Selective Reporter Expression in Mast Cells Using a Chymase Promoter*

(Received for publication, August 9, 1996, and in revised form, October 17, 1996)

Yongbo Liao Dagger , Taolin Yi §, Brian D. Hoit , Richard A. Walsh , Sadashiva S. Karnik Dagger and Ahsan Husain Dagger par

From the Departments of Dagger  Molecular Cardiology and § Cancer Biology, Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 and the  Division of Cardiology, University of Cincinnati Medical Center, Cincinnati, Ohio 45267

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Primate alpha -chymases are mast cell neutral proteases that are involved in regulating several regulatory peptides including angiotensin II. Because of significant substrate specificity differences among the chymase group of enzymes, animal models that overexpress primate chymases are crucial for delineating the in vivo function of these enzymes. Activation of alpha -prochymase requires processing enzymes and proteoglycans found in mast cell secretory granules. Thus, the development of models overexpressing active primate chymase requires a mast cell-specific promoter. We show that the 571-base pair (bp) 5'-upstream sequence of the baboon chymase gene, which encodes an alpha -chymase, coupled to the prokaryotic lacZ gene allows the targeting of beta -galactosidase to mast cells in transgenic mice. Tissue expression of the transgene is similar to the expression of the endogenous mouse alpha -chymase mouse mast cell protease-5. A mouse mast cell line that endogenously expresses mouse mast cell protease-5 (JKras mast cells) also selectively supports the expression of this transgene. In vitro transcription studies in JKras mast cells shows the critical role of a GATA cis-regulatory motif in baboon chymase promoter, located ~430-bp upstream of the transcription start site. These results suggest that the 571-bp domain of the baboon chymase promoter contains most, if not all, of the mast cell-specific region of the promoter. We describe here for the first time a promoter that directs expression of transgenes specifically to mouse mast cells. This promoter should be generally applicable for dominant expression of mast cell regulatory proteins.


INTRODUCTION

Mast cells originate from hematopoietic stem cells, but mature mast cells reside in mucosal and connective tissue (1). Through the release of proteases, cytokines, and other chemical mediators, mast cells participate in a variety of immunological, inflammatory, and cardiovascular responses. Mast cell proteases include chymases, tryptases, and carboxypeptidase A (2). Our recent studies and those of other investigators suggest that chymases can activate and inactivate regulatory peptides and thus can produce complex effects on the cardiovascular system. For example, some chymases, e.g. human, baboon, and dog chymases, form angiotensin II efficiently (3, 4), whereas other chymases, e.g. rat chymase-1, degrade angiotensin II (5, 6). Angiotensin II is a potent vasoconstrictor peptide (7) and a myocyte growth factor (8). Recent phylogenetic studies indicate that mammalian chymases occur in two distinct isoenzyme groups, alpha  and beta  (6). A single chymase gene (chm gene) encoding an alpha -chymase has been identified in all mammals studied, including human, baboon, dog, rat, gerbil, and mouse (6, 9-12). In contrast, beta -chymases are species-specific. Mice, rat, and gerbil contain four, two, and one beta -chymase-encoding chm genes, respectively (6, 11). beta -Chymases have not been demonstrated to be present in humans and baboons. Substrate specificity studies indicate that alpha - and beta -chymases differ in their specificities and that there are more subtle substrate specificity differences between some alpha -chymases (3-6, 13).

Although the overall function of mast cells can be explored in mast cell-deficient mice, the in vivo role of alpha -chymases cannot be delineated because highly specific inhibitors of these proteases are not currently available. Also, subtle differences in substrate specificity between mammalian alpha -chymases make questionable the use of rodent models for the study of primate alpha -chymase function. One approach toward understanding the in vivo role of primate alpha -chymases is to create transgenic models that overexpress functionally active forms of these enzymes. Our recent studies show that the chymase propeptide sequence is important in the correct folding of this protease within the cell (14) and activation of prochymase requires heparin and dipeptidylpeptidase I, which occur abundantly in mast cells (15). Therefore, to assess the effect of dominant expression, it is necessary to target the zymogen to mast cells to ensure processing and proper tissue localization of chymase. The unavailability of a promoter that selectively targets the mast cell in vivo has hampered investigation of the in vivo role of these mast cell proteases.

We recently cloned the baboon chm gene, including its ~2.5-kb1 5'-upstream sequence. The 5'-upstream sequence of this gene is highly homologous to the corresponding region of the human chm gene described by Caughey et al. (16). In this report we show that the 571-bp 5'-upstream sequence of the baboon chm gene coupled to the prokaryotic lacZ gene allows the targeting of the beta -galactosidase reporter to mast cells in transgenic mice. We also show that tissue expression of the lacZ gene directed by the baboon chm 571-bp 5'-upstream region is similar to the endogenous pattern of expression of the mouse alpha -chymase mmcp5 and that this promoter is highly active in JKras mast cells, a mouse mast cell line that endogenously expresses mmcp5. Furthermore, in vitro transcriptional studies in this mouse mast cell line demonstrate the critical role of a GATA cis-regulatory motif in baboon chm promoter-directed expression of the reporter gene. These studies provide a novel tool for the specific expression of genes in mast cells that is likely to be crucial in uncovering the in vivo role of primate chymases as well as other mast cell proteins and provide insight into cis-trans interactions that may be important in baboon chm gene expression in mast cells.


EXPERIMENTAL PROCEDURES

Size-selected Baboon Genomic Library Construction, Gene Cloning, and Primer Extension Analysis

Southern blot analysis indicated the presence of a single 7-kb EcoRI-digested band from baboon genomic DNA that hybridized to a 32P-labeled human chymase cDNA probe. EcoRI-digested 6-8-kb fragments of baboon genomic DNA were cloned into a phage lambda  gt10 vector system (Stratagene, La Jolla, CA) as per manufacturer's protocol. The 7-kb insert, which strongly hybridized to the human chymase cDNA probe, was subcloned into a pBluescript KS+ phagemid (Stratagene). The nucleotide sequence of the overlapping fragments was determined in both directions.

