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(Received for publication, December 8, 1994; and in revised form, January
30, 1995) From the
Monocyte-macrophage differentiation was used as a model system
for studying gene regulation of the human vacuolar
H The mammalian vacuolar H The overall structure of
the V-ATPase appears similar to that of the F The M Of the transmembrane polypeptides,
a cDNA clone has been obtained only for the M The function of other subunits
of the mammalian V-ATPase in catalysis has not been established. Those
subunits for which cDNA clones have been isolated include the E (M The role of V-ATPase subunits in the amplification and targeting of
the enzyme is less clear. V-ATPase expression is greatly amplified in a
cell-specific manner in several types of renal tubular epithelial
cells, allowing them to transport hydrogen ion efficiently. We showed
recently that individual B subunit isoforms are highly amplified in
specific kidney cell types present in different segments of the nephron (14) . ( Macrophages and other cells of the mononuclear phagocyte system
perform several functions that require a high degree of V-ATPase
expression. Mature macrophages develop a large reserve of lysosomes,
required for intracellular digestive processes, that are acidified by a
V-ATPase (reviewed in (27) ). Macrophages utilize a plasma
membrane V-ATPase, in part, to regulate their intracellular pH in
acidic environments(28, 29) . The osteoclast, a cell
type closely related to the macrophage, exhibits amplified expression
of the V-ATPase at its ruffled membrane upon attachment to
bone(4) , creating an acidic resorptive space required for
digestion of bone mineral and matrix proteins (reviewed in (30) ). To investigate expression of the V-ATPase in
differentiating macrophages, we have studied both human peripheral
blood monocytes and a related cell line, THP-1. The human monocytic
leukemia cell line, THP-1, has recently come into use as a model for
monocyte-macrophage differentiation (reviewed in (31) ). Like
their more widely used counterparts, HL-60 and U-937, THP-1 cells may
be induced to differentiate toward a macrophage-like state by culture
with phorbol esters(32) . The THP-1 line, however, is thought
to mimic more closely monocyte-derived macrophages in expression of
oncogenes and membrane proteins(31) . We have examined
V-ATPase subunit expression during monocytic differentiation and
present evidence for both transcriptional and post-transcriptional
control mechanisms. We show that increased expression of the B2 subunit
isoform is mediated primarily at the level of transcription. We have
isolated B2 gene fragments containing the first exon and 5`-flanking
region and present an initial characterization of the promoter elements
responsible for transcriptional control.
Densitometry of the autoradiograms was performed with a Hoefer
Scientific (San Francisco, CA) model GS300 scanning densitometer in
transmittance mode connected to a Spectra-Physics SP4270 integrator.
The B2 subunit cDNA probe used in
these studies for library screening and Southern hybridization was an
85-bp PCR-amplified cDNA spanning the region from -29 to +56
with respect to the translational start site. The oligonucleotides
designed to amplify this region were 18 mers of the sequences
5`-GCTGGGCCAGTCGGGACA-3` (sense direction) and 5`-ACGGGTAGCTCGGGTGCG-3`
(antisense direction). This probe was amplified from 50 ng of a B2
subunit cDNA clone ( DNA sequencing was performed using the TaqTrack sequencing system
from Promega. Gene-specific oligonucleotides were used to prime
sequencing extension reactions, and the sequencing ladder was
visualized by incorporation of
293 cells were transfected by the method of calcium phosphate
precipitation(39) . Each 100-mm plate (at about 80% confluence)
was incubated overnight with a buffered calcium phosphate mixture
containing 5 µg of the test plasmid and 2 µg of pRSVcat. The
medium was changed the following morning, and after an additional 24 h,
the cells were cultured in the presence or absence of 160 nM TPA for 5 h.
Luciferase activity was measured in an Optocomp II luminometer
system (MGM, Hamden, CT). Cell extracts were injected with 100 µl
of a luciferase assay reagent (Promega), and the resulting luminescence
was measured for 20 s.
Binding
sites for Sp1 and AP2 were detected using the Core DNase footprinting
system (Promega), purified Sp1, and either purified AP2 or an extract
from E. coli expressing recombinant AP2 (all from Promega).
Binding reactions and DNase I digestion were performed following the
manufacturer's instructions. The resulting products were
separated by electrophoresis in a 6% denaturing polyacrylamide gel next
to a sequencing ladder as a size marker.
Figure 1:
Immunoprecipitation of V-ATPase
complexes from monocytes and macrophages. V-ATPase complexes were
precipitated with monoclonal antibody E11 as described under
``Experimental Procedures'' and separated on 10 or 12.5%
SDS-polyacrylamide gels. For both native cells and THP-1 cultures,
precipitations were performed from 10
A pronounced increase in immunoprecipitable V-ATPase
was observed in the macrophages in comparison with the monocytes.
Densitometric measurements of these immunoprecipitates showed that in
the macrophages, the A subunit increased 4.3-fold, and the B subunit
increased 5.6-fold. Similar experiments in differentiating THP-1 cells
showed increases of 4.0- and 3.6-fold for the A and B subunits,
respectively (right panel). Immunoblots were performed on
peripheral blood monocytes and monocyte-derived macrophages as a
second, independent method for assessing changes in V-ATPase content
during monocyte differentiation (Fig. 2). Gel-separated proteins
from 5
Figure 2:
Immunoblots of B1, B2, and E subunits
during monocyte-macrophage differentiation. Total cell homogenates were
prepared from 5
These results indicate that THP-1 cells may
serve as a model system for the study of amplification of V-ATPase
expression during monocyte to macrophage differentiation. We next
examined whether the increase in V-ATPase content during
differentiation was a result of increases in steady-state mRNA levels
for any of the subunits.
Figure 3:
V-ATPase subunit steady-state mRNA levels
during monocyte-macrophage differentiation. Total cellular RNA was
isolated from (A) THP-1 cells treated for indicated times with
160 nM phorbol ester, or from (B) human monocytes (day 0) or from monocyte-derived macrophages (day 5 in culture), separated on agarose gels, and transferred to
membranes as described under ``Experimental Procedures.'' RNA
hybridization analysis was performed with a cDNA fragment specific for
the B2 subunit isoform, or with a probe for glyceraldehyde-3-phosphate
dehydrogenase (GPDH) as a control for RNA loading (upper
two panels). In the lower panel, poly(A)-enriched RNA was
isolated from THP-1 cells treated for 0 or 24 h with 160 nM phorbol ester, separated, and transferred to membrane as above,
and hybridization analysis was performed using cDNA probes for the
other V-ATPase subunits indicated, or glyceraldehyde-3-phosphate
dehydrogenase as a control.
