From the Department of Cell Biology, Neurex Corporation, Menlo Park, California 94025
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ABSTRACT |
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Cell death plays an important role in a number of
physiological processes in all complex multicellular organisms. One of
the molecules that regulates this process is BAX, an integral membrane protein, that promotes apoptosis. The function of BAX is countered by
BCL-2 and BCL-XL. The differential expression of these
proteins can influence the ability of the cell to die or survive. In
this paper, we describe the cloning, biochemical, and functional
characterization of a novel splice isoform of BAX, called BAX-.
Transient overexpression of BAX-
protein potentiates cell death at
levels comparable to that of BAX-
overexpression.
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INTRODUCTION |
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A family of BCL-2-related proteins has the effect of potentiating
or attenuating apoptotic cell death (1-4). This family is specifically
defined by four regions that share amino acid sequence homology
designated BH1, BH2, BH3, and BH4. These domains are essential either
for dimerization and/or the function of the different family members
(5-7). BAX (8), the prototypical family member involved in
potentiating apoptotic cell death, heterodimerizes with BCL-2 and
BCL-XL and when overexpressed counters the protective effect of these two family members (8, 9). Many of the family members,
including BCL-2 and BAX, have a putative transmembrane domain that
anchors the proteins to intracellular membranes including mitochondrial, endoplasmic reticulum, and nuclear membranes (10-12). The BCL-2 gene encodes two proteins (26 and 22 kDa) that
differ in their C termini as a result of alternative mRNA splicing
mechanisms (13, 14). The smaller form, designated BCL-2- which lacks the C terminus, lacks the transmembrane domain and thus represents a
soluble form of BCL-2 (14). Both the BCL-2-
and BCL-2-
proteins enhance tumorigenicity of fibroblast NIH-3T3 cells (15), and both are
able to malignantly transform rat embryo fibroblasts with the
ras oncogene (16). In contrast, BCL-2-
has been shown not
to prolong cell survival nor suppress apoptosis (17). Although membrane
attachment is not necessarily required for its protective effect (18),
the smaller form of BCL-2 does not possess all of the characteristics
of the longer form.
The BAX gene has also been shown to encode spliced variants
(8). Four members of the BAX family have thus far been characterized including BAX- that encodes for a 21-kDa protein; BAX-
that encodes for a 24-kDa protein lacking the C terminus due to a
termination codon within the coding region of intron 5; BAX-
, which
is missing exon 2 resulting in a 4.5-kDa protein that prematurely
terminates in exon 3 due to a translational frameshift; and BAX-
(19) that is missing exon 3 but retains the same translational frame, the BH1 and BH2 domains, and the putative transmembrane domain. The
function of these alternatively spliced variants is not yet known.
In this paper, we report the cloning, biochemical characterization, and
functional analysis of a novel splice isoform that we have called
BAX-. BAX-
has a structure distinct from BAX-
including the
absence of a putative transmembrane domain. BAX-
is found in every
tissue tested. Overexpression of BAX-
increases basal levels of cell
death but does not appear to potentiate death by other inducers.
Interestingly, mouse fibroblast L929 cells stably transfected with
BAX-
exhibit properties distinct from both wild-type and
vector-transfected cells when induced to undergo apoptosis in that the
cells appear to be more resistant to other inducers of cell death.
These data suggest that the putative function of this alternatively
spliced form of BAX is to induce apoptosis, but under conditions of
constitutive overexpression, BAX-
directly or indirectly protects
the cells from apoptotic cell death.