The 5'-end of baboon heart-derived chymase mRNA was defined by primer extension analysis (17). Poly(A)+ RNA, prepared from the baboon left cardiac ventricle, was incubated for 1 h at 42 °C in a reaction buffer containing Moloney murine virus reverse transcriptase (Boehringer Mannheim) and a 30-bp 32P-labeled oligonucleotide derived from the 3'-end of the first exon of the baboon chm gene. After phenol/chloroform extraction and ethanol precipitation, the sample was electrophoresed on a 6% polyacrylamide gel, and the size of the prominent radiolabeled DNA fragment extended onto the primer was determined.

Plasmid Construction and Generation of Transgenic Mice

The lacZ gene was released from a pSV-beta -galactosidase plasmid by SalI and BamHI digestion (the SalI site is created by PCR and located just before the ATG translation start site). Two different transgenes were constructed by ligation of 2,426- or 571-bp fragments of the baboon chm gene's 5'-upstream region with the lacZ gene. The linearized transgene constructs were microinjected (18); ~2 pl of the DNA solution (~1000 copies) was microinjected into one pronucleus of each mouse (FVB) F2 embryo. Injections were made by the Transgenic Core Facility of the Cleveland Clinic Foundation. Two-cell stage embryos were transferred to the oviducts of 0.5-day post-coitus pseudopregnant Swiss FBR females. The offspring were screened for transgene integration by Southern blot analysis of tail DNA using a 32P-labeled 3.34-kb prokaryotic lacZ gene as probe.

Histochemistry

A previously described histochemical staining procedure was used to assess beta -galactosidase activity (18). Three-week-old transgenic mice as well as their nontransgenic littermates were sacrificed. Tissue samples from each mouse were fixed for 40-60 min in 2% formaldehyde, 0.2% gluteradehyde, 0.02% Nonidet P-40, and 0.01% sodium deoxycholate in phosphate-buffered saline, embedded in the embedding medium OCT and frozen on dry ice. Serial 6-µm tissue sections were cut with a cryostat, loaded on charge-coated slides (Fisher), and refixed in the same solution for 10 min. After three washes in phosphate-buffered saline, sections were submerged in 0.1 M phosphate buffer, pH 7.3, containing 1 mg/ml 4-chloro-5-bromo-3-indolyl-beta -D-galactoside (Boehringer Mannheim), 2 mM MgCl2, 10 mM K3Fe(CN)6, and 10 mM K4Fe(CN)6. Incubation was carried out for 3-4 h at 37 °C. After 4-chloro-5-bromo-3-indolyl-beta -D-galactoside staining, sections were dehydrated and mounted in Permount (Fisher). Adjacent slides were stained with hematoxylin and eosin or with 1% toluidine blue (T-blue).

Northern Blot Analysis

Total RNA was isolated from 1 × 108 cells using an RNAzol kit (TEL-TEST, Friendswood, TX). 20-µg RNA samples were size-fractionated on a 1% formaldehyde-agarose gel and transferred to a nitrocellulose filter (19). After UV cross-linking, the blot was hybridized with 32P-labeled human chymase cDNA at high stringency.

Cell Lines and Tissue Culture

Mouse mastocytoma P815 cells, mouse erythroid leukemia (MEL) cells, and NIH3T3 (3T3) fibroblasts were obtained from the American Type Culture Collection. JKras mast cells (20) were obtained from Dr. James N. Ihle (St. Jude Children's Research Hospital, Memphis, TN). P815 mast cells and MEL cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum. JKras mast cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and murine interleukin-3 (20 units/ml). 3T3 fibroblasts were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cells were kept at 37 °C in an atmosphere of 5% CO2 and split (~1:4) every 5 days.

Construction of Recombinant Plasmids

Baboon chm promoter-CAT chimeras were made by fusing varying lengths of the baboon chm gene's 5'-upstream sequence to the coding sequences for CAT. These baboon chm promoters of varying lengths were generated by PCR amplification. For each construct two primers were used; each contained either a terminal PstI site or a SalI site to facilitate cloning into the PstI and SalI sites immediately upstream of the CAT reporter gene in the plasmid pSV40-CAT enhancer (Promega, Madison, WI). The number following the Delta  in Fig. 1 indicates the end of the 5'-upstream sequence with respect to the transcription-initiation site (designated +1) was determined by primer extension analysis. In addition to the specified amounts of the 5'-upstream sequence, these constructs contained the transcription-initiation site and 30 bp of the 5'-upstream region between the transcription-initiation site and the putative translation start site of the baboon chm gene.


Fig. 1. Organization of the baboon chm gene. The nucleotide sequence of the five coding blocks, four partial intervening sequences (italicized), and 5'- and 3'-flanking regions are shown. The transcription start site is numbered as +1. The deduced encoded amino acids of baboon preprochymase are indicated below the corresponding nucleotides. Numbers at the right refer to the nucleotides. Sequences, double underlined, in the 5'- and 3'-flanking region include a TATA and CAAT box, several consensus (A/T)GATA(A/G)-binding elements, and the consensus polyadenylation motif, AATAAA. In the 5'-flanking region, numbers preceded by down-arrow Delta show the start sites of the various promoter constructs used in this study.
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Transient Cell Transfection and CAT Activity Assays

Transient transfection of 3T3 fibroblasts was performed using the calcium/phosphate co-precipitation method (19). JKras mast cells and P815 mast cells were transiently transfected by the DEAE-dextran method (19). As an internal control for these transfections, the pSV-lacZ plasmid was co-transfected with the test plasmid (1:5). The cells were harvested and ruptured by three freeze-thaw cycles. One-half of the extract was heated for 10 min at 65 °C to destroy endogenous deacetylating activity. After removal of the cellular debris, the heat-treated extract was assayed for CAT activity. The remaining extract was assayed for beta -galactosidase activity. Based on beta -galactosidase activity, CAT activity in each sample was adjusted for between plate and between experiment variability in transfection efficiency. Transient transfection of COS-1 cells was performed using the DEAE-dextran method (19). The pSV-lacZ plasmid was co-transfected with the test plasmid (1:5) as an internal control. Human GATA-2 cDNA plasmid, a gift from Dr. S. H. Orkin (Harvard Medical School, Boston), was co-transfected with the Delta 571-bchm-CAT construct to determine transcriptional activation of this promoter construct by human GATA-2. CAT activity in each sample was adjusted for between plate and between experiment variability in transfection efficiency.

