From the Cardiovascular Research Institute and Departments of Medicine and Laboratory Medicine, University of California, San Francisco, California 94143-0911
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ABSTRACT |
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Tryptases are serine proteases implicated in
asthma and are very highly expressed in human mast cells. They fall
into two groups, Mast cell tryptases are trypsin-like serine proteases whose
distinguishing features include secretion as catalytically active, heparin-bound oligomers that resist inactivation by plasma
antiproteases (1). Extracellular targets of human tryptases suggested
by in vitro studies include neuropeptides, procoagulant
proteins, urokinase, stromelysin, and proteinase-activated receptor-2
(2-6). Isolated tissue and in vivo studies in animals (7,
8) and humans (9, 10) suggest a pathogenic role for tryptases in asthmatic airway inflammation and obstruction. Moreover, studies in
mice suggest that genetic determinants of asthma-like airway hyperresponsiveness include one or more genes in the vicinity of
tryptase loci on chromosome 17 (11).
Known members of the tryptase family are expressed selectively in mast
cells and basophils (12-14). Compared with amounts of granule-associated serine proteases in leukocytes, levels of tryptases in human mast cells are exceptionally high, comprising up to 25% of
cellular protein (15, 16). These levels are achieved by packaging and
storage in secretory granules. Purified human tryptases exhibit size
heterogeneity by SDS-polyacrylamide gel electrophoresis and can be
chromatographically separated into isoforms which in part may be due to
differences in glycosylation (17). Based on differences in
immunoreactivity and in amino acid sequence deduced from cloned
cDNAs, known human tryptases divide into two groups, Four different human tryptase cDNAs ( In mice, two different tryptases (mMCP-6 and
-7)1 have been identified
(12, 13). Structurally, human In addition to functional data linking tryptases to the pathogenesis of
asthma, recent genetic data suggest that chromosome 16 in some
populations contains determinants of susceptibility to asthma (28). In
work reported here, to explore a potential link between tryptases and
the genetics of asthma, we more precisely localize tryptase genes on
chromosome 16, and, to help resolve enigmas concerning the nature and
number of haploid human tryptase genes and identify new members of the
tryptase family, we characterize a locus of tryptase genes.
Screening of YAC and BAC Libraries--
Human genomic MegaYAC
libraries A, B, and C from Center d'Etude du Polymorphisme Humain (29)
and a human genomic BAC (30) library were screened by PCR using sets of
oligonucleotide primers spanning various portions of known tryptases
cDNAs and genes. When MegaYAC screening with these
tryptase-specific primers failed to identify positive clones, the BAC
library was screened. The primers that identified two BAC clones and
isolated two BAC clones amplify a highly conserved 184-bp region in the
3'-UTR of tryptase genes. This region contains repetitive elements (see
Fig. 1). These PCR primers (5'-ccaaaacaccactgcttcct and
5'-ccggtgcaggcgtcaggctt) were used at a final concentration of 200 nM in buffer F from the Optimizer kit (InVitrogen)
containing Taq Gold (Perkin-Elmer). Reactions were cycled as
follows: 95 °C for 10 min (one cycle); 95 °C for 1 min, 55 °C
for 30 s, 72 °C for 30 s (25 cycles); and 7 min at
72 °C (1 cycle). Products were separated on agarose gels.
Blotting and Hybridization--
Full genomic and BAC DNA was
digested with restriction endonucleases at 37 °C, size-fractionated
on agarose gels, transferred to Hybond N-Plus membranes (Amersham
Pharmacia Biotech), and then fixed by UV cross-linking or baking in a
vacuum oven. Blots were prehybridized for 1 h at 65 °C in 90 mM sodium citrate buffer containing 0.9 M NaCl,
2.5% SDS, 1 g/liter each of Ficoll 400, polyvinylpyrrolidone, and
bovine serum albumin, and 100 µg/ml of sheared salmon sperm DNA and
then probed with radiolabeled DNA prepared from full-length human
tryptase Fluorescence in Situ Hybridization--
BAC clones identified in
the present work and a Isolation and Sequencing of BAC Tryptase Genes--
The BAC
libraries were originally prepared from partial HindIII
digests of genomic DNA. To obtain smaller fragments of the BAC inserted
genomic sequences, the insertions were completely digested with
HindIII, and the resulting fragments were subcloned into
pBluescript (SK+) (Stratagene). Briefly, HindIII-digested BAC DNA ligated into pBluescript was electroporated into DH10B Escherichia coli (Life Technologies, Inc.). Bacterial
plaques containing cloned tryptase genes were identified by plaque
hybridization to labeled tryptase DNA probes. Plasmid DNA from
individual clones was purified, digested with HindIII, and
compared with the pattern of fragments generated by HindIII
digestion of DNA from the parent BAC clones. Relevant portions of
tryptase-positive subclones were sequenced by the Biomolecular Resource
Center of the University of California at San Francisco using tryptase-
or vector-specific oligonucleotide primers.
