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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 25-31
Protease-Activated Receptor Genes Are Clustered on 5q13
By
Véronique Guyonnet Dupérat,
Béatrice Jacquelin,
Pierre Boisseau,
Benoît Arveiler, and
Alan T. Nurden
From UMR 5533 CNRS, Hôpital Cardiologique, Pessac, France; and
Laboratoire de Pathologie Moléculaire et Thérapie
Génique, Université Victor Segalen Bordeaux 2, France.
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ABSTRACT |
The serine protease, thrombin, is both a potent agonist for platelet
aggregation and a mitogen inducing the proliferation of other cell
types. Many cellular responses to thrombin are mediated by a
G-protein-coupled thrombin receptor (protease-activated receptor-1, PAR-1). This represents the prototype of a new family of
proteolytically cleaved receptors that includes PAR-2 and the recently
identified PAR-3. Like PAR-1, PAR-3 is a potential thrombin receptor.
Their similar gene structure, mechanism of activation, and
colocalization to 5q13 raises the question of a common evolutionary
origin and of their belonging to a clustered gene family. Construction
of a physical map of the 5q13 region by pulsed-field gel
electrophoresis (PFGE) has allowed us to identify six potential CpG
islands and to establish a linkage of the PAR genes. Southern blot
analysis showed that they were in a cluster on a 560-kb Asc I
fragment, in the order PAR-2, PAR-1, and PAR-3. PAR-1 and PAR-2 genes
were contained within the identical 240-kb Not I fragment, thus
confirming a tight linkage between them. The localization of other CpG
islands suggested that more PAR-family genes may be present.
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INTRODUCTION |
THROMBIN is a serine protease that is a
potent agonist for platelet aggregation as well as being a mitogenic
agent for the proliferation of various cell types.1-3 Many
cell responses are mediated by a thrombin receptor (known as
protease-activated receptor-1, PAR-1) that is a member of a
G-protein-coupled receptor family. It contains seven transmembrane
helical domains and is activated by a two-step mechanism.4
Thrombin cleaves PAR-1 at a site within the amino-terminal extension.
Then, the newly exposed N-terminus acts as a tethered ligand that
interacts secondarily with other domains of the same receptor to
initiate signal transduction.5,6 PAR-1 is the prototype of
a new putative family of proteolytically cleaved receptors that
includes PAR-2 and a recently identified receptor,
PAR-3.7,8 PAR-2 is not expressed by platelets, but by
vascular endothelial cells, keratinocytes, and T cells. Trypsin and
other "physiological" proteases activate PAR-2 by proteolytic
cleavage. Like PAR-1, PAR-3 is expressed on human platelets and
megakaryocytes and is a new potential thrombin receptor.9
The human genes for PAR-1, PAR-2, and PAR-3 have been cloned and
localized to 5q13, by in situ hybridization (FISH) or using radiation
hybrid panels.7,10-12 This region is contiguous to the
proximal breakpoint in the 5q syndrome.13 Comparison
of the three human genes for PAR-1, PAR-2, and PAR-3 suggests a similar genomic organization. These genes are of limited complexity. They contain two exons, the majority of the coding sequence being encoded by
the larger second exon.7,11,14 In addition, PAR-2 is
structurally closely related to PAR-1 with 30% amino acid homology.
The similar gene structure, mechanism of activation of PAR-1 and PAR-2,
and the recent discovery of PAR-3, raises the question of whether the
three genes have a common evolutionary origin, and might be part of a
gene family whose members are clustered on the long arm of human
chromosome 5. The linkage of closely related genes has been shown on
several occasions as, for example, for members of the hematopoietic
growth factor,15 purinoceptor (P2Y),16 and
mucin families.17,18 Construction of a physical map of the
5q13 region by pulsed-field gel electrophoresis (PFGE) has allowed us
to establish a linkage of the PAR genes and to establish their order
within this PAR locus.
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MATERIALS AND METHODS |
Sources of human genomic DNA.
Lymphoblastoid (3360, 3362) and erythroblastic K562 cell lines exhibit
a normal karyotype for chromosome 5. White blood cells from a healthy
female volunteer were also used as a source of normal DNA (BJ).
