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(Received for publication, August 4, 1994; and in revised form, October 14, 1994) From the
The molecular basis for the commitment of multipotential myeloid
progenitors to the eosinophil lineage, and the transcriptional
mechanisms by which eosinophil-specific genes are subsequently
expressed and regulated during eosinophil development are currently
unknown. Interleukin-5 (IL-5) is a T cell and mast cell-derived
cytokine with actions restricted to the eosinophil and closely related
basophil lineages in humans. The high affinity receptor for IL-5
(IL-5R) is composed of an Interleukin-5 (IL-5), ( Prior studies based on
binding and cross-linking experiments on murine B cell lines suggested
a two-chain model for the IL-5 receptor (IL-5R): a unique Like other hematopoietic genes, expression of the
cytokine receptor genes are likely regulated in part at the
transcriptional level in a lineage-specific and temporal, developmental
manner. Regulation of IL-5R expression is extremely pertinent to an
understanding of the processes involved in the commitment and
differentiation of multipotential hematopoietic progenitors to the
eosinophil lineage. However, the mechanisms for the transcriptional
control and tissue-specific expression of human cytokine receptor genes
such as IL-5R
Figure 6:
Functional activity of the IL-5R
Figure 7:
Activity of IL-5R
Figure 1:
Structure of the 5` upstream region of
the IL-5R
Figure 2:
RT-PCR analysis of mRNA for the IL-5R
Figure 3:
Primer extension analysis; IL-5R
Figure 4:
RNase protection to locate the 5`-most end
of the IL-5R
Figure 5:
Nucleotide sequence of the IL-5R
Figure 8:
Lineage specificity of IL-5R
Figure 9:
Gel mobility shift analysis of the
IL-5R
Little is currently known regarding the mechanisms by which
human cytokine and growth factor receptor genes are expressed and
regulated during the commitment and differentiation of hematopoietic
progenitors to the myeloid lineages in general or the eosinophil
lineage in particular. In addition to genes encoding the murine (52) and human IL-5R Prior
studies indicate that IL-5 is a late-acting cytokine that demonstrates
maximum activity on an eosinophil progenitor pool expanded by the
earlier-acting, multipotential cytokines such as IL-3 or GM-CSF (10) . These observations suggest that IL-5, as a
tissue-specific cytokine, plays a crucial role in regulating the
development of the eosinophil lineage, and that the IL-5R The cDNA sequence and genomic structure of the human
IL-5R We have carefully mapped the transcriptional
start site of the IL-5R Activity of the promoter for
the IL-5R An analysis
of the lineage specificity of the IL-5R The
structure of the murine IL-5R Our current studies have served to identify a
functionally active promoter region for the human IL-5R
Volume 270,
Number 3,
Issue of January 20, 1995 pp. 1462-1471
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Subunit Gene (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunit (IL-5R) expressed by the
eosinophil lineage, that associates with a 
subunit
shared with the receptors for IL-3 and granulocyte-macrophage colony
stimulating factor (GM-CSF). As a prerequisite to studies of the
transcriptional regulation of the IL-5R subunit gene, we used
three different methods, including primer extension, RNase protection,
and 5`-RACE to precisely map the transcriptional start site to a
position 15 base pairs (bp) upstream of the 5` end of the published
sequence of IL-5R exon 1. To initially identify the IL-5R
promoter, 3.5 kilobases (kb) and 561 bp of the 5` sequence flanking the
transcriptional start site were subcloned into the promoterless
pXP2-luciferase vector. Transient transfection of these constructs into
an eosinophil-committed HL-60 subline, clone HL-60-C15, induced the
expression of 240-fold greater luciferase activity than the
promoterless vector, identifying a strong functionally active promoter
region within the 561 bp of sequence proximal to the transcriptional
start site and with activity equivalent to pXP2 constructs containing
the entire 3.5 kb of upstream sequence. To more precisely localize the cis-acting regulatory elements in this region important for
promoter activity, a series of 5` deletion mutants of the 561-bp region
were generated in the pXP2-luciferase vector. Deletion of the region
between bp -432 and -398 reduced promoter activity by more
than 80% in the HL-60-C15 cell line. Further analyses of the activity
of the IL-5R
promoter constructs in various other eosinophil,
myeloid, and non-myeloid cell lines indicated that the promoter was
relatively myeloid and eosinophil lineage-specific in its expression.
Consensus sequences for known transcription factor binding sites were
not present in the 34-bp region of the promoter required for maximal
activity, suggesting unique myeloid- and possibly eosinophil-specific
regulatory elements. Using electrophoretic mobility shift assays, we
have identified a nuclear factor(s) that binds to the 34-bp functional
region of the the promoter and that is expressed in the myeloid and
eosinophilic cell lines in which the promoter is active, but not in
non-myeloid or non-hematopoietic lines. This functional promoter
segment likely serves as the binding site for a myeloid- and possibly
eosinophil-specific transcription factor(s). Further study of the
IL-5R promoter should elucidate unique transcriptional features of
this gene whose expression is essential to the commitment and
differentiation of multipotential myeloid progenitors to the eosinophil
lineage and to the functional activation of the mature cell.
)produced primarily by
activated T cells (1) and mast cells(2, 3) ,
stimulates the proliferation and differentiation of murine activated B
cells and regulates the production of
eosinophils(4, 5, 6) . The proliferation,
differentiation, and maturation of eosinophils in the bone marrow and
their post-mitotic functional activation in tissues occurs in response
to a number of cytokines in addition to IL-5, including GM-CSF and IL-3 (7, 8, 9) . Both IL-3 and GM-CSF have
activities on other hematopoietic lineages, whereas IL-5 is more
eosinophil-specific and plays a crucial role in regulating the
differentiation and development of the eosinophil lineage(10) .
Although IL-3 and GM-CSF participate in the proliferation and
commitment of progenitors to the eosinophil lineage, IL-5 is both
necessary and sufficient for eosinophil development to
proceed(10, 11) . In humans, the high affinity
receptor for IL-5 is apparently restricted to eosinophils and
hematopoietically related basophils(12) ; in contrast to murine
B cells, the activity of IL-5 on human B cells is controversial (10, 13) and is still being delineated (14) .
