Note: Descriptions are shown in the official language in which they were submitted.
CA 02312266 2000-07-14
TITTLE OF THE INV)~'fION: -
The Use of HCaRG, a Novel Calcium-Regulated Gene Coding for a Nuclear
Protein for Regulating Celi Proliferation
BACKGROUND OF THE INVEt~'TION:
Ionized calcium concentration in plasma is maintained within a very narrow
range. The major
players maintaining extracellular calcium homeostasis are calciotropic
hormones, parathyroid
hormone (PTH),' 1,25 dihydroxyvitamin D, calcitonin and calcium itself.
Indeed, extracellular
calcium regulates its own concentration as an extracellular messenger by
acting on calcium
receptors or calcium sensors. The calcium-sensing receptor is linked to
several intracellular
second messenger systems via guanylyl nucleotide-regulating G proteins and
activates
phosphoinositide-specific phospholipase C, leading to accumulation of inositol
1,4,5
trisphosphate (IP3) and diacylglycerol (1-5).
'List of abbreviations
BN.Ix Brown-Norway rats
DMEM Dulbecco's modified Eagle's
medium
FBS Fetal bovine serum
FCS Fetal calf serum
GFP Green fluorescent protein
GST Glutathione S-transferase
HCaRG _ _Hypertension-Related, Calcium-Regulated
Gene
IP3 Inositol 1,4,5 trisphosphate
MTE Multiple tissue expression
nCARE Negative calcium-responsive
element
PAGE Polyacrylamide gel electrophoresis
PBS Phosphate-buffered saline
PCR Polymerase chain reaction
PHF Parathyroid hypertensive factor
PTC Parathyroid cells
PTH Parathyroid hormone
RACE Rapid amplification of cDNA
ends
RT Reverse transcription
SDS Sodium dodecyl sulfate
SHR Spontaneously hypertensive
rat
SSC Standard sodium citrate
VSMC Vascular smooth muscle cells
WKY Wistar-Kyoto rats
ZFP7 Zinc finger protein 7
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Cells of the parathyroid gland possess such a calcium sensor (6). Even slight
reductions in
extracellular ionized calcium concentration (in the order of I-2% or less)
elicit prompt increases
in the rate of PTH secretion and mRNA levels. Historically, research on the
parathyroid gland
has focused on the chemistry, regulation, synthesis and secretion of PTH.
There is growing
interest in other calcium-regulating proteins of this gland that are also
negatively regulated by
extracellular calcium, such as chromogranin A and Secretory Protein-I (7), as
well as a
hypertensive factor of parathyroid origin (PHF) (8,9).
Arterial hypertension is associated with numerous disturbances of calcium
metabolism
manifested not only in humans but also in genetic as well as acquired models
of hypertension
(10-14). Disturbances in renal and intestinal handling of calcium in
hypertension have been
reported by several investigators ( I S). Urinary calcium has generally been
shown to be increased
(so-called urinary leak) and intestinal calcium absorption diminished in
genetically hypertensive
or spontaneously hypertensive rats (SHR) (15,16). Cytoplasmic free calcium
concentration has
most often been found to be elevated in circulating platelets, lymphocytes,
erythrocytes, and
vascular smooth muscle cells (VSMC) from hypertensive animals and humans (for
review, see
( 17)). In SHR as well as in low-renin hypertensive patients, there seems to
be an inverse
relationship between extracellular and intracellular calcium ( 18). It has
been hypothesized that
certain genetic abnormalities might be responsible for the link between some
forms of
hypertension, calcium homeostasis and the parathyroid gland. To identify new
genes that might
be abnormally regulated by extracellular calcium in the parathyroid gland of
genetically
hypertensive rats, we prepared a cDNA library from the parathyroids of SHR. In
this study, we
describe the isolation and characterization of a novel gene, designated HCaRG
(for
Hypertension-related, Calcium-regulated Gene), negatively regulated by
extracellular calcium
with higher mRNA levels in SHR. HCaRG is a nuclear protein with putative
'leucine zipper'
motifs and is potentially involved in the regulation of cell proliferation.
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EXPERIMENTAL PROCEDURES
Cell cultures
Parathyroid cells (PTC) were isolated from SHR and Wistar-Kyoto (WKY) rats.
Primary
cultures were passaged in Dulbecco's modified Eagle's medium (DMEM) with 10%
fetal calf
serum (FCS), as described previously (9). They were then maintained in Ham F12
medium
containing a low (0.3 mM) or normal (2.0 mM) total calcium concentration for 2
or 48 h. COS-7
or HEK293 cells were cultured in DMEM containing 10% fetal calf serum. All
cell types were
maintained in 5% COZ at 37°C.
Ischemia-reperfusion
SHR were anesthetized with light flurane, and the right kidney was removed
through a mid-
abdominal incision. The left kidney was subjected to warm transient ischemia
by occlusion of
the left renal artery and vein with a micro-clip, as described previously
(19). The skin incision
was temporarily closed. After 60 min of occlusion, the clip was removed, and
the wound was
closed with a 2-0 suture. The rats had access to water immediately after
surgery.
SHIZ parathyroid cDNA library
Parathyroid glands were removed from 100 12-week-old SHR and frozen
immediately in liquid
nitrogen. The glands were added to a guanidinium thiocyanate solution and
homogenized. Poly
A RNA was isolated on an oligo(dT) column. The cDNA library was constructed
with Poly A
RNA as template and the ZAP-cDNA synthesis kit (Stratagene, La Jolla, U.S.A.).
A summary of
the protocol is as follows: mRNA was reverse-transcribed from an XhoI-linker
oligo(dT) primer
using Moloney-Murine leukemia virus reverse transcriptase. Second strand
synthesis was then
produced with DNA polymerase I in the presence of RNaseH. cDNA termini were
blunted by
incubation with the Klenow fragment of DNA polymerase I and dNTPs. EcoRI
adaptors were
added using T4 ligase, and the ends phosphorylated with T4 polynucleotide
kinase. This mixture
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CA 02312266 2000-07-14
was then digested with Xho I to release adaptors from the 3' end of the cDNA.
The resulting
mixture was separated on a Sephacryl S-400 column. cDNAs were ligated into the
Uni-ZAP XR
vector using T4 DNA ligase, and packaged into Gigapack II Gold packaging
extract. The
packaged products were plated onto XL1-Blue MRF'. To screen the eDNA library,
phages were
plated onto bacterial host plates (XL1-Blue MRF') and incubated overnight.
After chilling at 4°C
for 2 h, a nitrocellulose filter was overlaid for 2 min. The filter was then
denatured, neutralized
and DNA crosslinked to it with UV light. Hybridization was performed with
digoxigenin-dUTP
labeled probes (Roche Molecular Biochemicals, Laval, Canada) derived from 3'-
and S'-RACE
(rapid amplification of cDNA ends), products described below.
RNA and cDNA preparation
Total RNAs were prepared from rat cells and organs according to the standard
guanidinium
thiocyanate-phenol-chloroform method (20) and kept at -70°C until used.
mRNA was extracted
from total RNA with the PolyATtract system (Promega, Nepean, Canada). cDNAs,
unless stated,
were synthesized with random hexamers for first strand synthesis and reverse-
transcribed.
Radiolabeled DNA probes were prepared by the random priming technique or
polymerase chain
reaction (PCR) amplification with 32P-dCTP.
3' or S'-RACE
Four mixtures of degenerate oligonucleotide primers were initially designed
according to the
putative amino acid sequence of PHF with the following degenerate sequence: S'
TA(T/C) TCI
GTI TCI CA(T/C) TT(T/C) (A/C) G 3'. From initial RACE experiments (described
below), 1
unique sequence primer TAC TCC GTG TCC CAC TTC CG was selected for its ability
to
generate reverse transcription (RT)-PCR DNA fragments from PTC total RNA and
used
subsequently as candidate primer for 3'-RACE. In brief, for 3'-RACE, total RNA
from PTC was
reverse-transcribed with a hybrid primer consisting of oligo(dT) (17 mer)
extended by a unique
17-base oligonucleotide (adaptor). PCR amplification was subsequently
performed with the
4
t
a:
CA 02312266 2000-07-14
adapter, which bound to cDNA at its 3'-ends, and the candidate primer
mentioned above (21).