Cell-free Transcription Assays

JKras mast cell nuclear extracts were prepared as described previously (21). Nuclear extracts were dialyzed overnight at 4 °C against 20 mM HEPES buffer, pH 7.9, containing 100 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.5 mM dithiothreitol, 1 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, and 25% glycerol, and stored at -70 °C. In vitro transcription reactions were performed with 2 nM supercoiled template DNA and 10-15 µl of nuclear extract (10-15 mg/ml) in a 50-µl reaction mixture containing 12 mM HEPES buffer, pH 7.9, containing 50 mM KCl, 25 mM (NH4)2SO4, 7.5 mM MgCl2, 0.1 mM EDTA, 0.3 mM dithiothreitol, 2 units of RNasin, 12% glycerol, 600 µM ATP, 600 µM UTP, 600 µM GTP, 25 µM CTP, and 10 µCi of [alpha -32P]CTP (Amersham Corp.). Transcription reactions were allowed to proceed for 60 min at 30 °C and terminated by the addition of 0.2% SDS, 10 mM EDTA, and 100 µg of tRNA carrier/ml. RNA in the transcription mixture was extracted with 200 µl of phenol/chloroform/isoamyl alcohol (25:24:1), ethanol precipitated, and dissolved in diethyl pyrocarbonate-treated Tris/HCl buffer, pH 8.0, containing 1 mM EDTA. The RNA samples were separated on a 1% formaldehyde-agarose gel in 1 × MOPS buffer, transferred to a nitrocellulose filter, and subjected to autoradiography for 16 h.

Electrophoretic Mobility Shift Assays

Electrophoretic mobility shift assays were performed as described (22). Binding reactions were performed with 20 µg of JKras mast cell nuclear extract and ~0.5 ng of 32P-labeled oligonucleotide corresponding to a fragment (-417 to -441 bp) of the baboon chm promoter (100,000 cpm) in 20 mM HEPES buffer, pH 7.9, containing 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, 100 mg/ml bovine serum albumin, and 1 µg/ml poly d(I-C). To study the effect of competitor oligonucleotides on the binding of the 32P-labeled oligonucleotide to JKras mast cell nuclear trans-acting factors, a 10-, 30-, or 60-fold excess of competitor oligonucleotide was added to the reaction mixture 10 min before the addition of the radiolabeled probe. 32P-Labeled DNA-protein complexes were resolved from unbound radiolabeled DNA on 6% polyacrylamide gels run in 22.5 mM Tris borate buffer, pH 8.3. The gels were dried under vacuum and autoradiographed for ~16 h.

Reverse Transcriptase (RT)-PCR

Single-stranded cDNAs were prepared from total RNA from JKras mast cells, MEL cells, P815 cells, 3T3 fibroblasts, and mouse spleen and from tissues from the transgenic mice and their nontransgenic littermates using oligo(dT) primers and Moloney murine virus reverse transcriptase. The resulting single-stranded cDNAs were amplified using a 0.5 µM concentration of each primer. For mmcp5, 5'-TTCTGAGAACTACCTGTCGGCC-3' was used as the sense strand, and 5'-GAATTGTGTCGAAAACTGGTGAAGTG-3' was used as the antisense strand. For mouse GATA-1, 5'-CAACAGTATGGAGGGAATTCCTGGGGGCT-3' was used as the sense strand, and 5'-GGCCGGTTCTGACCATTCATTTTGTGATA-3' was used as the antisense strand. For mouse GATA-2, 5'-GACGGAGAGCATGAAGATGG-3' was used as the sense strand, and 5'-AGGCATTGCACAGGTAGTGG-3' was used as the antisense strand; these primers were based on the sequence of human GATA-2. For mouse GATA-3, 5'-CCTTGGAACCTCAGCCCTTTTTCCAAGACCTCCA-3' was used as the sense strand, and 5'-GGCCGGTTCTGACCATTCATTTTGTGATA-3' was used as the antisense strand. For lacZ, 5'-CCAGCGTTTCAACGCGCTGTATGGCG-3' was used as the sense strand, and 5'-AGACGTCACGGAAAATGCCGCTCATC-3' was used as the antisense strand. For GAPDH, 5'-CAAAGTTGTCATGGATGACCTTGGC-3' was used as the sense strand, and 5'-GTCTTCACCACCATGGAGAAGGCTG-3' was used as the antisense strand.

Statistical Analyses

Autoradiographic data were analyzed using a Molecular Dynamics PhosphorImager. RT-PCR agarose gel bands were quantitated using a densitometric image analyzer (densitometer) driven by a Macintosh computer. Quantitative data are presented as the means ± S.E., and differences between means were evaluated using a Student's t test, with p < 0.05 considered statistically significant.


RESULTS

Structure of the Baboon chm Gene

The nucleotide sequence and organization of the baboon chm gene is shown in Fig. 1. Based on a comparison with the baboon chymase cDNA structure, this gene has five coding blocks. The first coding block is 58 bp in length; it encodes, in an open reading frame, the first 19 amino acids (-21 to -3) of the preproenzyme. The second, third, fourth, and fifth coding blocks of the baboon chm gene encode amino acids -2 to 49, 50 to 94, 95 to 179, and 180 to 226. As assessed by primer extension analysis, the primary transcription-initiation site of the baboon chm gene is 30 bp upstream of the translation-initiation site (data not shown). The translation-initiation ATG codon of the baboon chm gene was predicted based on the structure of the baboon chymase cDNA. The 5'-upstream region of the baboon chm gene contains a CAAT and a TATA box, -108 to -105 bp and -29 to -25 bp upstream, respectively, of the transcription-initiation site. A consensus polyadenylation motif, AATAAA, which is followed by a cleavage site motif, consisting of a CA sequence located 15 bp downstream along with a GT cluster, is found in the 3'-untranslated region. The structure of the baboon chm gene's 5'-upstream region (-2,426 to -1 bp) is ~92% identical to that of the human chm gene. Baboon chymase is 97% identical to human chymase (9) and 76% identical to its mouse homolog mmcp5 (12).