Thus, the expression of the high-affinity receptor for IL-5 is an
important prerequisite and very early lineage-specific event in the
hematopoietic program for these granulocytes. Of interest, IL-5 is
active in vitro both in the production of eosinophils from
bone marrow and umbilical cord blood progenitors as well as in the
priming, activation, and enhanced survival of mature eosinophils.
Overexpression of IL-5 is observed in many eosinophil-associated
diseases(15, 16, 17) , and IL-5 transgenic
mice develop profound eosinophilia(18, 19) ,
indicating that IL-5 plays important roles in promoting the production
and function of eosinophils in vivo. subunit
(60-kDa component) corresponding to the low affinity IL-5 binding site (K = 10
M), and a
subunit (130-kDa component)
that is shared with the IL-3 and GM-CSF receptors and associates with
the chain to form the high affinity receptor (K = 5
10M) (20, 21, 22) . Only the
high affinity binding site is generated upon induction of eosinophilic
sublines of human promyelocytic HL-60 cells with butyric
acid(23, 24) . Recently, it has been found that the
high affinity IL-5R requires both
and subunits (25) for optimal signaling, and that the intracellular
cytoplasmic portion of the chain is essential to this process (64) . (
)The isolated subunit if the GM-CSFR
has likewise been shown to participate in signaling, albeit via a
phosphorylation independent pathway(26) . To clarify the role
of these two components of the IL-5 receptor in eosinophil
differentiation and biologic function, Tavernier et al.(27) and Murata et al.(28) have explored
the characteristics of the human IL-5 receptor (IL-5R) gene.
The gene encoding the IL-5R subunit is located on chromosome 3 in
the region 3p26(29) . The organization of this gene reflects
the functional domains of this protein and shares many characteristics
with other members of the cytokine/hemopoietin receptor gene
family(9, 24, 29) . Several alternatively
spliced transcripts have been identified in the mRNA and reflect the
membrane versus soluble isoforms(30) . Aside from its
ability to bind IL-5 in vitro(31) , the in vivo function(s) of the soluble form of the IL-5R have not been
elucidated. are currently unknown. To investigate the regulation
of human IL-5R expression, we first isolated the 5` upstream
region of the gene and mapped the transcriptional start site. We have
identified a functional promoter region upstream of the transcriptional
start site that is highly active in eosinophil-inducible myeloid
leukemic cell lines and is active in a myeloid- and eosinophil-specific
manner, and we have localized the minimum cis-acting sequence
required for promoter activity to a 34-bp region between bp -432
and -398 of the gene.
Genomic Cloning Procedure
Two human genomic
libraries, prepared from placental DNA in the FIX II phage vector
(Stratagene), were screened as described previously(29) . A KpnI fragment from the
ghIL5R
-2 clone (29) covering the 5` upstream region of the gene was subcloned
into the pGEM7ZF(-) plasmid (Promega) and sequenced on both
strands using the Sanger dideoxy method with Sequenase 2.0 (U. S.
Biochemical Corp.).Cell Culture
An eosinophil-committed subline of
the HL-60 promyelocytic leukemia cell line, HL-60-C15(32) , was
maintained in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented
with 10% fetal bovine serum (Hyclone Laboratories), 2 mML-glutamine, 100 units of penicillin, 100 µg of
streptomycin (Life Technologies Inc.) and passaged twice weekly.
HL-60-C15 cells (2 10
/ml) were induced with 0.5
mM butyric acid (Sigma), and total RNA was isolated after 1,
3, and 5 days of culture. Other myeloid and non-myeloid cell lines
utilized in these studies including the promyelocytic HL-60 line (ATCC
CCL 240), monocytic U937 line (ATCC CRL1593), BJA-B B lymphocytic cells (33) , REX T lymphocytic cells, and the cervical carcinoma
line, HeLa (ATCC CCL 2), were maintained by passage twice weekly in
RPMI 1640 (Life Technologies Inc.) (HL-60, U937, BJA/B, REX) or
Dulbecco's modified Eagle's medium (HeLa) supplemented with
10% fetal bovine serum (Hyclone) and 2 mML-glutamine
as described previously(34, 35) . In addition, two
recently described acute myeloid leukemia cell lines, AML14, a line
committed to the eosinophil lineage(36) , and AML14.eos, a
cytokine-induced fully differentiated eosinophilic myelocyte subline of
AML14 that continues to proliferate and maintain a differentiated
phenotype with cytokine supplementation every 4-6
weeks(37) , were cultured in RPMI 1640 (BioWhittaker) with 8%
fetal bovine serum (Life Technologies, Inc.), 2 mML-glutamine, 5 10M
-mercaptoethanol, and 1 mM sodium pyruvate as
described previously(36, 37) .RNA Isolation and RT-PCR Assay
Total RNA was
prepared from HL-60-C15 cells, HeLa cells, and peripheral blood
eosinophils obtained from a patient with hypereosinophilic syndrome
(HES) as described previously(38) . The reverse transcription
polymerase chain reaction (RT-PCR) method was used to investigate
whether there was an additional exon upstream of exon 1 of the
IL-5R cDNA. Briefly, 2 µg of total RNA was treated with
RNase-free DNase I. After removal of the DNase I by extracting twice
with phenol, cDNA was synthesized with 9 units of avian myeloblastosis
virus reverse transcriptase (Promega) using 0.1 µM oligo(dT) primer in a total volume of 20 µl. Three to five
µl of cDNA was added to a standard PCR mixture containing a 1
µM concentration of each primer. The PCR reaction was
performed on a thermal cycler using 40 cycles of 1 min, 45 s at 94
°C, 30 s at 48 °C, and 45 s at 72 °C. The final
polymerization step was extended an additional 10 min at 72 °C. Two
upstream primers were used for this experiment: one forward primer,
5`-GTCTTTTGAAAGGATCT-3`, was identical in sequence with the 5` end of
the IL-5R cDNA as described by Murata et
al.(28) , and the second, 5`-CCGCATTTCTCAGGCCAG-3`,
spanned the exon 1-2 boundary. The reverse primer for this
reaction, 5`-GGGAGAAGTGAAATCTTTTCATC-3`, spanned the exon 4-5
boundary.Primer Extension Assay
Primer extension was
performed essentially as described previously(39) . Briefly,
100 ng of a reverse primer spanning the exon 4-5 boundary (see
above) was labeled by T4 polynucleotide kinase. The labeled primer was
hybridized with 20 µg of RNA from either HL-60-C15 or HeLa cells at
33 °C overnight. After precipitation with 100% ethanol, the sample
was dried and resuspended in 10 µl of HO. The extension
reaction was performed with 40 units of avian myeloblastosis virus
reverse transcriptase under standard conditions at 42 °C for 90
min. The specific cDNA fragments were analyzed on a 6% polyacrylamide,
7 M urea sequencing gel.RNase Protection Assay
We generated a hybrid
cDNA/genomic DNA probe by PCR which consisted of a fusion between part
of exon 2 (95 bp), all of exon 1, and 194 bp of upstream genomic
sequence. This 325-bp DNA fragment was cloned into a pGEM-T-vector
(Promega). The complementary sense RNA (cRNA) probe for RNase
protection was synthesized with T7 polymerase using linearized plasmid.