For 5'-RACE, RT was undertaken with an internal primer derived from the
sequence of the
cDNA fragment generated by 3'-RACE. A dA homopolymer tail was then appended to
the first
strand reaction products using teminal deoxynucleotidyl transfeiase. Finally,
PCR amplification
was accomplished with the hybrid primer described previously and a second
internal primer
upstream to the first one (21 ).
Subcloning
The DNA fragments generated from the RACE experiments were separated by
electrophoresis,
isolated from agarose gel and extracted by the phenol-chloroform method (20).
pSP72 plasmid
(Promega) was digested at the Sma I site and ligated to blunt DNA fragments
with T4 DNA
ligase. Transformed DHSa E. coli bacteria were grown and recombinant bacteria
were selected
by PCR. Similarly, HCaRG was subcloned in pcDNAI/Neo (Invitrogen, Carlsbad,
U.S.A.).
To determine the subcellular localization of HCaRG protein in mammalian cells,
the coding
region of HCaRG was fused to green fluorescent protein (GFP) cDNA and was
transfected in the
cells. Briefly, the entire coding region of HCaRG was amplified by PCR with
the primers ATG
TCT GCT TTG GGG GCT GCA GCT CCA TAC TTG CAC CAT CCC and TAA TAC GAC
TCA CTA TAG GGA GAC, gel purified, and fused in-frame to GFP in pEGFP-C 1
(Clontech,
Palo Alto, U.S.A.) through a blunt Hind III site. pEGFP-HCaRG was then
sequenced. Similarly,
the coding sequence of HCaRG was fused in frame to glutathione S-transferase
(GST) in pGEX-
3X (Amersham Pharmacia Biotech, Baie d'Urfee, Canada) through a Sma I site and
a blunt
EcoR I site.
Sequencing
Double-stranded sequencing of cloned cDNA inserts was performed with Sequenase
Version 2.0
(United States Biochemical, Cleveland, U.S.A.). 5 pg of recombinant plasmid
template were
CA 02312266 2000-07-14
denatured, annealed with T7 or SP6 primers, and labeled with 35S-dATP by
extension, using the
chain termination method of Sanger according to the manufacturer's protocol.
Cloning of human HCaRG
A 439-by cDNA fragment of rat HCaRG was 3zP-labeled and served as a probe for
screening a
human VSMC cDNA library. DNA from positive phages was purified and the
fragments were
cloned in pBluescript. All fragments were sequenced. We obtained a 1355-by
fragment
containing the coding region of HCaRG.
Northern blot hybridization, dot blot hydridization and competitive RT PCR
2 ~ g of poly A RNA from PTC or 10 ~ g of total RNA from kidneys were
denatured at 68°C and
separated on denaturing formamide I% agarose gel. After transfer onto
nitrocellulose by
vacuum, hybridization was performed overnight using 32P-labeled probes
generated from cDNA
clones) by PCR or random labeling. 1 ~g of total RNA was used in dot blot
experiments. A
human multiple tissue expression (MTE~) array (Clontech) and human fetal and
tumor panel
Northern Territory RNA blots (Invitrogen, Carlstad, U.S.A.) were hybridized
with 32P-labeled
human HCaRG cDNA according to the manufacturer's specifications. For
quantitative
determinations of HCaRG mRNA, total RNA was extracted from PTC and reverse-
transcribed.
A HCaRG competitor was constructed using the PCR Mimic Construction Kit
(Clontech) with
the following composite primers: GCA CGA GCC ACA GCC AGC TAC CCC AGC CAC CCA
TTT GTA CC (sense) and TGT GAC TGT CAG CGG GAT GGA GTC CGA GAT GTA GAG
GGC (antisense). The 344-by DNA obtained was cloned into pSP72 and transcribed
with SP6
RNA polymerase. The resulting RNA was quantified by photometry and
subsequently used in
competitive RT-PCR. The competitive reaction contained 1 or 2 ug total RNA
with increasing
amounts of competitor cRNA along with 3zP-labeled nucleotide. Two primers TGT
GAC TGT
CAG CGG GAT GG and GCA CGA GCC ACA GCC AGC TACO flanking the HCaRG intron
were employed to amplify a 186-by cDNA fragment. PCR was performed: 15 sec at
95°C, 20
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CA 02312266 2000-07-14
sec at 68°C, 30 sec at 72°C, for 30 cycles, followed by a 5-min
elongation step at 72°C. 10 ~1 of
the PCR were loaded on 1.8% agarose gel, then dried and exposed in a
PhosphorImager cassette
for quantification.
In situ mRNA hybridization
Tissues from SHR and WKY rats were rinsed in phosphate buffer, fixed in 4%
paraformaldehyde and embedded in paraffin. 3- to 5-pm sections were cut and
mounted on
microscope slides pretreated with aminopropylthiethoxysilane. The slides were
first dried at
37°C, then at 60°C for 10 min prior to use. The probe applied
was a unique 300-by fragment (3r
290 in Figure 2A) which had been subcloned into the BamH I site of a pSP72
vector. The DNA
was transcribed using T7 or SP6 polymerases to create sense and antisense
riboprobes which
were labeled with digoxigenin-UTP. They were validated by dot blot
hybridization with template
DNA. Prehybridization of slides was undertaken after de-waxing in xylene,
followed by
progressive ethanol-water hydration (95% to 50%). The slides were rinsed in
phosphate-buffered
saline (PBS) and incubated with proteinase K (20 pg/ml) for 20 min at room
temperature. After
this digestion, they were rinsed successively in glycine buffer, PBS and then
dehydrated in
ethanol. Actual prehybridization was done with 50% formamide, 0.2% sodium
dodecyl sulfate
(SDS), 0.1% Sarcosyl, 5X standard sodium citrate (SSC: NaC1 (0.15M), sodium
citrate (0.015M,
pH 7.0)) and 2% blocking reagent (Roche Molecular Biochemicals) for 1 h at
60°C.
Hybridization was performed by adding the probe (200 ng/ml) to 50 p1 of 4X SSC
and 50%
formamide per section. The slides were incubated overnight at 60°C in a
humidified chamber.
During hybridization, a coverslip was placed over the tissue section. After
hybridization, it was
removed and the sections rinsed with 4X SSC, then washed with 4X SSC for 15
min and in 2X
SSC for 15 min, at room temperature. Finally, the sections were washed with
0.1% SSC for 30
min at 60°C. For coloration, they were washed with Buffers 1 and 2 of
the DIG Luminescent
Detection Kit (Roche Molecular Biochemicals). They were then incubated with
anti-DIG
alkaline phosphatase antibody ( 1:500) in Buffer 2 for 40 min, washed twice in
Buffer 1 for 15
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min and in Buffer 3 for 2 min. Incubation in the color solution (NBT/x-phos)
was earned out for
45 min, after which the slides were washed in distilled water and dry-mounted
with Geltol.
In vitro translation
The full length of the HCaRG coding sequence was synthesized by RT-PCR with
specific
primers and inserted downstream of the T7 promoter into the pSP72 vector. In
vitro transcription
and translation were performed using a TNT-T7-coupled reticulocyte lysate
system (Promega) in
the presence of 35S-methionine. A plasmid containing the luciferase gene
supplied by the
manufacturer was used as a control. The synthesized proteins were analyzed by
1 S% SDS-
polyacrylamide gel electrophoresis (PAGE) in the absence or presence of 13-
mercaptoethanol.
Radioactive protein bands were detected by scanning with a PhosphorImager.
Antibody production
E. coli cells transformed with pGEX-3X were grown in LB medium containing SO
pg/ml
ampicillin at 37°C until As9s nm = 0.5. Isopropyl-(3-D-
thiogalactopyranoside was added to a final
concentration of 0.1 mM, and the cells were cultured for 2 h. Purification of
GST-HCaRG was
performed according to the manufacturer's protocol. Polyclonal antisera with
antibodies
recognizing HCaRG were produced by immunization of rabbits with GST-HCaRG
protein.
