CA2256123A1 - A novel hypertension related calcium regulated gene (hcarg) - Google Patents

Abstract

This invention relates to a novel gene that shows tissue specific expression and increased expression in a low calcium concentration medium. Low renin hypertension is characterized by decreased levels of serum ionized calcium in the presence of increased levels of parathyroid hormone. It is hypothesized that hypertensive factor(s) are co-secreted with PTH in SHR, a model of low renin hypertension, the parathyroid hypertensive factor being one of them. This invention identifies the genes differentially expressed in humans and in rat models of low renin hypertension (SHR) and in normotensive rat models (WKY) that are induced by a low calcium levels in the parathyroid gland. A cDNA of a human gene and a cDNA of a rat gene expressed in parathyroid cells have been cloned and sequenced with no high score homology found in the gene banks. Their expression is higher in hypertensive models and their expression is enhanced by cell incubation in a low calcium concentration medium. In situ hybridization showed specific expression of the genes in adrenal medula and cortex, proximal tubules and parathyroid gland with very low levels in heart ventricles, kidney medulla and glomeruli and liver. These novel calcium regulated genes show tissue specific expression and increased expression in hypertension.

Description

' . ' WO 97/49807 PCT/CA971~00439 A Novel Hypertension Related Calcium Regulated Gene (HCaRG) Field of the Invention The present invention relates to a novel gene that shows tissue specific expression and increased expression in a low calcium concentration medium and in hypertensive animals.
Back4round of the Invention Calcium ion is an essential element of life with distinct extracellular and intracellular roles. Extraceilular functions of calcium include its role in blood clotting, intercellular adhesion, bone metabolism, maintenance of plasma membrane integrity whereas its intracellular roles include protein secretion, cellular contraction and division. The free extracellular calcium concentration is maintained within a narrow range (-- 1 to 1.3mM) and that of intracellular calcium is in the order of 1 OOnM: 10,000 fold lower than the extracellular free calcium concentration.
The first priority of the extracellular calcium homeostatic system is to maintain a normal extracellular ionized calcium concentration. This component represents approximately 45% of the total circulating calcium concentration.
Another 45% of total circulating calcium is bound to proteins (primarily albumin) and about 10% is bound to small organic anion. A major rote has been attributed to catctotropic hormones parathromone (PTH), 1,25 dihydroxyvitamin D and calcitonin in maintaining extracellular calcium homeostasis but the extracellular ca~ium regulates its own concentration by acting on cells involved in the control of extracellular calcium homeostasis such as parathyroid, bone, intestine and kidney cells (Brown, E. M., 1994). For example, parathyroid cells are key sensors of extracellular calcium in vertebrates responding with increases in PTH
secretion when there is a decrease in catcemia white high calcemia stimulates hormonal release of calcitonin from C cells of the thyroid gland. Even slight reductions in the extracellular ionized calcium concentration (in the order of 1-2% or Less) elicit prompt increases in the rate of PTN secretion. Renal responses to the increase in _1_ circulating levels of PTH relevant to mineral ion metabolism include phosphaturia and enhanced distal tubular reabsorption of calcium. The most rapid changes in calcium handling by the target tissues of PTH take place in the kidneys and skeleton.
The parathyroid gland is particularly well positioned to respond to hypocalcemic stresses. The parathyroid cells (and probably few other cell types) are capable of sensing the changes in the extraceilular calcium concentration.
The process of calcium sensing (that is a capacity to recognize and respond to physiologically meaningful changes in extracellular calcium), differs from simple calcium dependence. A parathyroid calcium receptor has been recently characterized. It is present on the cell surface and interacts not only with calcium but also with a variety of other divalent rations as well as with poiycations.
The receptor has probably at least two binding sites that confer positive cooperativity to it. The putative calcium receptor is linked to several intracellular second messenger systems via guanyiyi nucleotide regulatory G proteins and activate a phosphoinositide specfic phosphoiipase C leading to accumulation of inositol 1,4,5 trisphosphate (IP3) and diacyiglycerol. Such a receptor is also found in the proximal tubular cells consistent with a regulation of tubular function through a mechanism similar to that in parathyroid cells. Hypocalcemia promotes parathyroid cellular hypertrophy and increases levels of the mRNA for PTH.
1,25 dihydroxyvitamin D has a clear inhibitory effects on parathyroid cellular proliferation.
Historically, the study of the parathyroid gland has been focused on the chemistry, regulation, synthesis and secretion of parathyroid hormone, PTH.
