©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Natural Disruption of the Mouse Mast Cell Protease 7 Gene in the C57BL/6 Mouse (*)

(Received for publication, September 8, 1995; and in revised form, October 20, 1995)

John E. Hunt (§) Richard L. Stevens K. Frank Austen Juan Zhang Zhinan Xia Namit Ghildyal

From the Department of Medicine, Harvard Medical School, and the Division of Rheumatology and Immunology, Brigham and Women's Hospital, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The C57BL/6 mouse differs from the BALB/c mouse in that its ear and skin mast cells and its progenitor bone marrow-derived mast cells (mBMMCs) do not express mouse mast cell protease (mMCP) 7. We now report that, as detected by nuclear run-on analysis, the mMCP-7 gene is transcribed in C57BL/6 mBMMCs at a rate comparable to that in BALB/c mBMMCs. Reverse transcriptase-polymerase chain reaction analysis and sequencing of the product revealed that the ears of C57BL/6 mice contain small amounts of a mMCP-7 transcript that possesses a 98-base pair deletion. The deletion begins at a normally quiescent cryptic splice site (GTGAG), 98 base pairs upstream of the normal exon 2/intron 2 splice site (GTGAG), and introduces a premature stop codon in the alternatively spliced transcript. Thus, even if translated, the mature protein would consist of only 18 amino acids as compared to 245 amino acids in normal mMCP-7. Sequence analysis of the mMCP-7 gene in the C57BL/6 mouse revealed that the cryptic splice site is activated due to a G A point mutation at the first nucleotide of the normal exon 2/intron 2 splice site. This is the first report of a mutation of a gene that encodes a mast cell secretory granule constituent that leads to its loss of expression. Moreover, the mMCP-7 gene is the first found in any species that sequentially has undergone a splice site mutation to cause retention of an intron and then a second splice site mutation to cause activation of a cryptic splice site.


INTRODUCTION

Mast cell tryptases are major constituents of the secretory granules of mouse(1, 2, 3, 4) , rat(5, 6) , dog(7) , and human (8, 9, 10, 11, 12) mast cells, and there is evidence that these serine proteases participate in allergic and inflammatory reactions. As assessed by linkage analysis (13) , the two mast cell tryptase genes that reside on chromosome 17 in the mouse (14, 15) are candidate genes for the inheritance of airway hyperresponsiveness, one of the major features of asthma. Elevated blood levels of immunoreactive mast cell tryptase are observed in some individuals undergoing systemic anaphylaxis and in others with systemic mastocytosis(16) . Mast cell tryptases can induce airway smooth muscle hyperresponsiveness in dogs (17) and can reverse airway smooth muscle relaxation induced by vasoactive intestinal peptide in the ferret(18) . In vitro, mast cell tryptases are mitogens for fibroblasts (19) , degrade high molecular weight kininogen (20) and vasoactive intestinal peptide(21) , convert latent metalloproteinase-3 (22) and urinary-type plasminogen activator (23) to their mature, enzymatically active forms, and generate C3a from C3(24) .

Bone marrow-derived mast cell-committed progenitors are distributed to tissue sites in the mouse where they proliferate, differentiate, and mature into at least four phenotypically distinct populations of cells (1, 2, 3, 4, 25, 26, 27, 28, 29, 30, 31) . Depending on their tissue locations, mouse mast cells express varied combinations of at least eight neutral proteases. Based on the conserved Asp in the bottom of their substrate-binding pockets(2, 3, 4) , mouse mast cell protease (mMCP) (^1)6 and mMCP-7 are tryptases. Although their nucleotide sequences are homologous, the mMCP-7 transcript is larger than the mMCP-6 transcript due to retention of an intron in its 5`-untranslated region. In the BALB/c mouse, the immature mast cells (mBMMCs) generated by culturing bone marrow cells for 3 weeks in interleukin 3-enriched medium (2, 3) and the mature mast cells that reside in the skin and ear (30, 32) express both tryptases. In contrast, the mast cells that reside in the peritoneal cavity express mMCP-6 but not mMCP-7, and the mast cells that accumulate in the intestines of helminth-infected BALB/c mice lack both tryptases (1, 2, 3) .

