From the Division of Cell Biology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84132
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ABSTRACT |
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We have sought to develop
methodologies to identify genes that are preferentially expressed
during the differentiation of mast cells from their hematopoietic stem
cell precursors. By using a modified differential display protocol, we
compared a subset of transcripts expressed in bone marrow cells
differentiated into immature mast cells with the exogenous addition of
stem cell factor (SCF) or interleukin 3. One gene was identified that
was preferentially expressed in the SCF-derived cells and encodes a
novel murine integrin subunit-like molecule, dubbed Pactolus-1
(Pactolus). Two distinct forms of Pactolus mRNA were detected
which, via alternative splicing, are predicted to encode a
membrane-bound form and truncated version of the protein. The
full-length Pactolus gene product is very similar to a number of
subunit integrin chains, particularly
2, with the notable exceptions
of the apparent deletion of the metal-binding site within the putative
metal ion-dependent adhesion site-like domain of the
Pactolus gene product and a cytoplasmic domain that shares no obvious
homology to similar domains of the other
subunit integrin proteins.
Although the Pactolus sequence was first identified in immature mast
cell samples, screening of murine tissues indicated the highest level
of Pactolus expression was found in the bone marrow, suggesting that
the expression of Pactolus is confined to immature and maturing
bone marrow-derived cells, and that the SCF-derived mast cells are more
representative of this state than are the interleukin 3-derived mast
cells. Immunoprecipitation of Pactolus revealed a cell-surface protein
with an apparent molecular mass of about 95 kDa. Surprisingly, no
associating
integrin subunit could be identified suggesting that
either Pactolus does not associate with another integrin subunit or the
association is too weak to be identified. These data suggest that
Pactolus represents a gene and gene product related to those of the
integrin
subunits but whose function(s) may be quite distinct
from those of the integrin
subunits.
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INTRODUCTION |
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It has been the goal of many investigations to isolate those genes whose expression is intimately tied to hematopoietic cell differentiation and maturation (reviewed in Refs. 1 and 2). This goal has been approached using a variety of different experimental approaches. One of the most successful techniques used to isolate cell-specific gene products has been cDNA subtraction (3). This protocol has been used to identify and isolate a number of significant gene products, notably the T cell receptor genes (4, 5). There are, however, several drawbacks to the subtraction technique when it is applied to study hematopoiesis. Most conspicuously, it requires relatively large amounts of mRNA and is labor intensive. A successful subtraction normally requires several rounds of hybridization and physical dissociation. This protocol also leads to the preferential enrichment of abundant mRNA species over the rarer species.
More recently, an alternative approach to subtraction was introduced in which mRNA species are randomly amplified from total RNA preparations. This technique, known as differential display (DD)1 (6, 7), is based on the theory that every mRNA in the cell can be amplified, via a cDNA intermediate, with a specific combination of a poly(T) containing anchoring primer and a random decamer oligonucleotide. This protocol allows for the rapid comparison of transcript species between two closely related cell types such as normal and transformed or quiescent and activated cells. By using this protocol the total complexity of transcripts within a cell can be displayed, thus achieving transcript saturation. Any transcript product that is specific for the cell type in question can, with this protocol, be identified, isolated, and sequenced. The advantages of this protocol are many and include the requirement of much less RNA, the simplicity of PCR amplification, and the ease of product resolution (6-8). The primary difficulties with DD have been the large number of false positives generated and the requirement of closely matched cell types with which to compare.
Mast cells arise from the multipotent bone marrow stem cells. There are two types of tissue mast cells in the mouse (mucosal mast cells and connective tissue mast cells) that are believed to be derived from the same precursor cells. A variety of researchers have demonstrated that immature mucosal-like and connective tissue-like mast cells can be derived in vitro by culturing mouse bone marrow with either IL-3 or SCF, respectively (9-12). The molecular events that drive the differentiation of these two cell types have remained an enigma despite the recent characterization of mast cell progenitor cells (13). Since these two cell types are very similar to one another and can, given the correct circumstances, shift their phenotype back and forth, they are ideal candidates for the utilization of DD to identify those gene products specific for each cell phenotype.