Message levels for other V-ATPase
subunits were examined by RNA hybridization analysis of
poly(A)-enriched RNA from THP-1 cells before, and 24 h after, TPA
induction, using subunit-specific cDNA probes (Fig. 3A, bottom). mRNA levels for neither the E nor proteolipid subunit
showed any increase at the same time points used to analyzed B2 subunit
message. The mRNA level for the A subunit, however, increased by about
the same amount as the B2 mRNA. Because the A subunit message is
detectable only in poly(A)-selected mRNA, we were unable to probe for
its presence in native monocytes and monocyte-derived macrophages due
to constraints on cell numbers available. RNA hybridization analysis of
total cellular RNA from these cells, however, done with the other cDNA
probes indicated in Fig. 3A, showed that mRNA
transcripts for neither the E subunit nor the proteolipid subunit
increased in monocyte-derived macrophages (data not shown). Although
the B1 protein was not detectable in macrophages, mRNA for this subunit
was detected in both monocytes and macrophages by RNA blot (data not
shown) and nuclear transcription assays (see Fig. 4).
Figure 4:
Nuclear runoff analysis of V-ATPase
subunit transcripts in THP-1 cells before and after phorbol ester
induction. Nuclei were prepared from THP-1 cells before and 24 h after
induction with 160 nM phorbol ester as described under
``Experimental Procedures.'' Nascent transcripts were labeled
with [
The B2 subunit showed
the largest change in transcription rates of the several V-ATPase
subunits tested. The B2 subunit transcript was barely detectable in
control THP-1 cells and increased substantially by 24 h after TPA
treatment. Densitometric quantitation in multiple experiments revealed
an average increase of 3.5-fold (data not shown). This number compares
favorably with the average 3.1-fold increase calculated from the RNA
hybridization experiments and suggests that transcriptional regulation
accounts for most or all of the increase in steady-state message
levels. Nuclear runoff analysis performed at additional time points
after treatment of THP-1 cells with TPA have indicated that enhanced
transcription begins within 4 h (data not shown). In contrast, there
was little or no change in transcription of the proteolipid and E
subunits. This result was not unexpected, as there was no change in
steady-state mRNA levels for these subunits after treatment with TPA.
There was a slight increase in transcription of the A and B1 subunits,
but the changes were far less than that found in the B2 subunit
isoform. Because the B2 subunit appeared to be regulated primarily
at the transcriptional level, we proceeded to examine the gene
sequences that might control transcription by isolating and
characterizing the 5` end of the B2 gene.
The same human genomic library was rescreened with a
PCR-amplified fragment corresponding to the region of the B2 subunit
from -29 to +56 (described under ``Experimental
Procedures''). This probe was chosen because it would not
hybridize to the genomic clones isolated initially, and the probe had
virtually no sequence identity with the B1 isoform. Following primary
and secondary screens, two clones, each of >20 kb, were isolated.
From one of these clones, a 6.0-kb EcoRI fragment was found to
hybridize on Southern blots to the probe used for the initial library
screening. This fragment was subcloned into the EcoRI site of
pBluescript and designated pBSL217. Restriction mapping of this
fragment was performed by single and double digests of pBSL217 using
appropriate restriction enzymes. From these data, pBSL217 was found to
contain approximately 1.4 kb of 5`-flanking region upstream of the
translational start site. The original
Figure 5:
Map of the 5` regions of the B2 subunit
gene. Two overlapping genomic clones, pBSL217 and pBSL220 are
designated, along with their respective sizes and restriction enyzme
sites. The first exon of the B2 gene is
indicated.
Using gene-specific oligonucleotides as primers,
we sequenced 0.90 kb upstream, and >0.15 kb downstream of the
translational start site. This sequence included a region of 100%
identity to B2 subunit coding sequences, and is shown in Fig. 6.
This figure also indicates the major transcriptional start site, which
we have determined as discussed below. The promoter region exhibits a
number of characteristics common to recently studied genes. First, no
TATA or CAAT boxes are evident. TATA-less promoters generally may be
grouped in two categories, those exhibiting a pyrimidine rich initiator
(Inr) sequence (43) and those that are (G+C)-rich. While
the B2 subunit gene falls in the latter group, it is somewhat unusual
in using predominantly one initiation site (discussed below). Most
genes of this type exhibit multiple initiation points that are used at
similar frequencies. The (G+C)-rich character of the first exon
extends into the coding region and stops at
Figure 6:
Sequence of the B2 subunit gene first exon
and 5`-flanking region. The first exon is designated in boldface type;
the 5`-flanking sequences and first intron are in normal type
face. The start of transcription is indicated as the first base in bold type(-207), and the ATG translation start codon is
marked with an asterisk. GA/CT stretches are indicated by a
single underline. Sp1 and AP2 binding sites are noted. These sequence
data are available from EMBL/GenBank
Figure 7:
Primer extension mapping of the 5` end of
the B2 subunit transcript. Eight micrograms of total cellular RNA from
TPA-treated THP-1 cells were allowed to hybridize with each of two
end-labeled primers spanning from -16 to +2 (lane
1) or from +39 to +56 (lane 2).
Figure 8:
Ribonuclease protection mapping of the 5`
end of the B2 subunit transcript. A
Figure 9:
Promoter activity studies of B2 gene
fragments in THP-1 and 293 cells. B2 gene promoter constructs with a
luciferase reporter were transfected into either THP-1 (solid
bars) or 293 cells (open bars) and tested for
inducibility of luciferase activity with TPA. The region of the B2 gene
promoter included in each construct is indicated. The B1 isoform
construct contains 4.0 kb from the 5`-flanking region of the B1 subunit
gene. Induction is expressed as percent of control luciferase activity
following TPA treatment. Cells were cotransfected with pRSVcat, and all
activity measurements were normalized against CAT activity. Assays were
performed at least three times for each construct/cell combination.
Data bars indicate mean inducibility ±
S.E.