BAC Restriction Mapping--
BAC DNA was digested with a variety
of endonucleases, including NotI, EcoRI,
HindIII, BamHI, BglII,
SacII, PstI, AvaII, Msc I,
and AluI. When necessary because of the generation of large fragments, restriction digests were separated on a 1% agarose gel via
inversed field gel electrophoresis on a CHEF Mapper (Bio-Rad). Electrophoresed DNA was blotted and sequentially probed with tryptase- and BAC insertion end-specific probes. BAC end sequences for each clone
were determined by direct sequencing using primers specific for the BAC
cloning vector arms. BAC end-specific probes were then generated via
PCR, cloned into pCR 3.1, and then sequenced and labeled for use as probes.
PCR Screening of BAC Tryptase Genes--
Because analysis of
human tryptase cDNA and gene sequences revealed the presence of
nucleotide changes in exon 3, causing differences in susceptibility to
SacII digestion, we developed a PCR approach to screen BACs
for tryptase genes based on restriction polymorphisms. We generated a
242-bp amplimer bracketing potential SacII restriction sites
using the following primers: 5'-caggaggcccccaggagcaagtggc and
5'-ccctgggcagcggaggatcccactc. These primers were used at a concentration of 200 nM in buffer containing Vent (exo-)
DNA polymerase (New England Biolabs, Beverly, MA). Reactions were
cycled as follows: 95 °C for 5 min (1 cycle); 95 °C for 30 s, 58 °C for 45 s, 72 °C for 30 s (40 cycles); and then
7 min at 72 °C. Products were separated on agarose gels. Some of the
242-bp amplimers were sequenced after subcloning into pCR 2.1 (InVitrogen) or pBluescript (Stratagene).
Gene Comparisons and Phylogenetic Analysis--
Matrix plots,
multiple sequence alignments, and phylogenetic trees were generated
with GeneWorks 2.5.1 (Oxford Molecular). All sequences were also
aligned with those in the GenBankTM nonredundant data base
and in the expressed sequence tag data base using various BLAST
algorithms (32). Repetitive sequences were identified via matrix plots
and by analyzing sequences by RepeatMasker2.2
Identification of BACs Containing Tryptase Genes--
In matrix
analyses of previously cloned tryptase cDNAs (Fig.
1A), we noted that the 3'-UTR
of all human tryptases is highly conserved in a region containing
several different repeated sequences (Fig. 1B) that are
present to a much smaller degree in dog tryptase and are inapparent in
murine tryptase 3'-UTRs by matrix analysis (not shown). These repeated
sequences differ from those reported in mouse chymase (mMCP-1, -2, and
-4) 3'-UTRs, which modulate mRNA stability (33). However, the human
tryptase 3'-UTR repeats may play a similar role. The portion of the
human tryptase 3'-UTR bracketing the repeated sequences is unique to
tryptases based on the results of nucleic acid data base searches. PCR
primers amplifying the repeat region identified two clones from a BAC library but no clones from a YAC library.
Characterization of BAC Tryptase Genes by DNA Blotting--
To
confirm the presence of tryptase-like sequences in the identified BACs,
restriction endonuclease-digested BAC DNA was subjected to blotting and
probed separately with a full-length tryptase Chromosomal Location of BAC Inserts and the Characterization of BAC Inserts--
By inversed field gel
electrophoresis, the BAC A and B insertions are ~74 and 62 kb,
respectively. As shown in Fig.
4A, we developed a complete
EcoRI, NotI, and HindIII (and partial
BglII) restriction map of both BACs. Double digests of the
BACs with NotI and EcoRI generate a similar
~17.5-kb fragment from both BACs. Multiple
HindIII-generated bands also are similar. Furthermore, a
probe based on the SP6 end of BAC A hybridizes with a specific HindIII fragment of BAC B and a probe developed from the SP6
end of BAC B hybridizes with a specific small, internal
HindIII fragment of BAC A. However, probes prepared from the
T7 ends of both BACs hybridize solely with the BAC of origin. BAC A
ends in the middle of a 5' fragment of a tryptase gene, which has a
highly similar (but complete) counterpart "mMCP-7-like gene" (see
below) in BAC B. Alignment of the two BACs based on restriction maps
and cross-hybridizing end sequences and genes (Fig. 4A)
suggest that over half of each BAC is structurally homologous to the
other, although the inserted sequences are oriented in opposite
directions relative to the SP6 and T7 polymerase sites of the vector.