Multiple sources of DNA were chosen because of the variable degrees of
methylation of DNA in cultured cells.19
Probes for PAR-1, PAR-2, and PAR-3. Genomic DNA was prepared from whole
human blood according to a described QIAGEN procedure (QIAamp Blood
Kit, Courtaboeuf, France). Polymerase chain reaction (PCR)
amplification with Taq DNA Polymerase (Promega, Charbonnières, France) was performed for 40 cycles at 95°C for 30 seconds, at the
hybridization temperature (50°C for PAR-1 and PAR-3, 45°C for
PAR-2) for 30 seconds, and at 72°C for 1 minute using: (1) for
PAR-1, sense primer 5 -GAA TCA AAA GCA ACA AAT GCC-3 (Oli 42) and antisense primer 5-CTA AGT TAA CAG CTT TTT GTA-3
(Oli 43); (2) for PAR-2, sense primer 5-GAA CCA ATA GAT CCT CTA
AA-3 (Oli 23) and antisense primer 5-AAT AGG AGG TCT TAA
CAG T-3 (Oli 24); and (3) for PAR-3, sense primer 5-GTG
ACC CTG TGG ATG CTT TT-3 (Oli 38) and antisense primer
5-CAG CTA CTT GGG AGG CTG A-3 (Oli 39). Each pair of
oligonucleotides permitted the amplification of the coding sequence
within the second exon of the corresponding gene.4,7,8
These primers amplify a 1190-, 1109-, and 1041-nucleotide long fragment
of PAR-1, PAR-2, and PAR-3, respectively, according to the published
cDNA sequences of the human receptors.
After PCR amplification of genomic DNA, the PCR products were
electrophoretically separated on a 1% low-melting agarose gel. Bands
of appropriate size were cut out, purified (Promega), and cloned into
p-GEM T vector according to the manufacturer's recommendations (Promega). DNA from positive colonies was sequenced using the same
primers. The DNA corresponding to the probe was extracted from the
vector using restriction enzymes.
Probes were labeled with [ 32P] dCTP by random priming
using standard procedures, and purified by gel filtration on a Sephadex G50 column (Pharmacia Biotech, Saclay, France). PAR-3 DNA contains Alu
repeats.8 As a result, the purified labeled probe was
resuspended with 200 µg of sonicated human DNA in sodium phosphate
buffer, pH 7.2, to a final concentration of 0.1 mol/L. The mixture was incubated at 65°C for 90 minutes. The probe was used directly without further treatment.
Classical Southern blotting analysis.
Human genomic DNA was prepared from whole human blood using the QIAamp
Blood Kit as recommended by the manufacturer. DNA was cut with
EcoRI, BamHI, HindIII, Pst I, and
Xba I. Fragments were separated by conventional electrophoresis
in phosphate buffer (90 mmol/L Tris-phosphate, 2 mmol/L EDTA,
pH 8.0) in an 0.8% agarose gel and transferred to Hybond-N+ nylon
membrane (Amersham, Les Ulis, France) by capillary blotting.
Genomic DNA preparation and restriction enzyme digestion.
Heparinized blood (10 mL) was subjected to two incubations with 30 mL
red cell lysis buffer (155 mmol/L NH4Cl, 10 mmol/L
KHCO3, 1 mmol/L EDTA, pH 7.4). Each time, cells were left
for 15 minutes on ice, followed by centrifugation for 15 minutes at
1,800g. Cultured cells were washed in phosphate-buffered saline
(PBS).
High-molecular-weight human DNA was prepared in 1% low-melting-point
agarose plugs from a cell suspension (3.5 × 107
cells/mL) in PBS to give a final concentration of 106 cells
or 10 µg DNA per block.20 The plugs were treated with a
solution containing 0.5 mol/L EDTA, pH 8.0, 200 µg/mL proteinase K,
and 1% sarkosyl, at 50°C for 48 hours and then washed once with TE
buffer (10 mmol/L Tris-HCl, pH 7.0, 1 mmol/L EDTA) and twice
with TE buffer containing 40 µg/mL phenylmethylsulfonyl fluoride
(PMSF) at 50°C. Agarose blocks were equilibrated in a large excess
of the appropriate restriction enzyme buffer for 1 hour. Restriction
enzyme digestion was carried out on blocks (10 µg DNA) in a 400 µL
volume with rare cutting enzymes using the buffer conditions
recommended by the suppliers (Ozyme-Biolabs New England, Montigny le
Bretonneux, France; or Promega). For complete digestion, two
applications of 100 U and 50 U of restriction enzyme were used. Partial
digests were obtained by using variable amounts of restriction enzyme
(0.5 or 2 U/µg of DNA). Enzyme digestion was stopped by several
washes with cold TE. Each DNA preparation was tested for the absence of
nuclease activity by incubating a block without added restriction
enzyme.