Because the B2 subunit is
ubiquitously expressed in all cell types that have been examined in
this laboratory, it was not possible to create a negative control for
basal expression levels. It was found, however, that 293 cells (another
human cell line) transfected with the same reporter constructs showed
no induction of luciferase activity after treatment with TPA (Fig. 9, open bars). A luciferase construct containing
a 4.0-kb fragment of the human B1 subunit isoform promoter region (
Figure 10:
DNase
I footprinting of transcription factors Sp1 and AP2 in the first exon
of the B2 gene. A DNA probe spanning from -338 to +84 was
tested for its ability to bind purified Sp1 (left panels), and
either purified AP2 or an AP2-enriched E. coli extract (right panels). Amounts of protein added are indicated above
each lane. Schematics of the relative positions of factor binding sites
are shown.
The V-ATPase is a multisubunit complex, with a variable
composition in different tissues and subcellular membrane fractions (14, 49) . A multiplicity of genetic controls may be
requisite to accomplish this type of cell-specific amplification.
However, no studies of the mechanisms behind this cell specificity have
been reported to date. In this report, we have identified a model
system for examination of V-ATPase gene control mechanisms. We found
that regulation of the B2 isoform occurs primarily by changes in
transcription, in contrast to other V-ATPase subunits examined here.
This observation suggests that cells increase the content of proton
pumps with a specific structure by amplifying expression of a desired B
subunit isoform, while regulating the remaining subunits through other
means. Although we have not addressed B1 regulation in this study, we
found that kidney cells, which express a high level of B1 isoform mRNA,
may also increase the content of V-ATPases containing the B1 subunit by
enhancing its transcription. Our initial search for the B2 subunit
gene resulted in isolation of a clone containing an exon with 86%
identity to the B2 cDNAs already described. We were unable to detect a
corresponding mRNA transcript in a number of tissues tested. Although
we cannot rule out the possibility that this gene is expressed in a
tissue or cell type we did not test, our findings suggest that this
clone represents a pseudogene. V-ATPases are ancient enzymes, and their
coding sequences may have been prone to rearrangement or inactivation
during the course of evolution. Our laboratory also has identified a
number of pseudogenes of the E subunit during screens of genomic
libraries(21) . The structure of the first exon and
surrounding regions of the confirmed B2 isoform gene is typical of a
number of recently described ``TATA-less'' genes.
(G+C)-rich promoter regions of this type are found commonly in
``housekeeping'' genes, as well as those encoding cell
growth-related products, such as growth factor receptors and
oncogenes(50, 51, 52) . Less typical of
TATA-less genes, however, is the presence of a single, major
transcriptional start site in the B2 gene. (G+C)-rich TATA-less
promoters routinely initiate transcription at multiple start sites,
although initiation at a single site has been documented in a minority
of cases(53, 54) . Like many others in this class of
promoter, the B2 gene exhibits multiple potential Sp1 and AP2 binding
sites. The consensus binding sites for these transcription factors are
themselves rich in G+C content. More interesting from an
evolutionary standpoint, however, is the location of these consensus
sequences in the B2 gene. All of the potential Sp1 and AP2 binding
sequences reside within the first exon, rather than within upstream
sequences. Two AP2 binding sites reside just downstream from the
translational start site. The (G+C)-rich character of the
5`-untranslated region extends into the coding region, and its 3`
boundary corresponds precisely to the point at which the B1 and B2
isoforms begin to show a high degree of identity. Thus, the sequence
differences between these two isoforms at their amino termini may be
less important for the function of the proteins than for regulation of
their expression. Genomic sequence of only one other mammalian
V-ATPase subunit has been described in the literature(17) . The
230-bp region immediately 5` to the human proteolipid subunit coding
sequences similarly shows a high G+C content, with putative Sp1
elements, but no TATA or CAAT boxes. However, the transcription
initiation site was not mapped, so important elements may lie further
upstream from the region sequenced. Our laboratory has isolated the
promoter region of the human B1 isoform, and while it also is
TATA-less, the overall structure of this region shows little similarity
to the B2 isoform. We have begun to examine promoter
elements responsible for induction of B2 expression during monocytic
differentiation and have found that elements downstream of the
transcriptional start site are critical for B2 regulation. We found
that a 179-bp fragment surrounding the transcriptional start site
(-274 to -96) could mediate TPA induction as efficiently as
a fragment containing an additional 2.3 kb of 5`-flanking region.
Deleting all but 8 bp of the 5`-untranslated region abolished the
phorbol ester sensitivity. Because the B2 gene is expressed in all cell
types tested, we were unable to test these promoter fragments for
cell-specific basal expression levels. We were, however, able to show
that another human cell line, 293, was unable to mediate the phorbol
ester-induced expression by B2 promoter construct. Induction of B2
promoter activity may be mediated by a unique protein-binding sequence;
alternatively, the numerous potential AP2 binding sites present in the
5`-untranslated sequence may mediate this effect. No other known
sequences which mediate phorbol ester responses were found in this
region. AP2 is a well characterized transcription factor that mediates
responses to phorbol ester and cAMP(48) . It is also responsive
to retinoic acid stimulation of teratocarcinoma cells and may be
required for retinoic acid-induced differentiation in developing
vertebrates(55) . AP2 expression is amplified in a few tissues
during embryogenesis, including developing brain and kidney, two
tissues that express very high levels of B2 isoform mRNA. Expression of
AP2 in brain and kidney continues into adulthood(56) . More
experimentation will be required to determine whether AP2, or some
other factor is responsible for the effects of phorbol ester on B2
subunit transcription in THP-1 cells. In addition to AP2 sites, we
found two Sp1 binding sites in the 5`-untranslated region. Sp1 is a
ubiquitously expressed protein with binding sites in many promoters. It
is often found in clusters and is thought to be involved in linking
distant control elements(57, 58) . Unlike AP2, it is
not thought to regulate phorbol ester responsiveness of promoters. These studies represent the first attempts to examine mechanisms of
genetic regulation of the mammalian vacuolar ATPase. We have shown that
amplification of certain subunits occurs in differentiating monocytes,
and that transcriptional and post-transcriptional mechanisms are likely
to be involved. We have also isolated B2 subunit gene fragments that
mediate this amplification. Experiments like these in our monocyte
system and in other cell systems should provide us with an
understanding of mechanisms controlling tissue-specific expression of
the V-ATPase. These kinds of studies will be most illustrative for
tissue and cell types that express very high levels of this enzyme,
such as kidney, brain, and the osteoclast, a cell type related to the
macrophage.