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EXPERIMENTAL PROCEDURES |
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Cloning BAX---
Both human BAX-
and
BAX-
were cloned from human hippocampal cDNA
(CLONTECH) by polymerase chain reaction
(PCR)1 in a volume of 50 µl
using 1 µM forward (5'-GGAATTCGCGGTGATGGACGGGTCCGG) and
reverse (3'-TCTGGAAGAAGATGGGCTGAG) primers, 200 µM
deoxynucleotide triphosphates, 1.5 mM MgCl2, 75 mM KCl, 10 mM Tris, pH 9.2 (Stratagene PCR
Optimal Buffer 10) and 2.5 units of Taq polymerase
(Boehringer Mannheim). The brain cDNA was amplified for 25 cycles
as follows: denaturation at 94 °C for 1 min 20 s, annealing at
60 °C for 2 min, and extension at 72 °C for 1.5 min. Immediately
following the last cycle, the mixture was heated to 94 °C for 1.5 min, 60 °C for 2 min, and 74 °C for 10 min. The resulting PCR
product was end-filled with Klenow enzyme, blunt-ligated into the
SmaI site of pBluescript (Stratagene, La Jolla, CA), and
sequenced starting from the 5' end.
Screening a cDNA Library--
A human frontal
cortex-LambaZAP cDNA library (Stratagene) was screened (1 × 106 clones) with a probe corresponding to the first 452 nucleotides of BAX- radiolabeled with 32P by
random priming (Boehringer Mannheim). The screening used standard
hybridization protocols (21) followed by washing in 0.1× SSC, 0.1%
SDS at 55 °C.
RT/PCR Analysis-- Normal tissue was obtained from male Sprague-Dawley rats. Total RNA was isolated by the method of Chomczynski and Sacchi (22), and first strand cDNA synthesis was accomplished in a volume of 2.5 µl containing 150 ng of total RNA, Moloney murine leukemia virus reverse transcriptase (2 units; Boehringer Mannheim), and 1 µM complementary 3'-primer TCTGGAAGAAGATGGGCTGAGG (exon 6) at 42 °C for 30 min. Following first strand synthesis, 1 µM 5'-primer GAGCGGCTGTTGGGCTGGATCCAA (corresponding to sequence in exon 5), 200 µM dNTPs (dCTP was replaced with radiolabeled dCTP), and Taq DNA polymerase (10 units; Boehringer Mannheim) was added, and the reaction mix was heated to 95 °C for 3 min and amplified for 25 cycles with the following conditions: denaturation at 94 °C for 1 min, annealing at 63 °C for 1 min, and extension at 72 °C for 40 s. The products were electrophoresed on a 5% polyacrylamide/urea gel, and the gel bands were analyzed with a PhosphorImage Scanner (Molecular Dynamics, Sunnyvale, CA).
Ribonuclease Protection Analysis--
The RPA probe for
BAX transcripts was prepared using the MAXIscript SP6 kit
(Ambion, Inc., Austin, TX). The 400-bp BAX RNA probe was
synthesized from a BamHI-linearized plasmid cDNA
containing a 778-bp insert of human BAX cDNA in the
vector, pcDNA3 (Invitrogen, Carlsbad, CA), and was radiolabeled
with [-32P]CTP that was included in the reaction mix.
The control probe of 18 S rRNA was transcribed in vitro
from an 18 S rRNA plasmid cDNA. The radiolabeled probes were
purified on a 5% polyacrylamide urea gel. The full-length probes were
cut from the gel and eluted overnight.
Western Blot Analysis-- Normal brain, liver, lung, kidney, and heart tissues were obtained from male Sprague-Dawley rats. Approximately 2 g of each tissue were homogenized in 500 µl of ice-cold RIPA containing aprotinin, phenylmethylsulfonyl fluoride, and leupeptin at 4 °C. Following homogenization, the tissue lysate was sonicated for 1 min and then incubated on ice for an additional 30 min. The protein lysate was centrifuged at 2500 rpm for 10 min at 4 °C, and the resultant supernatant was subjected to the Bradford method of protein analysis (Bio-Rad). Following the Bradford analysis, equal amounts of protein were denatured by boiling for 10 min in RIPA containing 100 µM dithiothreitol, and the proteins were electrophoresed on a 12% polyacrylamide gel (SDS-PAGE). Prestained molecular weight markers were run in parallel. The gel was electroblotted to polyvinylidene difluoride membrane (Bio-Rad) for 1 h at room temperature. For immunoblot analysis of proteins, the membrane was blocked in 5% non-fat dry milk (milk solution) for 30 min followed by incubation with the primary antibody, NXrBAX-1, for 1 h in milk solution, washing, incubating with the secondary goat anti-rabbit antibody coupled to peroxidase in milk solution for 1 h, washing and visualizing using ECL (Amersham Pharmacia Biotech), followed by autoradiography.