Pulsed-field gel electrophoresis (PFGE).
After digestion, blocks were equilibrated in running buffer and loaded
onto 1% agarose gels (20 × 20 cm) in TBE buffer (90 mmol/L Tris-HCl, pH 8.3, 90 mmol/L boric acid, and 2 mmol/L EDTA) and
run in a contour-clamped homogeneous electric field apparatus (CHEF)21 at a constant temperature of 12°C and a
constant voltage of 170 V.
Three kinds of electrophoretic conditions were used: (1) molecular size
resolution optimal between 50 and 400 kb: pulse time of 30 seconds for
24 hours, ramp from 30 to 40 seconds for 24 hours, and then 40 seconds
for 24 hours; (2) molecular size resolution optimal between 300 and
1,000 kb: ramp from 110 to 140 seconds for 64 hours; (3) molecular size
resolution optimal between 800 and 2,000 kb: pulse time of 120 seconds
for 24 hours and 240 seconds for 36 hours. Size markers were  phage
concatemers, Midrange I PFG marker (Biolabs) and chromosomes of
Saccharomyces cerevisiae (strain AB 1380). The electrophoresis
gels were stained with ethidium bromide and photographed.
Southern blotting and hybridization.
Gels were depurinated for 15 minutes in 0.25 N HCl, denatured by two
30-minute treatments with 1.5 mol/L NaCl, 0.5 mol/L NaOH, and then
neutralized by two 30-minute treatments with 0.5 mol/L Tris-HCl, pH
7.5, 3 mol/L NaCl, before being blotted onto charged Hybond-N+ nylon
membrane overnight with 20× SSC buffer (1 × SSC = 0.15 mol/L
NaCl, 0.015 mol/L trisodium citrate). After transfer, DNA was fixed on
the membrane by drying for 1 hour at 37°C and then cross-linked
using UV light.
Prehybridizations were performed at 65°C in prehybridization buffer
(6× SSC, 5× Denhardt's, 0.5% sodium dodecyl sulfate
[SDS]) for 3 hours. Hybridizations were performed overnight at
65°C in hybridization buffer (6× SSC, 5× Denhardt's,
0.5% SDS, 10% dextran sulfate, 750 µg/mL ssDNA) for all probes.
Filters were washed twice in 2× SSC, 0.1% SDS, for 30 minutes at
65°C. Higher stringency washes of 0.1× SSC, 0.1% SDS for the
PAR-3 probe were performed at 70°C. Autoradiography of filters was
at 80°C between two intensifying screens.
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RESULTS |
Classical Southern blot analysis.
To verify the specificity of each probe, we first performed Southern
blot analysis of human genomic DNA digested with EcoRI, BamHI, HindIII, Pst I, and Xba I. Results showing hybridization with the PAR-1, PAR-2, and PAR-3 probes
are illustrated in Fig 1. Whatever the
enzyme used, a single cross-hybridizing fragment was seen for each
probe thereby establishing that PAR-1, PAR-2, and PAR-3 are present as
single-copy locus genes and that they do not cross-hybridize with each
other. For each of the EcoRI and Xba I digestions, the
three probes gave patterns similar to those reported by Schmidt et
al11,12 using total human genomic DNA. However, Schmidt et
al12 also found an additional cross-hybridizing ~5-kb
EcoRI fragment in YAC DNA hybridized to the PAR-1 probe, suggested by the investigators to represent either incompletely digested DNA, rearrangements within the YAC, or a homologous gene or
pseudogene. This 5-kb fragment was not found by us.
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| Fig 1.