Because the sequences at homologous loci are similar but not identical
in the two BACs, despite close organizational parallels, we conclude
that BACs A and B are most likely to be partially overlapping sections
of sister chromatids and that tryptase genes at homologous sites are
allelic variants of each other.
Characterization of BAC Characterization of BAC Characterization of BAC mMCP-7-like Genes--
As noted above, the
SP6 end of BAC A contains the 5' portion of a tryptase gene. This
sequence ends at the terminal HindIII site of BAC (the BAC
library was generated from HindIII partial digests). BAC B,
however, contains the full sequence of the apparent allelic partner of
the partial BAC A gene. Although the phase and placement of introns are
similar to those of other human tryptase genes (as shown in Fig.
6A), their size varies, particularly in intron 4, which is
much larger than the other introns, as is also true of intron 4 in the
gene encoding mMCP-7 (13). Among the protein-coding sequences, however,
only exon 5 closely resembles mMCP-7. The region of nucleotide sequence
similarity to mMCP-7 includes all of exon 5 and contiguous portions of
introns 4 and 5. However, there is no discernible similarity between
most of the large human intron 4 insertion and the intron of similar
size in the mMCP-7 gene. The closest matches in searches of the
GenBankTM data base (including expressed sequence tags),
using exon 5 as the query sequence, are mMCP-7 and the apparent
orthologs of mMCP-7 in gerbil (39) and rat (40). No human expressed
sequence tags are highly similar. Remarkably, the remaining exons of
the mMCP-7-like gene are closely related to Distinguishing Tryptase Genes by Sac II Digestion of PCR
Amplimers--
Sequencing of BAC A and B tryptase genes revealed
differences in SacII restriction sites in exon 3 (Fig.
6A). Tryptases Conclusion--
In this work we characterize a cluster of mast
cell tryptase genes encoding known as well as previously undescribed
members of the family extending over ~50 kb of chromosome 16p13.3.
Our findings establish that in the haploid genome there are at least two and
. Although several related tryptase
mRNAs are known, it is unclear which if any are transcripts of
separate haploid genes. The studies described here investigated the
nature and number of human tryptases and sought possibly novel members
of the family. To this end, two human bacterial artificial chromosome (BAC) clones containing tryptase genes were identified and mapped to
chromosome 16p13.3, of which ~2.2 megabases are syntenic with the
part of mouse chromosome 17 containing tryptase genes mouse mast cell
protease (mMCP)-6 and -7. Sequencing and restriction mapping suggest
that the BACs may partially overlap. Sequenced BAC genes correspond to
three known
-tryptases (
I,
II, and
III), an
-like gene,
and a pair of novel hybrid genes related partly to
/
-tryptases
and partly to orthologs of mMCP-7.
II and
III,
I and
II, as
well as the two mMCP-7-like genes, may be alleles at single loci; in
total, there are at least three nonallelic tryptase genes in the
isolated BAC clones. DNA blotting and restriction analysis suggest that
the BACs include most members of the immediate tryptase family. Thus,
chromosome 16p13.3 harbors a cluster of known and previously
undescribed members of the tryptase gene family.
INTRODUCTION
Top
Abstract
Introduction
References
and
(18-20).
-Tryptase appears to be the major type stored in mast cell
secretory granules and is the major form isolated from extracts of the
richest tissue source, which is the lung (21).
-Tryptase mRNAs
are also the main types identified in purified mast cells from human
lung and skin (22). On the other hand, by immunoassay,
-tryptase is
the major isoform in blood in normal subjects (23) and may be the
predominant type expressed by basophils (22). The basis for the cell
selectivity of tryptase expression and for the exceptionally high
expression of
-tryptase in human mast cells is not known.
,
I,
II, and
III)
have been generated from human lung (19, 20) and skin (18) mRNA,
and the organization and complete sequence of one human tryptase gene
(
I) has been determined (18). In addition, one or more tryptase
genes are expressed in cell lines, including Mono Mac 6 cells
(
-tryptase) (24), HMC-1 and U-937 cells (
I-tryptase) (24), and
KU812 cells (
- and
II-tryptases) (22, 25). In addition to
I-tryptase, HMC-1 cells also transcribe a second very closely
related mRNA (here designated
Ib-tryptase), whose product is
predicted to differ from
I-tryptase itself by one amino acid (26).
Because all of the described
-tryptases are 98-99% identical in
amino acid sequence, they have been considered to be possible allelic
variants of each other (18, 27).