Immunocytological reaction at the electron microscopic level
Rat tissues (liver, anterior pituitary, spleen, heart and adrenal gland) were
quickly removed and
fixed in 4% paraformaldehyde with 0.05% glutaraldehyde in phosphate buffer
solution for 90
min. A part of the specimens was cryoprotected in 0.4M sucrose phosphate
buffer solution for 30
min at 4°C, then frozen in a cold gradient of fuming nitrogen (Biogel,
CFPO, Saint Priest,
France) to -4°C, and immersed in liquid nitrogen, as described
previously (22). Ultrathin frozen
s
CA 02312266 2000-07-14
sections of 80 nm thickness were obtained using a dry sectioning method at -
120°C with an
Ultracut S microtome (Leica, Lyon, France). The other part of the specimens
was dehydrated
before embedding in Lowicryl K4M with the AFS system (Leica) (23). Sections
were mounted
on 400 mesh collodion-carbon-coated nickel grids. For ultrastructural
localization of HCaRG
protein, the grids were first placed in buffer containing 0.1 M phosphate
buffer, O.1S M NaCI,
and 1% albumin, pH 7.4, for 10 min. They were then incubated for 1 h with
polyclonal IgG
raised against HCaRG protein at concentrations of 1:1000 and 1:50 for
ultrathin frozen sections
and Lowicryl sections respectively. After 10-min washing in the same buffer,
antigen/antibody
complexes were revealed with anti-rabbit IgG conjugated with 10 nm gold
particles in buffer
containing O.OS M Tris, O.1S M NaCI, 1% albumin, pH 7.6, for 1 h. The grids
were washed in the
same buffer and fixed with 2.S% glutaraldehyde. The specificity of the
immunocytological
reaction was tested on sections with omission of primary antibody and
incubation of the primary
antibody with particle-adsorbed antigen. No signal was observed on these
tissue sections. Before
observation in a Philips CM 120 electron microscope at 80 kV, the cryosections
were contrasted
in 2% uranyl acetate, embedded in 8% methylcellulose, and the Lowicryl
sections were
contrasted for 20 min in S% uranyl acetate.
Transfection and subcellular localization
COS-7 cells were plated at ~30-SO% confluency 1 day prior to transfection
which was performed
with S ug/well of pEGP-HCaRG or pcDNAI/Neo HCaRG, according to the calcium
phosphate
method. After 24 h, the cells were fixed with 4% paraformaldehyde in PBS for
30 min at room
temperature. Following 3 washes with PBS, cells transfected with pEGFP-HCaRG
or
pcDNAI/Neo-HCaRG were mounted on coverslips. The cells were permeabilized with
0.3%
Triton X-100 for 12 min, blocked with 1% BSA-I% gelatin for 1S min, incubated
with HCaRG
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CA 02312266 2000-07-14
antibodies at 37°C for 1 h, washed in 0.5% BSA, incubated with anti-
rabbit FITC-labeled
antibodies and washed again. Fluorescence and immunofluorescence were detected
with a Zeiss
fluorescence microscope.
Stable transJection
HEK293 cells were plated in a 100-mm plate at a density of 0.5x 106
cells/plate. They were
transfected with the control plasmid pcDNAI/Neo (Invitrogen, Faraday, U.S.A.)
or with the
plasmid containing rat HCaRG using a standard calcium phosphate
coprecipitation method. 48 h
after transfection, the cells were plated in 150-mm plates in the presence of
400 pg/ml 6418
(Life Technologies, Burlington, Canada). After 2 weeks, the clones were picked
and the level of
ectopic HCaRG expression was determined by northern hybridization.
Cell counting and jH thymidine incorporation
The rate of stable clone cell proliferation was measured by counting the
number of cells after
plating. Cells were seeded at a density of 0.1x10' cells/6-well plate, with
triplicate plates for each
cell line. Every 24 h, the cells were trypsinized and counted in a
hemocytometer. HEK293 cells
which stably expressed either Neo control plasmid or HCaRG were used for the
estimation of
DNA synthesis by 3H-thymidine incorporation. The clones were trypsinised at
90% confluency,
counted in a standard hemocytometer and inoculated at an identical initial
cell density of 40,000
cells/ml in DMEM containing 10% FBS and 6418 at 400 p.g/ml. All cells were
inoculated in
Poly-D-lysine-pretreated 24-well plates in a volume of 1 ml/well (40,000
cells/well). They were
allowed to attach and grow for a period of 24-48 h. The growth media were then
replaced by
DMEM containing 0.2% FBS and 6418 (400 pg/ml) for a period of 48 h to
synchronise the
cells. After the synchronisation period, the cells were supplied with fresh
medium containing
10% FBS and allowed to grow for 48 h. [3H]-thymidine, 1 pCi/ml (ICN) was added
to the cells
1Q
CA 02312266 2000-07-14
d
for the last 4 h of the 48 h-growth period. At the end of incubation, the
medium was removed
and the monolayers washed twice with PBS. The cells were then fixed with
ethanol:acetic acid
(3:1, V:V), and DNA was digested/extracted with O.SN PCA. at 80-90°C
for 20 min.
11
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RESULTS
Isolation of a novel cDNA whose expression is negatively regulated by
extracellular calcium
in the SHR parathyroid gland
Using sense candidate primers (from a putative amino acid sequence of PHF
(24)) and a hybrid
oligo dT primer, 3'-RACE experiments, performed on total RNA extracted from
SHR PTC
cultured in low-calcium medium, generated 1 major 700-by fragment that was
digested and
cloned in the BamH I site of pSP72. As a BamH I site was present in the 700-by
fragment, a
recombinant plasmid containing a 300-by insert was isolated and sequenced.
This fragment was
used to screen the PTC library and to generate new oligonucleotide primers to
extend the cDNA
towards the 5'- and 3'-ends by RACE. From 7 overlapping DNA fragments isolated
in the above
experiments and from SHR PTC cDNA library screening, a 1100-by cDNA was
reconstituted
(Fig. 1A). The rat 1100-by reconstituted cDNA sequence contained an open
reading frame of
224 codons preceded by 2 in-frame stop codons and followed by the most
frequent variant of the
poly A tail (Fig. 1 B). A 342-by intron was localized at position -52 from the
translation initiation
site.
Poly A RNA was isolated as described and analyzed by Northern hybridization
with the 32P-
labeled 300-by fragment (Fig. 2A). Two bands were detected with this probe, at
approximate
lengths of 1.2 and 1.4 kb. These results suggest either the existence of 2
genes or differential
splicing. Furthermore, they indicate that the reconstituted 1100-by cDNA is
almost full length
cDNA, estimated at 1.2 kb by the major band in the northern hydridization
experiments.
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Regulation of the expression of this novel gene was investigated by
competitive RT-PCR assay
in PTC from WKY and SHR. Cells between 5 and 12 passages were tested in these
studies. In
WKY PTC, lowering of ambient calcium from 2.0 mM to 0.3 mM induced a rapid 2-
fold
increase in the mRNA levels of this novel gene at 2 h, which lasted up to 48 h
(Fig. 2B). This
calcium regulation was detected in WKY PTC up to about 12 passages but
disappeared in long-
term cultures. Lowering of calcium concentrations in the cell medium also
increased the mRNA
levels of this novel gene in SHR PTC but to a lesser extent than in WKY cells
(data not shown).
We then compared its mRNA levels between 2 normotensive rat strains (Brown
Norway, BN.Ix,
or WKY) and hypertensive animals (SHR). We observed that the mRNA levels of
this novel
gene were significantly higher in PTC derived from SHR (Fig. 2C left panel)
compared to
normotensive WKY rats at normal calcium. Similarly, when we extracted RNA
(Fig. 2C right
panel) or proteins (Fig. 2D) directly from the kidneys, we found significantly
higher levels of
this novel gene in hypertensive rats. These results clearly show that this
novel gene is negatively
regulated by extracellular calcium concentrations and that its levels are
significantly higher in
genetically hypertensive rats compared to 2 normotensive strains. We,
therefore, named this gene
Hypertension-related, Calcium-Regulated Gene (HCaRG).