Classical Southern blot analysis. Human genomic DNA was
digested by Xba I (X), HindIII (H), Pst I (P),
BamHI (B) and EcoRI (E). Successive hybridizations were
performed with PAR-1, PAR-2, and PAR-3 probes. The fragment size is
given in kilobases below each lane. Whatever the enzyme used, a single
cross-hybridizing fragment was seen for the three probes. For the
Pst I digest, PAR-1 hybridized with two fragments because of
the presence of a Pst I site in the PAR-1 coding sequence.
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General considerations.
PFGE analysis was used to establish a physical map of the region
containing the PAR family genes. High-molecular-weight DNAs from two
lymphoblastoid cell lines (3360, 3362), an erythroblastic cell line
(K562), and human lymphocytes (BJ) were analyzed. The selected
restriction enzymes fell into two categories: (1) those recognizing
(G+C)-rich sites included in CpG islands (Not I, Asc I,
Sgf I, BssHII, SacII) and (2) those mainly
recognizing sites outside CpG islands (Mlu I, Nru I,
Xho I, Sfi I). A series of 30 blots with single or
double digests allowed us to construct a restriction map of the region
containing the PAR cluster in relation to the putative CpG islands. The
sizes of the various restriction fragments detected by each of the
PAR-1, PAR-2, and PAR-3 probes are given in
Tables 1, 2 and
3.
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Table 1.
Summary of the Restriction Fragments Hybridized by the
PAR-1, PAR-2, and PAR-3 Probes Using Lymphoblastoid (L), Erythroblastic K562 (E), and BJ DNA
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Table 2.
List of the BssHII Fragments From the
Lymphoblastoid (L) and Erythroblastic K562 (E) Cell Lines and From BJ
DNA Hybridized by the Three PAR Gene Probes
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Table 3.
Summary of Restriction Fragments of BssHII,
Asc I, and Not I Digests (Tables 1 and 2) Used in the
Localization of the CpG Islands Surrounding PAR-1 and PAR-2 Genes
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Sites in the 5q13 region containing the PAR-1, PAR-2, and PAR-3 genes
were sequentially mapped using the data generated by the single and
double digests. Some enzymes gave little useful information because the
fragments were too large (Sgf I) or, in contrast, too small
(Sfi I and Xho I). Nevertheless, numerous variably
methylated sites for Asc I, Not I, BssHII,
SacII, Mlu I, and Nru I were found in the
different sources of DNA studied. The variable degree of DNA
methylation generated partial digestion fragments that were useful in
both the assignment and the confirmation of sites.
Physical linkage and relative positions of the PAR-1, PAR-2, and
PAR-3 genes.
The first Southern blot analysis demonstrated that large-sized DNA
fragments (>240 kb) were in most cases positive with cDNA probes for
PAR-1 and PAR-2 (Fig 2A). A summary of
these fragments is given in Table 1. The smallest common fragment
cohybridizing with PAR-1 and PAR-2 is a 240-kb fragment in the
Not I digest (N1N2) from the
lymphoblastoid cell line (also see Fig 3).
A 110-kb fragment was detected by PAR-1 and a 130-kb fragment by the
PAR-2 probe in samples digested by BssHII
(B6B7, B5B6) and
SacII (S3S4, S2S3) alone or in combination with Not
I (Tables 1-3, Fig 2). The sum of the size of these two
fragments is in good agreement with the estimated size of 240 kb for
the Not I fragment that houses the PAR-1 and PAR-2 genes and
indicates that these two fragments are contiguous.
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| Fig 2.
PFGE analysis of DNA from a lymphoblastoid cell line to
localize the PAR-1 and PAR-2 genes. Fragment size was assigned using the multimer ladders (). (A) The largest fragments obtained with
Asc I (A), Nru I (Nr), and Not I (N) are common
to PAR-1 and PAR-2 whereas the smallest obtained with SacII (S)
are specific for each probe. PFGE conditions: ramp from 110 to 140 seconds for 64 hours. (B) By varying the electrophoretic conditions
(pulse time of 30 seconds for 24 hours, ramp from 30 to 40 seconds for 24 hours and then 40 seconds for 24 hours), we achieved an improved separation of the different 110-kb and 130-kb fragments. Whichever the
combination of enzymes used (Not I, BssHII [B],
SacII), each probe gave an identical pattern, indicating that
the cutting sites for these enzymes are clustered.