-Tryptase is less closely related
(91% identical to
I) and is more likely to be a product of a
separate gene in the haploid genome (19, 20). Both
- and
-tryptases appear to reside on human chromosome 16 based on
amplification of DNA from hamster-human hybrid cells (20). However, the
number and proximity of haploid
- and
-genes as well as their
precise chromosomal location and genetic neighbors are not known.
- and
-tryptases are more closely
related to each other than to mMCP-6 and -7, which are much more
different from each other than any combination of known human tryptases
(27). These observations suggest 1) that the ancestors of the known
human tryptases diverged from each other after the point in evolution
when the known mouse and human tryptases shared a common ancestor, 2)
that human
- and
-tryptase therefore are not the equivalents,
respectively, of mMCP-6 and -7, and 3) that the human genes that are
true orthologs of mMCP-6 and -7 may await discovery. The latter
possibility is particularly likely to apply to orthologs of mMCP-7,
whose gene structure is distinct in several features from that of the
cloned human
I-tryptase gene (13). Among serine protease genes as well as other extended gene families, differences in intron-exon organization tend to suggest more remote phylogenetic relationships (27).
EXPERIMENTAL PROCEDURES
III cDNA (18) or with a
BamHI/AvaII tryptase
I gene fragment
containing exon 5 and portions of flanking introns as templates (18).
After labeling, probes were purified on a G-25 spin column (Amersham Pharmacia Biotech) and hybridized at 2 × 105 to
1 × 106 cpm/ml. After incubation overnight, blots
were washed twice in 30 mM sodium citrate buffer containing
0.3 M NaCl and 0.1% SDS for 15 min at 65 °C followed by
two washes in 1.5 mM sodium citrate buffer containing 15 mM NaCl and 0.1% SDS at 65 °C for 15 min. Hybridizing
bands were revealed by autoradiography.
clone containing the tryptase
I gene (18)
identified in prior work were localized to a specific human chromosomal
band by standard FISH techniques, as described previously (31).
Briefly, metaphase spreads of human peripheral blood lymphocytes were
denatured in 30 mM sodium citrate buffer containing 0.3 M NaCl and 70% formamide for 5 min at 72 °C and then
dried in graded solutions of ethanol. BAC and
probes incorporating
digoxigenin-11-dUTP were prepared by nick translation, and a human
chromosome 16-specific centromeric probe was labeled directly by
incorporating Texas Red dUTP. 10 µl of hybridization mixture
contained labeled probes, and Cot-1-digested DNA in 30 mM sodium citrate buffer containing 0.3 M NaCl,
50% formamide, and 10% dextran sulfate were applied to each slide. After incubation overnight at 37 °C, slides were washed to remove unbound probes, incubated with fluorescein-labeled anti-digoxigenin antibody, and then counterstained with 0.2 mM
4,6-diamino-2-phenylindole to reveal chromosomal bands.
RESULTS AND DISCUSSION
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Fig. 1.
Comparison of and
genes and identification of
conserved repeats in flanks and 3'-UTRs. A shows a
computer-assisted matrix plot of human
II and
I tryptase gene
nucleotide sequence. The two genes are highly similar, even in introns
and flanking regions, as manifested by the strong, nearly continuous
diagonal line of identity. This suggests that the two
nonallelic genes arose from recent duplications. The 5' and 3' termini
of the genes are indicated. Three prominent regions of repeats
(designated a-c) appear as lines parallel to the main line
of identity. Repetitive region a is near the end of the
sequenced 5' flanks. Repetitive region b is in the 3'-UTR.
Repetitive region c, in the 3' flank, is a short
interspersed element of the dimeric Alu family (subfamily
Jb) whose relatives are numerous in the human genome. B
shows the portion of
I 5' flank and 3'-UTR containing the repeated
elements identified above. The sequences are nearly identical in
II.
The 5' flank sequence contains three direct, G-rich, 19-bp repeats,
which are underlined. Data base searches suggest that the
repeated elements may be unique to tryptases, because they are not
detected elsewhere. Because of their location and conservation, they
are candidate transcriptional regulatory sites that can lie within
tandemly repeated sequences. In the 3'-UTR sequence, two types of
repeats are underlined, with or without italics.
All known human tryptase cDNAs and genes have these repeats, which
appear to be unique and to be present to a much lower degree or not at
all in other mammalian tryptase cDNAs, as revealed by further
matrix analysis (not shown). These repeated sequences potentially play
a role in regulation of human tryptase mRNA stability. Residues in
bold print correspond to primers used in PCR amplifications
to screen YAC and BAC libraries for genomic clones containing tryptase
genes.