In this work, we describe a modification of the original differential
display protocol designed to identify transcripts implicated in bone
marrow maturation. This protocol not only allows for a more rapid
progression of the protocol but also appears to increase the
specificity of the products such that the generation of false positives
is greatly diminished. By using this protocol we identified a
transcript preferentially expressed in SCF-derived mast cells but
absent in those derived with IL-3. The gene fragment identified in this
protocol was used to screen a mast cell cDNA library from which an
apparent full-length transcript was obtained. This cDNA predicts a
novel murine integrin subunit-like molecule, Pactolus, that, via
alternative splicing, would be expected to produce both a
membrane-bound and truncated form. The expression of this gene is most
pronounced in the murine bone marrow. The expression of Pactolus in
cells derived in SCF but not the IL-3 cells suggests that the
SCF-derived cells may represent a more immature mast cell type than
those derived in IL-3 culture.
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EXPERIMENTAL PROCEDURES |
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Mice and Tissue Culture-- 5-wk-old female NIH(s) mice were obtained from the National Institutes of Health. Mice were used at 5-10 weeks of age. Bone marrow-derived mast cells cultured in IL-3 or SCF were produced as described previously (14).
RNA Preparation and cDNA Synthesis-- Total RNA from various cells and tissues was isolated using the CsCl/guanidine method (15). cDNA was synthesized by mixing 5 µg of RNA, 10 µl of 5× first strand buffer, 5 µl of 5 mM dNTP, 5 µl of 0.1 M dithiothreitol, 2 µl of perspective anchoring primer (6, 7), 2 µl of Moloney murine leukemia virus reverse transcriptase, and water to a final volume of 50 µl. The reaction mixture was incubated at 37 °C for 1 h. 2 µl of DNase-free RNase (1 mg/ml) was then added, and the reaction mixture was incubated for an additional 5 min followed by phenol/chloroform extraction and ethanol precipitation.
Differential Display PCR Amplification-- PCR amplification was done by mixing 200 ng of cDNA (reversed transcribed with anchoring primers), 1 µl of 500 µM dNTP, 1 µl of 10× PCR buffer, 0.5 µl of 1 µg/µl decamer, 0.15 µl of Taq DNA polymerase (Life Technologies, Inc.), 0.25 µl of [32P]dCTP, and water to a total volume of 10 µl per reaction. Each reaction was done in triplicate. The reaction mixture was then put into a capillary tube (Idaho Technology) and amplified under the following conditions. For the first five cycles, the amplification was carried out at 94 °C for 1 s, 40 °C for 1 s, and 72 °C for 10 s. The following 35 cycles were carried out at 94 °C for 1 s, 50 °C for 1 s, and 72 °C for 10 s. The reaction was then quenched by adding 10 µl of stop buffer, and then 5 µl of the reactions was loaded onto a 6% sequencing gel. After 2 h of electrophoresis, the gel was dried followed by autoradiography.
Primers used in this study have been described previously (6-8) and are shown in the following: anchoring primer, 5' TTT TTT TTT TTT (ACG)G 3' and random decamer, 5' TGGATTGGTC 3'. Bands containing candidate DNA fragments were eluted with 450 µl of water and 25 µl of 5 M NaCl overnight. The supernatant was recovered followed by ethanol precipitation. The DNA pellet was resuspended in 20 µl of water. 2 µl was then used for the second round amplification. RT-RPCR was performed as described previously (16, 17). Pactolus specific primer sets are shown as follows: gap 1, 5' CTC TGG CTC TGC GCA AGG CC 3' and 5' AAG CAC CAG AAA TCG GGT CC 3'; gap 2, 5' TAG TAC TCG GAG CAG CGA TGG 3' and 5' CGA GTG CGA CAA TGT CAA CTG 3'; and full-length, 5' GAA AGA GGC CAC TGC TCC TG 3' and 5' CAC CTG GCA CAG GAG TAG TAC 3'. Primer sets for other adhesion molecules are as follows:Cloning and Sequencing-- Purified DNA fragment was ligated into pmm5 vector (a kind gift from Dr. Eric Kofoid, University of Utah) which has a single dT overhang on both ends. DNA sequence was generated by standard fluorescence DNA sequencing in the University of Utah core facility.