Volume 270,
Number 13,
Issue of March 31, 1995 pp. 7320-7329
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-ATPase B2 Subunit Gene in
Differentiating THP-1 Cells (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-ATPase (V-ATPase). We examined mRNA levels of
various V-ATPase subunits during differentiation of both native
monocytes and the cell line THP-1, and found that transcriptional and
post-transcriptional mechanisms could account for increases in cell
V-ATPase content. From nuclear runoff experiments, we found that one
subunit in particular, the B2 isoform (M
=
56,000), was amplified primarily by transcriptional means. We have
begun to examine the structure of the B2 subunit promoter region.
Isolation and sequencing of the first exon and 5`-flanking region of
this gene reveal a TATA-less promoter with a high G+C content.
Primer extension and ribonuclease protection analyses indicate a single
major transcriptional start site. We transfected promoter-luciferase
reporter plasmids into THP-1 cells to define sequences that mediate
transcriptional control during monocyte differentiation. We found that
sequences downstream from the transcriptional start site were
sufficient to confer increased expression during THP-1 differentiation.
DNase I footprinting and sequence analysis revealed the existence of
multiple AP2 and Sp1 binding sites in the 5`-untranslated and proximal
coding regions.
-ATPase, or V-ATPase, (
)performs a diversity of functions in establishing and
maintaining organellar, cytoplasmic, and extracellular pH. This
multisubunit enzyme is responsible for acidification of endosomal (1, 2) and lysosomal (3) compartments,
contributing to cellular processes such as protein transport and
degradation. In specialized cell types such as the osteoclast (4) or renal intercalated cell(5) , the V-ATPase
secretes acid from the cell in a vectorial manner by polarization of
the enzyme to a specific membrane domain.
-F ATPases in that the enzyme has a ``studlike'' or
``peg'' shape, with the head forming a catalytic cytoplasmic
domain, while the stem attaches to a membrane-spanning domain forming a
proton channel. The catalytic domain contains a M = 70,000 or ``A'' subunit, the site of ATP
hydrolysis, and a M = 56,000 or
``B'' subunit whose precise function is unknown. Three copies
of each polypeptide assemble to form the head structure(6) .
Although two A subunit isoforms have been identified in
plants(7) , until recently, only one mammalian form of the A
subunit was thought to exist. This form is ubiquitously expressed in
all tissue types examined, and cDNA clones have been isolated by our
laboratory and others(8, 9, 10) . A cDNA
clone for a second A subunit isoform was recently isolated from a human
osteoclastoma(11) . RNA hybridization analysis of multiple
mammalian tissues revealed no expression of this form in tissues other
than the osteoclastoma. = 56,000
or B subunit family includes at least two members whose cDNAs have been
isolated by our laboratory and
others(12, 13, 14) . One of these, the B2 or
``brain'' isoform, is expressed at moderate to high levels in
all tissues studied, while the B1 or ``kidney'' isoform is
amplified in kidney cortex and medulla, with lower levels of expression
in most other tissues. While different B isoforms predominate in
different tissues(14, 15) , it is not yet clear if
these isoforms vary in function. = 15,000 subunit, a proteolipid thought to be the major
structural component of the transmembrane proton
channel(16, 17) , although immunoprecipitation of
V-ATPase reveals the presence of multiple low molecular weight
polypeptides that may also reside in the membrane(18) . It is
believed that six proteolipid polypeptides assemble to form the
transmembrane channel (6) . = 31,000) (19) and the C (M = 44,000) (20) subunits.
Immunologic evidence exists for heterogeneity of the E
subunit(21) ; only a single form of the C subunit has been
reported(20) . Recent evidence indicates that while the C
subunit enhances activity of the V-ATPase, it is not required for
enzyme activity(22) , although gene deletion studies of the
homologous subunit in Saccharomyces cerevisiae indicate that
the subunit is essential(23) . An integral membrane protein of M = 95,000-120,000 has been
copurified with the V-ATPase from some mammalian, plant, and yeast cell
extracts (reviewed in (24) ). Mutation of the gene for this
subunit in Saccharomyces results in loss of
bafilomycin-sensitive ATPase activity and proton transport. However, a
requirement for the M =
95,000-120,000 subunit may not be universal among all species.
Active V-ATPase isolated from mammalian kidney (18) and
Golgi-enriched membranes lack this subunit(25, 26) . 
)Such variations in V-ATPase composition and
abundance among different cell types suggest that the expression of
V-ATPase subunits may be under tissue-specific control mechanisms,
allowing each subunit to be amplified or suppressed as required in
different cells. We were therefore interested to identify a model that
would allow us to examine how such regulation of V-ATPase subunits
occurs and found that cells of the monocytic lineage increase their
expression of V-ATPase during their differentiation into macrophages.
Materials
Unless otherwise indicated, all
reagents were obtained from Sigma and were reagent grade.Isolation and Culture of Cells
Peripheral blood
monocytes were isolated by countercurrent elutriation(33) .
Monocyte-derived macrophages were obtained by culturing purified
monocytes on plastic Petri dishes in RPMI 1640 (Sigma) with 12.5% human
AB serum (North American Biologicals, Inc., Miami, FL), 2 mML-glutamine, 1 mM sodium pyruvate, and 20
µg/ml gentamicin at 37 °C in a 5% CO incubator.
THP-1 cells were obtained from the American Type Culture Collection,
Rockville, MD, and were cultured in RPMI 1640 with 10% fetal bovine
serum (HyClone, Logan, UT), 2 mML-glutamine, and 20
µg/ml gentamicin in a 5% CO incubator. THP-1 cells were
induced to differentiate by addition of 160 nM 12-O-tetradecanoylphorbol-13-acetate (TPA). Upon addition
of TPA, THP-1 cells, which are propagated in suspension, stop dividing,
become adherent, and differentiate to a macrophage-like state. The
human embryonic kidney cell line 293 was obtained from the American
Type Culture Collection and cultured in modified Eagle's medium
with Earle's salts, 10% fetal calf serum, and 20 µg/ml
gentamicin.Antibodies
Monoclonal antibody E11 was used for
immunoprecipitation and immunoblot detection of the V-ATPase E (M = 31,000) subunit(21) .
