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J. Biol. Chem., Vol. 277, Issue 45, 42701-42705, November 8, 2002
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From the Division of Neuropathology, University of
Pennsylvania Medical School,
Philadelphia, Pennsylvania 19104-6100
Received for publication, July 3, 2002, and in revised form, August 22, 2002
The enhancement of RNA-mediated motor neuron
degeneration in transgenic mice by mutating a major mRNA
instability determinant in a light neurofilament (NF-L) transgene
implicates cognate RNA binding factors in the pathogenesis of motor
neuron degeneration. p190RhoGEF is a neuron-enriched guanine exchange
factor (GEF) that binds to the NF-L-destabilizing element, to c-Jun
N-terminal kinase-interactive protein-1 (JIP-1), and to 14-3-3 and may
link neurofilament expression to pathways affecting neuronal
homeostasis. This study was undertaken to identify additional RNA
species that bind p190RhoGEF and could affect interactions of the
exchange factor with NF-L transcripts. The C-terminal domain of
p190RhoGEF, containing the RNA-binding site, was expressed as a
glutathione S-transferase fusion protein and was used as an
affinity probe to isolate interactive RNAs in rat brain extracts. As
expected, NF-L mRNA was identified as an RNA specie eluted from the
affinity column. In addition, BC1 RNA was also found enriched in the
bound RNA fraction. BC1 is a 152-nucleotide RNA that is highly
expressed but untranslated in differentiated neurons. We show that BC1
and NF-L mRNA bind to a similar site in the C-terminal domain of
p190RhoGEF, and their bindings to p190RhoGEF are readily
cross-competed. Moreover, we identify a novel binding site in BC1 to
account for its interaction with p190RhoGEF. The findings suggest a
novel role of BC1 in differentiated neurons involving RNA-protein
interactions of p190RhoGEF.
Motor neuron degeneration occurs in transgenic mice expressing
high levels of a neurofilament light subunit
(NF-L)1 transgene (1) or
neurofilament heavy subunit transgene (2) but not in mice expressing
high levels of a neurofilament mid-sized subunit transgene (3). A more
severe form of motor neuron degeneration occurs in mice expressing low
levels of an NF-L transgene with a leucine-to-proline mutation in the
rod domain and a c-Myc tag appended to the C-terminal end of the
protein (4). A severe form of motor neuron degeneration also occurs
from low level expression of an NF-L transgene lacking the point
mutation but with the c-Myc tag at the end of the coding region (5).
The latter finding raised the possibility that neuropathic effects
result from expression of a mutant mRNA by the transgene. This
interpretation is based on the location of the c-Myc tag in a major
destabilizing element in NF-L mRNA and the ability of the c-Myc
insert to stabilize the transcript as well as alter the binding of
cognate binding factors in brain extracts to the NF-L mRNA
destabilizing element by gel-shift and cross-linking assays (5). More
recently, motor neuron degeneration was reproduced in transgenic mice
when the destabilizing element of NF-L mRNA was placed in the
3'-untranslated region of an enhanced green fluorescent protein
reporter transgene (6).
p190RhoGEF was identified as a potential component in RNA-mediated
motor neuron degeneration by virtue of its ability to bind to the
stability determinant and alter NF-L mRNA stability (7) as well as
its interaction with JIP-1 (8), microtubules (9), and 14-3-3 (10). More
recently, we have shown that overexpression of p190RhoGEF protects
Neuro-2a cells from stress-induced apoptosis, possibly by interacting
in the JIP-1/c-Jun N-terminal kinase
pathway.2 Moreover,
p190RhoGEF is a neuronal enriched exchange factor that is up-regulated
during postnatal development and highly expressed in large
differentiated neurons, including motor neurons in mouse spinal
cord.2 A similar expression profile is exhibited by NFs
(11) and by neuron-enriched isoforms of JIP-1 (12) and 14-3-3 (13-15).