Cell- and Tissue-specific Expression of Baboon chm-lacZ Transgenes

To identify the baboon chm promoter, two constructions were made. These constructs consist of 571- and 2,426-bp 5'-upstream sequences of the baboon chm gene linked to the prokaryotic lacZ gene, i.e. Delta 571-bchm-lacZ and Delta 2,426-bchm-lacZ, respectively. Delta 571-bchm-lacZ and Delta 2,426-bchm-lacZ constructions were used to generate transgenic founders. Germline transmission occurred with Delta 571-bchm-lacZ (two female and one male founder) and Delta 2,426-bchm-lacZ (1 male founder) constructs. A Delta 571-bchm-lacZ male founder and a Delta 2,426-bchm-lacZ male founder were used to generate F1 transgenic heterozygous mice. Subsequent tissue and cell expression of the lacZ gene was determined in F1 heterozygous transgenic mice.

Tissue expression of beta -galactosidase mRNA in transgenic heterozygous mice and mmcp5 expression in nontransgenic littermates was quantified using RT-PCR. Ratios of mmcp5/GAPDH in tissues from nontransgenic mice and lacZ/GAPDH mRNA in tissues from Delta 571-bchm-lacZ and Delta 2,426-bchm-lacZ transgenic mice are summarized in Fig. 2. The relative distribution of GAPDH-normalized lacZ expression in the stomach, intestine, spleen, heart, lung, and brain tissues was similar between the Delta 571-bchm-lacZ and Delta 2,426-bchm-lacZ constructs and also similar to that of mmcp5. To identify the cell types in transgenic mice that express the lacZ gene, we examined T-blue-staining cells (T-blue produces a deep purple stain in mast cells) and beta -galactosidase-containing cells in serial sections of the stomach from the Delta 571-bchm-lacZ and Delta 2,426-bchm-lacZ transgenic mice. Fig. 3 indicates the selective expression of beta -galactosidase in T-blue staining cells in the submucosa of the stomach from a Delta 571-bchm-lacZ transgenic mouse. Similar results were observed in the gastric submocosa and small intestine of other Delta 571-bchm-lacZ (n = 2) and Delta 2,426-bchm-lacZ (n = 2) transgenic mice (data not shown). Fig. 4 shows representative cells in the Delta 571-bchm-lacZ transgenic left cardiac ventricle staining for T-blue or beta -galactosidase. To determine if Delta 571-bchm promoter-driven lacZ expression occurs in circulating mast cell progenitors, leukocytes from Delta 571-bchm-lacZ transgenic mice were examined for beta -galactosidase activity. beta -Galactosidase activity was not observed in several leukocyte samples (data not shown).


Fig. 2. Tissue expression of beta -galactosidase mRNA in F1 heterozygous transgenic mice and mmcp5 mRNA in their nontransgenic littermates. Mmcp5, beta -galactosidase, and GAPDH mRNA levels were determined by RT-PCR. Data are presented as ratios of mmcp5/GAPDH in tissues (n = 3) from nontransgenic mice (A) and lacZ/GAPDH mRNA in tissues from Delta 571-bchm-lacZ (B) and Delta 2,426-bchm-lacZ (C) transgenic mice.
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Fig. 3.

Photomicrographs showing the expression of the Delta 571bchm-lacZ construct in the gastric submucosa of an F1 heterozygous transgenic mouse. Adjacent sections of the stomach of a transgenic mouse harboring the Delta 571-bchm-lacZ construct were stained with hematoxylin and eosin (a), T-blue (b), and beta -galactosidase (c). In b, purple-stained cells in the gastric submucosa indicated by arrows represent mast cells. In c, beta -galactosidase activity is found in cells located in the same position as the purple-stained mast cells shown in b. d and e show magnified views of stained cells in b and c, designated by-arrows with asterisks, respectively.


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Fig. 4. Photomicrographs showing the expression of the Delta 571-bchm-lacZ construct in the heart of an F1 heterozygous transgenic mouse. Adjacent sections of the cardiac left ventricle of a transgenic mouse harboring the Delta 571-bchm-lacZ construct were stained with T-blue (A) and beta -galactosidase (B). In A, purple-stained cells in the left ventricle represent mast cells. In B, beta -galactosidase activity is found in a mast cell-like cell located in the adjacent section but from a different grid than that shown in A.
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Baboon chm Gene 5'-Flanking Region Directs CAT Expression in JKras Mast Cells, but Not P815 Mast Cells

We examined several murine mast cell lines to see which ones endogenously express the mouse alpha -chymase mmcp5. Using a human chymase (an alpha -chymase-like mmcp5 (6)) cDNA probe, Northern blot analysis indicated chymase-like mRNA in mouse JKras mast cells but not in mouse mastocytoma P815 cells or mouse erythroid leukemia MEL cells (Fig. 5A). Using mouse chymase subtype selective PCR primers, Fig. 5B shows that JKras mast cells contain mmcp5 mRNA. Using mouse beta -chymase subtype-selective PCR primers showed the absence of beta -chymase mRNA in JKras mast cells (data not shown). JKras mast cells are interleukin-3-dependent Harvey sarcoma virus-infected immune mast cells from the mouse spleen (20).


Fig. 5. Chymase mRNA levels in different mouse cells. A, an RNA blot containing approx 20 µg/lane of total cellular RNA from mouse JKras mast cells, MEL cells, mouse mastocytoma P815 cells, and mouse 3T3 fibroblasts was probed under high stringency with human chymase cDNA. B, RT-PCR analysis of 1 µg of total RNA from JKras cells, MEL cells, P815 cells, and 3T3 fibroblasts using PCR primers specific for mmcp5 (mouse chymase 5) cDNA.
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Fig. 5 shows that mmcp5 is endogenously expressed in JKras mast cells but not P815 mast cells. To examine whether this discrimination in the expression of an alpha -chymase-encoding chm gene in these two mast cell lines is mimicked by the transcriptional activity of the Delta 571 baboon chm promoter-CAT construction, Delta 571-directed expression of the CAT gene was determined in JKras mast cells and P815 mast cells and compared with its transcriptional activity in 3T3 fibroblasts. Table I shows CAT activity in JKras mast cells, P185 mast cells, and 3T3 fibroblasts after transient transfection with the baboon chm-CAT construction Delta 571. The results of the transfection assays were expressed as a ratio of activity of Delta 571 to that of the nonspecific control plasmid (SV40-CAT control). The relative CAT activity directed by Delta 571 in JKras mast cells was 25-fold higher than in P815 mast cells and 51-fold higher than in mouse 3T3 fibroblasts.