Sequence and structure of HCaRG cDNA
The deduced protein contained 224 amino acids with a calculated molecular
weight of 22456 Da.
The estimated pI of the protein was 6Ø It comprised no known membrane-
spanning motif but
had an estimated 67% a-helix content. The absence of a putative signal peptide
sequence
suggested an intracellular protein. There were 2 cysteines in the sequence,
indicating possible
infra- or inter-molecular disulfide bridges (Cys 64-cys 218). The protein had
several putative
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CA 02312266 2000-07-14
phosphorylation sites for C- and A-kinases and 1 potential Asn-glycosylation
site (Asn 76). To
confirm that HCaRG mRNA encodes a peptide of expected size, the HCaRG cDNA
inserted into
pSP72 was incubated in vitro in a coupled transcription/translation labeling
system. It was
transcribed by T7 RNA polymerase, and translated in rabbit reticulocyte
lysate. As shown in
Figure 3 (lane 4), HCaRG mRNA directed the synthesis of a peptide with a
molecular mass of 27
kDa which closely corresponded to the molecular weight calculated from the
amino acid
sequence. PAGE analysis of the reaction product in the absence of the reducing
agent (3-
mercaptoethanol showed bands of 27 and 43 kDa (Fig. 3, lane S). These results
suggest possible
intramolecular or intermolecular disulfide bridges and the formation of
homodimers or
heterodimers with other proteins) present in the lysate.
Cloning of human HCaRG
We then used a 439-by cDNA fragment of rat HCaRG (+1 to +440 in Fig. 1) to
screen a human
VSMC cDNA library. We identified several positive clones that were purified,
subcloned in
pBluescript vector and sequenced. We obtained a 1355-by sequence containing
full length
human cDNA, while all other clones contained only partial sequences. A recent
sequence search
in GenBank revealed a region with complete DNA sequence homology within 3
cosmids
containing the zinc finger protein 7 (ZFP7) gene (accession numbers AF124S23,
AF146367 and
AF118808). Although the nucleotide sequence of human HCaRG could be found in
these
cosmids, we are the first to assign an expressed gene sequence to this DNA
region.
Sequence comparison between human HCaRG and rat HCaRG showed 80% identity at
the
nucleotide level (data not presented) and, similarly, 80% homology at the
amino acid level (Fig.
4). Analysis of protein structure with the PROSEARCH database revealed 4
overlapping putative
14
CA 02312266 2000-07-14
'leucine zipper' consensus motifs (Fig. 4 underlined). Further analysis
revealed homology to the
EF-hand calcium-binding motif (8 out of the 10 most conserved amino acids)
(Fig. 4 dashed
box). We also identified a nuclear receptor-binding motif (Fig. 4 bold and
italics). All these
motifs were conserved in the rat and human amino acid sequence'.
Subcellular localization ofHCaRG
We expressed GFP-HCaRG in COS-7 cells. Fluorescence study showed that GFP-
HCaRG
localized in the nucleus while cytoplasmic fluorescence was very faint (Fig.
5B). GFP, on the
other hand, had a very diffuse localization (Fig. 5A). This result was
confirmed by
immunofluorescence using antibodies specific to HCaRG (Fig. SC) and by
electron microscopy
(Fig. SD). Electron microscopy was also performed on different tissues. In all
tissues studied,
HCaRG was found in the nucleus with some labeling in protein synthesis sites.
HCaRG expression in various human tissues
A human MTE~ array was hybridized with human 3zP-labelled HCaRG cDNA as a
probe. The
array contained 76 polyA RNAs from various adult tissues, cell lines, fetal
tissues and cancerous
cell lines. These arrays were normalised to 8 different housekeeping genes.
Analysis of the array
showed that HCaRG was expressed preponderantly in the heart, stomach, jejunum,
kidney, liver
and adrenal glands. Comparison of HCaRG expression in fetal to adult organs
revealed that
HCaRG mRNA was less expressed in all fetal tissues compared (Fig. 6A),
particularly in the
heart, kidney and liver. Northern blots confirmed the lower abundance of HCaRG
in the fetal
heart compared to all regions of the adult heart (Fig. 6B). We also compared
HCaRG mRNA
levels in various cancerous cell lines to normal tissues (Fig. 6C). HCaRG mRNA
levels were
decreased in all cancerous cell lines studied. They were also much lower in a
glioblastoma, a
CA 02312266 2000-07-14
partly-differentiated renal cell carcinoma and a moderately differentiated
hepatocellular tumor
compared to the same amount of normal RNA of adjacent tissues excised from the
same
operational site (Fig. 6D).
In situ hybridization ofHCaRG mRNA in the kidney and adrenal
HCaRG expression was determined in SHR tissues by in situ hybridization. The
labeled
antisense riboprobe hybridized to the medulla and zona fasciculata of the
adrenal cortex (Fig. 7).
In the kidney, labeling was almost exclusively located in the cortex and
concentrated in the
tubular component, contrasting with virtual absence of the signal in glomeruli
(Fig. 7). In these
organs, the signal was clearly greater in hypertensive rats compared to their
normotensive
controls (Lewanczuk et al.; unpublished data). The sense probe was used as a
negative control
and appropriately revealed a low signal under our hybridization conditions,
demonstrating
specificity of the reaction (Fig. 7 lower panels).
HCaRG mRNA levels after ischemia-reperfusion
The process of kidney injury and repair recapitulates many aspect of
development. It involves
de-differentiation and regeneration of epithelial cells, followed by
differentiation (25-27). Since
we observed that HCaRG mRNA levels are lower in fetal than in adult organs, we
evaluated
HCaRG expression after unilateral renal ischemia in uninephrectomized rats
(19) as contralateral
nephrectomy has been shown to stimulate cell regeneration (28-31). We noted
that HCaRG
mRNA declined rapidly to its lowest levels at 3 h and 6 h of reperfusion (Fig.
8A). These values
then increased steadily to higher than baseline at 48 h of reperfusion. This
was observed in both
the kidney medulla (Fig. 8A) and cortex (Fig. 8B). In contrast to the decline
in HCaRG mRNA
16
CA 02312266 2000-07-14
levels, the proto-oncogene c-myc expression, which is correlated with
hyperplastic response in
mammalian cells, was rapidly increased following renal ischemia and
reperfusion (31). c-myc
mRNA levels were low in control kidneys and increased dramatically in the post-
ischemic
kidney at 3 h of reperfusion, at a time when HCaRG mRNA levels were already
reduced (Fig.
8A and 8C)
Overexpression of HCaRG inhibits cell proliferation
HEK293 cells were stably transfected with either plasmid alone or with plasmid
containing rat
HCaRG. After transfection, several clones were examined for the determination
of rat HCaRG
mRNA levels. Four clones (HCaRG clones l, 5, 8 and 9) expressed variable
amounts of rat
HCaRG mRNA, as detected by northern blots, while no HCaRG mRNA levels were
found in
clones transfected with the plasmid alone (Fig. 9). Clones expressing the
highest levels of
HCaRG (clones 8 and 9) were selected for cell proliferation studies. For these
studies, cells that
were transfected with the vector alone or polyclonal HCaRG-transfected cells
served as controls.
The proliferation rates of the HCaRG-transfected cell lines and vector control
cells were
examined under normal growth conditions (10% FCS and G-418) by counting cell
numbers
every day for a period of 8 days after plating. Cell lines transfected with
the vector alone (Neo
clones 1 and 6) showed a similar growth rate as non-transfected cells (not
presented). Clones 8
and 9 expressing high levels of rat HCaRG revealed a much lower proliferation
rate than vector
control cells while polyclonal cells expressing intermediate values of HCaRG
fell in between
(Fig. 10A). Consistent with a lower proliferation rate, stable HCaRG
transfection clones 8 and 9
showed much lower 3H-thymidine incorporation than clones transfected with the
empty vector
(Fig. 10B).