cDNA Library Preparation-- Total cellular RNA was extracted from bone marrow-derived CTMC as described previously (15). Poly(A)+ RNA was isolated by using the Oligotex mRNA Kit (Qiagen). cDNA library was constructed according to the instruction manual of lambda ZAP II Cloning Vector Kit (Stratagene). Greater than 500,000 independent clones were screened for gene-specific inserts.
In Vitro Transcription and Translation-- In vitro transcription was performed according to the manufacturer's manual of RNA Transcription Kit (Stratagene). mRNA was then translated in vitro using the rabbit reticulocyte lysate system (Amersham Pharmacia Biotech) in the presence of 35S-labeled methionine.
Generation of Pactolus-specific Antisera-- Two rabbits were injected with a peptide derived from the Pactolus cytoplasmic domain (CGTQKAAKLPRKG) using the keyhole limpet hemocyanin/bovine serum albumin conjugation protocol and reagents from Pierce. The first injection was done with an equal volume of Freund's complete adjuvant, whereas the subsequent injections were with Freund's incomplete adjuvant. Antisera from one of the rabbits was utilized.
Immunoprecipitation--
Bone marrow cells were harvested from
mice, and red blood cells were lysed with red blood cells lysis
solution (0.15 M NH4Cl, 1.0 mM
KHCO3, and 0.1 mM EDTA, pH 7.2) for 5 min at
room temperature. EL-4 cells were maintained in RPMI media supplemented
with 5% fetal bovine serum. Before labeling, cells were washed two
times with phosphate-buffered saline. Cells were labeled by incubating with 0.5 mg/ml sulfo-NHS-LC-biotin (Pierce) for 30 min at room temperature. After washing with ice-cold phosphate-buffered saline three times, cells were resuspended in lysis buffer (0.5% Nonidet P-40, 0.5% sodium deoxycholate 50 mM NaCl, 2 mM CaCl2, 25 mM Tris, pH 7.5, 1%
bovine serum albumin, 0.2 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, and 1 µg/ml pepstatin A) at 5 × 107 cells/ml, and the reactions were incubated on ice for
1 h. Lysates were immunoprecipitated with either polyclonal rabbit
antisera against Pactolus cytoplasmic peptide or anti-mouse 2
monoclonal antibody (PharMingen). After incubating the lysates with
respective antibody for 1 h at 4 °C, the protein-antibody
complex was absorbed with either protein A-Sepharose (for rabbit
antibody) or protein G-Sepharose (for rat antibody). After the
absorption, the Sepharose beads were washed 4 times as follows: once
with lysis buffer, twice with lysis buffer with 150 mM
NaCl, and once with 0.05 M Tris, pH 6.8. The samples were
boiled in 1× SDS loading buffer for 5 min before they were loaded on
to 10% SDS-polyacrylamide gel. After the electrophoresis, proteins
were then transferred to a polyvinylidene difluoride membrane
(Millipore). The membrane was then incubated with 1:10,000 dilution of
horseradish peroxidase-avidin D (Vector) for 1 h at room
temperature. The biotin-labeled protein was visualized by
chemiluminescence (MENTM Life Science).
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RESULTS |
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Isolation of Differentially Expressed Mast Cell Products-- To differentiate transcript products specific for mast cells derived in either IL-3 or SCF, we utilized the differential display protocol. This protocol was altered for use in the air thermocyler which differs from conventional PCR thermocyclers in reaction volumes (10 µl versus 50-100 µl in conventional machines), cDNA requirements (100 ng versus 1 µg or more for conventional machines), cycle times (23 s per cycle versus 2-6 min in conventional machines), and reagent vessels (glass capillary tubes versus plastic Eppendorf tubes in conventional machines). The specifics of the modified DD protocol are detailed under "Experimental Procedures."