III cDNA and with
a BamHI/AvaII
I gene fragment containing exon 5 and portions of flanking introns (18). The results shown in Fig.
2 in comparison with similarly treated
blots of human full genomic DNA suggest that both BACs contain more
than one tryptase gene. Although the banding patterns of BACs A and B
differ, several bands appear identical, suggesting either that the BACs
partially overlap or that they contain genes that are sufficiently
similar that they yield similar restriction patterns. Together, as
shown in Fig. 2, the bands in the two BACs account for all of the bands seen in the full genomic blot, suggesting that the BACs collectively include most or all of the tryptase genes of the genome.
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Fig. 2.
Genomic DNA blots. This figure shows
autoradiograms generated by hybridization of a radiolabeled fragment of
a tryptase gene with blots of full genomic and BAC DNA digested with
AvaII, BalI (MscI), or
BamHI). The probe is a 345-bp
AvaII/BamHI fragment of tryptase I comprising
portions of exons 5 and 6 and all of intron 5. The left and
right panels show blots of full genomic and BAC DNA,
respectively. The position of size markers in kb are shown to the
left. Note the similar size of many bands in the full
genomic and BAC blots. Although BAC patterns partly differ from each
other, in combination they appear to account for all bands generated by
full genomic DNA, suggesting that BAC A and B together may include most
of the tryptase genes of the genome. Differences in band intensity
arise from disparities in the number of genes contributing to a band of
a given size and from differences in the extent of homology to the
probe. For example, the BamHI digests of full genomic and
BAC B DNA appear identical, containing a strong ~0.8-kb band due to
multiple
/
tryptase genes and a weak ~1.6-kb band attributable
to an mMCP-7-like gene, which contains a ~0.8-kb intron 4 insertion
(see Fig. 4A) in comparison to
and
genes. This
~1.6-kb band is absent from the BAC A lanes because the BAC A insert
contains only part of the mMCP-7-like gene and lacks the 3' portion
homologous to the probe.
I Tryptase
Gene--
By FISH, both BACs, as well as a previously isolated
phage clone containing the tryptase
I gene (18), localize to a site near the end of the short arm of chromosome 16 (band 13.3). There is no
discernible difference in the location of any of these clones by this
technique, suggesting that all map to the same or closely adjacent
sites. These data confirm and extend prior data assigning
and
tryptase genes to chromosome 16 using panels of hamster human hybrids
(19, 20). Because no YACs containing tryptase genes were identified, it
is likely that the
I-tryptase gene and the identical tryptase-rich
BACs map to the small portion of chromosome 16p13.3 that is not
"covered" by YACs (34). As shown in Fig.
3, part of the 16p13.3 YAC-poor region is
syntenic with a small portion of mouse chromosome 17 containing mMCP-6 and -7 genes (35). This region of synteny corresponds to <0.1% of the
human genome.
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Fig. 3.
Chromosomal location of human tryptase
genes. A diagram of human chromosome 16 in metaphase is shown at
the top of the figure. The solid arrow indicates
the approximate location of the FISH signal generated by probes
prepared from of a bacteriophage clone, containing the human
tryptase
I gene, and from BACs A and B, containing multiple tryptase
genes. In all cases, selective hybridization was seen near the end of
the short arm on band 16p13.3, suggesting that the DNA cloned within
the
and BAC vectors map to the same or highly similar locations in
the genome. The remainder of the figure demonstrates that
tryptase-hybridizing human chromosome 16p13.3 is syntenic with a
portion of mouse chromosome 17 containing tryptase genes. After
identification of the general location of tryptase genes by FISH, we
identified a small portion of the telomeric side of band p13.3 as being
a likely site of the tryptase gene cluster because this particular ~3
megabase region has no coverage by YACs in existing chromosome 16 maps.
Because we find no tryptase-positive clones in screens of human YAC
libraries, we reason that a "YAC-poor" region harbors tryptase
genes. Existing genomic maps reveal that 2.2 megabases of the candidate
region are syntenic with a fragment of mouse chromosome 17 containing
genes encoding mouse tryptases mMCP-6 and -7, as indicated. Other genes
shared by humans and mice in this regions include polycystic kidney
disease type I (Pkd1) and tuberous sclerosis type 2 (Tsc2) genes. The congruence of the human FISH and mouse
synteny data allow more precise prediction of the location of a
tryptase gene locus than is possible with FISH alone. Based on these
data, we predict that BAC tryptase genes lie within ~1 megabase of
Tsc2 and Pkd1 genes on 16p13.3.