For Western analysis of cultured cells, L929 cells, which were either transfected with vector alone or with BAX-Transfection of Cells--
Mouse fibroblast L929 cells
were grown in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum (HyClone), referred to as complete medium, in an
environment of 5% CO2 at 37 °C. Exponentially growing
cells were seeded at 5 × 105 cells per 10-cm tissue
culture plate (Falcon) in 10 ml of complete medium the day prior to
transfection. The cells were electroporated in Dulbecco's
phosphate-buffered saline containing 20 µg of
BAX--pcDNA3 (twice purified by CsCl2)
with 250 volts and a capacitance of 250 microfarads, grown for 2 days
in growth medium, and then selected in G-418 (Life Technologies, Inc.).
Drug-resistant clones were transferred to 24-well dishes and grown to
confluence with drug selection.
Measurement of Cell Death-- Exponentially growing L929 cells were seeded at 5-10 × 105 cells per 10-cm tissue culture plate (Falcon) in 10 ml of complete medium the day prior to the induction of cell death. Cell death was initiated by replacing the complete medium with Opti-MEM (Life Technologies, Inc.) containing 40 ng/ml TNF and 10 µg/ml cycloheximide. After various times, the cells were scraped from the plates, centrifuged at 1500 rpm for 5 min, and resuspended in Dulbecco's phosphate-buffered saline containing 0.2% trypan blue. The cells were incubated in the trypan blue solution for 5 min, transferred to a hemacytometer, and the number of viable (phase bright) and nonviable (blue) cells were recorded. For each sample, five fields were counted. We and others (23) found >95% death by 20 h.
In Vitro Protein Interactions--
Plasmids were constructed for
coupled in vitro transcription/translation. Human
BAX- was subcloned into pcDNA3 (Invitrogen) from the
PCR product obtained from the human hippocampal cDNA (as described
above). Human BCL-2 cDNA was obtained from
human hippocampal cDNA using the following primers:
5'-ggaattcgcggtgatggacgggtccgg-3' and
5'-ggaattctcagcccatcttcttccaga-3', and the resultant product was
subcloned into pcDNA3. A partial BAX-
clone, obtained
from the library screen (see above), was ligated into
PstI-digested pBluescriptSKII containing BAX-
,
resulting in a complete clone. This full-length BAX-
construct was then subcloned into EcoRI-digested pcDNA3.
HA-truncated BAX-
(lacking the last 18 residues) and HA-BAX-
were constructed into pcDNA3 by ligating a
EcoRI/SalI or EcoRI, respectively,
fragment containing the HA coding sequence in-frame with truncated
BAX-
or full-length BAX-
from the yeast expression vector pAS2-tBAX-
or pAS2-BAX-
into EcoRI/XhoI or EcoRI-digested
pcDNA3. Truncated BAX-
was constructed into pAS2 (CLONTECH) by standard PCR reactions.
BAX-
was constructed into pAS2 by ligating the
BamHI/SalI fragment from a partial
BAX-
clone obtained from the library screen (see above)
into BamHI/SalI-digested pAS2.
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RESULTS |
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Cloning of a Novel BAX Splice Variant from Human Brain--
Human
hippocampal cDNA was used to screen for brain-specific
BAX homologues using the technique of reverse
transcriptase/polymerase chain reaction (RT/PCR). By using synthetic
primers, we obtained three different splice variants of BAX,
including the previously described (8) BAX- and
BAX-
. Additionally, we obtained a novel splice variant
that we have called BAX-
. As shown in Fig. 1, BAX-
is identical to
BAX-
through the region of the protein encoded by exon 5;
at this point, BAX-
is generated as a result of the
splice donor site on exon 5 joined to a site 49 base pairs 5' to the
BAX-
acceptor site on exon 6 (Fig.