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| Fig 3.
Restriction map of the region 5q13 containing the PAR-1,
PAR-2, and PAR-3 genes. Sites used were Asc I (A),
BssHII (B), Mlu I (M), Not I (N), Nru I
(Nr), SacII (S). The majority of the fragments shown in Tables
1-3 are presented in the form of arrows below the restriction
map. Their sizes are given in kilobases. Some partial fragments for
SacII are not illustrated. A map of the CpG islands is shown at
the top of the figure.
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With the K562 cell line, the smallest BssHII restriction
fragments were monospecific for each probe (Table 2): the 110-kb (B6B7), 130-kb (B5B6)
fragments recognized by PAR-1 and PAR-2, respectively, were the same as
those seen with the lymphoblastoid cell line (Fig 3). A 280-kb band
(B9B11) was now recognized by the PAR-3 probe.
Partial fragments specific for the PAR-2 probe were also detected.
Strikingly, no partial restriction fragment common to PAR-1 and PAR-2
was obtained, indicating a preferential cutting-site for BssHII
between the PAR-1 and PAR-2 genes. With these partial digests,
fragments above 480 kb reacted identically with PAR-1 and PAR-3 probes.
This finding allows us to deduce that PAR-3 resides on a 280-kb
BssHII fragment distal to the 110-kb BssHII fragment
carrying PAR-1. Both probes are also linked on a common 590-kb
BssHII partial digestion product
(B6B11). In this partial digest, PAR-1 alone
recognized a 310-kb BssHII partial hydrolysis fragment
(B6B9), which therefore must be juxtaposed to
the 280-kb fragment (B9B11) recognized by
PAR-3. By deduction, the PAR-1 310-kb BssHII
(B6B9)-specific fragment is located between the
PAR-2 130-kb and PAR-3 280-kb specific fragments.
In conclusion, the above results imply that PAR-2 is contained in a
130-kb fragment adjacent to the 110-kb fragment on which PAR-1 lies. In
contrast, the PAR-1 and PAR-3 genes are not contiguous, they are at
least 200-kb apart (Fig 3).The order of these genes is PAR-2,
PAR-1, PAR-3.
Additional restriction sites.
The smallest common DNA fragment cohybridizing with PAR-1, PAR-2, and
PAR-3 probes is a 560-kb Asc I fragment
(A2A3) for the lymphoblastoid cell line (Table
1, Fig 3). Asc I/Mlu I digests generated small 310-kb
(A2M2) and 390-kb
(A1M2) fragments only hybridizing with PAR-1
and PAR-2 probes using lymphoblastoid cell line and BJ DNA,
respectively. An additional 700-kb fragment
(A1A4) is also identified by these two probes
in BJ DNA (Table 1, Fig 4). PAR-3
hybridizes to this fragment, as well as to smaller 140-kb (A4M3) and 310-kb
(A4M2) fragments. The sum of the 310-kb
(A4M2) PAR-3 and of the 390-kb
(A1M2) PAR-1/PAR-2 hybridizing fragments suggests that they are contiguous within the 700-kb
(A1A4) fragment.
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| Fig 4.
PFGE analysis of Asc I and Asc
I/Mlu I fragments from BJ DNA. The Asc I fragments (700 and 560 kb) are common for the three probes showing the existence of a
PAR cluster. The double digest gave a partial 700-kb Asc I
fragment, the smaller 560-kb fragment was not detected. Asc I
shows marked site preference, and we obtained variable amounts of
cleavage at this site in different digests. The Asc
I/Mlu I digest shows that PAR-3 is located at one end of the
PAR cluster. PAR-1 and PAR-2 are located on a 390-kb fragment while
PAR-3 is seen on a separate 310-kb fragment (and on a shorter 140-kb
fragment). PFGE conditions: ramp from 110 to 140 seconds for 64 hours.