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Fig. 4.
Restriction map, alignment, and PCR analysis
of BACs containing human tryptase genes. BACs A and B (74 and 62 kb, respectively, plus 7.5 kb of vector) were identified by PCR
screening of a human genomic BAC library. Restriction maps of the
inserted genomic sequence were constructed based on patterns of
digestion with EcoRI (E), NotI
(N), HindIII (H), and BglII
(B, partial). In A, the location of -,
-,
and mMCP-7-like genes in the two BACs are indicated. Note that the
BACs, although inserted in opposite orientations relative to the T7 and
SP6 polymerase sites on vector arms, align in a manner suggesting
homology and partial overlap. The
- and mMCP-7-like genes are
similar but not identical between BAC A and B. The SP6 end of the BAC A
insert is comprised of a 5' portion of a gene highly homologous to the
complete mMCP-7-like gene found in BAC B. Exons of each of the three
genes sequenced so far correspond to known
tryptase cDNAs
I-III. BAC A
exons differ in a few nucleotides from published
cDNA, and the BAC A gene is therefore designated
II. The
simplest explanation of these data is that BACs A and B overlap
together comprising a mini-contig of ~93 kb. The alignment predicts
that
II and
III, mMCP-7-like genes I and II, and
II and
Ia
are allelic partners and that there are a minimum of 3 tryptase genes per haploid genome. Arrows indicate direction
of transcription. B is an ethidium-stained polyacrylamide
gel showing results of amplification and SacII digestion of
a 242-bp fragment covering a portion of exon 3 exhibiting sequence
variations among human tryptase genes. The lanes (from
left to right) contain products amplified from
the following sources: a 942-bp subclone of mMCP-7-like tryptase II
(serves as control for completeness of SacII digestion), BAC
B, BAC A, full genomic DNA, a 12-kb HindIII subclone of BAC
B containing the
I gene, a 10-kb HindIII subclone of BAC
B containing the
III gene, a 7.4-kb HindIII subclone of
BAC B containing the mMCP-7-like II gene, a 7.4-kb HindIII
subclone of BAC A containing the N-terminal portion of the mMCP-7-like
I tryptase gene, a 20-kb HindIII subclone of BAC A
containing the
II tryptase gene, and a 12-kb HindIII
subclone of BAC B containing
II tryptase gene. The location of these
HindIII fragments are given in A. The sizes of
the predicted bands, as estimated from size markers (not shown) are
indicated. These results support the hypothesized homology between BACs
and suggest that no genes of different types (e.g.
and
) are found in the individual HindIII subclones that span
much of the two BACs.
-Tryptase Genes--
BAC A contains a
-tryptase gene whose exons match previously described
II
cDNAs. BAC B contains two identified
-like genes whose exons
correspond to cDNAs
I and
III (18). The organization (see
Fig. 6A) of the BAC
genes is identical to that of a
previously characterized
I gene (GenBankTM accession
number M33494) (18). It should be noted that the BAC B
I gene
represents the Glu99 version of tryptase (
Ia) rather
than the Lys99 version (
Ib, which may be another allele
at this locus) also transcribed in HMC-1 cells (26). The
III gene
resides at a separate site. The reason for the existence of two loci of
highly similar genes is unclear.
III-Tryptase differs from
Ia at
only three of 245 catalytic domain amino acids, none of which (based on
preliminary modeling) are in sites thought to be critical determinants of function. One potential explanation for the presence of two very
similar genes is a recent gene amplification event, perhaps in response
to evolutionary pressure to increase production of tryptase mRNA
and protein. Related events are observed in connection with other
genes. For example, gene amplification is a known genetic adaptation to
selective pressure to increase the product of the mammalian
dihydrofolate reductase gene and is a mechanism that can occur more
readily than regulatory region mutations as a means of increasing
transcription (36). Another possible explanation is that the
duplication of
genes was remote rather than recent and that near
identity at the two loci has been maintained by gene conversion (37),
which is a postulated mechanism for maintaining similarity of tandemly
arrayed genes. Intriguingly, the three residues by which
III differs
from
I tryptase are all
-like residues, suggesting that there may
have been genetic exchange between
and
genes. Our complete
I
and
III tryptase gene sequences (including 5' and 3' flanks) and
II partial sequences, including 3' flank, are deposited in
GenBankTM (AF099144, AF099143, AF099145 and AF099146, respectively).
-Tryptase Genes--
BAC A contains a
gene (
II) with an exon sequence similar but not identical to that
reported for
cDNA (here designated
I) (19). The deduced
amino acid sequences of
I and II, as shown in Fig.