2). We have confirmed that the extra
49-bp insert found in BAX-
is derived from the 3' end of
intron 5 by sequencing a genomic clone of BAX (data not
shown). The addition of 49 bp results in a translational frameshift and
a protein distinct from BAX-
. In order to obtain the complete coding
sequence for BAX-
, it was necessary to use 3'-rapid
amplification of cDNA ends as described under "Experimental
Procedures." The complete coding sequence for BAX-
is shown in
Fig. 2. While BAX-
encodes a 21-kDa protein, the novel transcript
predicts a larger 24-kDa protein. An interesting characteristic of this
novel protein is that it contains no putative transmembrane domain,
suggesting a cytosolic rather than membrane localization. BH1, BH2, and
BH3 domains are almost entirely conserved, however.
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BAX- Is Widely Distributed--
Ribonuclease protection
analysis was performed to evaluate the tissue distribution of
BAX-
versus BAX-
. RNA from human brain, liver, heart, lung, and kidney were hybridized with an RNA probe synthesized from a cDNA clone containing a portion of the
BAX-
gene corresponding to nucleotides 300-600, which
includes the unique 49-bp insert of BAX-
. The resultant
RNA probe was designed to recognize all of the known splice variants of
BAX. As shown in Fig. 3, we
observed three main transcripts that correspond to the predicted sizes
for BAX-
, BAX-
, and BAX-
. Bgl
II was used to digest the resultant bands to confirm their
identity (data not shown). While BAX-
is the predominant
transcript in human lung and kidney, both BAX-
and
BAX-
are expressed at levels comparable to
BAX-
in human brain, liver, and heart.
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Interaction of BAX- with Other BCL-2 Family
Members--
Full-length BAX-
, BAX-
,
BCL-2, and a hemagglutinin protein of human
influenza virus (HA)-tagged form of full-length BAX-
(HA-BAX-
) and a HA-tagged form of truncated
BAX-
(lacking its transmembrane domain,
HA-tBAX-
) were prepared by in vitro
transcription and co-translation and assayed for their ability to
co-immunoprecipitate. Translation of HA-BAX-
produces a
major product that migrates slightly higher than the untagged protein
(Fig. 5, lower panel, compare
1st and 4th lanes). Lower molecular weight bands
are also apparent, probably resulting from internal initiations at the first BAX-
methionine and an internal methionine at amino acid residue 20. A similar pattern of one major product and several internal
initiations is seen with the translation of HA-tBAX-
(Fig. 5, upper panel, 1st lane).
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Expression of BAX- in Mammalian Cells--
An expression
plasmid derived from pcDNA3 (Invitrogen) was constructed placing
BAX-
under the control of the CMV promoter. The resultant
pcDNA3-BAX-
plasmid was stably transfected into the
mouse fibroblast cell line, L929, by electroporation. Twenty four
clonal cell lines were obtained, and 12 were evaluated by Western
analysis for the expression levels of BAX-
protein using the BAX-
antibody, BAX (P-19) (Santa Cruz Biotechnology). The translational
product of BAX-
migrated at approximately 28 kDa in
SDS-polyacrylamide gel electrophoresis as shown in Fig.
6. Two other bands are detected with this
antibody that migrate at approximately 42 and 60 kDa. Although we do
not know the nature of these bands, we can say that the 42-kDa band
appears infrequently and randomly and, thus, appears to be nonspecific.
The 60-kDa band is frequently seen but with a number of different
antibodies, as well as with preimmune serum, suggesting that it too is
a nonspecific band. Several of the transfected cell lines express
BAX-
protein (Fig. 6). Although all of the cells lines have
detectable BAX-
mRNA levels as shown by RT/PCR
analysis (data not shown), some of the cell lines express no detectable
levels of the protein using this antibody. One possibility for this
difference is that the BAX-
protein expressing cell lines express a
more stable form of the protein. Another possibility is that the
cellular milieu of some of the transfected cell lines is more amenable to the expression of or stability of BAX-
protein (i.e.
depending on where the gene inserts). To evaluate the function of
BAX-
, we evaluated its role in a model of apoptotic cell death,
namely TNF-induced cell death.