Twenty µg of DNase I-treated total RNA from HL-60-C15 and HeLa cell
lines were hybridized overnight at 46 °C with the
[P]UTP-labeled cRNA probe in 40 µl of 80%
formamide, 40 mM PIPES, pH 6.5, containing 400 mM NaCl and 1 mM EDTA. After hybridization, unhybridized
cRNA probe was removed by digestion for 20 min at 37 °C with 40
µg/ml RNase A and 1 µg/ml RNase T1 in 350 µl of 10 mM Tris-HCl, pH 7.5, containing 300 mM NaCl and 5 mM EDTA, with 5 µg of carrier RNA added. Protected fragments were
ethanol-precipitated and analyzed on a 6% polyacrylamide sequencing gel
as above.
Rapid Amplification of 5` cDNA End Assay
The
IL-5R cDNA was reverse-transcribed from 10 µg of total RNA
isolated from the butyrate-induced HL-60-C15 cell line using the same
exon 4-5 boundary primer used for primer extension. The cDNA was
diluted to 1 ml with water, and excess primers were removed by two
rounds of centrifugal ultrafiltration on a Centricon 100 filter
(Amicon) for 20 min at 1,000 g. The retentate was
further reduced to 10 µl by evaporation and treated with 30 units
of terminal transferase (Life Technologies, Inc.) and 200 µM dATP in a final volume of 20 µl at 37 °C for 13 min,
followed by incubation at 67 °C for 5 min. The tailed cDNA was
diluted to 300 µl with 10 mM Tris buffer, pH 7.5, with 1
mM EDTA, and 10 µl were amplified by PCR as described
above, using 7 pmol of an oligo(dT)-adapter primer and 25 pmol of both
the short adapter primer and a specific reverse primer,
5`-GGCGAGGACCGTGTCTGTCGTGTCTAT-3`, from exon 2 of the IL-5R
sequence. The PCR products were cloned into a pBluescript-T-vector and
sequenced with T3 or T7 universal primers.Construction of Plasmids for Promoter Analysis
For
analysis of the putative promoter region of the human IL-5R
subunit gene, three reporter constructs in the promoterless
pXP2-luciferase vector were prepared as shown in Fig. 6; a
2.9-kb BamHI/KpnI DNA fragment from the
ghIL5R
-2 clone(29) , a 612-bp fragment covering all
of exon 1 and 561 bp of upstream sequence, and a 3.5-kb fragment
including both the 2.9-kb and 612-bp regions were cloned into the
promoterless pXP2-luciferase vector in the BamHI/KpnI, KpnI/XhoI, and BamHI/XhoI sites, respectively (Fig. 6A). Deletion mutants from the 5` end of the
-561/IL-5R-pXP2 plasmid were generated using PCR
amplification of the appropriate region with oligonucleotide primers,
cloning into a pBluescript-derived T-vector, followed by subcloning
into the pXP2-luciferase vector at BamHI and XhoI
sites. Deletion mutants containing 561, 517, 469, 432, 398, 179, and
-561/-377 bp of upstream sequence were prepared; all
deletion mutants were sequenced in their entirety to identify potential
PCR-generated errors and to confirm the 5` end of each mutant.
promoter. A, construction of the IL-5R promoter
constructs in the promoterless pXP2 luciferase expression vector. A
3.5-kb fragment, comprised of 2.9 and 0.6 kb of the 5`-flanking region
of IL-5R including all of exon 1, was subcloned into pXP2 and
analyzed for functional activity (see B and Fig. 7). B, functional activity of the longest IL-5R-pXP2 promoter
constructs. Constructs of IL-5R upstream sequence (A)
were transiently transfected along with a cytomegalovirus-human growth
hormone plasmid (CMV-hGH) as transfection control into uninduced
HL-60-C15 cells by electroporation. Luciferase activity was measured by
luminometry in cell lysates prepared 5 h post-transfection. Relative
light units (RLU) were corrected based on the concentration of hGH
(ng/ml) released into the culture supernatants. The mean ± S.D.
for three or more replicate experiments is
shown.
promoter deletion
mutants in uninduced HL-60-C15 cells. 5` deletion mutants of the
IL-5R promoter in the pXP2-luciferase vector were generated as
shown on the left and co-transfected with a CMV-hGH control
plasmid as in Fig. 6B. Relative promoter activity of
each mutant is shown in comparison to the wild type, -561-bp
IL-5R/pXP2 plasmid (100%).
Transient Transfections of Leukemic Cell Lines
The
promoterless luciferase plasmid pXP2 was used for all promoter studies (40) . A cytomegalovirus-human growth hormone (CMV-hGH)
plasmid, for use as an internal control in transfections, was provided
by Dr. Leonard Zon (Children's Hospital and Harvard Medical
School, Boston, MA). Plasmid DNA was prepared, and cells were
transfected as described previously using 1-1.5 10
cells per transfection with minor
modifications(34, 35, 41) . Transfection of
plasmid DNA was carried out by electroporation (35) using 5
µg of test plasmid, 25 µg of carrier plasmid, and 0.5-1
µg of the reference plasmid (CMV-hGH) expressing human growth
hormone. The HL-60-C15 subline and parental HL-60 cells were
electroporated at 310 and 300 V, 960 µF, respectively, and the
AML14.eos subline and AML14 parental cells at 350 V, 960 µF,
conditions optimized for these cell lines. The U937, BJA-B, REX, and
HeLa lines were electroporated at 300, 220, 220, and 150 V, 960 µF,
respectively, optimal conditions previously utilized for these
lines(34, 41, 42, 43, 44) .