5, differ in only three residues. The
alignment shown in Fig. 4A suggests that the BAC A
II
gene and the BAC B
Ia gene may be allelic partners, a possibility
further supported by the very high homology between the two genes and
their respective extended flanking regions, as revealed by the matrix
analysis in Fig. 1A. The gene corresponding to previously
reported
I cDNA also may be an allele at this locus or may be
the product of another
gene not detected in the BACs. This is the
first report of
-tryptase gene structure, which, not surprisingly,
is similar to that of
-genes (Fig.
6A), containing a 6-exon,
5-intron pattern, including the placement of a phase-0 intron
immediately upstream of the initiator Met codon, which separates the
site of transcription initiation from protein coding sequence. This
feature is characteristic of tryptases but is unusual in other genes.
The
gene described here also has an 11-bp deletion in intron 4 compared with
-tryptase genes. Another difference between
and
genes is the presence of a SacII restriction site in
exon 3 (see Figs. 4B and 6A). These differences
provide ways to measure
versus
DNA in PCR-generated amplimers containing tryptase gene products of mixed heritage. The
predicted protein products of both new
-tryptases in this work
contain Gln residues in the
3 position of the propeptide. It has been
suggested that this residue impairs
-tryptase activation from its
zymogen form by replacing the usual Arg that is present in this
position in
-tryptases, rendering the
enzyme amenable to
autolytic activation (38). Our complete
II tryptase gene sequence,
including 5' and 3' flank, is deposited in GenBankTM
(AF098328).
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Fig. 5.
Amino acid sequence alignment. The
deduced amino acid sequence of human prepro-mMCP-7-like tryptase is
compared with that of - and
-tryptases. Regions of sequence
identity among all of the tryptases are boxed. Note that the
region of greatest sequence disparity corresponds to exon 5 (residues
167-221), which is the mMCP-7-homologous portion of the human
mMCP-7-like gene. The remainder of the sequence is nearly equally
related to human
- and
-tryptases, although Gln28 of
the propeptide (in the
3 position relative to the IVGG N terminus of
the mature catalytic domain) is a key
-like feature that may limit
autolytic trimming of the propeptide by hydrolysis at the basic residue
that occupies this position in most tryptases.
View larger version (26K):
[in a new window]
Fig. 6.
Organization and origins of human tryptase
genes. A compares intron-exon structures. Genes so far
obtained from BAC clones are of three types: -,
-, and
mMCP-7-like, as shown. Each has a 6-exon (boxes), 5-intron
(lines) structure, drawn to scale. UTRs are hatched
boxes. Exons encoding prepro- and mature enzyme are lightly
shaded and black boxes, respectively. Codons of
catalytic His (H) Asp (D), and Ser (S)
residues common to serine proteases are indicated. Human gene intron 1 separates 5'-UTR from protein-coding sequence, an idiosyncratic feature
of tryptase genes.
- and
-like genes are nearly identical in
intron size and phase, except for an 11-bp deletion in intron 4. The
mMCP-7-like human gene is distinguished by insertions in introns 4 and
5 and by a 5th exon more closely related to the mouse tryptase mMCP-7
than to any human tryptase, as indicated by stippling of exons. The
other exons are closely related to human
- and
-tryptases
(slightly more closely to the former than the latter). The 0.8-kb
intron 4 insertion is similar in size but not in sequence to that of
mMCP-7. Thus, the novel mMCP-7-like human tryptase gene appears to be a
chimera arising recently in mammalian evolution from an
-like gene
and a relative of mMCP-7, the remainder of which may have been lost to
the human genome during recombination. It remains to be seen whether
the human mMCP-7-like chimera is expressed and active. B
displays dendrograms based on deduced amino acid sequence of tryptase
exons 4 and 5. These trees, which were prepared using the unweighted
pair group with arithmetic mean (UPGMA) multiple sequence alignment
algorithm in GeneWorks, help to clarify the origins of the novel human
mMCP-7-like tryptase. The number accompanying each branch is
the fraction of mismatched amino acids in pairs of aligned sequences.
Human
-tryptases I and II are identical in amino acid sequence in
exons 4 and 5, as are
I, II, and III; therefore known
- and
-tryptases appear as single, separate branches. The mMCP-7 branches
of the tree are bold lines. The exon 4 tree suggests that
the human mMCP-7-like exon 4 is most closely related to the
corresponding exon in
-tryptase, whereas exon 5 is most closely
related to exon 5 in mMCP-7 and related rodent genes. These data
support the proposed hybrid nature of the human mMCP-7-like gene and
also suggest that the known human
- and
-tryptases (as well as
known dog and cow tryptases) are orthologs of the mouse tryptase
mMCP-6.