17
CA 02312266 2000-07-14
~.: _...
DISCUSSION
The cloning of a novel extracellular calcium-responsive gene (~ICaRG) in the
rat parathyroid
gland from SHR is described here. HCaRG mRNA and protein levels were higher in
cultured
PTC and in several organs of SHR, compared to their normotensive counterparts.
They were
negatively regulated by extracellular calcium, i.e. lowering extracellular
calcium led to increases
in HCaRG mRNA. The identification of an extracellular calcium-sensing receptor
from the
parathyroid gland has provided novel insights into the mechanisms of direct
action of
extracellular calcium on several cell types. The calcium sensor has also been
localized in the
cerebral cortex and cerebellum, in the tubular region of the kidney cortex,
the thyroid, adrenal
medulla, lung, and blood vessels (1,32,33). As shown here, HCaRG mRNA levels
are also
detected in several of these tissues. The calcium receptor is a member of the
superfamily of G
protein-coupled receptors activating phospholipase C (34,35). In the
parathyroid gland, it is a key
mediator of inhibition of PTH expression by high calcium (36). The calcium
sensor has been
shown, in the kidney, to be directly related to inhibition of tubular
reabsorption of calcium and
magnesium in the thick ascending loop (for review, see (34)). In PTC cultures
prepared from
human or bovine parathyroids, low extracellular calcium (0.3 mM) has been
demonstrated to
increase PTH secretion and mRNA levels whereas augmentation of calcium in the
incubation
medium reduces PTH mRNA. Similar regulation was observed for PHF in rat
parathyroid cells
(9). We show here that HCaRG expression is regulated in a manner similar to
PTH and PHF in
PTC isolated from the rat.
To date, very few extracellular calcium-negative responsive genes have been
cloned.
Parathormone was the first gene described to possess a negative calcium-
responsive element
(nCARE) in its 5'flanking region (37). Several types of nCARE have been
reported: Type 2 is a
regulatory element consisting of a palindromic core sequence and several
upstream T nucleotides
18
CA 02312266 2000-07-14
originally described in the PTH gene. Its transcriptional inhibitory activity
is orientation-specific.
The nCARE core is present in an Alu-repeat in 111 copies in the human genome,
suggesting the
possibility that other genes may possess functional nCARE (38). With the
properties described in
the present study, HCaRG may be one of them.
HCaRG is not only expressed in the parathyroid gland but also in most organs
tested, although at
highly variable levels. Elevated HCaRG levels have been noted consistently in
the tissues of
genetically hypertensive animals, suggesting abnormalities of HCaRG regulation
in several
organs of SHR that could be due to either: 1) decreased extracellular calcium
levels; 2) an
abnormal response to extracellular calcium; 3) abnormal
transcription/stability of HCaRG
mRNA in hypertensive rats, or 4) a combination of these. A state of negative
calcium balance
has been described in SHR that could support the first possibility. On the
other hand, 2-fold
higher HCaRG mRNA levels were observed in PTC from SHR than from WKY at normal
calcium concentration (Fig. 2C). Thus, the modest reduction of calcemia in
hypertension will not
be the sole explanation of increased levels, suggesting increased expression
or decreased
degradation of this gene product in hypertension.
No homologous protein sequence to the HCaRG open reading frame was found in
the
SWISSPROTEIN database. The HCaRG coding sequence contains 1 consensus motif
known as
the EF-hand or HLH Ca motif (Fig. 3 dashed box). This motif generally consists
of a 12-residue,
Ca-binding loop flanked by 2 a-helices. Eight of the 10 most conserved amino
acids are present
in HCaRG protein. Usually, the basic structural/functional unit consists of a
pair of calcium-
binding sites rather than a single HLH motif. The HCaRG coding sequence
contains only 1 EF-
like motif but it is possible that its high a-helix content favours coiled-
coil interactions and
dimerization of the protein. Pairing of the 2 EF-hand motifs may enhance its
calcium function.
Hodges and collaborators (39,40) have demonstrated that domain III of troponin
C (a synthetic
19
CA 02312266 2000-07-14
34-residue calcium-binding domain) can form a symmetric 2-site homodimer in a
head-to-tail
arrangement in the presence of calcium (41 ). Similarly, a 39-residue
proteolytic fragment
containing calcium-binding site IV of troponin C was shown to form a dimer
(42). These studies
and others (43-45) have demonstrated that dimerization of single HLH
structures controls
calcium affinity and that even homodimers can bind 2 calcium molecules with
positive
cooperativity (40). Hydrophobic interactions at the interface between calcium-
binding sites
appear to stabilize the calcium domains. Our in vitro translation studies
showed the appearance
of a protein band of about 43 kDa under non-reducing conditions. HCaRG protein
might form
reductant-sensitive, non-covalent homodimers compatible with its putative high
a-helix content,
but the existence of a functional calcium domain in HCaRG protein remains to
be established.
Several characteristics of HCaRG are similar to those of S 100A2 protein, a
calcium binding
protein of the EF-hand type that is preferentially expressed in the nucleus of
normal cells but
down-regulated in tumors (44). As with HCaRG, S 100A2 expression is down-
regulated by
calcium (46,47).
We also cloned the human homolog of HCaRC~ from a VSMC cDNA library, using a
437-by
fragment of rat HCaRG as a probe. The coding sequence was found to be 80%
homologous to
the rat sequence and to contain the putative EF-hand domain. A restriction
fragment length
polymorphism permitted us to localize the HCaRG locus on chromosome 7 of rats
(Tremblay et
al, unpublished). The gene was assigned within a 4.4-cM region on the long arm
of chromosome
7 between Mit 3 and Mit 4 genes. By analogy, we suggested the assignment of
HCaRG on
human chromosome 8q21-24. In a recent search of HCaRG homologous sequences in
Genbank,
homologies were found with 3 chromosome 8 clones containing ZFP7. It was,
therefore, possible
to localize HCaRG on chromosome 8q24.3, confirming our initial assignment.
This region
contains loci involved in several bone diseases, including osteopetrosis and
multiple exostosis
and several human neoplasms (48,49).
CA 02312266 2000-07-14
Many DNA-binding proteins utilize zinc-containing motifs to bind DNA. Other
classes of DNA-
binding proteins have a DNA-recognition domain at their N terminus that
dimerizes to form a 2-
chain coiled-coil of a-helices, also known as a 'leucine zipper'. We
identified 4 overlapping
'leucine zipper' regions conserved in the rat and human sequence, and the high
a-helix content
of HCaRG makes it a possible DNA-binding protein. We are currently
investigating this
possibility. It has been shown that nuclear receptors require the ligand-
dependent recruitment of
co-activator proteins to effectively stimulate gene transcription (50). The
nuclear receptor
interaction domain of these factors is highly conserved and contains the
consensus sequence
LXXLL (where X is any amino acid). This motif is sufficient for ligand-
dependent interaction
with nuclear receptors (5 I ). We have identified 1 of these motifs in HCaRG.
Nuclear localization
of HCaRG protein makes this gene a potential transcription regulator.
Recently, a new transcription factor from the rat kidney (Kid-I) was
identified (52-SS). It was
reported that Kid-1 mRNA levels declined after renal injury secondary to
ischemia (55).
Similarly, decreased HCaRG mRNA levels are seen when epithelial cells are de-
differentiated
and proliferate (following ischemia and reperfusion). In the model of
unilateral ischemic injury,
it was shown that contralateral uninephrectomy attenuates apoptotic cell death
and stimulates
tubular cell regeneration (28-31). We demonstrate here that HCaRG mRNA levels
decreased 3
and 6 h after ischemia in contrast to c-myc expression which is correlated
with hyperplastic
responses (31). We also observed that its levels are lower in all fetal organs
tested when
compared to adult organs, and lower in tumors and the cancerous cell lines
tested. It is possible
that the gene product may exert a negative effect on growth. This was
confirmed by the stable
expression of HCaRG in HEK293 cells. We found that HCaRG overexpression had a
profound
inhibiting effect on HEK293 cell proliferation. This was shown not only by
lower cell number
__
21
CA 02312266 2000-07-14
but also by lower DNA synthesis, suggesting that the effect seen was not due
to a death-
promoting effect of HCaRG.