Recently, there has been a growing interest in other calcium-regulated proteins of the gland such as chromogranin A or Secretory Protein I (SP-I). Chromogranin A
is also found in the endocrine and neuroendocrine systems (Cohn, D. et ai., 1994).
An hypertensive factor of parathyroid origin has been recently documented with simiiaritices to an intracellular calmodulin-PDE activator, described in hypertensive tissues and organs (Pang et al, 1989; Huang et al., 1988). This factor increases blood pressure when injected into anesthetized rats and has been shown to ' WO 97/49807 PCT/CA97/00439 potentiate the action of pressor agents (norepinephrine) on the contraction of vascular smooth muscle (Lewanczuk et al.. 1989).
Diseases associated with hypertension include arteriosclerosis, hypertensive renal failure, stroke, heart failure and myocardial infarction, to name a few. llVhile methods to treat hypertension are available, the etiology of hypertension, for the most part, remains unknown.
A number of persons have attempted to purify the active component of parathyroid hypertensive factor in an attempt to improve methods of treating patients with diseases which involve extracellular calcium elevation, such as hypertension. In one patent application, PCT 93US5626, the inventors describe a purified and isolated parathyroid hypertensive factor component including a polypeptide linked to a phospholipid. This component produces a delayed onset of an increase in blood pressure of a normotensive rat to which it is administered.
The increase in blood pressure is said to temporarily correlate with an increase in extracellular calcium uptake by vascular smooth muscle. However, this factor, when highly purified, is not greatly increased in hypertensive states.
Similarly, other hypertensive factors derived from parathyroid gland are described in other patent applications, such as Japanese patent application 4134098 and PCT 90US1577. The factors are obtained by culturing, dialysing, ultrafiltering, refrigerating drying plasma component and separating the active fraction by gel filtration column chromatography. Again, these factors are not greatly increased in hypertensive states.
Despite the work that has been done in the area of hypertensive factors, a need still exists to identify a mammalian gene which is increased in hypertensive states. This gene could be used (1 ) to treat diseases related to modulation in calcium levels, (2) to screen pharmaceutical components which are effective in treating diseases related to modulation in calcium levels, or (3) for the diagnosis of the presence of diseases related to modulation in calcium levels. Diseases related to modulation in calcium levels include hypertension, hyperthyroidism, osteoporosis, osteopetrosis, heart failure, insulin dependent and independent diabetes, cancer (including breast, thyroid, colon, kidney and leukaemia), WO 97/49807 PCTlCA97I00439 disorders of the central nervous system including stroke, artheroscierosis, gastrointestinal diseases. inflammatory bowel disease and asthma.
Summa,rr of the Invention The present inventors have recently identified a new gene expressed in the parathyroid gland. The expression of this gene is regulated in a way similar to that of PTH that is: hypocalcemia increases its mRNA levels. E~cperiments involved spontaneously hypertensive rats (SHR), models of low renin hypertension, and nomnotensive counterparts Wistar-Kyoto (WKY). The expression of this novel gene was higher in the SHR parathyroid cells than in cells from WKY. In situ hybridization studies showed that this gene has a specific pattern of expression. ft is highly expressed in the tubular fraction of the renal cortex, in the medulla and the inner part of the adrenal cortex, in the intestine and in the brain with low levels of expression in the heart. the liver and the lung.
Therefore, the present invention relates to a nucleic acid molecule isolated from parathyroid of a mammal and whose expression is regulated by extraceilular calcium concentration. In one case, the mammal is a human and the molecule has the sequence set out in Figure 4. In another case, the mammal is a rat and the molecule has the sequence set out in Figure 3. The invention includes a nucleotide molecule of a human, and having a homology of 60% or greater to all or part of the sequence set out in Figure 4. The molecule may have a 60% or greater homology to the translated portion of the sequence.
The invention also includes a purified and isolated protein encoded by the nucleic acid molecule of this invention. Mimetics of and antibodies to this protein are included within this invention as are proteins having a homology of 60% or greater to the proteins encoded by the nucleic acid molecules of this invention.
A pharmaceutical composi~on of this invention would include at least a portion of the protein encoded by the nucleic acid molecules of this invention or would include a mimetic of such a protein. The portion of the protein or the mimetic could include at least the portion of the protein or mimetic responsible for biological activity. The composition would also include a pharmaceutically acceptable carrier, auxiliary or excipient.