- and
-tryptases. The
-like features include a predicted Gln at the
3 position of the
propeptide and a SacII site in exon 3. However, this
/mMCP-7-like gene has a second, novel SacII site in the
same exon; it also has a
-like PstI site in exon 6. Finally, a stop codon that is 40 amino acid residues premature relative
to that in other tryptase genes is identified. Together, the above
findings suggest that this mMCP-7-like gene is a hybrid that may have
arisen from one or more homologous recombination events involving
-like genes and the putative ortholog of mMCP-7. The similarity of
exons 1-4 to
-tryptase suggests that the formation of this hybrid
was recent on the time scale of mammalian evolution. At present, there
is no evidence that other exons of the mMCP-7-homologous gene, which
may have become lost following unequal crossing over events, persist in
the human genome. A dendrogram summarizing the possible phylogenetic
relationship between different exons of the
/mMCP-7-like gene is
shown in Fig. 6B, which also suggests that known
- and
-tryptases may be orthologs of another mouse tryptase, mMCP-6. The
sequence of the BAC B gene (mMCP-7-like I), encompassing all
protein-coding exons and intervening introns, and that of the 5'
portion of the BAC A gene (mMCP-7-like II) up to the HindIII
site at the SP6 terminus of the BAC insert, are deposited in
GenBankTM (AF099147 and AF098327, respectively).
I,
II, and
III have no
SacII sites in this region. Therefore, as shown in Fig.
4B, the 242-bp fragment of the gene amplified using the
primers described above is unaffected by SacII. The
II
gene, however, has a single site, generating two fragments, of which the 113-bp fragment is unique to known
-genes. The mMCP-7-like gene
in BAC A has a single SacII site, which differs from the
site and generates fragments of 51 and 191 bp. The mMCP-7-like gene in
BAC B has two sites, one the same as the BAC A gene and the other
identical to the
-gene site, resulting in 51-, 62-, and 129-bp
fragments. Thus, the 51- and 62-bp fragments are characteristic of
mMCP-7-like genes. As predicted, based on the genes we characterized, BAC A amplimers restricted with SacII generate fragments of
242, 191, 129, 113, and 51 bp but are missing the 62-bp fragment found only in the BAC B mMCP-7-like tryptase. BAC B, however, has the 51-bp
fragment, as well as all of the BAC A fragments. Similar PCR/SacII analysis of individual HindIII BAC
subclones from which
II,
I,
II, and
III genes were obtained
identify only the restriction pattern of the gene already identified in
the subclone, suggesting that only one type of gene lies within a given
subclone. No
-like genes were identified in the BAC B-derived
subclones; however, the 113-bp band in the BAC B digest suggests the
possible presence of an
-like gene not yet subcloned. PCR of full
human genomic DNA yields all of the bands seen in BAC B. Overall, the
PCR results suggest that both BACs contain
,
, and mMCP-7-like
genes and are consistent with the hypothesis that the BACs contain
overlapping portions of the same locus in distinct haploid chromosomes.
An alternative, although less likely interpretation is that each BAC
contains separate parts of the genome that are highly homologous because of past, large scale duplication.
/
tryptase genes that appear to be orthologs of mMCP-6. Homologous positions of
II and
Ia genes suggest that one or more
/
pairs may be alleles at the same locus. Additionally, we
identify a novel gene with one of six exons being closely related to
the mouse tryptase, mMCP-7. Duplication of
/
-like genes, via a
gene dosage effect, may contribute to the high levels of tryptase
expression in human mast cells. Whether the identified unique 3'-UTR
repeated sequences and regulatory sequences flanking the
and
genes contribute to differences in gene transcription and whether
"locus control" elements exert more global influence over
expression of members of the tryptase gene cluster remain to be determined.
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FOOTNOTES |
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* This work was supported in part by Grants HL-54774 and HL-24136 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) AF098327, AF098328, AF099143, AF099144, AF099145, AF099146, and AF099147.
To whom correspondence should be addressed: Cardiovascular
Research Inst., University of California, San Francisco, CA 94143-0911. Tel.: 415-476-9920; Fax: 415-476-9749; E-mail:
ghc{at}itsa.ucsf.edu.
The abbreviations used are: mMCP, mouse mast cell protease; YAC, yeast artificial chromosome; BAC, bacterial artificial chromosome; FISH, fluorescence in situ hybridization; UTR, untranslated region; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s); contig, group of overlapping clones.
2 http://ftp.genome.washington.edu/cgi-bin/RepeatMasker.
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REFERENCES |
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