The involvement of the NF-L-destabilizing element in RNA-mediated motor
neuron degeneration raised the possibility that additional RNA-protein
interactions of the C-terminal domain of p190RhoGEF may be instrumental
in modulating RNA-mediated neuropathic effects. To pursue this line of
study, the C-terminal domain of p190RhoGEF (p190RhoGEF-C) containing
the RNA-binding site was fused to GST protein and used as affinity
matrix for identifying additional interactive RNA species in soluble
brain extracts. To our surprise, we identified BC1 as an RNA with high
affinity for the RNA-binding site in p190RhoGEF. BC1 is an untranslated
152-nucleotide polymerase III transcript that is up-regulated during
postnatal development (16) and highly expressed in large neurons of rat
brain and spinal cord (17, 18). The importance of BC1 in neuronal
function is not yet fully defined, but the favored view is that BC1
serves as a molecular scaffold for regulating transport and translation of neuronal mRNAs at dendritic sites (19, 20). The binding of BC1
and NF-L mRNA to the same binding site in p190RhoGEF, as reported
herein, suggests that BC1 may play a role in RNA-protein interactions
of p190RhoGEF and in modulating the effects of the exchange factor in
large differentiated neurons.
DNA Constructs--
A cDNA-encoding sequence between amino
acids (aa) 1276-1582 in the C-terminal domain of p190RhoGEF was fused
in-frame to glutathione S-transferase (GST) in pGEX-6P
(Amersham Biosciences) and is referred to as GST/p190RhoGEF-C. The same
cDNA was cloned into pHM6 (Roche Molecular Biochemicals) for HA
tagging and was modified by PCR to generate N- and C-terminal
truncations. All cDNA constructs were verified by DNA sequencing.
Isolation of RNA from Brain Extracts Using GST Fusion
Proteins--
GST/p190RhoGEF-C and unfused GST proteins (100 µg)
were bound to 40-µl glutathione-Sepharose beads and equilibrated with
RNA-binding buffer (RBB) containing 10 mM Hepes, pH 7.5, 50 mM KCl, 3 mM MgCl2, 2 mM dithiothreitol, and 5% glycerol. High speed supernatant
(200 µl) of rat brain homogenate was precleared by passage through an
unfused GST column and then applied to a column of immobilized GST
fusion proteins. The column was washed with RNA-binding buffer containing heparin (1 mg/ml), and RNA was recovered by boiling the
beads in 0.1% SDS. Supernatants were phenol/chloroform-extracted, and
RNAs were ethanol-precipitated.
cDNA Library Construction from Extracted RNA--
cDNA
libraries were constructed from total RNA (50 ng) using the SMART
cDNA library construction kit (Clontech).
Individual phage plaques were amplified, Preparation of RNA Probes--
RNA polymerase templates for
full-length and truncated BC1 probes were prepared as PCR products by
incorporating T7 promoter sequence into the upstream primer. A probe to
the 68-nucleotide destabilizing element in NF-L (NF68) was obtained
from a linearized pSK+ plasmid (7). Probes were uniformly labeled with
[ Dot-Blot Assay--
Fused or unfused GST proteins or BSA (150 pmol) were spotted onto nylon membrane, preincubated in hybridization
buffer (1 M Denhardt's, 1 mM dithiothreitol, 1 mM EDTA, 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1 mg/ml heparin) for 1 h at room
temperature, and then incubated for 1 h following the addition of
32P-labeled probes (5 × 104 cpm/ml).
Membranes were washed extensively with hybridization buffer and
subjected to autoradiography.
Gel-shift Studies--
Gel-shift studies were conducted by
incubating 10 µg of protein and 5 × 104 cpm/ml
radioactive probe in 20 µl of RBB at room temperature for 10 min,
then adding 2 µl of RNase T1 (2 units/µl), and incubating for an
additional 10 min. Five µl of heparin (50 mg/ml in 50% glycerol) was
added, and the total sample was loaded onto a 10% acrylamide gel and
electrophoresed at 100 V in 0.5× TBE.
Cell Culture and Transfection--
Neuro-2a cells were obtained
from ATCC and maintained in Dulbecco's modified Eagle's medium
containing 10% fetal calf serum. Cell transfections were performed
using the FuGENE6 reagent (Roche Molecular Biochemicals).