Table I.

Relative activity of a baboon chm gene 5'-flanking region in different mouse cells

Relative CAT expression directed by pSV40 and the 571-bp 5'-flanking region of the baboon chm gene (Delta 571). The pSV40 contains the SV40 promoter and its enhancer. For each cell type (mean ± S.E.; n = 3), CAT activity is expressed relative to that of the pSV40-CAT control construction, which is defined as 1. The cell specificity ratio represents the relative CAT activity obtained in the JKras mast cell (JKras) relative to that obtained in P815 mast cells (P815) or 3T3 fibroblasts (3T3 cells).
Construction Relative CAT activity
Cell specificity ratio
JKras P815 3T3 cells JKras/P815 JKras/3T3 cells

pSV40-CAT 1 1 1
 Delta 571 8.2 ± 1.6 0.32 ± 0.052 0.16 ± 0.017 25.6 51.3

A GATA-binding cis-Regulatory Element in the Baboon chm Promoter Is Essential for Transcription in JKras Mast Cells

We studied the in vitro regulation of the baboon chm promoter in JKras mast cells. To map the location of cis-acting elements within the baboon chm promoter, transcription studies were carried out in JKras mast cell nuclear extracts with deletion mutants of the 571-bp baboon chm promoter-CAT construct (Delta 571-bchm-CAT). The results are summarized in Fig. 6. The transcriptional activity of the Delta 453-bchm-CAT construct was 11-fold greater than of the Delta 426-bchm-CAT construct but similar to that of Delta 571- and Delta 506-bchm-CAT constructs, indicating the presence of a positive regulatory element between -426 and -453 bp of the baboon chm gene's 5'-upstream region. Motif analysis indicates that this region contains a GATA-binding element, situated at -432/-427 bp upstream of the transcription-initiation site. To determine if the putative -432/-427 GATA-binding site is a necessary for transcription, we examined transcriptional activities of the Delta 435-bchm-CAT construct and its mutant [A-430right-arrowC]-Delta 435-bchm-CAT containing a defective GATA-binding site (Fig. 7). Transcriptional activity of [A-430right-arrowC]-Delta 435-bchm-CAT in JKras mast cell nuclear extracts was ~12-fold lower than that of Delta 435-bchm-CAT, indicating the importance of this single GATA cis-regulatory site in the transcriptional activation of the baboon chm gene.


Fig. 6. Transient expression of bchm-CAT fusion constructions. A, schematic diagram of bchm-CAT deletion constructions representing -571 to -292 bp of the bchm 5'-upstream region used in B and C. The position of each deletion is denoted by the vertical lines. The sites of the GATA, CAAT, and TATA boxes in the baboon chm 5'-upstream region are indicated. The large shaded boxes indicate sites 3' to the transcription start site denoted by +1 and represent the coding sequences for the CAT gene, the SV40 small T antigen intervening sequence, and the SV40 polyadenylation signal. B, CAT mRNA accumulation, relative to that of SV40, during transient expressions of baboon chm-CAT fusion constructions (shown in A) in a cell-free transcription system using JKras mast cell nuclear extracts. Histogram represents the mean of three to five separate experiments. S.E. values, not shown here, were <15% of the mean for each bchm-CAT construction. C, a representative experiment showing CAT mRNA accumulation during transient expression of bchm-CAT fusion constructions.
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Fig. 7. Transient expression of wild type (construct A), GATA-defective (construct B), and GATA-deleted (construct C) bchm-CAT constructions. The nature of the mutations in constructs B and C is shown at the bottom of the figure. Data represent CAT mRNA accumulation, relative to that of SV40, during transient expressions of the constructs in a cell-free transcription system using JKras mast cell nuclear extract (n = 3). Relative transcription activity for constructs B and C was 12 and 10%, respectively, of that of the wild type construct (p < 0.01).
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Previous studies have indicated synergistic interactions between GATA and other transcription factors in gene regulation (23, 24). To identify putative cis-acting elements in the Delta 571-bchm-CAT construct that could synergize with the GATA cis-regulatory element, transcriptional activity of additional Delta 571-bchm-CAT mutants was examined. Deletion of the 139-bp sequence in the Delta 571-bchm-CAT construct 5'-upstream of the GATA site (i.e. Delta 432-bchm-CAT) did not reduce the transcriptional activity of the mutant promoter construct in JKras mast cell nuclear extract (construct 2, Fig. 8). However, replacement of the 41-bp sequence immediately 3' to the GATA site in the Delta 432-bchm-CAT construct with an indifferent sequence produced a ~50% decrease (p < 0.05) in transcription (construct 3, Fig. 8). This indifferent sequence, shown in Fig. 8, was 41% homologous to the wild type. Deletion of a 333-bp sequence, -386 to -53 bp upstream of the transcription-initiation site, in the Delta 432-bchm-CAT construct did not affect transcriptional activity (construct 4, Fig. 8).