In conclusion, we have cloned the cDNA of a novel gene that is regulated
negatively by
extracellular calcium and presents greater expression in several organs of the
genetically
hypertensive rat model which is known to demonstrate negative calcium balance.
HCaRG
mRNA levels are rapidly regulated by calcium, perhaps via the action of
calcium receptor
signaling. Comparison of HCaRG mRNA levels in fetal to adult organs and normal
and tumour
cells showed that HCaRG is more expressed in all adult normal tissues tested.
We also report
that HCaRG mRNA levels are modulated during ischemia-reperfusion injury which
mimics
kidney ontogeny. Furthermore, its nuclear localisation, identified motifs and
patterns of
expression make this gene a potential regulator of cellular proliferation.
22
CA 02312266 2000-07-14
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CA 02312266 2000-07-14
FIGURE LEGENDS:
FIGURE 1. cDNA cloning of HCaRG. A. Reconstitution scheme of HCaRG cDNA.
Overlapping fragments leading to the reconstitution of rat HCaRG 1100-by cDNA
are shown.
cDNA fragments were initially obtained using 5'-RACE and 3'-RACE strategies as
well as by
screening a SHR parathyroid cDNA library. The first cDNA fragment was by 3'-
RACE (3r 290).
This initial fragment served to screen the SHR parathyroid cDNA library.
Fragments HCaRG 2c-
t3 + 2c-t7, HCaRG 825, HCaRG 10-ic, and HCaRG 10-174 were isolated from the
cDNA
library. Fragments Sr 285 and Sr 260 were obtained by 5'-RACE. This
reconstitution was
confirmed by sequencing a 860-by PCR product with nested primers in Sr 260 and
HCaRG 825
and containing the complete open reading frame. B. Nucleotide and deduced
amino acid
sequences of HCaRG. The translation initiation start site codon is at position
1 and the
termination codon is at position 675. The deduced amino acids are indicated
below the
nucleotide sequence. The localization of a 482-by intron is indicated at
position -52 by a
triangle.
FIGURE 2. Identification of a novel gene negatively regulated by extracellular
calcium. A.
Northern blot analysis of Poly A RNA isolated from parathyroid cells (PTC).
HCaRG mRNA
appears as a doublet of approximately 1.2 and 1.4 kb. The positions of
ribosomal RNAs and
GAPDH transcript are indicated. B. PTC extracted from normotensive rats (WKY)
(from
passages 8 to 10) were incubated in low (0.3 mM) or normal (2 mM) calcium-
containing
medium for 2 and 48 h. Total RNA was extracted and analysed by RT-PCR as
described in the
Experimental Procedures section. Incubation of PTC for 2 h in 0.3 mM (L)
calcium significantly
increased HCaRG mRNA compared to 2 mM (N) calcium; this increase lasted up to
48 h. C.
26
CA 02312266 2000-07-14
Significantly higher basal HCaRG levels were found in PTC from hypertensive
rats compared to
the normotensive rat strain WKY (left panel). This was confirmed with RNA
(right panel) and
proteins (D) extracted directly from the kidneys of SHR and BN.Ix, another
normotensive strain.
The figure represents the mean ~ S.E.M. of 2 independent experiments performed
in duplicate.
** indicatesp<0.02, * indicatesp<0.05 as evaluated by the unpaired t-test.
FIGURE 3. In vitro translation of HCaRG cDNA. cDNA was cloned into pSP72
vector and
used for coupled transcription/translation in the presence of 35S-methionine.
Lane 1: molecular
weight markers; lane 2: translation products of the control luciferase gene;
lane 3: translation
products without the insert; lane 4: translation product from HCaRG cDNA; lane
5: translation
products of HCaRG cDNA. The proteins were separated by 15% PAGE in the
presence (lanes 1
to 4) or absence (lane 5) of (3-mercaptoethanol. Transcription/translation of
HCaRG cDNA
yields a protein of 27 kDa (lane 4). In the absence of (3-mercaptoethanol, a
product of 43 kDa
was also observed (lane 5), suggesting intramolecular or intermolecular
disulfide bridges and the
formation of homodimers or heterodimers with other proteins) present in the
lysate.
FIGURE 4. Sequence comparison between human HCaRG and rat HCaRG. The deduced
amino acid sequences of rat HCaRG (rHCaRG) and of human HCaRG (hHCaRG) are
aligned.
Identical amino acids are boxed while homologous amino acids are shaded. We
calculated 80%
homology between these 2 sequences. Analysis revealed homology to the EF-hand
motif, with 8
out of the 10 most conserved amino acids (dashed box). Further analysis using
the
PROSEARCH database revealed 4 overlapping putative 'leucine zipper' consensus
motifs
(underlined). We also identified a nuclear receptor-binding domain (bold and
italics).
27
CA 02312266 2000-07-14
FIGURE 5. Subcellular localisation of HCaRG in cultured cells. COS-7 cells
were
transfected with GFP-HCaRG. 24 h later, the cells were fixed and observed.
Cells transfected
with pEGFP vector alone show diffuse fluorescence (A) while'cells transfected
with pEGFP-
HCaRG present nuclear fluorescence (B). Nuclear localization was confirmed by
immunofluorescence on COS-7 cells transfected with pcDNAI/Neo-HCaRG (C), and
by
electron microscopy (D) on pituitary.
FIGURE 6. Tissue distribution of HCaRG mRNA. A. Comparison of HCaRG expression
in
fetal versus adult human organs. HCaRG mRNA is expressed less in all fetal
tissues compared,
particularly in the heart, kidney and liver ( / adult; ~ fetal). B. Northern
blot containing 2 pg of
polyA+ RNA from fetal and adult human hearts. HCaRG is more expressed in all
regions of the
adult heart (L: left, R: right). C. Comparison of HCaRG expression in adult
human organs
versus cancerous cell lines. HCaRG mRNA is expressed less in most cancerous
cell lines
compared. Lymphocyte ( normal; ~Burkitt's lymphoma Raji; ~ Burkitt's lymphoma
Daudi).
Leukocyte (~ normal;0 leukemia HL-60; ~ leukemia K-562; ~ leukemia MOLT-4).
Rectum
( normal; Ocolorectal adenocarcinoma SW480). Lung (/ normal; lung carcinoma
A549).
D. Northern blot containing 20 pg of total RNA isolated from 3 different human
tumours (T) and
normal tissue (N) excised at the same operational site. HCaRG expression is
decreased in brain,
kidney and liver tumours.
FIGURE 7. In situ hybridization of HCaRG mRNA in the kidney and adrenal. In
situ
hybridization of HCaRG mRNA in the rat adrenal shows specific detection in the
zona
2B
CA 02312266 2000-07-14
fasciculata and medulla. Specific hybridization in the kidrrey is restricted
to proximal tubules,
contrasting with virtual absence in the glomeruli (G). (Upper panels:
antisense probe, lower
panels: sense probe).
FIGURE 8. Analysis of kidney mRNA of HCaRG and c-myc obtained after ischemia
and
various periods of reperfusion. A. Dot blot of total RNA taken from the
medulla of kidneys
which underwent 60-min ischemia and reperfusion for various time periods (full
lines) or from
contralateral control kidneys (dotted lines). HCaRG mRNA declined rapidly to
its lowest levels
at 3 h and 6 h of reperfusion. It then increased steadily to exceed baseline
at 48 h of reperfusion.
In contrast, c-myc mRNA levels rose dramatically by 12 h and returned below
HCaRG mRNA
levels at 48 h of reperfusion. B. Representative northern blots of HCaRG and c-
myc mRNA from
the cortex of kidneys which underwent 60-min ischemia and 3 h, 6 h, 12 h, 24 h
or 48 h
(HCaRG) or 12 h or 24 h (c-myc) of reperfusion (I/R) or from contralateral
control kidneys (C).