Luciferase activity in cell lysates prepared 5 h post-transfection was
measured as relative light units (RLU) using a Monolight 2010
luminometer (Analytical Luminescence Laboratory, San Diego, CA) as
described previously(34, 41) ; cell extracts were
prepared in 500 µl of 1% Triton X-100, 25 mM Gly-Gly, 15
mM MgSO
, 4 mM EGTA, 1 mM dithiothreitol, and 100 µl of the extract (equivalent to
3
10
cells) was analyzed for luciferase
activity. The CMV-hGH co-transfection provided for standardization
among the different myeloid and non-myeloid lines, different plasmid
DNA preparations, and individual transfection experiments(43) ,
and the carrier DNA decreased variability due to differences in the DNA
preparations for the individual IL-5R promoter-pXP2
constructs(45) . An enhancer-containing vector, CMV-pXP2, was
used as a positive control for each transfection as well. Growth
hormone production was measured by assaying the culture supernatants
from transfected cells with a commercially available radioimmunoassay
kit (Nichols Institute Diagnostics, San Juan Capistrano, CA). The RLU
from individual transfections were normalized for transfection
efficiency based on the number of nanograms of growth hormone produced
per ml of culture supernatant. Individual transfection experiments were
repeated a minimum of three times with at least two different
preparations of plasmid DNA, and the results are reported as mean
RLU/ng/ml hGH (± S.E.) or as the mean percentage (± S.E.)
of the activity of the -561/luc construct in each experiment.Nuclear Extracts
Nuclear extracts were prepared
from HL-60, HL-60-C15, AML-14, AML-14.eos, U937, BJA-B, and HeLa cell
lines, essentially according to the method of Dignam et al.(46) with minor modifications. Since nuclear extracts were
prepared from protease-rich promyelocytic and eosinophilic cell lines,
we included two protease inhibitors, diisopropylphosphofluoridate
(Sigma) and phenylmethylsulfonyl fluoride (Sigma), at final
concentrations of 1 mM in the preparation of all nuclear
extracts.Gel Mobility Shift Assay
The probe for mobility
shift analysis in this study was a 93-bp fragment located in the bp
-469 to -377 region of IL-5R promoter. This fragment
was generated by PCR amplification. Prior to amplification, one of the
primers was labeled with T4 polynucleotide kinase and
[-
P]ATP. The PCR fragments were separated
from free primer by gel electrophoresis. The gel shift assay was
performed essentially as described by Singh et
al.(47) . Each binding reaction contained 2 µg of
crude nuclear extract with 4 µg of poly(dI-dC) and 1
10 cpm of P-labeled DNA fragment in 20 µl
of a buffer containing 20 mM Hepes (pH 7.9), 50 mM KCl, 5 mM EDTA, 1 mM dithiothreitol, 3 mM MgCl
, and 5% glycerol. After a 20-min incubation at
room temperature, the binding reaction was separated on a native 4%
polyacrylamide gel (19:1 bisacrylamide) in 0.25 TBE buffer at 5
V/cm for 4 h at room temperature. The gels were dried prior to
autoradiography using Kodak X-Omat XAR film and Lightning Plus
enhancing screen. Competition for nuclear factor binding to the
radiolabeled probe was performed by adding a 50-fold molar excess of
cold competitor DNA probe to the binding reaction prior to adding the P-labeled DNA fragment, followed by analysis on
nondenaturing, native gels as above.
RT-PCR Analysis of a Putative Exon 0 in the IL-5R
The cDNA sequence and genomic structure of the human
IL-5R
mRNA chain gene has been reported previously (27, 29) (Fig. 1). However, the structure of
the 5`-untranslated and upstream regulatory regions of the IL-5R
gene was not completely characterized. The 196-bp 5`-untranslated
region of the mRNA is encoded by exons 1-3 and 3 bp of exon
4(27, 29) . However, Murata et al.(28) reported an additional 18-bp sequence 5` of exon 1
(referred to here as ``putative exon 0'' for clarity) that
was not identified in the cDNAs cloned by Tavernier et
al.(27) , as well as the absence of exon 3 in their cDNA
sequence(28) . These conflicting findings made the precise
localization and characterization of the transcriptional start site a
prerequisite for further analysis of the regulatory promoter region of
the gene. To address this issue, we first isolated the 5`-untranslated
sequence by RT-PCR using RNA isolated from both butyrate-induced
HL-60-C15 cells and blood eosinophils purified from a patient with HES.
Oligonucleotide primers designed to hybridize to either the boundary of
exon 1 to 2 or the entire putative exon 0 sequence (Fig. 1) were
used in combination with a reverse primer spanning the exon 4-5
boundary in RT-PCR (Fig. 2). After 40 cycles of amplification,
no specific PCR DNA products were detectable using the putative exon 0
primer. In contrast, only the exon 1 primer was able to generate an
appropriately sized 252-bp fragment. Sequence analysis of this 252-bp
fragment amplified from both of the RNA samples tested from
butyrate-induced HL-60-C15 cells or HES patient eosinophils showed it
to be identical with that reported previously (27, 28) , except for the absence of exon 3. A 3.5-kb
fragment from the ghIL5R
-2 genomic clone (29) covering the entire upstream region of the gene was also
screened with the 18-bp putative exon 0 oligonucleotide primer by both
Southern blotting and sequencing; no homologous sequence could be
detected by either method (data not shown). These findings indicated
that the putative exon 0 sequence was not present in either the
5`-untranslated region of the IL-5R mRNA or 3.5 kb of upstream
genomic sequence.
gene. Oligonucleotide primers and the primer extension,
RNase protection and 5`-RACE strategies, and anticipated fragment sizes
for mapping of the transcriptional start site are indicated. Exons
1-3 encode the 261-bp 5`-untranslated region. The 15-bp dashed line for the primer extension and 5`-RACE procedures
refers to the additional 15 bp of upstream sequence identified by these
techniques. The ``putative exon 0'' represents the 18 bp of
5`-untranslated sequence published by Murata et
al.(28) , which was not identifiable in the IL-5R
cDNA by RT-PCR using oligonucleotides A and D or by 5`-RACE nor present
in 3.5 kb of upstream genomic DNA by Southern blotting. The 5`-RACE
utilized oligonucleotide D for the initial reverse transcription step
and the internal oligonucleotide C for subsequent amplification of the
resultant cDNAs.
subunit for detection of putative exon 0 in mRNA from eosinophils
purified from a patient with HES or from HL-60-C15 cells induced with
butyrate for 5 days. Reverse transcription used oligo(dT) priming and
PCR amplification for 40 cycles with primers identical in sequence with
putative exon 0 or the exon 1/2 boundary and an exon 4/5 boundary
reverse primer (primers A, B, and D, Fig. 1). 10 µl of the
PCR products were electrophoresed on a 2% agarose gel and stained with
ethidium bromide. A 252-bp PCR fragment was obtained with the exon
1-2 boundary primer, but not the putative exon 0
primer.