Fig. 8. Transient expression of a wild type Delta 571-bchm-CAT construct (construct 1), a Delta 432-bchm-CAT construct (construct 2), a Delta 432-bchm-CAT mutant construct in which the 41-bp region 3' to the GATA site was replaced with an indifferent sequence (construct 3), and a Delta 432-bchm-CAT mutant construct with a 333-bp deletion between -386 and -53 bp (construct 4). The data represent CAT mRNA accumulation, relative to that of SV40, during transient expressions of the constructs in a cell-free transcription system using JKras mast cell nuclear extract (n = 3). Relative transcription activity for constructs 2 and 4 was not different from that of wild type baboon chm promoter construct, but activity of construct 3 was 47% of that of the wild type construct (p < 0.05). The indifferent 41-bp sequence used in construct 3, i.e. 5'-ACAACAATTTTGTAGTATTAGTATGTCTCATTCAATATTTG-3', was 41% homologous to the wild type. The sequence of the DNA fragments that were deleted is shown in Fig. 1.
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GATA-2 Is Present in JKras Mast Cells and Supports Transcription of the Baboon chm Promoter

Electrophoretic mobility shift analysis using an end-labeled DNA fragment comprising the sequence -441 to -417 bp of the baboon chm promoter containing a GATA-binding element indicated the presence of a trans-acting factor in JKras mast cell nuclear extracts (Fig. 9). To characterize the specificity of this binding, competition assays were performed using mutated forms of the -441/-417 baboon chm gene DNA fragment. Mutation of the GATA sequence to G<UNL>c</UNL>TA or <UNL>c</UNL>ATA produced a loss of binding to the JKras mast cell trans-acting factor (Fig. 9). The sequence specificity illustrated by these experiments is similar to that previously shown for GATA-1, GATA-2, and GATA-3 binding proteins (25).


Fig. 9. DNA binding activity of JKras nuclear proteins. A 25-bp DNA fragment spanning the AGATAA-site from the bchm promoter comprising the region -444 to -417 bp was used as the probe in electrophoretic mobility shift assays. The arrow indicates the position of the specifically labeled band. Competitor DNA fragments include the unlabeled 25-bp fragment used as the probe (GATA), the G-431 to C mutant of the GATA 25-bp fragment (cATA), and the A-432 to C mutant of the GATA 25-bp fragment (GcTA). Lane 1 is competitor-free. Lanes 2, 5, and 8, lanes 3, 6, and 9, and lanes 4, 7, and 10 contain a 10-, 30-, and 60-fold excess of competitor DNA, respectively.
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RT-PCR analysis with specific primers for GATA-1, GATA-2, and GATA-3 was used to examine expression of these transcription factors in JKras mast cells. Fig. 10 shows that GATA-2 but not GATA-1 or GATA-3 mRNA is present in JKras mast cells and P815 mast cells. The presence of GATA-1 but not GATA-2 or GATA-3 mRNA in MEL cells and GATA-2 mRNA in P815 cells has previously been shown. These observations of GATA-binding protein subtypes in MEL and P815 cells indicates the specificity of these PCR primers (Fig. 10). Because GATA-3 mRNA could not be identified in these cells as a positive control, RT-PCR analysis was also performed with total RNA from mouse spleen, which is known to contain lymphoid cells expressing GATA-3. The absence of GATA-1, GATA-2, and GATA-3 mRNA in 3T3 fibroblasts also indicates the specificity of the PCR primers.


Fig. 10. Identification of GATA-1, GATA-2, and GATA-3 mRNA in different mouse cells by RT-PCR. RT-PCR analysis of 1 µg of total RNA each from JKras mast cells, MEL cells, P815 cells, NIH-3T3 fibroblasts, and mouse spleen using PCR primers specific for GATA-1 (A), GATA-2 (B), and GATA-3 (C) cDNA. Arrows indicate the expected size of the amplified products.
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To determine if ectopically expressed GATA-2 can support Delta 571-bchm-CAT expression in a heterologous cell, we determined transcriptional activity of the Delta 571-bchm-CAT construct in COS-1 cells transfected with a human GATA-2 cDNA construct (26). Fig. 11 shows an ~5-fold increase in transcription of the Delta 571-bchm-CAT construct in COS-1 cells transfected with human GATA-2 cDNA compared with mock transfected COS-1 cells.


Fig. 11. Transcriptional activity of the Delta 571-bchm-CAT promoter construct in mock-transfected COS-1 cells (-) or COS-1 cells transiently transfected with a human GATA-2 cDNA construct (+). Relative promoter activity increased by 4.9-fold in GATA-2 transfected COS-1 cells (p < 0.01, n = 3).
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DISCUSSION

Chymase occurs in numerous human (10) and baboon (27) tissues including the alimentary tract, heart, lungs, blood vessels, and reproductive tissues is implicated in diverse functions and represents a differentiation-specific marker that identifies a population of mature tissue resident mast cells (28). Using a transgenic approach, we show that the 571-bp 5'-upstream region of the baboon chm gene is a promoter that allows restricted mast cell expression of a reporter gene. These findings, as well as our identification of a critical cis- and trans-acting positive regulatory element within this promoter, shed light on how chm genes are transcriptionally regulated.

Introduction in mice of constructs containing 0.571- or 2.426-kb fragments of the 5'-upstream region of the baboon chm gene coupled to the prokaryotic lacZ gene, i.e. Delta 571-bchm-lacZ or Delta 2,426-bchm-lacZ, respectively, allows the restricted expression of beta -galactosidase in T-blue staining mast cells resident in the gastric submocosa of transgenic mice. Cells containing beta -galactosidase with mast cell-like granules were also detected in the heart and intestine of mice containing the Delta 571-bchm-lacZ transgene; however, because the beta -galactosidase-containing cells from the heart did not cluster like mast cells of the gastric submucosa, it was not possible to confirm from T-blue staining of adjacent sections whether these cells were mast cells. Circulating progenitor mast cells must normally be resident in tissue before ultrastructural characteristics and protease expression patterns associated with fully differentiated mast cells are observed (1). We did not observe any cells with beta -galactosidase activity in circulating leukocyte samples from mice harboring the Delta 571-bchm-lacZ transgene. The relative lack of expression of the lacZ gene in brain tissue is also relevant to tissue-specific expression of the chm gene. Blood cells cannot readily penetrate the blood-brain barrier, and thus progenitor mast cells cannot get into brain tissue. These observations illustrate the high fidelity of the 571-bp 5'-upstream region of the baboon chm gene in conferring development-specific in vivo expression of the lacZ gene in mast cells. Previously, our in situ hybridization and electron microscope immunohistochemical studies showed the presence of human chymase and its mRNA in some vascular endothelial cells (10). In all tissues studied from mice expressing the Delta 571-bchm-lacZ or Delta 2,426-bchm-lacZ transgenes, beta -galactosidase activity was not detected in endothelial cells. This finding suggests that either mouse endothelial cells do not contain trans-activating factors necessary for chymase expression or cis-regulatory sequences other than those present in the ~2.5-kb 5'-upstream fragment of the baboon chm gene are required for chymase expression in endothelial cells.