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When using lymphoblastoid cell line or BJ DNA, PAR-1 and PAR-2 probes
identified a 380-kb Nru I fragment
(Nr1Nr2), whereas the PAR-3 probe detected a
370-kb band (Nr2Nr3) (Fig 3). Double digests
performed with Asc I and Not I or Asc I and
Nru I gave a 320-kb (A3N2 or
A3Nr2) fragment specific to the PAR-3 probe, whereas the PAR-1 and PAR-2 probes hybridized to a shorter 240-kb fragment (A2Nr2), corresponding to the fragment
Not I (N1N2).
These results with additional sites supplement the construction of the
most probable restriction map shown in Fig 3. The map extends about
1,200 kb and satisfies all of the restriction data obtained.
Identification of CpG islands surrounding the PAR-1, PAR-2, and PAR-3
genes.
Rare cutting restriction enzymes were selected according to their
ability to cut into CpG islands in a specific manner.22 The
enzymes selected were Not I, Asc I, BssHII, and
SacII. Lymphoblastoid cell line DNA was first cut by
Not I, Sac II, and BssHII. Whatever the
combination of enzymes used, PAR-1 and PAR-2 genes are always seen on
the same 110-kb and 130-kb fragments (Table 3, Fig 2B). This indicates
that the cutting sites for these enzymes are clustered and that they
show the presence of three CpG islands, one on each side of the PAR-1
and PAR-2 genes and one located between them. With SacII and
BssHII, we observed the same 220-kb fragment
(S1S3 and B4B6)
hybridizing to the PAR-2 probe, superimposing sites A1,
B4 and S1 and so indicating the presence of a
fourth CpG island. The close vicinity of the A4 and
B10 sites localizes a fifth, while the A3 site
suggests a sixth CpG island, since 93% of Asc I sites are
within CpG islands.22 Therefore, the entire 1,200-kb map
potentially contains at least six putative CpG islands (Fig 3).
Remark.
The human PAR-3 cDNA was initially cloned by screening a human small
intestine cDNA library.8 In our study, the PCR and sequencing of PAR-3 were performed on human genomic DNA with Oli 38 and
39 primers covering the majority of the cDNA sequence. This implies
that the major part of the coding sequence is contained in a single
exon, just as for PAR-1 and PAR-2.
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DISCUSSION |
The protease-activated receptors are closely related with 45% sequence
homology. Previous studies have shown that the PAR-1, PAR-2, and PAR-3
genes have a similar genomic organization and reside on the long arm of
chromosome 5.7-12,14 Preliminary studies on radiation
hybrids have shown that PAR-3 is colocalized with the other PARs in the
same region of this chromosome.11 We have now used PFGE to
identify potential CpG islands and to produce a map of the 5q13 region.
Evidence has been obtained that the three PAR genes are arranged in a
cluster within a 560-kb region and in the order PAR-2,
PAR-1, PAR-3.
Central to our long-range mapping strategy was the use of several cell
lines with different methylation status as a source of DNA, because
"natural" partial digestions could be obtained. The detection of
partial restriction fragments in common confirmed that the PAR-1,
PAR-2, and PAR-3 genes were in the same genomic region (for example,
see Table 1 for Asc I digests). In addition, the analysis of
partial digests helped us define the order of the three genes along the
chromosome. Finally, this strategy allowed us to extend the restriction
map on both sides of the PAR-1, PAR-2, and PAR-3 gene cluster.