Mapping of the Transcriptional Start Site
To
identify the functional promoter region that regulates IL-5R gene
expression, we have carefully mapped the transcriptional start site
using three different techniques: primer extension, RNase protection,
and 5`-RACE. For primer extension, a specific exon 4-5 boundary
reverse primer (Fig. 1) was labeled and hybridized to the RNA
templates, and IL-5R cDNA was synthesized using avian
myeloblastosis virus reverse transcriptase. Two different transcripts
were identified in the RNA from butyrate-induced HL-60-C15 cells (Fig. 3), with no transcripts identified when total RNA from
HeLa cells was used as a control. Two cDNA fragments were obtained
including a major species of 291 bp and a minor one of 332 bp in
length. Expression of both transcripts was up-regulated in response to
butyrate-induced eosinophilic differentiation of the HL-60-C15 cell
line (Fig. 3). According to the published cDNA
sequence(27) , the distance between the starting point of the
reverse primer and the first nucleotide of exon 1 should be 276 bp when
exon 3 is skipped. This size was in close agreement with the 291-bp
size of the major transcript shown in the primer extension analysis,
but suggested that the previous cDNA sequence was not full-length and
contained additional sequence upstream of the 5`-most end of exon 1.
These extra nucleotides were either from an additional short exon or
part of the genomic (``intronic'') sequence immediately
upstream of exon 1. To distinguish these possibilities, we performed an
RNase protection analysis, as well as cloned and sequenced the 5` end
of the IL-5R mRNA by RACE. For RNase protection, we generated a
hybrid cDNA/genomic DNA probe consisting of a fusion between part of
exon 2 (95 bp), all of exon 1, and 194 bp of upstream genomic sequence.
The RNA samples from butyrate-induced HL-60-C15 cells protected a
series of fragments from this ``cRNA'' that clustered around
148 bp in length relative to the DNA standards (Fig. 4). No
protected fragments were found when RNA from HeLa cells was used as a
negative control. The protected size of the longer fragment precisely
matched the major start site identified in the primer extension
analysis, which was 15 bp upstream of the first nucleotide of exon
1. An additional minor protected species was also observed which was
about 24 bp shorter than the major 148-bp fragment. Since primer
extension and RNase protection did not provide the exact sequence of
the transcriptional start site, the 5` end of the mRNA was
reverse-transcribed from total RNA of butyrate-induced HL-60-C15 cells
(5-day induction), and the cDNA was amplified and subcloned into
pBluescript for sequencing using the 5`-RACE procedure. Plasmid DNA
from 7 individual clones was isolated and purified for sequence
analysis; sequences from 4 of these clones localized the
transcriptional start site to a position 15 bp upstream of exon 1, a
perfect match to the major transcriptional start site suggested by both
primer extension and RNase protection. No longer species were found
even though 5 additional clones were analyzed (data not shown).
However, 3 shorter species were found; 1 started at the middle of exon
1, 1 at the first nucleotide of exon 2, and 1 at the fifth nucleotide
of exon 2 (Fig. 5). Taken together, the data from the above
experiments unequivocally localize the major transcriptional start site
of the hIL-5R
gene to a position 15 bp upstream of the published
end of exon 1 ( Fig. 1and Fig. 5).
mRNA
from HL-60-C15 cells induced toward eosinophil differentiation with
butyrate for 5 days. The primer for reverse transcription, located at
the exon 4-5 boundary region of the gene (primer D, Fig. 1), was kinased using [-P]dATP.
The primer was hybridized to the RNA samples, and cDNA was synthesized
with avian myeloblastosis virus reverse transcriptase. The major 291-bp
product (arrow) corresponds to a transcriptional start site
15 bp upstream of the published exon 1 sequence (Fig. 1).
transcript. A plasmid clone containing a 325-bp
hybrid cDNA/genomic DNA fragment (Fig. 1) was generated and
transcribed with T7 RNA polymerase and
[-P]UTP. The probe was hybridized to total
RNA from butyrate-induced HL-60-C15 cells and HeLa cells and treated
with RNase, and protected fragments were analyzed on a 6%
polyacrylamide sequencing gel. The major, largest protected fragment of
148 bp (arrow) corresponds to a transcriptional start site
consistent with the primer extension analysis ( Fig. 1and
3).
promoter. Open boxes show the positions of exon 1 and part of
exon 2 as previously reported. The transcriptional start sites as
identified by primer extension, RNase protection, and 5`-RACE are
indicated. The consensus start site based on results from all three
methods is labeled as +1. Several consensus sequences are
indicated for putative TFIID/TBP, and other potential transcription
factor binding sites are boxed and shaded.
Functionally active promoter regions ( Fig. 7and Fig. 8)
are boldly underlined. This genomic sequence has been
deposited in the GenBank data base under Accession No.
U18373.
promoter
activity. The -3.5-kb and -561-bp IL-5R
pXP2-luciferase constructs were transfected into eosinophil-inducible
(HL-60-C15, AML-14, AML-14.eos), myeloid (HL-60), lymphoid (BJA-B
B-cell, REX T-cell), and non-myeloid (HeLa) cell lines. Luciferase
activities for each construct and cell line from at least two
experiments were measured, and values were corrected using hGH levels
to control for differences in transfection efficiency among the various
cell lines.