Immunoprecipitation Assays--
Transfected N2a cells in 100-mm
cell culture plates were detached and collected by centrifugation,
washed with PBS, and suspended in 0.6 ml of cell lysis buffer (1× PBS,
1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented
with 0.01% (v/v) of Protease Inhibitor Mixture (Sigma), dispersed by
centrifugation in a QIA shredder Mini column (Qiagen). Extracts were
mixed with 20 µl of anti-HA antibody-agarose conjugate (Santa Cruz
Biotechnology) on a shaker for 1 h at 4 °C, and beads were
precipitated by centrifugation, washed 4 times with PBS, suspended in
20 µl of 0.1% SDS, and boiled for 3 min to release bound components.
The supernatant was then used as a template for RT-PCR detection of BC1 RNA.
Northwestern Blot--
GST fusion proteins (10 µg) were
electrophoresed in 8% SDS-PAGE gel and electroblotted to Hybond-C
membrane (Amersham Life Science). Membranes were preincubated with
Blotto A (10 mM Tris-HCl, pH 8.0, 150 mM NaCl,
5% milk) on shaker at 4 °C overnight and incubated with 5 × 104 cpm/ml probe in hybridization buffer for 1 h at
room temperature. Membranes were extensively washed with hybridization
buffer, and dried membranes were subjected to overnight exposure for autoradiography.
BC1 and NF68 Bind to and Cross-compete for p190RhoGEF--
The C
terminus of p190RhoGEF (p190 RhoGEF-C) was isolated from a rat
brain cDNA library by virtue of its binding to a 68-nucleotide RNA
probe (NF68) comprising a destabilizing element in mouse NF-L mRNA
(7). To identify additional RNA targets of p190RhoGEF-C, the cDNA
was fused to GST, and fusion protein was used as an affinity matrix to
screen a high speed supernatant fraction of rat brain. Bound RNAs were
recovered from the beads, reverse-transcribed into cDNAs, and the
cDNAs cloned and sequenced. The resulting cDNAs not only
included NF-L but also revealed more frequent recovery of short
elements matching the sequence of BC1. The structure and partial
sequence of BC1 is shown in Fig. 1.
To verify the binding of BC1 to p190RhoGEF-C, GST/p190RhoGEF-C (150 pmol) was spotted on nylon membrane, along with equimolar amounts of
unfused GST protein (GST) and bovine serum albumin (BSA), and reacted
with uniformly labeled full-length BC1 probe (Fig.
2). BC1 bound to p190RhoGEF-C but not to
BSA or to unfused GST protein. A similar-sized RNA probe to sequence
adjoining the T7-binding site in pSK+ (SK+) did not bind to
GST/p190RhoGEF-C, GST, or BSA. Moreover, binding of BC1 was more than
10-fold greater than binding of NF68 to p190RhoGEF-C by comparing the
respective amounts of bound radioactivities. Similar disparity in
binding affinities were observed using probe to the ID sequence of BC1
(data not shown), suggesting that the binding site on BC1 was situated
within the ID region of BC1 RNA (see below).
Gel retardation assays were undertaken to assess the abilities of BC1
and NF68 probes to alter the electrophoretic migration of
GST/p190RhoGEF. Fig. 3 shows that
incubation of GST/p190RhoGEF-C with 32P-labeled NF68 or BC1
probes generates similar radioactive bands (arrow). The
radioactive gel-retarded band formed with NF68 (3rd lane)
was competed by 100-fold excesses of unlabeled NF68 (4th lane) or unlabeled BC1 (5th lane) but not by an
unlabeled nonspecific SK+ probe (data not shown). Likewise, the
gel-retarded band formed with BC1 (6th lane) was competed by
100-fold excesses of unlabeled NF68 (7th lane) or unlabeled
BC1 (8th lane) but not by an unlabeled SK+ probe (data not
shown). Incubation of GST/p190RhoGEF with 32P-labeled SK+
probe did not generate a gel-retarded band (9th lane).