FIGURE 9. Characterization of stable cell lines. A. HEK293 cells transfected
with
pcDNAI/Neo or pcDNAI/Neo rat HCaRG were examined for expression of rat HCaRG
by
northern blot using rat HCaRG as a probe. Rat HCaRG was undetectable in cells
transfected with
the empty vector while different levels of expression were observed in cells
tranfected with the
vector expressing HCaRG. B. The levels of ectopic expression were determined
by densitometric
measurement and normalized to GAPDH.
FIGURE 10. HCaRG expression inhibits cell proliferation. A. Stable clones
Neol, Neo6, Neo
Poly, HCaRGB, HCaRG9, and HCaRG Poly were plated at low density. For each time
point,
29
CA 02312266 2000-07-14
triplicate plates were counted, and average cell number- was recorded. The
level of DNA
synthesis was monitored by measuring [3H] thymidine incorporation (B).
Representative
experiment performed in triplicate.
CA 02312266 2002-O1-17
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: TREMBLAY, Johanne
HAMET, Pavel
SOLBAN, Nicolas
LEWANCZUK, Richard
(ii) TITLE OF INVENTION: The Use of HCaRG, A Novel
Calcium-Regulated Gene Coding for a Nuclear Protein, for
Regulating Cell Proliferation
(iii) NUMBER OF SEQUENCES: 4
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: GOUDREAU GAGE DUBUC
(B) STREET: Stock Exchange Tower, Suite 3400
(C) CITY: Montreal
(D) STATE: Quebec
(E) COUNTRY: Canada
(F) ZIP: H4Z 1E9
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,312,266
(B) FILING DATE: 14-JUL-2000
(C) CLASSIFICATION: C12N-15/10
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: DUBUC, Jean H.
(C) REFERENCE/DOCKET NUMBER: DH/12725.24
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (514) 397-7607
(B) TELEFAX: (514) 397-4382
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1100 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Rattus rattus
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 132..803
31
CA 02312266 2002-O1-17
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GCACGAGCCA CAGCCAGCTA CCGCGGCTAG GTTCCTCCAG GTGCAGAGGG CGGTAAAGGC 60
TTGGTTTGTA TTTGTAATGC AACTGTGGTT AGGACCTTCT CTTCGGACTG GTCAAGAAAC 120
GGGAAGAAAG G ATG TCT GCT TTG GGG GCT GCA GCT CCA TAC TTG CAC CAT 170
Met Ser Ala Leu Gly Ala Ala Ala Pro Tyr Leu His His
1 5 10
CCC GCT GAC AGT CAC AGT GGC CGG GTC AGT TTC CTG GGT TCC CAG CCC 218
Pro Ala Asp Ser His Ser Gly Arg Val Ser Phe Leu Gly Ser Gln Pro
15 20 25
TCT CCA GAA GTG ACG GCC GTG GCT CAG CTC TTG AAG GAC TTA GAC AGG 266
Ser Pro Glu Val Thr Ala Val Ala Gln Leu Leu Lys Asp Leu Asp Arg
30 35 40 45
AGC ACC TTC AGA AAG TTG TTG AAA CTT GTA GTC GGG GCC CTG CAT GGG 314
Ser Thr Phe Arg Lys Leu Leu Lys Leu Val Val Gly Ala Leu His Gly
50 55 60
AAA GAC TGC AGA GAA GCT GTG GAG CAA CTT GGT GCC AGC GCC AAC CTG 362
Lys Asp Cys Arg Glu Ala Val Glu Gln Leu Gly Ala Ser Ala Asn Leu
65 70 75
TCA GAA GAG CGT CTG GCC GTC CTG CTG GCG GGC ACA CAC ACC CTG CTC 410
Ser Glu Glu Arg Leu Ala Val Leu Leu Ala Gly Thr His Thr Leu Leu
80 85 90
CAG CAG GCT CTC CGG CTG CCC CCT GCT AGT CTA AAG CCA GAT GCC TTC 458
Gln Gln Ala Leu Arg Leu Pro Pro Ala Ser Leu Lys Pro Asp Ala Phe
95 100 105
CAG GAA GAG CTC CAG GAA CTT GGC ATT CCT CAG GAT CTA ATT GGA GAT 506
Gln Glu Glu Leu Gln Glu Leu Gly Ile Pro Gln Asp Leu Ile Gly Asp
110 115 120 125
TTG GCC AGT TTG GCA TTT GGG AGT CAA CGC CCT CTT CTC GAC TCT GTA 554
Leu Ala Ser Leu Ala Phe Gly Ser Gln Arg Pro Leu Leu Asp Ser Val
130 135 140
GCC CAA CAG CAG GGA TCC TCG CTG CCT CAC GTG TCT TAC TTC CGG TGG 602
Ala Gln Gln Gln Gly Ser Ser Leu Pro His Val Ser Tyr Phe Arg Trp
145 150 155
CGGGTGGAC GTGGCCATC TCA AGCGCT CAGTCCCGC TCCCTGCAA 650
ACC
ArgValAsp ValAlaIle SerThrSerAla GlnSerArg SerLeuGln
160 165 170
CCGAGTGTT CTCATGCAG CTGAAGCTCACA GATGGATCT GCACACCGC 698
ProSerVal LeuMetGln LeuLysLeuThr AspGlySer AlaHisArg
175 180 185
TTCGAGGTG CCCATAGCC AAATTTCAGGAG CTGCGGTAC AGTGTAGCC 746
PheGluVal ProIleAla LysPheGlnGlu LeuArgTyr SerValAla
190 195 200 205
TTGGTCCTT AAGGAGATG GCAGAACTGGAG AAGAAGTGT GAGCGCAAA 794
LeuValLeu LysGluMet AlaGluLeuGlu LysLysCys GluArgLys
32
CA 02312266 2002-O1-17
210 215 220
CTG CAG GAC TGACTGAACC CTGGTACTGT GGGTGCTGAA GCTGGTACCA 843
Leu Gln Asp
GAACACAGCCCCCCACTGGTGATGAGCCCAACTCCATTGAGGTCCTGCAT GTGAGAACGT903
ATTTTAAGTGAAAAGACAGCGGGACTTTCAGGTTTTGTTTTATGAGTCAA CAGCTGGGCA963
GGGTGGCACAGTTTATAATCTCAGCCCTTGGAAGTCTGAGGCTGGAGAAT GGGAAGTGTA1023
AGCTGGGCCTGGCTTTCATAGTGAGGCTCAGTGTCGAATTAAAGAGGTAA AGCAACTATT1083
p~~~AAAAAAAAAAAAAA 110
0
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 224 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Ser Ala Leu Gly Ala Ala Ala Pro Tyr Leu His His Pro Ala Asp
1 5 10 15
Ser His Ser Gly Arg Val Ser Phe Leu Gly Ser Gln Pro Ser Pro Glu
20 25 30
Val Thr Ala Val Ala Gln Leu Leu Lys Asp Leu Asp Arg Ser Thr Phe
35 40 45
Arg Lys Leu Leu Lys Leu Val Val Gly Ala Leu His Gly Lys Asp Cys
50 55 60
Arg Glu Ala Val Glu Gln Leu Gly Ala Ser Ala Asn Leu Ser Glu Glu
65 70 75 80
Arg Leu Ala Val Leu Leu Ala Gly Thr His Thr Leu Leu Gln Gln Ala
85 90 95