Sequence of the IL-5R
Two
human genomic placental DNA libraries in the Promoter RegionFIX II phage vector
were screened as described previously(29) . A 4.3-kb KpnI fragment from the
ghIL5R
-2 clone (29) was subcloned into the pGEM7ZF(-) plasmid (Promega),
and the region from -561 to +51 relative to the
transcription start site was sequenced in its entirety on both strands (Fig. 5). The consensus position of the major transcriptional
start site as mapped by primer extension, RNase protection, and
sequencing of the 5`-RACE products is indicated as +1. Two
potential TATA boxes (TFIID/TBP binding sites) were identified by
searching the Ghosh transcription factors' data
base(48) , as were a number of putative consensus binding sites
for GATA-1 (49) immediately upstream of the transcriptional
start site. Putative binding sites (consensus sequences) for AP-1 (50) and GATA-1 (51) were likewise identified in
regions further upstream, some of which are in regions of the promoter
that are functionally active in experiments characterizing the promoter
region of the gene (see below).Localization and Characterization of Functionally Active
Promoter Regions
To identify the regulatory sequence elements
required for expression of this gene in the eosinophil lineage, we
first analyzed promoter activity using transient transfections of
reporter (luciferase) gene constructs in the eosinophil-committed
HL-60-C15 cell line (Fig. 6). Three constructs were initially
prepared in the promoterless pXP2 luciferase (luc) expression vector (Fig. 6A). Transient transfection of the IL-5R
promoter construct containing 3.5 kb of upstream sequence directed the
expression of 240-fold greater luciferase activity than the
promoterless pXP2 plasmid alone in HL-60-C15 cells (Fig. 6B). This marked promoter activity was
essentially ablated when the sequence from bp -561 to +51
was deleted. In contrast, the sequence from bp -561 to +51
reproducibly showed equal or greater promoter activity than the longest
3.5-kb fragment analyzed in the HL-60-C15 cell line. Transient
transfection of the series of 5` deletion mutants localized the most
functionally active element of the IL-5R
promoter to the 34-bp
region between bp -432 and -398, such that 78% of activity
was lost when this sequence was deleted (Fig. 7). Of interest,
the bp -561 to -377 promoter fragment itself expressed only
20% of the activity of the full-length promoter in the absence of
sequence more proximal to the transcriptional start site, suggesting
that the proximal region is required for full promoter activity (Fig. 7).Analysis of the Myeloid and Eosinophil Lineage
Specificity of the IL-5R
The specificity of the
IL-5R Promoter promoter for the eosinophil and other myeloid lineages was
assessed by transiently transfecting the -3.5 kb/luc and
-561 bp/luc promoter constructs into a variety of leukemic cell
lines committed to the eosinophil (HL-60-C15, AML14, AML14.eos),
lymphoid (BJA-B, REX), myeloid (HL-60), and non-myeloid (HeLa) lineages
as shown in Fig. 8. Both promoter constructs were significantly
less active in the parental multipotential HL-60 line than in the
eosinophil-committed HL-60-C15 subline. Among the various other cell
lines tested, the IL-5R promoter showed the greatest activity in
the AML14.eos acute myeloid leukemia subline that has been
differentiated into eosinophils by culture with IL-3, IL-5, and GM-CSF,
is comprised of eosinophilic myelocytes and mature
eosinophils(36, 37) , and expresses markedly increased
amounts of IL-5R mRNA. (
)Both IL-5R promoter
constructs were also active in the undifferentiated AML14 parental
line, which likewise expresses mRNA for the IL-5R subunit and a
functional IL-5 receptor(36) . The IL-5R promoter
constructs showed significantly reduced activity that was comparable to
the parental HL-60 line, in both lymphoid (T and B) and non-myeloid
(HeLa cervical carcinoma) cell lines, indicative of basal promoter
activity. These results suggest that the region of the IL-5R gene
required for maximal expression of promoter activity possesses both
myeloid and possibly eosinophil lineage specificity.The Region between bp -432 and -377 of the
IL-5R
As indicated above,
the majority of promoter activity was localized to the region between
bp -432 and -398. This promoter activity is likely derived
from the interaction between DNA binding cis-elements in this
region and lineage-specific transcriptional activators expressed by the
myeloid and eosinophilic cell lines, but not the non-myeloid or
non-hematopoietic lines. To identify the transcription factors in these
lines that may form DNA-protein complexes with this region of the
promoter, we performed gel mobility shift assays by using a Promoter Binds a Nuclear Factor(s) Present in Myeloid and
Eosinophilic, but Not Non-myeloid Cell LinesP-end-labeled 93-bp DNA fragment which encompasses the
region from bp -469 to -377. Two major DNA-protein
complexes, termed C1 and C2, were detected in all nuclear extracts
isolated from myeloid and eosinophil lineage cells, including the
promyelocytic HL-60 parental line, undifferentiated
eosinophil-committed AML-14, eosinophil-committed HL-60-C15, fully
differentiated AML-14.eos, and myeloid U937. In contrast, these two
complexes were not detected using nuclear extracts from the non-myeloid
BJA-B B cells and non-hematopoietic HeLa lines (Fig. 9). In
addition, a third complex (termed C3) was detected using nuclear
extracts from all the myeloid cell lines tested, from non-hematopoietic
HeLa, but not from the BJA-B line (Fig. 9). Formation of all
three DNA-protein complexes could be specifically inhibited by excess
unlabeled DNA probe. In addition, a longer 184-bp probe (bp -561
to -377) and a shorter 55-bp probe (bp -432 to -377)
both produced the identical pattern obtained with the 93-bp probe used
in Fig. 9(data not shown). These results suggest that the
sequence element(s) involved in DNA binding is likely located in the
region between bp -432 and -377.
promoter sequence using eosinophil, myeloid, and non-myeloid
nuclear extracts. A 93-bp PCR-generated probe from bp -469 to
-377 of the IL-5R promoter was labeled with T4
polynucleotide kinase and used for gel shift analysis. Two µg of
crude nuclear extract from the indicated cell lines were mixed with 4
µg of poly(dI-dC) and 1 10 cpm of P-labeled DNA fragment and incubated 20 min at room
temperature. Two DNA-protein complexes (C1 and C2, arrows) were identified using nuclear extracts from HL-60-C15,
HL-60, AML14, AML14.eos, and U937 cell lines, but not with extracts
from BJA-B or HeLa cells. A third specific complex (C3, arrow) was identified using nuclear extracts from all cell
lines including HeLa, with the exception of BJA-B. Labeled free DNA
probe is indicated (F). A 50-fold molar excess of the
identical unlabeled DNA probe added as cold competitor completely
inhibited the formation of the C1, C2, and C3
complexes.