Whereas these differences reflect tissue-based regulation of tryptase expression, the C57BL/6 strain of mouse has been shown to be deficient in transcripts and immunodetectable protein for mMCP-7 in its mBMMCs and ear mast cells(32) . The failure of this mouse strain to express mMCP-7 could be caused by an abnormality in an accessory cell that produces a factor that instructs the mature skin/ear mast cell and its committed progenitor to produce mMCP-7 or by an abnormality in the mast cells themselves. Both mouse mast cell tryptase genes reside on chromosome 17(14, 15) . As assessed by restriction enzyme mapping of genomic DNA, no large insertion or deletion has occurred in the mMCP-7 gene of the C57BL/6 mouse(32) .

We now show that the mMCP-7 gene is transcribed at a normal rate in C57BL/6 mBMMCs. However, because of a point mutation at the exon 2/intron 2 splice site, the mMCP-7 pre-mRNA is improperly processed and the resulting mRNA is rapidly degraded. As a consequence of this genetic defect, the secretory granule tryptase, mMCP-7, is selectively absent in the C57BL/6 mouse strain. The mMCP-7 gene is unique in that it has undergone two sequential splice site mutations during its evolution in the C57BL/6 mouse.


EXPERIMENTAL PROCEDURES

Nuclear Run-on Assay

mBMMCs (1-5 times 10^7) developed with WEHI-3 cell-conditioned medium (33) from BALB/c mice and from C57BL/6 mice were harvested, washed with ice-cold phosphate-buffered saline, and lysed in 5 ml of ice-cold lysis buffer (10 mM NaCl, 3 mM MgCl(2), 0.5% Nonidet P-40, and 10 mM Tris-HCl, pH 7.4). After centrifugation of the lysates at 800 times g for 5 min at 4 °C, the nuclei recovered in each bottom fraction were resuspended in 200 µl of 40% glycerol, 5 mM MgCl(2), 0.1 mM EDTA, and 50 mM Tris-HCl, pH 7.5(34) . Each nuclear run-on reaction was initiated in 400 µl of reaction buffer (30 mM Tris-HCl, pH 8.0, containing 20% glycerol, 5 mM MgCl(2), 150 mM KCl, 1 mM dithiothreitol, 10 units of RNase inhibitor, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, and 100 µCi of [P]UTP). After a 30-min incubation at 30 °C, the samples were digested with DNase I (25 µg/ml, RNase-free) for 5 min and the radiolabeled transcripts were purified using Tri Reagent(TM) (Molecular Research Center, Cincinnati, OH). Each slot blot, containing 5 µg of pBluescript (pBS) DNA and gene-specific probes for mMCP-6(2) , mMCP-7(3) , and beta-actin (35) immobilized onto separate regions of the nylon membrane, was placed in a small vial containing 1-2 ml of hybridization buffer (10 mM TES, 10 mM EDTA, 0.2% SDS, 0.6 M NaCl, 1 times Denhardt's solution, and 0.3 mg/ml salmon sperm DNA) and 1 to 5 times 10^6 cpm/ml P-labeled RNA. After a 36-48-h incubation at 65 °C, the slot blot was treated with RNase A for 20 min at 37 °C, washed three times (20 min each) with 2 times SSC at 65 °C, and subjected to autoradiography.

RNA Blot and Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analyses

RNA was isolated from the ears of euthanized female BALB/c, C57BL/6, and C3H mice (Jackson Laboratories, Bar Harbor, ME). Tissues were surgically excised, immediately frozen in liquid nitrogen, ground in liquid nitrogen with a mortar and pestle, and homogenized briefly with an electric blender in 4 M guanidinium thiocyanate, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol, and 25 mM sodium citrate. Total RNA (10 µg), isolated according to the method of Chomczynski and Sacchi(36) , was denatured in formaldehyde/formamide, electrophoresed in 1.3% formaldehyde/agarose gels, and transferred to MagnaGraph membranes (Micron Separations Inc., Westover, MA)(37) . The resulting blots were incubated at 68 °C for 1.5 h in QuikHyb solution (Stratagene, La Jolla, CA) containing 100 µg/ml salmon sperm DNA and a radiolabeled 168-base pair (bp) gene-specific fragment that corresponds to the 3`-untranslated region of the mMCP-7 transcript and nucleotides 2151 to 2317 of the gene(3) . The RNA blots were washed at 60 °C in 15 mM NaCl, 1.5 mM sodium citrate, and 0.1% SDS, and then analyzed by autoradiography and/or by Betascope (Betagen). Blots were stripped and reanalyzed with a gene-specific probe for mMCP-6(2) .