The RNA to be analyzed was isolated from four different mast cell sources. Bone marrow cells were cultured in the presence of either IL-3 or SCF for 21 days to develop into mast cells phenotypically similar to immature mucosal (MMC) or connective tissue mast cells (CTMC), respectively. RNA was isolated from such cells. Additionally, RNA was isolated from IL-3-derived cells that had been transferred into SCF without IL-3 for 24 h (MMC + SCF) and SCF-derived cells transferred into IL-3 without SCF for 24 h (CTMC + IL-3). The isolated total RNA was reverse-transcribed with a specific anchoring primer and expanded by PCR-based amplification with randomly designed decamers. Each sample was amplified independently in triplet (three 10-µl reactions) and resolved by denaturing acrylamide gel electrophoresis (Fig. 1). As shown, this procedure provides consistent and reproducible band patterns comparable with those obtained from the conventional DD protocols. The two products found to be differentially expressed in this experiment with a single decamer/primer oligonucleotide set are marked by the arrows A and B.
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Identification of a Novel Integrin Subunit-like
Sequence--
When the nested primers derived from the fragment B
sequence were used to re-analyze the primary mRNA samples (using
the standard RT-RPCR protocol) the expression of the fragment B gene
was primarily confined to the SCF-derived cells, not those initially
derived in IL-3 (Fig. 3). Accordingly, a
cDNA library constructed with RNA isolated from mast cells derived
in SCF was screened with the fragment B probe. Twelve clones were
isolated from this screening. The sequence of the largest of these
cDNA clones possessed an insert with 2,585 nucleotides. A single
large open reading frame was determined to start at an ATG at base 64 and terminate at a stop codon at base 1686, encoding a protein with 540 amino acids. This sequence thus predicted a long 3'-untranslated
sequence of 901 nt. This predicted amino acid sequence suggested a
secreted protein possessing a signal sequence but lacking a
transmembrane domain for membrane anchoring (see below). A
GenBankTM search with this cDNA sequence (and its
derived amino acid sequence) indicated it was very similar to those
within the
integrin subunit family of genes. The highest degree of
homology was found with the murine and human
2 integrin subunits.
The gene and gene product was named Pactolus-1.
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Alternative Pactolus Transcripts Predict a Membrane-bound and
Truncated Form of the Protein--
When the nucleotide sequence of
Pactolus was plotted against that for murine 2 (18) using a homology
matrix analysis, two features were striking. First, two distinct gaps
were noted between the two sequences (Fig.
4) that denoted apparent insertions in the
2 sequence compared with that of Pactolus. And second, a significant level of homology remained between the two sequences after
the putative stop codon of the Pactolus sequence (base 1692). This
region corresponded to the 3'-untranslated sequence of the Pactolus
transcript and extracellular coding sequences (amino-terminal of the
transmembrane sequence) of the
2 integrin. DNA sequence analysis of
the other Pactolus cDNA clones isolated from the CTMC cDNA
library confirmed the sequence of the Pactolus-1 cDNA at these
sites (data not shown).
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mRNA Expression of Pactolus-- As shown in Figs. 3 and 5, Pactolus transcripts are evident in bone marrow-derived mast cells (cultured in SCF) and in bone marrow and spleen. Due to the extreme sensitivity of RT-PCR, the expression of Pactolus transcripts in the bone marrow was further confirmed by Northern blot analysis. As shown in Fig. 8, panel A, a 2.8-3.0-kilobase pair band was evident when 5 µg of total RNA from mouse bone marrow was hybridized with the Pactolus cDNA sequence. The size differential between the two Pactolus mRNA isoforms is too small to detect two distinct bands with this type of assay.