Anti-peptide rabbit antiserum rB1-CT was utilized for detection of the
B1 isoform subunit. The B2 isoform was detected with rabbit
antiserum rB2-NT, derived to a fusion protein construct encompassing
the N-terminal 120 amino acid residues added to the carboxyl terminus
of the Escherichia coli maltose binding protein.Immunoprecipitation
For metabolic labeling
of monocytes or macrophages, one 100-mm tissue culture plate of cells
was incubated with 100 µCi of Tran
S-label (ICN) in 4
ml of methionine-free medium overnight. 1 
10
human
peripheral blood monocytes or macrophages were solubilized by 14 passes
through a motorized Teflon-glass homogenizer (Wheaton, Millville, NJ)
in solubilization buffer (20 mM Tris-Cl, pH 7.4, 5 mM sodium azide, 1 mM EDTA, 1 mM dithiothreitol, 1%
Triton X-100, and 0.1% SDS). Insoluble material was pelleted at 150,000
g for 1 h at 4 °C, and H-ATPase
complexes were precipitated from the supernatant using monoclonal
antibody E11 coupled to protein A-Sepharose as described
previously(18) . After a 2-h incubation at 4 °C, the
E11-coupled beads were pelleted briefly in a microcentrifuge and washed
three times with ice-cold Net-gel buffer(34) . The supernatants
from the first immunoprecipitations were removed to separate tubes, and
a second precipitation was performed to verify quantitative recovery of
V-ATPase complexes. Immunoprecipitated proteins were solubilized in
SDS-PAGE gel-loading buffer, and one-third of each sample was loaded
onto a 10% SDS-polyacrylamide gel. Following electrophoresis, the gel
was fixed, treated in Fluorohance (RPI, Mount Prospect, IL), and
juxtaposed to film.Immunoblots
For whole cell extracts, 0.5
10 human peripheral blood monocytes or macrophages were
solubilized by heating for 10 min at 95 °C in SDS-PAGE gel sample
buffer, and samples were applied directly to lanes of a
SDS-polyacrylamide gel. After separation by SDS-PAGE, proteins were
transferred to Immobilon-P membrane (Millipore, Bedford, MA), and the
membrane was incubated overnight at 4 °C in blocking
solution(34) . Primary antibody E11, as tissue culture
supernatant, or rB1-CT or rB2-NT antiserum diluted 1:300 in blocking
solution, was incubated with the membranes for 2 h at room temperature.
Following washing of the membrane using standard conditions,
horseradish peroxidase-conjugated secondary antibodies were incubated
with the membrane(34) , and bound proteins were detected by
chemiluminescence using the ECL reagents and protocol from Amersham
Corp.cDNA Probes and Antibodies
A 1.1-kb cDNA clone for
the human V-ATPase M = 15,000 (proteolipid)
subunit was the kind gift from S. Reeders (Harvard University). A
0.27-kb ApaI-EcoRI fragment from the 3` end of this
clone was used as a probe for RNA hybridization (Northern) analysis,
while the intact cDNA in a pBluescript vector (Stratagene, La Jolla,
CA) was used as a probe for the nuclear runoff assay. The cDNA for the
human E subunit was isolated as reported previously(21) . The
full-length 1.3-kb fragment in pBluescript was used for the nuclear
runoff assay, while a 0.43-kb EcoRI-KpnI fragment
from the 5` end was used for the RNA hybridization analysis. A cDNA
clone for the human V-ATPase B1 (kidney) isoform cDNA was isolated by
polymerase chain reaction from a human kidney cDNA library (a generous
gift from Dr. Graham Bell, University of Chicago) using
oligonucleotides derived from the published sequence(12) . A
400-base pair fragment from the 5` end was used directly for RNA
hybridization analysis, and was subcloned into pBluescript for the
nuclear runoff analysis. A cDNA clone for the human V-ATPase B2 (brain)
isoform encompassing the start codon was isolated
previously(14) . From this clone, a 0.56-kb EcoRI-ClaI fragment from the 5` end was used for both
RNA hybridization and nuclear runoff analysis. Although the cDNA clones
for the B1 and B2 subunit isoforms are highly homologous in some
internal regions(14) , the 5` regions used here as
isoform-specific probes exhibit an identity of only 65%, and therefore
specific hybridization to a single isoform was easily attained under
high stringency conditions. A 2.5-kb partial cDNA clone for the human
V-ATPase A subunit was also isolated in this laboratory. (
)This clone, which extends from nucleotide 840 in the
coding region to the poly(A) tail, was used directly for RNA
hybridization and was subcloned into pBluescript for the nuclear runoff
assay. A cDNA probe for the human enzyme glyceraldehyde-3-phosphate
dehydrogenase, obtained from Clontech (Palo Alto, CA), was used for
normalization of mRNA levels in the RNA hybridization analysis. Isolation of RNA and Hybridization Analysis
Total
cellular RNA was isolated from cells by treatment with guanidinium and
acid-phenol(35) . Poly(A)-selected mRNA was prepared with a
FastTrack commercial isolation kit (Invitrogen, San Diego, CA), using
the manufacturer's protocol. RNA was electrophoretically
separated in a formaldehyde-containing agarose gel under standard
conditions, and was transferred to a Nytran membrane (Schleicher &
Schuell, Keene, NH). Probes were radiolabeled with
[
-
P]dCTP using the Prime-a-Gene random
priming labeling kit from Promega (Madison, WI), and were allowed to
anneal bound RNA using a hybridization solution recommended by the
manufacturers of Nytran. Final washes of the membrane prior to exposure
to film were usually in 0.2 SSPE, 0.1% SDS at 55 °C, unless
specified otherwise in the text.Nuclear Runoff Analysis
Preparation of THP-1
nuclei and radiolabeling of nascent transcripts were performed as
described by Greenberg(36) . Radiolabeled RNA was isolated by
the guanidinium thiocyanate acid-phenol method described above.
Concentrations of radiolabeled RNA samples from different time points
were adjusted to equal counts/min and were added to nylon membranes to
which 10 µg of linearized denatured plasmid containing the
appropriate cDNA probe had been applied in a slotted filtration
manifold (Bio-Rad). Hybridization and washing of the membrane strips
were performed as described by Greenberg(36) .Southern Analysis, Polymerase Chain Reaction (PCR), and
DNA Sequencing
DNA hybridization (Southern) analysis was
performed by standard procedures. Following electrophoretic separation
of DNA, the DNA fragments were transferred to a Nytran membrane
(Schleicher & Schuell) using a Vacu-Blot vacuum transfer apparatus
(Hoefer). Hybridization of P-labeled probes to
membrane-bound DNA was performed under standard
conditions(37) . Final washes of the membrane prior to exposure
to film were usually in 0.2 SSPE, 0.1% SDS at 55 °C, unless
specified otherwise in the text.