Using field inversion gel electrophoresis (FIGE) and YAC clones,
Schmidt et al12 found that PAR-1 and PAR-2 genes are
contained within identical Xho I (~90-kb) and Not I
(~120-kb) restriction fragments. Using CHEF and genomic DNA, we found
that PAR-1 and PAR-2 genes are contained within two specific
Xho I (~70- and 50-kb, respectively; data not shown) and
within the same Not I (~40-kb) restriction fragments. These
differences may be explained by the fact that YAC DNA, when compared
with genomic DNA, particularly that from cultured cells,19
is unmethylated but is subject to rearrangements. In addition, if there
is a restriction site for Not I in the CpG island between PAR-1
and PAR-2 genes, this site may be methylated in genomic DNA and the
site would not be cut. An explanation could be that the 240-kb fragment
observed by us effectively corresponds to the sum of two bands of
~120-kb when YAC unmethylated genomic DNA was the substrate and which
were not resolved using FIGE. With this technique, mobility decreases with size until an inflection point above which longer molecules travel
faster than shorter ones. This complex relationship makes it difficult
to obtain accurate sizes.23 Thus, for optimal separation and accurate sizing we used the CHEF procedure. By varying the electrophoretic conditions we were able to separate different 110-kb
and 130-kb fragments with BssHII and SacII, which for
others were detected as a single ~120-kb fragment.12
It is interesting that genes encoding other G-protein-coupled
receptors, for example certain adrenergic receptor
subtypes24 and P2Y receptors,16 are clustered
in the genome. Conservation of structural and/or functional
domains among these G-protein-coupled receptors suggests that they may
have a common ancestor.25 The PAR-1 and PAR-3 genes were
located on BssHII fragments that were separated by at least 200 kb and PAR-1 and PAR-2 were on contiguous BssHII fragments of
combined size of 240 kb. The proximity of these three related genes on
human 5q13 supports the possibility that they may have evolved by
recent gene duplication and that the close linkage, particularly
between PAR-1 and PAR-2, may be important in the regulation of their
expression. However, further studies will be required to determine the
mechanisms leading to their transcription in cells. In our study, we
found SacII fragments in the same size range (315 and 400 kb)
hybridized by the three probes. However, each fragment is too small to
contain all three genes (Table 2). Given the results obtained with
other restriction enzymes, it is probable that each probe recognizes an
SacII fragment (315 kb) containing one PAR gene. This suggests
that a symmetry exists in the distribution of these
sites.18 This observation reinforces the previous
suggestion that a single ancestral PAR gene may have given rise by
successive duplications to several distinct PAR genes.
PFGE analysis of large DNA fragments is a powerful tool to identify CpG
islands that are frequently found in the promoter regions of genes and
that constitute valuable markers to locate genes.22 If
these PAR genes are members of a distantly related gene family, the
possibility exists that other as yet unidentified members of the family
exist within the cluster. It is possible that they are associated with
one of the six potential CpG islands that we have identified, bearing
in mind that associations with PAR-1, -2, or -3 can account for a
maximum of three of them. Studies are planned in our laboratory to
examine the DNA surrounding these islands and to determine if other PAR
genes are also localized in this region of the chromosome.
The PAR genes are localized to 5q13, a region that is contiguous to the
proximal breakpoint (q13  q15) identified in the 5q-
syndrome.13 Because of the known effect of thrombin on
multiple cell types, including megakaryocytes, Boultwood et
al26 and Demetrick et al13 suggested that the
PAR-1 gene may be involved in the dysmegakaryocytopoiesis observed in
this syndrome. Our results suggest that the recently cloned second
thrombin receptor, PAR-3, could also be a potential candidate gene
implicated in this disorder. Using dual-label FISH and complementary
techniques involving interphase and metaphase nuclear analysis of cells
from seven patients with a del (5)(q13q33), Demetrick et
al13 showed that the PAR-1 gene is centromeric to the
proximal breakpoint and therefore uninvolved in the region encompassed
by the interstitial deletion. However, it is possible that PAR-2,
PAR-3, and other potential PAR genes would be involved. In addition, it
should be noted that the breakpoints in the 5q anomaly vary
somewhat among patients and that the 5q deletion is also often
associated with other chromosomal abnormalities.27 So it
would be interesting to analyze the rearrangements in the 5q13 region
of DNA of patients with the 5q syndrome by FISH and PFGE to
clarify the involvement of PAR genes in the 5q syndrome and
related disorders.
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FOOTNOTES |
Submitted February 17, 1998;
accepted April 9, 1998.
Supported by the CNRS, Université Bordeaux II, the Conseil
Régional d'Aquitaine, and the Ministère de l'Enseignement
Supérieur et de la Recherche (ACC-SV No. 9). V.G.D. was a
recipient of postdoctoral fellowships from the Association Sanofi
Thrombose pour la Recherche and from l'ARC.
Address reprint requests to Véronique Guyonnet Dupérat,
PhD, UMR 5533 CNRS, Hôpital Cardiologique, 33604 Pessac, France.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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Abstract
Introduction
Abstract