Distribution of beta -galactosidase mRNA in tissues from mice containing the Delta 571-bchm-lacZ or Delta 2,426-bchm-lacZ transgenes was similar to the distribution of mmcp5 mRNA (mmcp5 is the mouse alpha -chymase). Given that the same observations were made in two independently generated groups of transgenic mice, we believe that transcriptional specificity in transgene expression is likely to be due to baboon chm regulatory sequences rather than to integration of the constructs near a strong endogenous mast cell regulatory domain. These studies and the observation that Delta 571-bchm-driven CAT expression is high in a mouse mast cell line that endogenously expresses the mmcp5 gene (JKras mast cells) but low in a mouse mast cell line that does not (P815 mast cells) implies that this baboon promoter could show in vivo transcriptional specificity between mmcp5-expressing connective tissue mast cells and mucosal mast cells that do not express mmcp5. Additional in vivo studies are necessary to prove that Delta 571-bchm-driven reporter expression is limited to mmcp5-expressing mast cells in vivo; however, the use of this promoter for generating transgenic mouse models with dominant mast cell expression of active primate chymases is also likely to be dependent on proteoglycan composition of the mast cells. Mmcp5 is found in all populations of mouse mast cells containing heparin sulfate proteoglycans but not in chondroitin sulfate-containing mucosal mast cells (29). Because heparin sulfate but not chondroitin sulfate is an important cofactor in alpha -prochymase activation (15), Delta 571-bchm-driven zymogen expression in vivo is likely to lead to alpha -prochymase activation in connective tissue mast cells but not in mucosal mast cells.

Transcriptional regulation of a gene is achieved through interactions of cis-regulatory regions uniquely present in a gene and cell-specific trans-activating factors with components of the basal transcriptional complex assembled at the initiation site. We used the Delta 571-bchm-CAT promoter construct to identify cis-regulatory regions of the baboon chm promoter critical for transcriptional activation in JKras mast cells. Deletional and site-specific mutational analyses revealed a strong cis-acting positive regulatory GATA element between -453 to -426 bp upstream of the transcription-initiation site. A GATA-binding site is also present ~430 and ~100 bp upstream of the transcription-initiation site of the human chm (16, 30) and mmcp5 (12) genes, respectively, and therefore, GATA-binding proteins could be involved in transcriptional activation of these alpha -chymase encoding chm genes in mast cells.

We found GATA-2 but not GATA-1 or GATA-3 DNA-binding proteins in JKras mast cells. The further observation that CAT activity in COS-1 cells increases by ~5-fold after transfection with human GATA-2 cDNA shows the importance of this transcription factor in trans-activation of the baboon chm promoter. GATA-1, GATA-2, and GATA-3 transcription factors have markedly differing patterns of cellular expression, but they recognize the same cis-regulatory motif (A/T)GATA(A/G) (31). In cell-dependent expression of genes requiring a GATA cis-trans-interaction, a high level of transcriptional activation can be achieved through synergistic interactions with other transcription factors. For example, GATA-1 and Krüppel family factors cooperate from a distance in directing expression of the globin gene in erythroid cells (23). A similar cooperative interaction has been described between GATA-2 and AP1 in trans-activation of the endothelin-1 gene (24). In these examples, synergistic interactions between GATA and Sp1, AP1, or EKLF produce a substantial effect, i.e. a >10-fold increase in transcription over a simple additive effect. Using motif analysis, we were unable to locate cis-regulatory motifs for AP1, Sp1, or EKLF in the 571-bp baboon chm promoter. Furthermore, deletion or replacement of DNA sequences 5' or 3' to the GATA cis-regulatory element produced small decreases (<50%) in transcriptional activation of the Delta 571-bchm-CAT construct in JKras mast cell nuclear extracts. These data indicate an absence of strong synergistic interactions between GATA-2 and other trans-acting factors in the transcriptional activation of the baboon chm promoter in JKras mast cells.

In summary, we show that the 571-bp 5'-upstream sequence of the baboon chm gene can be used to selectively target mouse mast cells in vivo and in vitro and thus provides an important tool in the generation of transgenic mice where proteins need to be targeted to mast cells. We also show that a GATA cis-trans-interaction is both necessary and sufficient for transcriptional activation of the baboon chm promoter in vitro in JKras mast cells. However, given that GATA-2 is found in differentiated mast cells but also has an important role early in hematopoietic cell development (31-33), it is clear that engaging the GATA cis-regulatory element must be one of multiple cis-trans-interactions necessary for transcriptional specificity. The finding that the P815 mast cell contains abundant GATA-2 but does not support a high level expression of CAT with the Delta 571-bchm-CAT promoter construct suggests that suppression of dominant negative interactions as well as enhanced GATA-2 expression could coordinate chm gene expression as progenitor mast cells differentiate into mature tissue resident mast cells. The availability of a mast cell-specific promoter now makes it possible to study how chm gene expression is suppressed in primate mast cell progenitors as well as in other GATA transcription factor-expressing hematopoietic cells.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant HL44201. 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) U38463[GenBank] and U38521[GenBank].


par    To whom correspondence should be sent: Dept. of Molecular Cardiology, FF30, Research Inst., Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-2057; Fax: 216-444-9410; E-mail: husaina{at}cesmtp.ccf.org.
1    The abbreviations used are: kb, kilobase(s); mmcp5, mouse mast cell protease-5; RT, reverse transcriptase; bp, base pair(s); PCR, polymerase chain reaction; T-blue, toluidine blue; MEL, mouse erythroid leukemia; MOPS, 4-morpholinepropanesulfonic acid; CAT, chloramphenicol acetyltransferase.

Acknowledgments

We thank Dennis Wilk and Xiaopu Liu for excellent technical assistance and Christine Kassuba for manuscript editing.