)derived from a human fetal brain
library (Clontech). PCR was performed under standard conditions using
the following cycling temperatures and times: 94 °C, 40 s; 58
°C, 1 min; 72 °C, 40 s. After 30 cycles, the product was run in
2.0% low melting-point agarose. The gel slice was excised and melted,
and PCR was performed from 5 µl of the gel slice as above, except 5
µCi of [-P]dCTP (3,000 Ci/mM)
were added to the reaction mixture. After 15 cycles, the product was
purified over a Sephadex G-50 spin column for use in hybridization. S-dATP into the extended
products.Isolation of Promoter Fragments
1 10 plaques from a WI38 human lung fibroblast genomic DNA 
phage
library (courtesy of V. Sukhatme, Harvard University) were screened
with an 85-bp PCR-amplified cDNA probe corresponding to a 5` region of
the B2 subunit mRNA (described above). Hybridization and washing
conditions were the same as those described for Southern analysis,
above. Following two rounds of screening, two clones were
isolated. From one of these, two overlapping subclones, pBSL217 and
pBSL220 were isolated. Further details are described in the text.Primer Extension and Ribonuclease Protection
Assays
A commercial kit (Promega) was used for the primer
extension analysis. Eight micrograms of total cellular RNA from resting
or induced THP-1s were annealed with either of two P-end-labeled antisense primers: 5`-ATCTTGTCTCCTCTGTCC-3`
(+2 to -16); or 5`-ACGGGTAGCTCGGGTGCG-3` (+56 to
+39). The primer extension reactions were performed with avian
myeloblastosis virus reverse transcriptase according to the
manufacturer's instructions. Extension products were separated in
a 6% polyacrylamide gel containing 1 TBE (0.09 M Tris
borate, 2 mM EDTA, pH 8.3) as a buffer, and using P-labeled 
X174-HinfI fragments as markers.
The gel was fixed in 25% ethanol, dried, and exposed to film.
Ribonuclease protection assays were performed using the RPA II kit
(Ambion, Austin, TX). 0.5-1.0 µg of poly(A)-selected mRNA
from TPA-induced THP-1s or 293 cells were hybridized overnight at 45
°C with a P-labeled RNA probe made using a Riboprobe
(Promega) transcription system and T7 RNA polymerase. The probe
contained 222 bp of 5`-untranslated and flanking regions (between a NarI site at -96 and an MluI site at
-317) plus 33 bp of pBluescript polylinker. Following RNase
digestion according to the manufacturer's protocol, protected
fragments were separated in a 6% polyacrylamide gel with 1 TBE
buffer. The gel was fixed in 25% ethanol, dried, and exposed to film.Promoter Fusion Constructs
For construction of
promoter-luciferase fusion plasmids, the 5`-flanking regions from
pBSL217 and pBSL220 were first spliced together in pBluescript at a
common SpeI site. The resulting plasmid, pBSL222, contained
approximately 4.5 kb of B2 subunit 5`-flanking region plus 30 bp of
coding sequence. From this spliced promoter region, smaller fragments
were isolated and cloned into pGL2-basic (Promega) upstream of the
luciferase coding sequences.Transfection of Cells
For optimal transfection
efficiency, cells were cultured at a density of 2-5
10
/ml, and were grown continuously for no more than 6
weeks. Transfections of THP-1 cells were performed essentially as
described by Mackman et al.(38) . Each 100-mm plate of
cells was transfected with 5 µg of the test plasmid and 2 µg of
pRSVcat as an internal control. Forty-six h after transfection,
cultures were divided in half, and each half was incubated an
additional 5 h in the presence or absence of 160 nM TPA. Luciferase and Chloramphenicol Acetyltransferase
Assays
Cell extracts for luciferase and chloramphenicol
acetyltransferase activity measurements were prepared by standard
procedures(40) . Chloramphenicol acetyltransferase activity was
measured by a modification of the two-phase diffusion assay of Neumann et al.(41) , except the final reaction mixture
contained 20 mM Tris-Cl (pH 8.0), 1 mM chloramphenicol, 30 µM acetyl-CoA, and 0.4 µCi of
[H]acetyl-CoA (ICN, 5-15 Ci/mmol). DNase I Footprinting
A genomic EcoRV-SacI fragment spanning from -338 to
+84 with respect to the start of translation was subcloned into
pGEM-3Z (Promega). The fragment was radiolabeled at one end with
[
-P]ATP and T4 polynucleotide kinase
(Promega) and was purified from pGEM-3Z following restriction enzyme
digestion and agarose gel electrophoresis(38) . The probe was
further purified with an Elutip-D column (Schleicher & Schuell),
following the manufacturer's instructions, except the DNA was
eluted in 0.4 ml of a high salt buffer containing 2.0 M NaCl,
20 mM Tris-Cl (pH 7.4), and 1 mM EDTA.
Characterization of V-ATPase Protein Levels in
Monocytic Cells
Since macrophages develop an extensive lysosomal
system as part of their capacity to digest pathogens and present
antigen, we examined whether differentiating monocytes could serve as a
model system for studies of V-ATPase gene regulation. Fig. 1shows an immunoprecipitation of assembled V-ATPase
complexes from fresh human peripheral blood monocytes and from
macrophages obtained by culturing monocytes in vitro for 5
days (left panel). The immunoprecipitates contained several
subunits known to be present in the mammalian vacuolar
H-ATPases, including the A (70 kDa), B (56 kDa), C
(
40 kDa), E (31 kDa), and proteolipid (15 kDa) subunits. ()
10 cells. The
A (M = 70,000), B (M=56,000), E (M = 31,000), and proteolipid (M = 15,000) subunits are indicated. Two additional
polypeptides at 45 and 40 kDa are present in the immunoprecipitates.
Immunoprecipitation in the presence of excess E11 peptide demonstrated
that the 40-kDa polypeptide is precipitated specifically with the
other V-ATPase subunits and probably represents the C subunit; the
45-kDa polypeptide is precipitated nonspecifically (data not
shown).