The cDNA Cycle kit from Invitrogen (San Diego, CA) was used in the RT-PCR analyses. In each reaction, the first strand cDNA was synthesized from 5 µg of total RNA using the avian myeloblastosis virus reverse transcriptase primed with oligo(dT). The reaction was carried out at 42 °C for 1 h, and the enzyme was then inactivated by a 3-min incubation at 95 °C. The cDNA, recovered after phenol/chloroform extraction, was resuspended in 20 µl of distilled water. A 2-µl portion was added to 48 µl of PCR buffer (50 mM KCl, 2.5 mM MgCl(2), 10 mM Tris-HCl, and 0.01% gelatin) containing 2 units of Taq polymerase and 0.5 mM deoxynucleotide triphosphates. The PCRs were carried out with sense (5`-GCACTACTCCTCACTGTG-3`) and antisense (5`-CGCATTTTATTGAGGCATAGCAGA-3`) primers (250 ng each) specific for mMCP-7. Control PCRs were performed with primers specific for mMCP-6 (sense, 5`-GCACATCAAAAGCCCACAGC-3` and antisense, 5`-TAGACAGGGGAGACAGAGGAC-3`), mouse mast cell carboxypeptidase A (mMC-CPA) (sense, 5`-ATCGCAGGCACGCACAGTTAT-3` and antisense, 5`-AACCCAGTCTAAGGAAGAGCC-3`) and beta-actin (sense, 5`-GTGGGCCGCTCTAGGCACCAA-3`, and antisense, 5`- CTCTTTGATGTCACGCACGATTTC-3`). Each of the 30 cycles of the PCR consisted of a 1-min denaturing step at 94 °C, a 2-min annealing step at 55 °C, and a 3-min extension step at 72 °C. At the completion of the PCR, 15-µl samples were electrophoresed in separate lanes of a 1% agarose gel and photographed. In some instances, the RT-PCR products were purified by electrophoresis on a 1% low melting point agarose gel. The appropriate bands were excised and then purified with Geneclean(TM) (BIO 101, Inc., Vista, CA). The purified products were sequenced, either in our laboratory using standard dideoxy sequencing methods or at a core facility with Taq cycle sequencing methods (Biocore Facility, Dana Faber Institute for Cancer Research, Boston, MA).

Isolation, Cloning, and Sequencing of the mMCP-7 Gene from the C57BL/6 Mouse

A DASH-II library (Stratagene, La Jolla, CA) prepared from C57BL/6 mouse genomic DNA was screened under high stringency conditions with the gene-specific mMCP-7 probe described above(3) . As assessed by restriction enzyme mapping, one of the isolated clones contained the mMCP-7 gene. The relevant nucleotide sequence of this clone was determined in both directions using numerous oligonucleotide primers that correspond to various regions in the BALB/c mMCP-7 gene. The region residing 100 bp upstream to 100 bp downstream of the normal exon 2/intron 2 splice site of the mMCP-7 gene was also amplified from the genomes of the BALB/c mouse and the C57BL/6 mouse by PCR technology as described above with sense and antisense primers that correspond to residues 416-436 and 592-615, respectively, of the gene. The correctly sized PCR products obtained after 25 cycles were liberated from the low melting point agarose gels, purified using Geneclean, and inserted into a PCR cloning vector using a commercially available cloning kit (5 Prime 3 Prime, Inc., Boulder, CO). Transformed bacteria were amplified in Luria-Bertani medium. Plasmid DNA was isolated using a plasmid purification kit (Qiagen), and the inserts were sequenced with standard dideoxy sequencing techniques.


RESULTS AND DISCUSSION

As assessed by a nuclear run-on assay, the relative rates of transcription of the tryptase mMCP-6 and mMCP-7 genes and the beta-actin gene in C57BL/6 mBMMCs were comparable to those in BALB/c mBMMCs (Fig. 1), even though the C57BL/6 mouse strain does not contain high steady-state levels of the mMCP-7 transcript in its ear mast cells(32) . RT-PCR analysis revealed that the appropriately sized product was amplified from the BALB/c, C57BL/6, and C3H mouse strains with the mMCP-6 primer set. In contrast, whereas the appropriately sized RT-PCR product was amplified from the BALB/c and the C3H mouse strains with the mMCP-7 primer set (Fig. 2), the RT-PCR product generated from the C57BL/6 mouse was 100 bp smaller.