WO 97l4981Y1 PCTlCA97/0043~
In the aftemative, a pharmaceutical composition could include a nucleic acid molecule of this invention, or a portion thereof, for use in gene therapy. The composition could be used to treat a patient suffering from a condition caused by the abnormal intracellular or extracellular modulation of calcium comprising administering an effective amount of the sense molecule of Figure 4, for example, (to upreguiate the molecule's expression) or the antisense molecule of Figure 4, for example, (to downregulate the molecule's expression) to the patient.
The molecule could be delivered as part of a recombinant vehicle, or in liposomes, for example. In one case, the molecule would include a sense or an antisense sequence to ail or part of the nucleic acid sequence of a human gene sequence as set out in Figure 4. The sense sequence would enhance the effect of the sequence set out in Figure 4. The antisense sequence would suppress the effect of the sequence set out in Figure 4.
InGuded within this invention is a kit for the detection of a disease, disorder or abnormal physical state caused by abnormal modulation of calcium levels in a patient. The kit could include, as a target or as a marker, all or part of the nucleic aad molecule of this invention, for example, the sequence of a human gene set out in Figure 4. In another case, a kit could include as a marker or as a target all or part of a protein encoded by a nucleic acid molecule of this invention, a mimetic of such a protein or an antibody to such a protein. The kit could be used to help diagnose hypertension, hyperthyroidism, osteoporosis, heart failure, insulin dependent and independent diabetes, disorders of the central nervous system including stroke, cancer. (including breast, thyroid, colon, kidney and leukaemia), artherosclerosis, gastrointestinal diseases, inflammatory bowel disease and asthma. Once diagnosed, patients may wish to regulate extracellular calcium uptake by increasing dietary calcium levels or taking calcium supplements.
Also included within this invention is the use of the pharmaceutical compositions of this invention to treat a patient having a disease, disorder or abnormal physical state related to abnormal intracellular or extracellular calcium levels. Also included is the use of the protein of this invention or the mimetics of such protein to screen for inhibitors to such protein.

WO 97/49807 PCTlCA971a0439 A method for assaying for abnormal intracellular or extracellular calcium levels would include (a) reacting a sample of a patient with a nucleic acid molecule of this invention, or a portion thereof, under conditions where the sample and the molecule, or a portion thereof, are capable of forming a complex; (b) assaying for complexes, free molecule, or a portion thereof; and (c) comparing with a control.
In one case, the molecule is a sense or an antisense sequence to all or part of the human gene sequence as set out in Figure 4.
In another assay for abnormal intracellular or extracellular calcium levels, the assay includes (a) reacting a sample of a patient with a protein of this invention, or a portion or a mimetic thereof, or an antibody thereto, under conditions where the sample and the protein, or a portion or a mimetic thereof, or an antibody thereto, are capable of forming a complex; (b) assaying for complexes, free protein, or a portion or a mimetic thereof; or an antibody thereto, and (c) comparing with a control.
In yet another assay for screening for efficacy of products modulating (enhancing or inhibiting) abnormal calcium levels, the assay includes (a) reacting a sample of a patient with a protein of this invention, or a portion or a mimetic thereof, or an antibody thereto, under conditions where the sample and the protein, or a port'on or a mimetic thereof, or an antibody thereto, are capable of forming a complex; (b) assaying for complexes, free protein, or a portion or a mimetic thereof; or an antibody thereto, and (c) comparing with a control.
Furthermore, another assay for screening for efficacy of a product for modulating (enhancing or inhibiting) abnormal intracellular or extracellular calcium levels could include (a) reacting the product with a protein of this invention, or a portion or a mimetic thereof, or an antibody thereto, under conditions where the product and the protein, or a portion or a mimetic thereof, or an antibody thereto, are capable of forming a complex; (b) assaying for complexes, free protein, or a portion or a mimetic thereof; or an antibody thereto, and (c) comparing with a control.
This invention includes a method for screening for efficacy of a product for use in modulating (enhancing or inhibiting) abnormal intracellular or extracellular _ ~ WO 97149807 PCTICA97N043°
calcium levels, the assay includes (a) reacting the product with a nucleic acid molecule of this invention. or a portion thereof, under conditions where the product and the molecule, or a portion thereof, are capable of forming a complex; (b) assaying for complexes, free molecule, or a portion thereof; and (c) comparing with a control.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of example only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
Descrj~tion of the Figures The invention will now be described in relation to the figures in which:
Figure 1: This figure describes the reconstitution of HCaRG rat cDNA including the putative full length open reading frame. The 1130 by cDNA
sequence was initially obtained using 5'-RACE and 3'-RACE strategy as welt as by screening a SHR parathyroid cDNA library. Black boxes represent overlapping sequences. The first cDNA fragment was obtained by 3'-RACE (3r290). This fragment was used to screen the SHR parathyroid cDNA library. Fragments (hcarg 2c-t3 + 2c-t7), hcarg 825, hcarg 10-ic, hcarg 10-174 were cloned from the cDNA
library. Fragments 5r 285 and 5r 260 were obtained by 5'-RACE.