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BAX- Expression Decreases TNF-induced Cell Death--
The
BAX-
-transfected L929 cells were used to determine
whether BAX-
has an activity similar to or distinct from BAX-
.
Vector-transfected L929 or BAX-
-transfected cells were
treated with TNF (40 ng/ml) and cycloheximide (10 µg/ml) for 12 h. Cell death was measured using the technique of trypan blue
exclusion. As shown in Fig. 7, of the
three cell lines tested, pcDNA3BAX-
1,
pcDNA3BAX-
3, and pcDNA3BAX-
8 all
showed increased viability relative to the vector-transfected control.
These data indicate that BAX-
has a function opposite to that of
BAX-
. To determine how long after treatment with 40 ng/ml TNF
BAX-
remained protective, a time course of treatment was done. As
shown in Fig. 8, BAX-
was protective up to 12 h following treatment. After 20 h, a majority of the cells died, probably because cycloheximide prevented the ongoing translation of all proteins. These data suggest that constitutive overexpression of BAX-
protects L929 cells from TNF-induced cell death.
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Transient Overexpression of BAX- Increases Basal Levels of Cell
Death in Monkey E5 Cells--
The small number of transfectants
obtained from the BAX-
-transfected L929 cells, relative
to BCL-2-transfected cells, suggested to us that BAX-
may
have been deleterious to the cells. To address this question, we
evaluated cell survival in cells transiently expressing BAX-
.
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DISCUSSION |
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Cell death is important to protect cells from uncontrolled cell growth, as a mechanism to protect surrounding cells from stress or trauma, and is a normal feature of embryonic or postnatal development of major tissues. The prototypical mammalian programmed cell death pathway gene is BCL-2 (2). BCL-2 functions as a repressor of cell death (24-26) and is the best understood member of this gene family.
The BCL-2 family members interact with each other as shown by the yeast
two-hybrid (27) or co-immunoprecipitation analysis. The BH1, BH2, BH3,
and BH4 (6) domains have all been shown to be critical for these
protein-protein interactions (5, 28). BAX protein homodimerizes or
heterodimerizes with BCL-2 or BCL-XL to affect cell death
(5, 29). Overexpression of BAX- counteracts the cell death
repressing activity of BCL-2 and BCL-XL (5, 9). It has been
proposed that the ratio of BCL-2 to BAX determines survival or death
following an apoptotic signal (30). Site-directed mutagenesis studies
(5) have shown that mutations that disrupt the heterodimerization
interaction of BCL-2 with BAX but that still maintain the ability of
BCL-2 to homodimerize, completely abrogate the death repressor action
of BCL-2, suggesting that BAX alone or as a homodimer is sufficient to
trigger death. Recently it has been demonstrated that the BH3 domain is
critical for both homo- and heterodimerization between BCL-2,
BCL-XL, and BAX (31, 32).
Dimerization of molecules is a mechanism that cells utilize to control their environment and destiny. For example, how does one regulate molecules that are themselves regulators? One way to do this is to change the phosphorylation state of the molecule, which in turn changes the partnering of the molecule. Partnering between BCL-2 family proteins may be dependent on the phosphorylation state of the different members since it has been demonstrated that phosphorylation of BCL-2 interferes with its ability to inhibit apoptotic cell death (33). It has been suggested that activation of Raf 1 kinase may be responsible for its phosphorylation, based on inhibitor studies (34).
BAX-, BCL-2-
, and BCL-XL-
all possess a C-terminal
hydrophobic domain that is thought to target the proteins to internal membranes. Interestingly, BRAG-1 (35), BFL-1 (36), or A1 (37), BCL-2
family members as well as BCL-XL-
(38), a naturally
occurring splice variant of BCL-XL, and BCL-2
C22 (6),
an artificially constructed form of BCL-2, all lack their hydrophobic
domains and thus do not contain within their primary structure the
conventional transmembrane hydrophobic domain and yet are still capable
of repressing cell death.