REFERENCES

  1. Schwartz, L. B. (1993) in The Mast Cell in Health and Disease (Kaliner, M. A., and Metcalfe, D. D., eds), pp. 219-236, Marcel Dekker, New York
  2. Schwartz, L. B. (ed) (1990) Neutral Proteases of Mast Cells, Karger, Richmond, VA
  3. Urata, H., Kinoshita, A., Misono, K. S., Bumpus, F. M., and Husain, A. (1990) J. Biol. Chem. 265, 22348-22357 [Abstract/Free Full Text]
  4. Kinoshita, A., Urata, H., Bumpus, F. M., and Husain, A. (1991) J. Biol. Chem. 266, 19192-19197 [Abstract/Free Full Text]
  5. LeTrong, H., Neurath, H., and Woodbury, R. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 364-367 [Abstract]
  6. Chandrasekharan, U. M., Sanker, S., Glynias, M. J., Karnik, S. S., and Husain, A. (1996) Science 271, 502-505 [Abstract]
  7. Peach, M. J. (1977) Physiol. Rev. 57, 313-370 [Free Full Text]
  8. Sadoshima, J., Xu, Y., Slayter, H. S., and Izumo, S. (1993) Cell 75, 977-984 [Medline] [Order article via Infotrieve]
  9. Urata, H., Kinoshita, A., Perez, D. M., Misono, K. S., Bumpus, F. M., Graham, R. M., and Husain, A. (1991) J. Biol. Chem. 266, 17173-17179 [Abstract/Free Full Text]
  10. Urata, H., Boehm, K. D., Phillip, A., Kinoshita, A., Gabrovsek, J., Bumpus, F. M., and Husain, A. (1993) J. Clin. Invest. 91, 1269-1281 [Medline] [Order article via Infotrieve]
  11. Itoh, H., Murakumo, Y., Tomita, M., Ide, H., Kobayaski, T., Maruyama, H., Horii, Y., and Nawa, Y. (1996) Biochem. J. 314, 923-929 [Medline] [Order article via Infotrieve]
  12. McNeil, H. P., Austen, K. F., Somerville, L. L., Gurish, M. F., and Stevens, R. L. (1991) J. Biol. Chem. 266, 20316-20322 [Abstract/Free Full Text]
  13. Powers, J. C., Tanaka, T., Harper, J. W., Minematsu, Y., Barker, L., Lincoln, D., Crumley, K. V., Fraki, J. E., Schechter, N. M., Lazarus, G. G., Nakajima, K., Nakashimo, K., Neurath, H., and Woodbury, R. G. (1985) Biochemistry 24, 2048-2059 [Medline] [Order article via Infotrieve]
  14. Urata, H., Karnik, S. S., Graham, R. M., and Husain, A. (1993) J. Biol. Chem. 268, 24318-24322 [Abstract/Free Full Text]
  15. Murakami, M., Karnik, S. S., and Husain, A. (1995) J. Biol. Chem. 270, 2218-2223 [Abstract/Free Full Text]
  16. Caughey, G. H., Schaumberg, T. H., Zerweck, E. H., Butterfield, J. H., Hanson, R. D., Silverman, G. A., and Ley, T. J. (1993) Genomics 15, 614-620 [CrossRef][Medline] [Order article via Infotrieve]
  17. Murasawa, S., Matsubara, H., Urakami, M., and Inada, M. (1993) J. Biol. Chem. 268, 26996-27003 [Abstract/Free Full Text]
  18. Flenniken, A. M., and Williams, B. R. (1990) Genes & Dev. 4, 1094-1106 [Abstract]
  19. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  20. Rein, A., Keller, J., Schultz, A. M., Holmes, K. L., Medicus, R., and Ihle, J. N. (1985) Mol. Cell Biol. 5, 2257-2264 [Medline] [Order article via Infotrieve]
  21. Tolunay, H. E., Yang, L., Kemper, W. M., Safer, B., and Anderson, W. F. (1984) Mol. Cell Biol. 4, 17-22 [Medline] [Order article via Infotrieve]
  22. Carthew, R. W., Chodosh, L. A., and Sharp, P. A. (1985) Cell 43, 439-448 [Medline] [Order article via Infotrieve]
  23. Merika, M., and Orkin, S. H. (1995) Mol. Cell Biol. 15, 2437-2447 [Abstract]
  24. Kawana, M., Lee, M. E., Quertermous, E. E., and Quertermous, T. (1995) Mol. Cell Biol. 15, 4225-4231 [Abstract]
  25. Ko, L. J., and Engel, J. D. (1993) Mol. Cell Biol. 13, 4011-4022 [Abstract]
  26. Visvader, J. E., Crossley, M., Hill, J., Orkin, S. H., and Adams, J. M. (1995) Mol. Cell Biol. 15, 634-641 [Abstract]
  27. Hoit, B. D., Shao, Y., Kinoshita, A., Gabel, M., Husain, A., and Walsh, R. (1995) J. Clin. Invest. 95, 1519-1527 [Medline] [Order article via Infotrieve]
  28. Craig, S. S., Schechter, N. M., and Schwartz, L. B. (1989) Lab. Invest. 60, 147-157 [Medline] [Order article via Infotrieve]
  29. Springman, E. B., and Serafin, W. E. (1995) in Mast Cell Proteases in Immunology and Biology (Caughey, G. H., ed), pp. 169-201, Marcel Dekker, New York
  30. Caughey, G. H., Zerweck, E. H., and Vanderslice, P. (1991) J. Biol. Chem. 266, 12956-12963 [Abstract/Free Full Text]
  31. Orkin, S. H. (1992) Blood 80, 575-581 [Medline] [Order article via Infotrieve]
  32. Zon, L. I., Gurish, M. F., Stevens, R. L., Mather, C., Reynolds, D. S., Austen, K. F., and Orkin, S. H. (1991) J. Biol. Chem. 266, 22948-22953 [Abstract/Free Full Text]
  33. Tsai, F. Y., Keller, G., Kuo, F. C., Weiss, M., Chen, J., Rosenblatt, N., Alt, F. W., and Orkin, S. H. (1994) Nature 371, 221-226 [CrossRef][Medline] [Order article via Infotrieve]

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