This reconstitution was confirmed by sequenang a 860 by PCR
product obtained with nested primers of 5r 260 and hcarg 825.
Figure 2: Detection of the mRNA levels of the novel calcium-regulated gene in parathyroid cells from SHR and in aortic smooth muscle cells from 8N
and SHR incubated 48 hours in low (0.3 mM) or normal (2 mM) extraceilular calcium condition. Upper: Ethidium bromide gel.
Middle: HCaRG expression. Lower: Hybridization with '2P-labeled 18 S rRNA probe.

Figure 3: Rat cDNA sequence of HCaRG. The translation initiation start site and the termination codon are indicated in bold. The coded amino acids are indicated in italic.
Figure 4: Comparison of HCaRG nucleic acid sequences between rat and human. Alignment of rat cDNA and human DNA sequences of HCaRG. A 75% homology was calculated. Both the initiating codon and the EF-hand like motifs are conserved. No intron is found in this region. A one base pair insertion in the human sequence at position 46 could cause a frameshift in the coding region of the human HCaRG protein.
Figure 5: Detection of HCaRG expressions in tissues of WKY and SHR rats by in situ hybridization. Higher signal was found in the SHR than in the WKY parathyroid gland with the antisense labelled probe. Only low signal could be detected with the non sense probe. Figure 5A - SHR
Parathyroid Antisense (X400);SHR Parathyroid Sense (X400); Figure 58 - WKY Parathyroid Antisense (X400);WKY Parathyroid Sense (X400).
Figure 6: HCaRG was expressed in the medulla and zona fasciculate of the adrenal gland with higher signal in the gland from SHR rat. Figure 6A
- SHR Adrenal Antisense (X32~SHR Adrenal Sense (X32); Figure 6B
- SHR Adrenal Antisense (X125);SHR Adrenal Sense (X125); Figure 6C - WKY Adrenal Antisense (X125);WKY Adrenal Sense (X125).
Figure 7: The expression of HCaRG in the kidneys is concentrated in the tubular fraction with no signal in the giomenrli. Figure 7 - SHR Kidney Cortex Antisense (X125);SHR Kidney Cortex Sense (X125).
Figure 8: Localization of HCaRG on rat chromosome 7. A HCaRG Bgl ll polymorphism was used as marker on genomic DNA from SHR and BN.1 x rat inbred strains with mu~iple well characterized SDPs.
HCaRG cosegregated with D7Cebrp187s31D7Cebr77s1 of rat chromosome 7 in 31 out of 33 strains. Two recombinations mapped the HCaRG between Cyp11 biz and Myc genes. cM represents ~WO 97149807 PCT/CA97I00439 distance in centimorgans on rat chromosome 7. On the right side, a possible linkage position of human homologous gene is depicted as based on conserved linkages on rat chromosome 7 and human chromosome 8.
Detailed Description of the Invention The inventors have cloned and sequenced a novel hypertension related calcium-regulated gene (HCaRG) that shows a tissue specific pattern of expression with higher levels in the genetic rat model of hypertension. The expression of this gene is regulated by extracellular calcium concentration in glands possessing a calcium sensor such as the parathyroid gland.
Included within this invention are nucleic acid sequences having 60% or greater homology to ail or part of the sequence of the gene for HCaRG of the rat as shown in Figure 3. Furthermore, this invention includes nucleic acid sequences having 60% or greater homology to all or part of the translated portion of the gene for HCaRG of the rat. ?his would include nucleic acid sequences whose codon usage has been mod~ed to suit a particular host. Sense, antisense and mRNA
sequences are encompassed by the term "nucleic acid sequences".