Leu Arg Leu Pro Pro Ala Ser Leu Lys Pro Asp Ala Phe Gln Glu Glu
100 105 110
Leu Gln Glu Leu Gly Ile Pro Gln Asp Leu Ile Gly Asp Leu Ala Ser
115 120 125
Leu Ala Phe Gly Ser Gln Arg Pro Leu Leu Asp Ser Val Ala Gln Gln
130 135 140
Gln Gly Ser Ser Leu Pro His Val Ser Tyr Phe Arg Trp Arg Val Asp
145 150 155 160
Val Ala Ile Ser Thr Ser Ala Gln Ser Arg Ser Leu Gln Pro Ser Val
165 170 175
33
CA 02312266 2002-O1-17
Leu Met Gln Leu Lys Leu Thr Asp Gly Ser Ala His Arg Phe Glu Val
180 185 190
Pro Ile Ala Lys Phe Gln Glu Leu Arg Tyr Ser Val Ala Leu Val Leu
195 200 205
Lys G1u Met Ala Glu Leu Glu Lys Lys Cys Glu Arg Lys Leu Gln Asp
210 215 220
(2) INFORMATION FOR SEQ ID N0:3:
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1355 basepairs
(B) TYPE: nucleic
acid
(C) STRANDEDNESS: le
doub
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: cDNA
(vi)ORIGINAL SOURCE:
(A) ORGANISM: Homo iens
sap
(ix)FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 295..966
(xi)SEQUENCE DESCRIPTION:EQ ID
S N0:3:
GGGCAGGC AG TTGAGGTGGA TTAAACCAAACCCAGCTACGCAAAATCTTA 60
GCATACTCCT
CAATTACC CA CATAGGATGA ATAATAGCAGTTCTACCGTACAACCCCGGA 120
GGCGCAGACC
GTCCACAC GG GACAGGGACG CCCGCTCGGCTCCGCTCCGCGCTGATCCTC 180
AAGGTCTCGC
GGTTTCCC CG CCGCCCACCC GGACGCCGACGAAAGCCAGCGAGCTCCTCA 240
GCCTCAGGCA
TCTGCATCTG CTGATCAAAGAGGAAGCAGC ATG 297
GGACCGACCT AGCA
CCTGGGCTGG
Met
1
TCTGCTGTG GGG GCT GCA ACT TAC CTG CATCCT GGTGAT AGT 345
CCA CAT
SerAlaVal Gly Ala Ala Thr Tyr Leu HisPro GlyAsp Ser
Pro His
5 10 15
CACAGTGGC CGA GTG AGT TTC GGG GCC CTTCCT CCAGAG GTG 393
TTG CAG
HisSerGly Arg Val Ser Phe Gly Ala LeuPro ProGlu Val
Leu Gln
20 25 30
GCAGCAATG GCC CGG CTA CTA GAC CTA AGGAGC ACGTTC AGA 441
GGG GAC
AlaAlaMet Ala Arg Leu Leu Asp Leu ArgSer ThrPhe Arg
Gly Asp
35 40 45
AAGTTGCTG AAG TTT GTG GTC AGC CTG GGGGAG GACTGC CGA 489
AGC CAG
LysLeuLeu Lys Phe Val Val Ser Leu GlyGlu AspCys Arg
Ser Gln
50 55 60 65
GACGGTGTG CAG CGT CTT GGG AGC GCC CTGCCG GAGGAG CAG 537
GTC AAC
AspGlyVal Gln Arg Leu Gly Ser Ala LeuPro GluGlu Gln
Val Asn
70 75 80
34
CA 02312266 2002-O1-17
CTG GGT GCC CTG CTG GCA GGC ATG CAC ACA CTG CTC CAG CAG GCC CTC 585
Leu Gly Ala Leu Leu Ala Gly Met His Thr Leu Leu Gln Gln Ala Leu
85 90 95
CGT CTG CCC CCC ACC AGC CTG AAG CCT GAC ACC TTC AGG GAC CAG CTC 633
Arg Leu Pro Pro Thr Ser Leu Lys Pro Asp Thr Phe Arg Asp Gln Leu
100 105 110
CAG GAG CTC TGC ATC CCC CAA GAC CTG GTC GGG GAC TTG GCC AGC GTG 681
Gln Glu Leu Cys Ile Pro Gln Asp Leu Val Gly Asp Leu Ala Ser Val
115 120 125
GTA TTT GGG AGC CAG CGG CCC CTC CTT GAT TCT GTG GCC CAG CAG CAG 729
Val Phe Gly Ser Gln Arg Pro Leu Leu Asp Ser Val Ala Gln Gln Gln
130 135 140 145
GGG GCC TGG CTG CCG CAT GTT GCT GAC TTT CGG TGG CGG GTG GAT GTA 777
Gly Ala Trp Leu Pro His Val Ala Asp Phe Arg Trp Arg Val Asp Val
150 155 160
GCA ATC TCC ACC AGT GCC CTG GCT CGC TCC CTG CAG CCG AGC GTC CTG 825
Ala Ile Ser Thr Ser Ala Leu Ala Arg Ser Leu Gln Pro Ser Val Leu
165 170 175
ATG CAG CTG AAG CTT TCA GAT GGG TCA GCA TAC CGC TTT GAG GTC CCC 873
Met Gln Leu Lys Leu Ser Asp Gly Ser Ala Tyr Arg Phe Glu Val Pro
180 185 190
ACA GCC AAG TTC CAG GAG CTG CGG TAC AGC GTG GCC CTG GTC CTA AAG 921
Thr Ala Lys Phe Gln Glu Leu Arg Tyr Ser Val Ala Leu Val Leu Lys
195 200 205
GAG ATG TGT GAG AGA CTG GAC 966
GCA GAT CGC CAG
CTG GAG
AAG AGG
Glu Met Asp Leu u Lys Cys Glu Arg Leu Asp
Ala Gl Arg Arg Gln
210 21 5 220
TGACCCCTCACTTGACCAGTCCCATTCAGATCCGGCTTGGACAGGCACCTGAGATGGTGC1026
CAAAGTGCAGCTGACTCTTCCCACGACAGCCCTGGCCTTCCCATCAGGCAGGCTCTTCAG1086
TGAGTGTTTGAACGTAATTATGTAGTTTTCTGTTTAATTGAAAAAGAGAGCTATGCCTTT1146
TTTTCTTTTTGGAAGTAAAGCAGCTAAAACATGTTTCTATAGGTGAGTGTTGGACCTTCA1206
CACCTCCCCTTCCCTGTACATTTGTCTTTGGTGCTGGACGTGGCCATGTGAGGCCAGGTT1266
GAGGCCCTTTGTAGACAACATACAGTTGCTCAGCCTGGCCCCATGTAGCCAGGTGCTTTT1326
GTAGATCTTGTGTTTCAGGCAGGGCCCGG 1355
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 224 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
CA 02312266 2002-O1-17
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Ser Ala Val Gly Ala Ala Thr Pro Tyr Leu His His Pro Gly Asp
1 5 10 15
Ser His Ser Gly Arg Val Ser Phe Leu Gly Ala Gln Leu Pro Pro Glu
20 25 30
Val Ala Ala Met Ala Arg Leu Leu Gly Asp Leu Asp Arg Ser Thr Phe
35 40 45
Arg Lys Leu Leu Lys Phe Val Val Ser Ser Leu Gln Gly Glu Asp Cys
50 55 60
Arg Asp Gly Val Gln Arg Leu Gly Val Ser Ala Asn Leu Pro Glu Glu
65 70 75 80
Gln Leu Gly Ala Leu Leu Ala Gly Met His Thr Leu Leu Gln Gln Ala
85 90 95
Leu Arg Leu Pro Pro Thr Ser Leu Lys Pro Asp Thr Phe Arg Asp Gln
100 105 110
Leu Gln Glu Leu Cys Ile Pro Gln Asp Leu Val Gly Asp Leu Ala Ser
115 120 125
Val Val Phe Gly Ser Gln Arg Pro Leu Leu Asp Ser Val Ala Gln Gln
130 135 140
Gln Gly Ala Trp Leu Pro His Val Ala Asp Phe Arg Trp Arg Val Asp
145 150 155 160
Val Ala Ile Ser Thr Ser Ala Leu Ala Arg Ser Leu Gln Pro Ser Val
165 170 175
Leu Met Gln Leu Lys Leu Ser Asp Gly Ser Ala Tyr Arg Phe Glu Val
180 185 190
Pro Thr Ala Lys Phe Gln Glu Leu Arg Tyr Ser Val Ala Leu Val Leu
195 200 205
Lys Glu Met Ala Asp Leu Glu Lys Arg Cys Glu Arg Arg Leu Gln Asp
210 215 220
36
