed from internal
translational initiation sites. Under certain conditions, we have
observed a 24-kDa polypeptide (see Fig. 3B), in addition to
the 26-kDa polypeptide. We believe that the 24-kDa polypeptide is
translated from an internal initiation codon corresponding to
Met16 since a BCL-2 mutant UTR/M16L does not produce the
24-kDa band (not shown). The mutant M16L (Met16
Leu)
still induces cell death, suggesting that the cell death activity
observed in our studies may be primarily contributed by the 26-kDa
BCL-2. However, we cannot fully rule out the role of other truncated
versions generated during high levels of expression being
pro-apoptotic. A hypothesis would be that higher levels of BCL-2 might
result in formation of dimers and oligomers that may be pro-apoptotic.
Under conditions of relatively lower levels of protein expression,
BCL-2 may function as a monomer with anti-apoptosis activity. In this
context, it has been shown that BCL-xL folds as a monomer
in solution (32). Recent studies indicate that both BCL-xL
(32) and BCL-2 form membrane pores (33, 34), and BCL-2 has been shown
to block leakage of cytochrome c into the cytosol (35, 36).
BCL-2 has been shown to form channels with larger conductances,
suggesting the formation of BCL-2 dimers and oligomers in the membrane
(33). It is possible that, above a threshold level, BCL-2 may cause
constitutive loss of cytochrome c or other apoptotic factors
from the mitochondria by forming megapores. BCL-2 retains the BH3
domain essential for the death activity of the pro-apoptotic proteins
of the BCL-2 family (37-39). Our mutational analysis suggests that
pro-apoptotic and anti-apoptotic activities of BCL-2 are separable. It
remains to be determined if the cell death activity of BCL-2 is
mechanistically similar to the known pro-apoptotic proteins. Recent
studies from several laboratories suggest that CED-9 promotes cell
survival by simultaneously sequestering the cell death proteins CED-4
and CED-3 (reviewed in Refs. 40-42). If BCL-2 also functions by a
similar mechanism, it is possible that homodimerization of BCL-2 (at
higher levels of expression) may result in unsequestered BCL-2
interacting pro-apoptotic proteins resulting in cell death.
The two opposing activities of the BCL-2 gene appear to be similar to the activities of other oncogenes, such c-myc and E1a, which promote cell proliferation as well as cell death. Interestingly, two pro-apoptotic proteins of the BCL-2 family, BAK (43) and BAX (44) have been reported to suppress apoptosis under certain conditions (45, 46). As suggested by Evan (47), coupling the two opposing effects induced by a single oncogene gene may be a defense against cancer. It would be expected that the loss of the cell death activity of BCL-2 as a result of somatic mutations may also play a role in oncogenesis in addition to other activating mutations. In a sense, mutations that inactivate the apoptosis activity of BCL-2 could be considered as gain-of-function mutants that contribute to enhanced oncogenesis. Consistent with this prediction, at least one of the somatic mutations expressed at high levels that we have examined results in reduced apoptosis activity compared with comparable levels of wild type.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank S. Korsmeyer, C. Croce, and T. Ito for the gifts of BCL-2 cDNAs.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health NCI Research Grants CA-73803 and CA-33616 and American Cancer Society Grant VM-174.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.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: Institute for
Molecular Virology, Saint Louis University, 3681 Park Ave., St. Louis,
MO 63110. Tel.: 314-577-8416; Fax: 314-577-8406.
1
The abbreviations used are: PCR, polymerase
chain reaction; ORF, open reading frame; UTR, untranslated region;
DMEM, Dulbecco's modified Eagle's medium; X-gal,
5-bromo-4-chloro-3-indolyl -D-galactopyranoside; CMV,
cytomegalovirus; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; bp, base
pair(s); kb, kilobase pair(s).
2 E. J. Uhlmann and G. Chinnadurai, unpublished results.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|