Also included within this invention are nucleic acid sequences having 60%
or greater homology to all or part of the sequence of the gene for HCaRG of the human as shown in Figure 4. Although Figure 4 shows the partial gene sequence for HCaRG of the human, those skilled in the art would appreciate how to sequence the entire gene using conventional methods. Furthermore, this invention includes nucleic acid sequences having 60% or greater homology to ail or part of the translated portion of the gene for HCaRG of the human. This would include nucleic acid sequences whose codon usage has been modified to suit a particular host. Again, sense, antisense and mRNA sequences are encompassed by the tens "nucleic acid sequences".
Furthermore, proteins encoded by all or part of the nucleic acid sequences of the gene for HCaRG of the rat and of the human are within this invention.
One protein would include the amino acid sequence for the HCaRG protein of the rat as shown in Figure 4 (at the top of each line of Figure 4). Another protein would _g.

WO 9'1149807 PCTlCA97100439 include the amino acid sequence for the HCaRG protein of the human as shown in Figure 4 (at the bottom of each line of Figure 4). Again, proteins having 60%
or greater homology to all or part of these proteins are within this invention.
It will be appreciated that a protein encoded by the genes of this invention may be modified by substituting amino acids for tike amino acids. For example, a basic amino acid may be substituted with a difFerent basic or non-basic amino acid. The substitutions would be chosen so as not alter the properties of the protein encoded by the genes of this invention.
Mimetics of the protein may also be used in the methods and compositions of the invention. The term "mimetic" refers to compounds which have a related three dimensional structure i.e. compounds which have the characteristic structure of the protein encoded by the DNA sequences of this invention. Mimetics may be based on the biologically active portion of the proteins of this invention and may try to mimic the three dimensional structure of that active portion.
There is abnormal calcium transport, concentration and binding in patients with hypertension inGuding calcium leak in cortical tubules. This invention provides additional solutions for patients having hypertension and other diseases caused by abnormal calcium levels.
In addition to hypertension, abnormal modulation of calcium levels can lead to a number of other diseases, disorders or abnormal physical states including hyperthyroidism, osteoporosis, osteopetrosis, heart failure, insulin dependent and independent diabetes, disorders of the central nervous system including stroke, cancer {including breast, thyroid, colon, kidney and leukemia), arteriosclerosis, gastrointestinal diseases, inflammatory bowel disease and asthma. The nucleic acid sequence of this invention could be used (1 ) for the treatment of diseases related to the modulation in calcium levels, (2) to develop pharmaceutical compositions for the treatment of diseases related to the modulation in calcium levels, or (3) to diagnose diseases related to the modulation in calcium levels. As certain types of cancer are characterized by an increase in intracellular free calcium, the nucleic acid sequence could be used to generate immunological WO 97/49807 PGT/CA97100d3'' assays (or markers) for these types of cancers and to develop pharmaceutical compositions to treat these types of cancers.
Similarly, ail or part of the proteins encoded by the nucleic acid sequences of this invention or antibodies to the proteins could be used to generate immunoiogical assays (or markers) to test for diseases, disorders or abnormal physical states associated with abnormal modulation of calcium levels. The assays could be screening assays to determine whether a product enhances or inhibits calcium levels or whether a product has had its intended effect in enhancing or inhibiting calcium levels.
In the assays of this invention, the complexes may be isolated by conventional methods known to those skilled in the art, such as isolation techniques, for example, chromatography, electrophoresis, gel filtration, fractionation, absorption, poiyacrylamide gel electrophoresis, or combinations thereof. The complexes or free protein or mimetics may be assayed using known methods. To facilitate the assay, antibody against the protein or mimetic may be labelled or a labelled compound may be used. Detectable markers or labels which would serve to identify the complexes could include fluorescein, HRP and biotin.
The invention also relates to pharmaceutical compositions to treat patients having abnormal modulation of calcium levels. The compositions could include {1) nucleic acid sequence for use in gene therapy in which the sense sequence of the HCaRG gene is used in liposomes or a recombinant vehicle, for example, to enhance the gene. (2) nuGeic acid sequence for use in gene therapy in which the antisense sequence of the HCaRG gene is used in liposomes or a recombinant vehicle, for example, to suppress the gene, (3) a protein or mimetic which competes with the protein encoded by the nucleic acid sequences of this invention thus suppressing the native protein's effect. (4) a protein encoded by the nucleic acid sequence of this invention to enhance the native protein's effect. The composition could include an acceptable carrier, auxiliary or excipient.
The pharmaceutical compositions may be used as an agonist or antagonist of the interaction of a protein encoded by HCaRG and a receptor. The compositions can be for oral, topical, rectal, parenteral, local, inhalant or _11_ VldO 97/49807 PCTlCA97I00439 intracerebral use. There may be in solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules.
gels, membranes, tubelets. The compositions of the invention may also be conjugated to transport molecules to facilitate transport of the molecules.