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Identification of the Pactolus Protein-- To analyze the protein product of the Pactolus gene, a peptide corresponding to the Pactolus cytoplasmic tail was synthesized, and polyclonal rabbit antisera were generated. To determine whether the antisera recognized Pactolus protein, 35S-labeled protein was generated by in vitro translation of Pactolus mRNA in the presence of [35S]methionine and immunoprecipitated with either preimmune or post-immune serum. As shown in Fig. 9, panel A, only the post-immune serum specifically recognized the Pactolus gene product. To identify Pactolus protein in vivo, mouse bone marrow cells were surface-labeled with biotin, and the lysate was immunoprecipitated with either preimmune or post-immune serum. As shown in Fig. 9, panel B, a 95-kDa protein was readily identified by this approach. However, in identically labeled splenocytes, this protein was not evident (data not shown). The increase in apparent molecular weight in the Pactolus gene product between the two panels is presumably due to post-translational modification of the protein in the bone marrow sample (i.e. glycosylation) and charge differences via the addition of the biotin complexes.
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DISCUSSION |
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In this report, we describe the isolation of a novel murine gene
(Pactolus) that shares a high degree of homology with the family of
adhesion molecules known as the integrin subunits. The Pactolus
sequence was obtained from a differential display (DD) analysis of
murine bone marrow-derived mast cells in which transcripts derived from
cells cultured in IL-3 were compared with those derived in SCF. Based
upon our data and data from others (11, 14, 32), those cells derived
solely in IL-3 possess a phenotype associated with mast cells found in
the intestinal mucosa, whereas those derived in SCF possess
characteristics of mast cells found in the skin and peritoneal cavity
of the animal. Following this cell culture rationale and utilizing a
modified DD protocol, we isolated a novel sequence from bone marrow
cells cultured in SCF which was lacking, as an apparent transcript, from those cells cultured in IL-3. Subsequent cloning and sequence analysis indicated this novel gene transcript was very similar to the
members of the integrin
subunit gene family.
The protein predicted by the Pactolus sequence is related to the
integrin subunits but is clearly divergent in two important domains. The first of these is within the proposed I (inserted) domain-like structure (also termed A for activation domain)
which is also present within the majority of the
integrin subunits (reviewed in Ref. 33). This region resides at the amino terminus of the
protein (thus extracellular) and is implicated in ligand binding,
heteroduplex formation, and metal ion binding. Single amino acid
mutations in this site abrogate stable expression of the integrin
heterodimers and block ligand binding. Structural analysis of the I
domain of the CD11b chain of the CR3 integrin complex suggested the
presence of a MIDAS motif (metal ion-dependent adhesion
site) that was formed in a three-dimensional fold utilizing a conserved
DXSXS-(65 amino acids)-T-(25 amino acids)-D
sequence, where the X residues and spacer amino acids are
not conserved (26). The analagous sequence present in Pactolus differs
from that of the
and
integrin subunits. Not only does Pactolus lack the amino-terminal Asp residue, but the spacing between the Ser
and Thr residues, which presumably is critical for the ternary folds of
the protein, is 21 amino acids shorter than the consensus sequence. Of
the more than 40 gene sequences that possess the proposed MIDAS motif,
the shortest distance between the analogous Ser and Thr residues is 61 amino acids, compared with 44 for Pactolus. This deletion would, with
respect to the proposed MIDAS motif structure, apparently delete the
2,
3 helices, thus placing the Thr residue in opposite orientation
to the Mg2+ ion within the MIDAS structure. These two
alterations in this region of Pactolus suggest that this site may not
be functionally similar to those of the integrin
subunits.
The other site of high homology within the integrin subunit family
is the cytoplasmic domain. Sequences have been defined in the
cytoplasmic tail of these subunits that are implicated in the binding
of the
chain to cytosolic proteins including
-actinin, talin,
paxillin, and others (reviewed in Ref. 34). Pactolus is lacking these
conserved residues in its proposed cytoplasmic domain suggesting it may
bind to a different set of cytoplasmic proteins than those described
for the integrin
subunits.