Figure 1: Nuclear run-on analyses. P-Labeled nuclear transcripts obtained from C57BL/6 mBMMCs (left lane) and BALB/c mBMMCs (right lane) were examined for their ability to hybridize to gene-specific DNA probes for beta-actin, mMCP-7, and mMCP-6 that had been each immobilized on separate lanes of a nylon membrane slot blot. Because the beta-actin, mMCP-7, and mMCP-6 probes were inserted in pBS, pBS-derived DNA was used as a negative control. Similar findings were obtained in another experiment.




Figure 2: RT-PCR analyses of mast cell tryptase transcripts in the ears of BALB/c mice and C57BL/6 mice. Depicted are ethidium bromide-stained agarose gels of the RT-PCR products amplified from total RNA isolated from the ears of BALB/c, C3H, and C57BL/6 mice. The RT reactions were primed with oligo(dT); PCRs were performed with primers specific for beta-actin, mMC-CPA, mMCP-6, and mMCP-7. The arrow on the left of each panel indicates the product that was expected. The arrow on the right of the mMCP-7 panel indicates the smaller, 905-bp product generated from the C57BL/6 mouse sample. Similar findings were obtained in two other experiments.



The failure to detect any mMCP-7 transcript in the ears of the C57BL/6 mouse and its progenitor mBMMCs by RNA blot analysis, even though the gene is efficiently transcribed, indicates that the truncated transcript must be quickly degraded inside the mast cell. To determine the basis of the short half-life of the mMCP-7 transcript, the nucleotide sequence and deduced amino acid sequence of the RT-PCR product generated from the ears of C57BL/6 mice with the mMCP-7 primer set were determined (Fig. 3). Relative to the mMCP-7 transcript obtained from the BALB/c mouse, the mMCP-7 transcript obtained from the C57BL/6 mouse had a 98-nucleotide deletion corresponding to the 3` end of exon 2. Based on these cDNAs, it is predicted that the first 15 amino acids of mature mMCP-7 are identical in the C57BL/6 and BALB/c mouse strains. However, in the C57BL/6 mouse the next sequence is ``frameshifted'' and a premature stop codon is introduced only 9 nucleotides 3` of the deletion. Thus, even if the truncated transcript was translated in the C57BL/6 mouse, the mature protein would consist of only 18 amino acids and would not be enzymatically active. Premature translation termination can dramatically impact the steady-state level of a transcript(38, 39) , especially when the stop codon is present in the initial two-thirds of the transcript (40, 41) as in the defective mMCP-7 mRNA. Thus, the inability to translate a normal-sized protein might explain why the C57BL/6 mouse rapidly degrades its defective mature mMCP-7 transcript. Because the C57BL/6 mouse represents a natural disruption of the mMCP-7 gene, this mouse strain should be valuable for evaluating the physiologic and pathophysiologic role of a tryptase.


Figure 3: Sequence analysis of a mMCP-7 transcript obtained from the C57BL/6 mouse. The nucleotide sequence of the RT-PCR product generated with the mMCP-7 primers from total RNA isolated from the ears of C57BL/6 mice is compared to the previously published sequences of the mMCP-7 transcript in BALB/c mBMMCs(3) . , identical nucleotide; *, deleted nucleotide. The stop codon (STP) in each transcript is indicated. The deduced amino acid sequences of the two transcripts are depicted in the top and bottom lines. The numbers on the right correspond to the specific amino acids in the mature tryptases. The regions in the transcript that correspond to the boundaries of the exons in the normal gene are indicated.



The truncation of the mMCP-7 cDNA isolated from the C57BL/6 mouse could have been caused either by a 98-bp deletion in exon 2 of the gene or by defective splicing of the pre-mRNA. To distinguish between these two possibilities, the mMCP-7 gene was isolated from a C57BL/6 mouse genomic library, and the sequence of exon 2, intron 2, and part of exon 3 of this gene were determined (Fig. 4). Relative to the corresponding gene in the BALB/c mouse, the mMCP-7 gene in the C57BL/6 mouse possesses a point mutation at its exon 2/intron 2 splice site. Thus, the transcript has a 98-bp deletion because the point mutation induces a cryptic splice site in exon 2 to be used during the processing of the pre-mRNA. The utilized cryptic splice site obeys the GTGAG consensus sequence for such sites(42, 43) .