subunits, the promoters for a number
of other hematopoietic growth factor receptor genes including the M-CSF
(CSF-1)(53, 55, 65) ,
G-CSF(56, 57, 66) , and GM-CSF (58, 67) receptors are currently being analyzed. Our
isolation of the 5` upstream region of the human IL-5R gene and
identification of functional promoter sequences provides an opportunity
to elucidate the cis-acting regulatory elements and possibly
unique trans-acting factors that regulate tissue- and
differentiation-specific transcription in the eosinophil as opposed to
other granulocyte or macrophage/monocyte myeloid lineages. subunit,
as an essential and specific component of this differentiation pathway,
is expressed very early in response to GM-CSF, IL-3, or possibly IL-5
itself. ()For these reasons, analysis of how expression of
the IL-5R gene is regulated, and what inducible and
tissue-specific transcription factors are involved in this process, is
extremely pertinent to understanding the mechanisms regulating
eosinophil development. Since eosinophil differentiation, maturation,
and activation are all regulated in part by IL-5, regulation of the
gene encoding the IL-5R subunit is likewise pertinent to the
multiple activities of this and other cytokines on eosinophil function (59) , especially with regard to the mechanisms for specific
receptor-mediated signal transduction pathways in eosinophil activation (9) . Studies of the IL-5R promoter will hopefully lead to
the identification of transcription factors uniquely expressed by the
eosinophilic granulocyte and should add to our understanding of the
molecular basis for the complex cytokine- and growth factor-mediated
processes that occur during the commitment of multipotential myeloid
progenitors to the eosinophil lineage and subsequent eosinophil
development and maturation. These regulatory mechanisms are also likely
to be important both to the development of eosinophilia and to the
functional activation of eosinophils in tissues in
eosinophil-associated allergic, parasitic, inflammatory, and other
diseases. subunit gene were reported
previously(27, 28, 29) . However, the
complete sequence of the 5`-untranslated region, transcriptional start
site, and promoter region had not been determined. The 5`-untranslated
region of the gene was originally reported to be derived in its
entirety from exons 1-3 (29) . In contrast, Murata et
al.(28) reported an additional 18-bp sequence at the
5`-most end of their eosinophil-derived cDNA that was not present in
our own HL-60-C15-derived cDNA clones(27) , as well as
alternative splicing which removes exon 3 in all cDNA clones we
obtained from RNA of HES patient eosinophils or HL-60-C15 cells. These
conflicting results made the identification of the transcriptional
start site an imperative prior to any attempt to localize and
characterize the promoter region of the gene, especially since there
was a possibility of the existence of an additional exon (putative exon
0). To resolve this issue, we first isolated the IL-5R
5`-untranslated sequence from RNA samples obtained from both HL-60-C15
cells and eosinophils purified from a patient with HES using RT-PCR.
These experiments showed that only an oligonucleotide primer identical
in sequence with the 5`-most end of exon 1 (27, 29) and not putative exon 0 (28) was able
to generate an appropriately sized DNA fragment. In addition, we also
failed to detect any homology of the putative exon 0 within 3.5 kb of
the upstream sequence of the IL-5R genomic DNA. These findings are
consistent with the conclusion that there is no putative exon 0
sequence in the 5`-untranslated region of the IL-5R gene as
transcribed in either the butyrate-induced eosinophilic HL-60-C15 cell
line or HES patient eosinophils. Sequence analyses also indicated that
exon 3 was missing entirely from the 5`-untranslated region in mRNA
samples from both of these sources. Tavernier and co-workers (29) also noted a cDNA variant from butyrate-induced HL-60-C15
cells in which there was an absence of exon 3; however, only 1 in 8 of
the fully characterized cDNA clones isolated from their
butyrate-induced HL-60-C15 library was missing this exon(27) .
One potential explanation for these observations may be variability in
the copy number of the exon 3-containing RNA isoform originally
reported (27) or differences in the patterns of alternative
splicing in HL-60-C15 cells maintained and induced with butyrate in
different laboratories. gene using three complementary techniques.
Primer extension produced two cDNA fragments of 291 bp and 332 bp in
length. Both species were consistent with additional sequences 5` of
the published end of exon 1 (27) although the longer 332-bp
fragment was likely a primer extension artifact, perhaps resulting from
the 3` end of the cDNA forming a loop by folding back on its own
template and serving as a primer for (limited) second strand synthesis.
These extra nucleotides were either from an additional short exon
further upstream or from the genomic sequence immediately upstream of
exon 1. To distinguish these possibilities, we performed both RNase
protection and 5`-RACE; RNase protection used an artificial probe
containing half of exon 2, all of exon 1, and the genomic sequence
immediately upstream of exon 1 (Fig. 1). Results from RNase
protection were consistent with an additional 15 bp of sequence
upstream of the published end of exon 1, as suggested by primer
extension. However, these results could not exclude the possibility of
an additional small exon further upstream in the gene. To address this
issue, we used RACE to specifically amplify the 5` ends of IL-5R
cDNAs prepared from RNA of butyrate-induced HL-60-C15 cells. Sequences
from the 4 longest clones obtained by the RACE procedure were identical
with the 15 bp of genomic sequence immediately upstream of the
published end of exon 1, providing confirmation of the major
transcriptional start site suggested by both primer extension and RNase
protection; no longer cDNA species were found. Taken together, these
results map the major transcriptional start site to a position
approximately 15 bp upstream of exon 1, albeit at a site that lacks a
canonical CANYYY CAP site(60) . gene was analyzed in the eosinophil-inducible HL-60-C15
cell line. Transient transfections with the IL-5R promoter
constructs suggested that most of the sequence elements required for
maximum promoter activity were located within a 561-bp region
immediately upstream of the transcriptional start site. A series of
smaller, 5` deletions in this region were generated using the
-561/luc plasmid to more closely map the minimal sequence
elements required for promoter activity. Results from transient
transfection of these mutants in HL-60-C15 cells indicated that the
region between -561 and -398 bp contributed 75% of
maximal promoter activity. Of interest, when a bp -561 to
-377 fragment was cloned into the promoterless pXP2-luciferase
vector, it expressed only 20% of total promoter activity suggesting
that both the distal and proximal regions of the promoter are required
for full promoter activity. Two potential TATA boxes (TFIID/TBP binding
sites) located in the bp -23 to -79 region (Fig. 5)
were identified by searching the transcription factor data
base(48) ; a TFIID site is critical for RNA II polymerase
activity (61, 62) . Since the distal region of the
IL-5R
promoter contains the DNA sequences required for functional
activity, it is likely that the promoter requires transactivation by
transcription factors which bind either directly or indirectly to both
this and the more proximal TFIID binding regions
independently(62) . In this regard, the functional cis-elements in the distal region of the promoter are likely
present within the small 34-bp region from bp -432 to -398 (Fig. 7), for which the nucleotide sequence does not contain any
previously identified transcription factor binding sites(48) .