In Vivo Binding of BC1 RNA to p190RhoGEF Protein--
Lysates from
Neuro-2a cells transfected with HA-tagged p190RhoGEF-C protein or pHM6
vector alone were immunoprecipitated with agarose-conjugated anti-HA
monoclonal antibody. The immunoprecipitates were washed extensively and
solubilized by boiling in 0.1% SDS. RT-PCR assays were conducted on
the solubilized fractions to assess the presence of BC1 RNA in the
respective immunoprecipitates. Fig. 4
shows that BC1 RNA (arrow) was recovered from Neuro-2a cells
transfected with HA-tagged p190RhoGEF-C (lane 1) but not from Neuro-2a cells transfected with the empty pHM6 vector (lane 2). The size of the BC1 RT-PCR fragment was 175 bp, including T7
promoter sequence in upstream primer, and had the appropriate electrophoretic migration when compared with the 100-bp electrophoretic standard markers (lane M).
Binding Site on p190RhoGEF Protein for BC1 RNA--
Full-length
and truncated GST/p190RhoGEF-C were constructed for localizing the
NF68-binding site by Northwestern
blots.3 The same full-length
and truncated GST fusion proteins were used to localize and compare the
BC1- and NF68-binding site (Fig.
5A). GST fusion proteins were
expressed in bacteria and purified by glutathione affinity
chromatography. Purified proteins were electrophoresed in SDS-PAGE
(Fig. 5B) and transferred to nylon membranes, and replicates
of transferred proteins were hybridized with BC1 (Fig. 5C)
and NF68 (Fig. 5D) probes by Northwestern blots. Full-length and N-terminal truncations (M4 and M5 constructs) of GST/p190RhoGEF-C bound 32P-labeled BC1 (Fig. 5C) and NF68 (Fig.
5D) probes. Binding of both BC1 and NF68 probes was lost in
C-terminal truncations of GST/p190RhoGEF-C. Whereas binding was
retained when the C terminus of GST/p190rhoGEF-C was truncated from aa
1582 to 1524 (M3 construct), binding of NF68 and BC1 probes was
eliminated upon C-terminal truncations to aa 1493 (M2 construct) and
1461 (M1 construct). The findings indicate that BC1 and NF68 bind to
similar sites in GST/p190RhoGEF and that sequence encoded between aa
1493 and 1524 (QLQEYQQSLERLREGQRMVERERQKMRVQQGL) is required for the
binding of both RNA probes.
Binding Site on BC1 RNA for GST/p190RhoGEF-C--
GST/p190RhoGEF-C
was separated by SDS-PAGE and transferred to nylon membrane for
Northwestern blots to determine the sequence in BC1 required for
binding to the fusion protein. Membrane containing fusion protein was
hybridized with full-length BC1 and with a series of truncated probes
lacking 5' or 3' sequences (Fig.
6A). 32P-Labeled
probes to full-length BC1 (BC1), to ID sequence (ID), and to the
full-length BC1 probe lacking the initial 10 nucleotides of the
dendrite-targeting motif or DTM (BC1 Novel binding features of BC1 are herein identified which expand
the prospective role of BC1 in neuronal metabolism. Binding of BC1 and
the destabilizing element in NF-L mRNA to a similar site in the
C-terminal domain of p190RhoGEF links BC1 to NF-L expression and to
RNA-mediated motor neuron degeneration (7). The recognition of
p190RhoGEF as an RNA-binding protein also links NF-L expression with
signal transduction pathways and with potential modifications of the
neuronal cytoskeleton in differentiated neurons (21-23). Moreover, the
identification of a novel binding site outside of the
dendrite-targeting motif (DTM) supports the likelihood that BC1 has
additional functional properties beyond its putative role in regulating
movement, localization, and translation of neuronal transcripts in the
dendritic compartment (19, 20).
There is presently much more information as to the derivation than
function of BC1. For example, BC1 is recognized as a master gene for
generating and amplifying short interspersed repetitive elements in the
rodent genome (24-26) analogous to the Alu repeats in the primate
genome (27-29). BC1 and other progenitor genes are believed to have
arisen by reverse transcription and by fortuitous integration of
cDNAs into a favorable site of the genome downstream of a pol III
promoter sequence. BC1 is, however, an atypical pol III transcript in
that it is highly and exclusively expressed in differentiated neurons
(30). Neuron-specific transcription is regulated by positive and
negative upstream elements that react with brain-specific and
nonspecific factors (18). Competitive binding of Pur protein to the BC1
transcript and to cis-acting enhancer elements in the
BC1 gene provides a potential feedback mechanism for
regulating BC1 transcription by its own gene product (31).