10 cells were transferred to a membrane and
probed with antibodies to the B1, B2, and E subunits. B1 subunit
protein (M = 58,000) was not detectable
either in monocytes or in macrophages. In contrast, the B2 (M = 56,000) and E subunit (M = 31,000) were detectable on immunoblots
of both cell types, and levels of both subunits increased significantly
in the macrophages.
10 human monocytes (lane
2), monocyte-derived macrophages (lane 3), or 10 µg
of bovine kidney microsomes as a positive control (lane 1).
Proteins were fractionated and transferred to membranes as described
under ``Experimental Procedures,'' and probed with antibodies
to the V-ATPase B1 (M = 58,000), B2 (M = 56,000), or E (M = 31,000) subunits as indicated. Size of immunoreactive
polypeptides is indicated as M
10
.
Steady-state mRNA Levels of Only the A and B2 V-ATPase
Subunits Increase during Monocytic Differentiation
In order to
examine the mechanisms regulating changes in V-ATPase subunit
expression during monocyte differentiation, we performed an RNA
hybridization analysis on total cellular RNA isolated from THP-1 cells
at different time points after TPA treatment. Fig. 3A (top) shows an RNA blot probed with a partial cDNA
fragment specific for the B2 brain isoform. The experiment demonstrates
that steady-state levels of the B2 subunit message increase after TPA
treatment, reaching maximal levels at 24 h (further time points not
shown). To determine whether this regulation occurs in native monocytes
as well as the THP-1 cell line, we analyzed mRNA levels for the B2
subunit in total RNA from human peripheral blood monocytes and from
monocyte-derived macrophages after 5 days in culture (Fig. 3B). As shown, mRNA levels for the B2 subunit
were similarly increased following differentiation of native monocytes.
In both experiments, the membranes were also probed with a cDNA to
glyceraldehyde-3-phosphate dehydrogenase (GPDH) as a control
for applied mRNA. In several experiments, densitometry measurements
yielded an average 3.1-fold increase (3.1 ± 0.4, n = 4) in the B2 subunit message in THP-1 cells when
normalized for glyceraldehyde-3-phosphate dehydrogenase. Similarly, the
mRNA level for the B2 subunit increased 3.1-fold in human
monocyte-derived macrophages as compared to peripheral blood monocytes (Fig. 3B).
-P]UTP and allowed to hybridize to
cDNA probes for the indicated V-ATPase subunits and controls bound to
nitrocellulose filters.
The B2 Brain Isoform Is Regulated Primarily at the
Transcriptional Level
To determine whether the increases in
steady-state mRNA levels of the A and B2 subunits were due to
transcriptional or post-transcription mechanisms, we performed a
nuclear runoff analysis on nuclei from THP-1 cells before, and 24 h
after, TPA treatment. Radiolabeled transcripts were allowed to
hybridize to cDNA probes for several V-ATPase subunits, and to the
cloning vector pBluescript and plasmids containing 
-actin and
c-fos cDNAs as controls. Although c-fos is known to
be activated transcriptionally immediately upon initiation of monocytic
differentiation, transcription returns to baseline levels by a few
hours after induction in THP-1 cells and can therefore be used as a
standard for transcript levels (42) .Isolation of the B2 Subunit First Exon and 5`-Flanking
Sequences
1 10 plaques of a human genomic
library were initially screened with a restriction fragment of the B2
subunit cDNA corresponding to nucleotides -19 to +530 with
respect to the translational start(14) . Because this probe
displayed only 65% identity with the corresponding region of the B1
isoform, the isoforms could be distinguished using high stringency
hybridization and washing conditions. After three rounds of screening,
two overlapping clones of >20 kb were isolated. From one of
these isolates, a 5.0-kb XbaI fragment that hybridized to the
original probe on a Southern blot was subcloned. Sequencing of this
fragment using primers corresponding to the B2 subunit coding region
revealed a region of homology with the B2 subunit coding region that
extended from +67 to +259. However, this sequence showed only
86% identity with the B2 subunit cDNA sequence. This sequence may
represent a third B subunit isoform or a pseudogene. This fragment was
used as a probe for RNA hybridization analysis on poly(A) mRNA from multiple human tissues (Clontech), but no hybridization
was detected. clone was digested with
several restriction enzymes and analyzed by DNA hybridization, using
the 5` 620 bp of pBSL217 as a probe. A hybridizing 4.0-kb SpeI fragment was found which overlapped pBSL217 by
approximately 0.8 kb. This fragment was subcloned into the SpeI site of pBluescript and designated pBSL220. A restriction
map of the 5`-flanking regions spanned by pBSL217 and pBSL220 is shown
in Fig. 5.
+100. Over this
region, the exon sequences are 78% G+C. Notably, the region
between the transcriptional start site and +100 corresponds
precisely to the sequences of the B2 subunit cDNA that exhibit no
significant homology to the B1 isoform (14) . Downstream of
+100, the percent G+C of the first exon drops to 60%; in this
region the two isoforms show >70% identity at the nucleotide level.
This suggests that the (G+C)-rich character of the B2 subunit
first exon may play a specific role in regulation of this gene. A
second notable feature of the B2 subunit gene is the presence of two
GA/CT stretches, from -653 to -626, and from -313 to
-220. These regions are similar to previously described
``GAGA boxes'': purine- or pyrimidine-rich sequences present
in the promoters of many genes (44) . The protein that binds to
these stretches, ``GAGA factor'' is a transcriptional
activator that probably acts by rearranging chromatin
structure(45) . Finally, the (G+C)-rich region of the
5`-untranslated region and coding sequences contain multiple sites for
Sp1 and AP2 binding (discussed below), although the 5`-flanking region
does not.
/DDBJ under accession
number Z37165.
Determination of the Transcriptional Start
Site
Because the sequence data from the regions 5` to the B2
subunit coding region showed no apparent TATA box or transcription
initiator (Inr) sequences, both primer extension and ribonuclease
protection assays were performed to determine the transcriptional start
site(s) of the B2 subunit message. Primer extension analysis was
performed on total cellular RNA from both resting (data not shown) and
induced THP-1s (Fig. 7). Two primers were used for each RNA
sample. Primer 1 (lane 1) corresponds to the region from
+2 to -16, and primer 2 (lane 2) corresponds to
+56 to +39. Fig. 7shows that extension from each of
these primers results in one major product, differing in size by 54 bp,
as expected from differences in the annealing sites of the two primers.