Using electrophoretic mobility shift assays, we identified a nuclear
factor or factors that bind specifically to this region of the promoter
and that are present in the myeloid and eosinophilic cell lines in
which the promoter was functionally active, but absent in the
non-myeloid and non-hematopoietic lines for which the promoter was
significantly less active. Thus, this functional segment likely serves
as a binding site for lineage-specific transcription factor(s) required
for optimal expression of the IL-5R gene. Whether the two
DNA-protein complexes consistently observed in electrophoretic mobility
shift experiments with this promoter region (Fig. 9) represent
distinct nuclear factors, proteolytic cleavage or post-translational
modification of a single factor, or binding by a heterodimer is unclear
pending further biochemical characterization or cloning. promoter in eosinophilic
and other myeloid and non-myeloid leukemic cell lines (Fig. 8)
suggests that the 561-bp upstream region of the gene required for
maximal expression of promoter activity possesses both myeloid and
possibly eosinophil specificity. Among the various eosinophil cell
lines tested, the IL-5R promoter showed greatest activity in
AML14.eos, a subline of AML14 that has been differentiated into
eosinophilic myelocytes and mature eosinophils by induction with a
combination of IL-3, IL-5, and GM-CSF(37) , expresses increased
amounts of IL-5R mRNA, and continues to proliferate in culture and
maintain the differentiated phenotype with cytokine
supplementation. Similarly, the IL-5R promoter showed
greater activity in the eosinophil-inducible HL-60-C15 line (in which
5-20% of the cells spontaneously become granulated in
culture (38) ) than the undifferentiated parental HL-60 line (Fig. 8). Of interest, the IL-5R
promoter also showed high
levels of activity in U937, a myelomonocytic cell line, with mean
levels of 13,631 ± 4,700 RLU/ng/ml hGH. Other myeloid promoters
analyzed thus far in our laboratories including those for the
eosinophil peroxidase (35) and CLC protein (34) genes
have likewise shown particularly high levels of activity in the U937
line for reasons that are as yet unclear but may relate to a
particularly high transfection efficiency for this monocytoid leukemic
cell line. Further, the U937 cell line contained the same nuclear
factor(s) that formed specific protein-DNA complexes with the
IL-5R promoter in the gel shift analyses (Fig. 9). subunit gene was recently
published(52) . In contrast to the human IL-5R
subunit's unique expression and activity in only the eosinophil
lineage(10) , the murine gene is also expressed and functional
in B cells(52) . Comparison of the promoter regions and
transcription factors regulating the differential expression of the
human versus murine genes in eosinophil only versus eosinophil and B cell lineages(10, 63) ,
respectively, could provide insights into their respective mechanisms
of expression and regulation. While the upstream, 5`-flanking region of
the murine IL-5R gene contains consensus sequences for Ap1, AP-1,
GATA-1, and PU.1, these sequences have not as yet been analyzed for
functional activity(52) . In the limited functional analysis of
the murine promoter published thus far(52) , 256 bp of the
5`-flanking region (-96 to +160 bp) exhibited minimal
promoter activity in a pCAT vector in fibroblast (NIH3T3), FDC-P1, and
IL-5-dependent pre-B cell (Y16) lines. Of interest, analysis of a
larger 1.5-kb construct (-1371 to +160) to find a region
that directs B cell-specific expression, failed to detect any promoter
activity in any of the murine lines tested, suggesting suppressive
elements in this region, in marked contrast to the results we have
obtained for the human IL-5R promoter with up to 3.5 kb of
upstream sequence. The murine gene also contains a 52-bp AC-rich Z-type
DNA sequence (-445 to -394), which is also found in the
IL-2R upstream region, but is absent in the human IL-5R gene
in the first 576 bp of upstream DNA we have sequenced thus far.
Alignments we have performed on the human and murine upstream regions
did not show any significant sequence similarities within the
functionally defined region of the human IL-5R promoter, i.e. bp -561 to +51. A comparison of the human IL-5R
upstream sequence to the 5`-flanking sequences of the human
GM-CSFR gene (58) also failed to identify any regions of
significant similarity in the functionally active segments of the
IL-5R promoter. These observations indicate important differences
in the regulatory regions of the human versus murine
IL-5R and human GM-CSFR genes that are likely relevant to
their differential expression in eosinophil versus B cell
lineages in the two species and granulocyte/macrophage lineages in
humans, respectively. Finally, the promoters for a number of other
genes preferentially expressed in the myeloid series, including
CD11b(54) , M-CSF(53) , and CD14 (44) have been
shown to require either PU.1 and/or Sp1 (44) binding for
myeloid-specific expression. However, the functionally active region of
the IL-5R promoter we have identified lacks consensus PU.1 or Sp1
binding sites, and the bp -561 to +51 region of the promoter
does not show any binding of in vitro-transcribed PU.1
transcription factor in electrophoretic mobility shift assays. ()However, these analyses do not exclude the presence of a
nonconsensus, weak, but functionally significant PU.1 binding site in
the promoter. gene that
is myeloid- and relatively eosinophil lineage-specific in its
expression in eosinophil-inducible leukemic cell lines in vitro. In addition, we have shown that this region binds a nuclear
factor(s) expressed in the same myeloid and eosinophilic lines in which
the promoter is functionally active. Studies to more precisely map the
functional elements in this region and to characterize and clone the
cognate transcription factors that bind to this region of the
IL-5R promoter and are required for optimal and
eosinophil-specific activity in vitro and in vivo are
currently in progress.
),
IL-5 receptor subunit; CLC, Charcot-Leyden crystal protein; RLU,
relative light units; bp, base pair(s); kb, kilobase(s); RT, reverse
transcription; PCR, polymerase chain reaction; WT, wild type; hGH,
human growth hormone; CMV, cytomegalovirus; HES, idiopathic
hypereosinophilic syndrome.))))
We thank Dr. René Devos for
scientific input and editorial suggestions and Mary Singleton for
secretarial and administrative assistance in the preparation of this
manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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