Recently, the predicted three-dimensional structure of
BCL-XL was solved (39). An arrangement of -helices in
BCL-XL that mimic the membrane translocation domain of
bacterial toxins, such as diphtheria toxin, was demonstrated. The
predicted membrane pore-forming domain is quite N-terminal to the
transmembrane domain, suggesting that proteins lacking a conventional
transmembrane domain, nevertheless, may be localized to membranes. Zha
et al. (7) have shown in fact that a construct of
BAX lacking its transmembrane domain (BAX
TM) retained
its cell death function, dimerized with wild type BAX-
and localized
to mitochondria. Thus, a conventional transmembrane domain is not
essential for either organellar localization nor for function. We now
describe another BCL-2 family member that is missing a hydrophobic
domain and acts to potentiate cell death. BAX-
, which lacks a
transmembrane domain, retains the ability to bind to both BCL-2 and
BAX-
proteins (and possible other death-effecting proteins) and
potentiates basal cell death in BAX-
-transfected cells.
Overexpression of BAX-
in the transient-transfected cells show
that BAX-
increases cell death comparable to BAX-
but that it
does not potentiate cell death effected by other inducers such as TNF
or staurosporine. BCL-2 transfectants were protected from
TNF and staurosporine-induced cell death. Together these data suggest
that BCL-2 and the two BAX isoforms converge with the death pathways of
TNF and staurosporine.
Our results with the stably transfected cell lines, on the other hand,
suggest that constitutive BAX- overexpression can protect a cell
from further insult, possibly through the constitutive up-regulation of
a protective protein. The absence of such a protein in a particular
cell may have deleterious effects. For example, experiments with a
BAX knock-out mouse (40) suggest a role for BAX (either
BAX-
, BAX-
, or both) in cell survival. In these mice,
BAX
/BAX
male mice
were infertile and showed increased apoptosis in the testes with
clusters of apoptotic germ cells, whereas thymocytes and B cells
displayed hyperplasia. This study suggested that, depending on the cell
type, the action of BAX may be more anti-apoptotic than pro-apoptotic.
It may be that BAX-
is the predominant or, at least, an
essential splice transcript in the testes, so that elimination of
BAX eliminates not only BAX-
but
BAX-
. Thus, BAX deficiency is manifested as an increase
or decrease in cell death depending on the cellular context.
The present set of investigations demonstrates that another form of
BAX exists in most cell types. It will be interesting to
determine under what conditions the ratio of the two forms of
BAX change and how those changes affect the state of the
cells. This work and the work of other labs suggest that RNA splicing may be an important form of control that the cell utilizes to express
proteins with different functions (not necessarily opposing functions).
Further investigations will be necessary to distinguish between these
two forms of BAX and to determine under what conditions each of these
molecules contributes to cell death and how they affect cell death
within a cell. It will also be interesting to determine whether cells
undergoing degeneration modify their expression of BAX- and BAX-
proteins by differential splicing, and what molecules are involved in
that control. This has important implications for cancer, male
fertility (as described above), as well as stroke, Alzheimer's
disease, and other neurodegenerative processes.
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ACKNOWLEDGEMENTS |
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We thank Dr. Robert Sapolsky (Stanford
University) for the expression vector, p22
gal-BCL-2, and Dr. Dora
Ho (Stanford University) for help and advice. We thank Mr. Frank Chang
for DNA sequencing and analysis. We thank Dr. Richard Scheller
(Stanford University) and Dr. William Hopkins (Neurex Corp.) for
critically reading the manuscript and for thoughtful comments.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed.
1 The abbreviations used are: PCR, polymerase chain reaction; RT/PCR, reverse transcriptase/polymerase chain reaction; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; TNF, tumor necrosis factor; RPA, ribonuclease protection analysis.
2 C. M. Bitler, M. Dimant, and M. Zhou, unpublished observations
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REFERENCES |
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