Figure 4: Nucleotide sequence of the mMCP-7 gene in the C57BL/6 mouse. In panel A, the nucleotide sequence of the mMCP-7 gene in the C57BL/6 mouse is compared to the corresponding gene in the BALB/c mouse(3) . Exon and intron nucleotides are indicated in uppercase and lowercase, respectively. A dash indicates an identical nucleotide. The arrow indicates the point mutation that has occurred in the exon 2/intron 2 junction of the mMCP-7 gene in the C57BL/6 mouse strain. The normally cryptic splice site is shown in the inset box. The nucleotides are numbered in the right-hand column according to the published sequence(3) . In panel B, the sequencing ladder around the exon 2/intron 2 junction of the mMCP-7 gene of the two mouse strains is depicted. The relevant nucleotide sequences are indicated; the mutated nucleotide is circled.



The genetic abnormality described in this study represents the second instance of an exon/intron splice site mutation in the mMCP-6/mMCP-7 gene locus on chromosome 17. The mMCP-7 gene in the BALB/c mouse (3) differs from the homologous mMCP-6 gene (2) and the human mast cell tryptase I gene (12) in that it consists of 5 exons rather than 6 due to a point mutation in the region equivalent to the intron 1/exon 2 splice site of the homologous mMCP-6 gene. Because exon 1 corresponds precisely to the 5`-untranslated region of the transcript, mMCP-7 pre-mRNA is processed in the BALB/c mouse, and the resulting transcript is translated even though, relative to other tryptase transcripts(2, 10, 11, 12, 44) , it possesses an unusually large 5`-untranslated region of 195 nucleotides. Since the transcripts that encode mMC-CPA and the chymase (mMCP-1, mMCP-2, mMCP-4, and mMCP-5) family of serine proteases all possess 5`-untranslated regions that are analogous in size to the 5`-untranslated region in mMCP-6, the mutation in the mMCP-7 gene that caused retention of intron 1 must have occurred after the divergence of the two mouse mast cell tryptase genes from a primordial tryptase gene. Approximately 90 vertebrate genes have been identified that have undergone splice site mutations(45, 46) . Of the four categories of splice site mutations, 51, 32, 11, and 6% of the mutations result in exon skipping, activation of a cryptic splice site, creation of a pseudo-exon within an intron, and intron retention, respectively. The mMCP-7 gene is the only mammalian gene of which we are aware that has undergone two splice site mutations, one causing the retention of an intron and the other causing activation of a cryptic splice site. The G A mutation at the exon 2/intron 2 splice site of the mMCP-7 gene is not present in the CBA, C3H/HeJ, BALB/cJ, A/J, AKR, RF, SWR, and SJL mouse strains but is present in all non-outbred strains of black mice from Miss Lathrop's albino lineage (e.g. C57BL/6J, C57BL/10J, C57BL/6By, C58, C57L, C57Br/cd) (data not shown). As the C58, C57BL, and C57BR lines diverged in the 1920s(47) , this mutation in the mMCP-7 gene must have occurred prior to the 1920s. In all mouse strains so far examined, the mMCP-7 gene possesses the point mutation that has resulted in the retention of the ancestral intron 1. Thus, it is likely that the mutation that caused retention of the intron most likely occurred before that which caused activation of the cryptic splice site. The discovery of two splice site mutations in the mMCP-7 gene suggests that there is a novel structural feature in this gene or in its site of residence on chromosome 17 that predisposes it to exon/intron splice site mutations.


FOOTNOTES

*
This work was supported by Grants AI-07306, AI-23483, AI-22531, AI-31599, AR-07530, AR-36308, and HL-36110 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank(TM)/EMBL Data Bank with accession number(s) U42405 [GenBank]and U42406[GenBank].

§
Recipient of a C. J. Martin fellowship from the National Health and Medical Research Council of Australia.

(^1)
The abbreviations used are: mMCP, mouse mast cell protease; bp, base pair(s); mBMMC, mouse bone marrow-derived mast cell; mMC-CPA, mouse mast cell carboxypeptidase A; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Drs. Nakai and Sakamoto (The National Institute for Basic Biology, Okazaki, Japan) for their data base on known splice site mutations in genes.


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