The up-regulation of BC1 during the late phase of
neuronal differentiation and high level expression in large
differentiated neurons (16) coincides with the expression profiles of
NF-L (11) and p190RhoGEF2 and is consistent with a
functional interaction of these components in large differentiated
neurons. A similar expression profile is also characteristic of
interactive partners of p190RhoGEF, including neuron-enriched isoforms
of JIP-1 (12) and 14-3-3 (13-15). It is possible that binding of one
or more of the interactive partners to the C-terminal domain could be
responsible for unmasking inherent GDP/GTP activity required for
activation of p190RhoGEF in vivo (9). It is also possible
for interaction of BC1 with p190RhoGEF to account for axonal transport
or localization of BC1 in discrete sites along the surface
membrane of goldfish Mauthner axons (32). It should be
noted, however, there are still large regions in the
N-terminal half of p190RhoGEF containing potential interactive sites of
unknown function, including zinc finger and leucine-rich
motifs (33).
The putative function of BC1 in regulating dendritic
expression is based on the properties of specific BC1-bound proteins. Likewise, our fortuitous identification of additional BC1-binding proteins suggests that BC1 may have additional interactive roles in
neuronal gene expression. In both instances, it is proposed that BC1
functions as a RNA-protein complex and that RNA binding confers,
alters, or modifies properties of the complex. Such RNA-protein interactions resemble the interactions of multiple RNAs and proteins in
RNP complexes that mediate the processing of RNA (34, 35). More
recently, it is also recognized that short single-stranded RNAs can
regulate gene expression by hybridizing with specific mRNAs as
small interfering or small temporal RNAs (36-38).
The potential involvement of p190RhoGEF in RNA-mediated
motor neuron degeneration is quite unexpected because the function of
p190RhoGEF has been attributed to regulation of the neuronal cytoskeleton (8). It should be noted, however, that another GEF,
i.e. RCC1, plays a critical role in regulating the
translocation of protein and RNA across the nuclear pore complex by
promoting the formation of Ran-GTP on the nuclear face of the complex
(39). More recently, a GEF protein with close homology to RCC1 was
identified as the mutant gene product in a recessive form of familial
motor neuron degeneration (40, 41). Why dysfunction of a GEF should lead to motor neuron degeneration is also unexpected. The role of
RNA-protein interactions on p190RhoGEF and their potential linkage to
RNA-mediated motor neuron degeneration could provide some insights into
the nature of motor neuron disease.
*
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.
Published, JBC Papers in Press, September 4, 2002, DOI 10.1074/jbc.M206635200
2
R. Cañete-Soler, unpublished observations.
3
J. Wu, unpublished data.
The abbreviations used are:
NF-L, neurofilament
light subunit;
GEF, guanine exchange factor;
JIP-1, c-Jun N-terminal
kinase-interactive protein-1;
GST, glutathione
S-transferase;
BSA, bovine serum albumin;
PBS, phosphate-buffered saline;
aa, amino acids;
HA, hemagglutinin;
RT, reverse transcriptase;
DTM, dendrite-targeting motif.
Binding of p190RhoGEF to a Destabilizing Element on the Light
Neurofilament mRNA Is Competed by BC1 RNA*
, and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
DNAs extracted, and
cDNAs sequenced.
-32P]UTP and fractionated by electrophoresis, and
gel-excised probes were diluted in RBB to 5 × 104
cpm/µl immediately prior to use.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (10K):
[in a new window]
Fig. 1.
Schematic diagram of BC1. The
152-nucleotide (nt) RNA is composed of ID, A-rich, and
unique sequences. Nucleotide composition of the proximal
(A), middle (B), and distal (C)
segments of the ID sequence is shown. The initial 10 nucleotides of the
ID sequence consist of a dendritic targeting motif
(DTM).
View larger version (42K):
[in a new window]
Fig. 2.