Primer 1 yielded a product of 209 bp; primer 2 gave a product of 263
bp. From the length of the extension products, the 5`-untranslated
region of the B2 mRNA is calculated to be 207 bp. The same results were
obtained when using RNA from resting THP-1 cells (data not shown). To
confirm that the cloned genomic sequences contain the transcriptional
start site, we performed a ribonuclease protection assay using an RNA
probe containing sequences from -96 to -317 (NarI
to MluI), plus 33 bases of pBluescript polylinker, for a total
of 255 bases. We allowed the probe to hybridize with poly(A)-selected
mRNA from either induced THP-1 cells, 293 cells, or a negative control. Fig. 8shows that a major fragment was protected for both THP-1
and 293 cells. When these same reactions were run on a sequencing gel
with a sequencing ladder marker, the size of the protected fragment was
determined to be 111 bases (data not shown), confirming the location of
the transcriptional initiation site obtained from the primer extension
assays. However, in addition to the major band, a minor protected
fragment of 36 bases was also seen in the ribonuclease protection assay (Fig. 8). This result suggests a possible minor, alternative
transcriptional start site at -131. However, this result was not
confirmed by the primer extension analysis.
P-Labeled X174-HinfI fragments are shown as
size markers. The single major bands in each lane differ in size by 54
bases, as expected, and indicate a 5`-untranslated region of 207
bp.
P-labeled RNA probe
spanning from -317 to -96 was allowed to hybridize to
0.5-1.0 µg of poly(A)-enriched RNA from either THP-1 (lane 3) or 293 cells (lane 4), and digested with
RNase. Lane 2 shows the probe incubated with no RNA to control
for probe self-hybridization. Lane 1 is an overnight exposure
of undigested probe; lanes 2-4 are 5-day exposures of
the hybridizations indicated. The major protected fragment of 111 bp
and minor protected fragment of 36 bp are
indicated.
Transfection of Promoter-Reporter Fusion Constructs in
THP-1 Cells
To determine whether the THP-1 model of monocyte
differentiation may be used for dissecting control elements of the B2
subunit, we prepared several constructs with different promoter
fragments ligated to the 5` end of a luciferase reporter gene, and
transfected them into THP-1 cells. The initial promoter fragments
terminated at -96 (NarI site) and therefore contained
the start of transcription, and about one-half of the 5`-untranslated
region. Upstream boundaries of the promoter fragments ranged from
-274 to -2.4 kb. THP-1 cells showed similar levels of basal
luciferase activity when transfected with each of the constructs (data
not shown). In transfected THP-1 cells incubated for 5 h in the
presence of TPA, luciferase activity increased between 3- and 3.5-fold
for each of the constructs tested (Fig. 9, solid bars).
These values correlate well with the increase in transcription rates
found in the nuclear runoff analysis for the B2 subunit (3.5-fold; see Fig. 4). This indicated that the response element(s) for TPA
inducibility lie proximal to the first exon, within the range of
-274 to -96. Because the sequences in this region upstream
of the transcriptional start site contained only GAGA sequences and a
short (12 bp) (G+C)-rich stretch, it was surmised that sequences
downstream of the start site may contain important regulatory elements.
A promoter-luciferase construct from -274 to -199 was
therefore prepared, which lacked all but 8 bp of the 5`-untranslated
region. In contrast to all other B2 promoter fragments tested, this
construct did not exhibit any induction of luciferase activity by
phorbol ester treatment. These results demonstrate the importance of
the sequences downstream of the transcriptional start site in phorbol
ester induction of promoter activity.
)was also tested in THP-1 cells and was unable to mediate
induction of luciferase activity by TPA (Fig. 9, solid
bars).Transcription Factor Binding Sites in the 5`-Untranslated
and Coding Regions
Sequence analysis of the 5`-untranslated
region of the B2 promoter revealed several potential Sp1 and AP2
binding sites. Both of these factors bind to consensus sequences with a
high G+C content. To determine whether these sequences could
function as actual binding sites, we performed DNase I footprinting
analysis on a promoter fragment extending from -338 to +84 (EcoRV-SacI fragment) incubated in vitro with AP2 and Sp1 proteins. Results are shown in Fig. 10. In
the left panel, the promoter fragment was incubated with
purified Sp1 protein obtained from a commercial source. Two clear Sp1
binding sites (GC boxes) were found, centered around -110 and
-77. Both sites conform well to the weight matrix analysis
prediction of Bucher (46) for GC boxes. In the right
panel, the same fragment was tested for the presence of AP2
binding sites, using both purified AP2 and recombinant E. coli AP2 extracts. Five AP2 sites were found, including two in the
coding region of the B2 mRNA, centered around -123, -63,
-24, +7, and +16 (see Fig. 6for details). With
the exception of the site at -24, all conform to the consensus
GSSGNNGSS. This is in good agreement with the palindromic consensus
sequence proposed by Williams and Tjian (47) for the core AP2
binding site, GCCNNNGGC. The remaining site at -24 is a
palindromic sequence, CTGGGCCAG, with similarity to the high affinity
AP2-binding site in the human growth hormone gene(48) .
We have isolated 5`-flanking
regions from the human B1 gene, and are currently in the process of
analyzing the control elements responsible for enhanced transcription
through the use of promoter-reporter constructions in transfected cell
systems. From our immunoblot analysis, we showed that
monocytes and macrophages express only the B2 isoform of the B subunit.
Although B1 protein was undetectable, the message for this isoform was
found in both monocytes and macrophages. Translation of the B1 protein
may be repressed in these cells. On hybridization analysis of
poly(A)-enriched RNA, B1 isoform levels were comparable to those of the
A subunit, yet the A subunit protein is easily detected in both
monocytes and macrophages.
)-ATPase; TPA,
12-O-tetradecanoylphorbol-13-acetate; PCR, polymerase chain
reaction; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s);
kb, kilobase(s).))))-ATPase represents the V complex that has
dissociated from the membranes.)
We are indebted to Dr. Howard Welgus, Washington
University, for suggesting the use of the THP-1 cell line and for many
valuable suggestions. We thank Suzanne Pontow and Dr. Philip Stahl for
providing the human monocytes used in these studies, Dr. Raoul Nelson
and Xiaoli Guo for helpful discussions, Dr. Kenneth Murphy for use of
the luminometer, Bisola Ojikutu for aid with the transfections, and
Irina Krits for technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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