The Pactolus gene transcripts also differ from members of the integrin
subunit family in predicting two distinct forms of the protein,
plus or minus the transmembrane and cytoplasmic domains. Attempts to
generate an antisera specific for the truncated form of the Pactulus
protein have so far been unsuccessful; thus we cannot conclude whether
the protein produced by the truncated transcript is stably expressed in
mammalian cells. Some integrin
subunits utilize alternative
splicing to produce variant isoforms of the proteins. In particular, at
least four distinct alternative cytoplasmic domains have been described
for integrin subunit
1 which alter the functional characteristics of
the protein (35-38). However, we do not know of any
integrin
subunit that would, like Pactolus, predict a secreted form of the
protein.
The expression of the Pactolus gene appears to be limited to immature
cells of bone marrow derivation. The murine tissue demonstrating the
highest level of expression is the mouse marrow. Based upon -actin
transcript levels, the quantity of Pactolus transcripts in the splenic
sample was 10% or less than that of the bone marrow. Since the spleen
is primarily populated by mature cells of bone marrow origin (B cells,
T cells, and macrophages), the absence of appreciable Pactolus
transcripts in the spleen may suggest the down-regulation of this gene
during cellular maturation.
Two key findings were provided in the analysis of the Pactolus protein.
First, the protein is expressed on the surface of the bone marrow cells
with an apparent molecule mass of 95 kDa. Second, we cannot detect any
significant association of Pactolus with any other cell-surface
protein, as might be expected if Pactolus was to function as a integrin subunit. Based upon the sequence of the Pactolus gene product
and the lack of an apparent heterodimer complex, it is likely that
Pactolus does not function as the typical
integrin subunit despite
its sequence homology with
2. Therefore placing Pactolus within the
integrin gene/protein family would imply a functionality of the protein
that it may not possess.
The major question left open by this study is the function of Pactolus.
Its expression pattern suggests it is expressed by immature and
maturing cells of bone marrow derivation. Its similarity in structure
to the integrin subunit gene family suggests it may be a receptor
mediating adhesion of such cells within the marrow stroma. Previously
the
4 integrin has been shown to be critical in marrow maintenance
(39, 40). Alternatively, Pactolus may be playing a signaling role for
cells within the marrow. For example, one of the alternative
cytoplasmic domains for the integrin
1 subunit (
1c) acts to
directly inhibit cell cycle progression (38). The marrow consists of
many cell types, some of which are held in a state of low replicative
activity. The two different forms of the Pactolus protein may, upon
ligation with ligand, send two quite distinct signals into the cell.
This model might be especially appropriate if a modulation of splicing
between the two forms is evident during bone marrow cell maturation and if we can detect the stable expression of the truncated form of the
Pactolus protein.
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ACKNOWLEDGEMENTS |
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We thank Ina Karbegovic for excellent technical assistance and the preparation of the mast cell cultures. We also thank Dr. Eric Kofoid for the T overhang cloning vector and Dr. David Stillman for a clean bench in his lab. We would also like to thank all the members of the Weis laboratories for their valuable comments and discussions.
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FOOTNOTES |
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* This work was primarily funded by National Institutes of Health Grant DK49219 (to J. H. W.) from the Center of Excellence in Hematology. Additional funds were provided by National Institutes of Health Grant AI-24158 (to J. H. W.). The project described was also supported in part by an award from the American Lung Association (to J. H. W.). Support was also obtained by National Institutes of Health Grants AR43521 and AI32223 (to J. J. W.). Support was also provided by the Huntsman Cancer Institute and NCI Grant 5 P30 CA42014 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) BankIt 175798 and AF051367.
To whom correspondence should be addressed: Dept. of Pathology,
University of Utah School of Medicine, 50 N Medical Dr., Salt Lake
City, UT 84132. Tel.: 801-581-7054; Fax: 801-581-4517; E-mail: john.weis{at}path.med.utah.edu.
1 The abbreviations used are: DD, differential display; CTMC, connective tissue mast cells; MMC, mucosal mast cells; RT-RPCR, reverse transcriptase-rapid polymerase chain reaction; PCR, polymerase chain reaction; SCF, stem cell factor; IL-3, interleukin 3; n, nucleotides; bp, base pairs; aa, amino acids; MIDAS, metal ion-dependent adhesion site.
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REFERENCES |
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