Binding of RNA probes comprising the
destabilizing element in NF-L mRNA (NF68),
full-length BC1 (BC1), or nonspecific sequence in pSK+
(SK+) to a GST fusion protein containing the
C-terminal domain of p190RhoGEF (GST/p190RhoGEF-C), to
unfused GST protein (GST), or to
BSA. Proteins (150 pmol) were spotted onto nylon
membranes and incubated with 32P-labeled (5 × 105 cpm/ml) probes.
View larger version (53K):
[in a new window]
Fig. 3.
Gel retardation assay showing similar
retardation of radioactive bands (arrow) when
GST/p190RhoGEF-C protein is electrophoresed with
32P-labeled probes to the destabilizing element in NF-L
mRNA (NF68) or full-length BC1
(BC1). The retarded radioactive NF68 band
(3rd lane) is competed with 100-fold excesses of cold NF68
(4th lane) or BC1 (5th lane) probes. Likewise,
the retarded radioactive BC1 band (6th lane) is competed
with 100-fold excesses of NF68 (7th lane) or BC1 (8th
lane) probes. Electrophoresis of GST/p190RhoGEF-C with a
32P-labeled probe to nonspecific pSK+ sequence
(SK+) did not generate a retarded radioactive band
(9th lane).
View larger version (57K):
[in a new window]
Fig. 4.
Immunoprecipitation assay of lysates from
Neuro-2a cells transfected with pHM6 vector containing HA-tagged
p190RhoGEF-C protein or from Neuro-2a cell transfected with empty
HA-tagged pHM6 expression vector. Immunoprecipitates from cells
transfected with p190RhoGEF-C (lane 1), but not from cells
transfected with the empty expression vector (lane 2),
contained a template for generating a BC1 band (arrow) by
RT-PCR. RT-PCR should generate a 175-bp BC1 fragment, including T7
promoter sequence in the upstream primer, and is shown with a 100-bp
marker standard (lane M).
View larger version (39K):
[in a new window]
Fig. 5.
Similar RNA-binding sites in p190RhoGEF for
BC1 and the NF-L-destabilizing element (NF68). A,
schematic diagram of p190RhoGEF and the 307 aa (1276-1582)
segment of the C-terminal domain that was mutated (M1-M5),
fused to GST, electrophoresed, and transferred to nylon membrane and
stained for protein (B) or hybridized with
32P-labeled probes to BC1 (C) or NF68
(D). Both probes bound to the full-length (full),
to N-terminal truncations (M4 and M5), and to a
C-terminal truncation (M3) to aa 1524. Binding of BC1 and
NF68 probes was lost when C-terminal truncations were extended to aa
1493 (M2) and 1461 (M1), indicating that amino
acid sequences encoded between aa 1493 and 1524 are required for the
binding of both RNA probes.
DTM) bound to the fusion protein
(Fig. 6B). Binding of 32P-labeled probes to the
fusion protein was lost when the 5' deletion was extended to include
the initial 25 (BC1ID-A) or 50 (BC1ID-A/B) nucleotides of BC1.
The findings indicated that sequence within the ID element of BC1 was
sufficient for binding and that sequences in the initial 25 nucleotides
of BC1, excluding the DTM, are necessary for binding of BC1 to
GST/p190RhoGEF-C.
View larger version (22K):
[in a new window]
Fig. 6.
Location of binding sites on BC1 for binding
to GST/p190RhoGEF-C fusion protein. A, schematic
diagram of BC1 RNA showing location of probes and their abilities to
bind to the fusion protein. B, Northwestern blot of
GST/p190RhoGEF fusion protein showing binding of
32P-labeled probes to full-length BC1 (BC1), to
ID sequence (ID), and a BC1 lacking the initial 10 nucleotides to the dendritic targeting motif (BC1 DTE).
Binding was abolish when 5'-truncations extended to include the initial
25 (BC1ID-A) and 50 nucleotides (BC1ID-A/B)
of the ID sequence. The findings suggest a novel binding in BC1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FOOTNOTES
To whom correspondence should be addressed: 609C Stellar Chance
Laboratories, 422 Curie Blvd., University of Pennsylvania Medical
School, Philadelphia, PA 19104-6100. Tel.: 215-662-7372; Fax:
215-573-2059; E-mail: wws435jp@mail.med.upenn.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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