The pharmaceutical composition can be administered to humans or animals. Dosages to be administered depend on patient needs, on the desired effect and on the chosen route of administration.
The pharmaceutical compositions can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.
Suitable vehicles are described, for example in Remington's Pharmaceutical Silences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton. Pa., USA 1985).
On this basis, the pharmaceutical composfions include the active compound or substance in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids. The methods of binding the compound to the vehicles or combining them with diluents is well known to those skilled in the art. The composi~on could include a targeting agent for the transport of the active compound to speilfied sites within cells, tissues or organs.
Compounds could be targeted to cells such as vascular smooth muscle, renal or cardiac cells, for example.
The invention also relates to a composfion for use in gene therapy.
Liposomes or a recombinant molecule, for example could contain a sense or antisense sequence of the nucleic add molecule of this invention. In the case of a recombinant molecule, the molecule would contain suitable transcriptional or translational regulatory elements.
Suitable regulatory elements may be derived from a variety of sources, and they may be readily selected by one or ordinary skill in the art. If one were to upregulate the expression of the gene, one would insert the sense sequence and i~VO 97!49807 PCTlCA9710043~
the appropriate promoter into the vehicle. If one were to downregulate the expression of the gene, one would insert the antisense sequence and the appropriate promoter into the vehicle. These techniques are known to those skilled in the art.
Examples of regulatory elements include: a transcriptional promoter and enhancer or RNA poiymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the vector employed, other genetic elements, such as selectable markers, may be incorporated into the recombinant molecule. The recombinant molecule may be introduced into cells of a patient using in vitro delivery vehicles such as retroviral vectors, adenovirai vectors. DNA vinrs vectors and liposomes. They may also be introduced into such cells in vivo using physical techniques such as microinjection and electroporation or chemical methods such as coprecipitation and incorporation of DNA into liposomes. The compositions may also be delivered in the fom~ of an aerosol or by lavage.
The present invention also provides for methods in which a patient suffering from a condition requiring modulation of calcium levels is treated with an effective amount of a composition.
Examale 1 - Isolation of cDNA fragments From sense primers and hybrid primer 3'-RACE experiments generated a 700bp fragment that was digested and cloned in the Bam H1 site of pSP72. A
recombinant plasmid containing a 300bp insert was isolated. This insert was sequenced and no highly homologous sequence was found in Genbank. This sequence was used subsequently for Northern blots, in situ hybridization, to screen libraries and to generate new oligonucleotide primers to extend the cDNA
towards the 5'- and 3'-ends. In the following 3'-RACE experiments, cDNA
fragments were cloned in a blunt Smal site of pSP72. From several overlapping DNA fragments isolated from above experiments, a 1140bp cDNA was reconstituted (Fig. 1 ).

~wo 9ma9so~ pcrcAS~,oo~
Example 2 - Calcium rec ul~ ation Parathyroid cells from spontaneously hypertensive rats (SHR) and aortic smooth muscle cells from normotensive Brown Norway (BN) rats or SHR were cultured in normal or low calcium medium. Total RNA was extracted and separated on a denaturing formamide agarose gel. After transfer, an RNA
transcript of about 1.4kb was detected in each lane (Fig. 2). The mRNA levels were found about 40 to 60% higher in cells incubated in low calcium medium for SHR. Because this new mRNA appeared regulated by extracellular calcium in the absence of a yet known activity, but expressed more in cells of hypertensive animals, we have named this gene: an hypertension gene-related calcium-regulated gene (HCaRG).
Example 3 - Seouence of the novel calcium-reQUlated cDNA from rat The rat 1140bp cDNA reconstituted sequence contained an open reading frame of 224 codons preceded by 2 in frame stop codons (Fig. 3). The cDNA
codes for a 224 amino-acid protein with a deduced molecular weight of 22456 Da.
The protein has several putative phosphorylation sites and one possible Asn-glycosylation site and 67% of alpha-helix content: A homology to the calcium binding EF-hand motif was found in this sequence. A low homology (12%) to Pseudomonas syringae omega-3 fatty acid desaturase was also found.
The 5'-end part of the rat HCaRG has homology to a cDNA extracted from a 73 days post-natal female (Genbank access #R20463). This cDNA was selected randomly by its discoverers and the coding sequence is incomplete. No function was attributed to this cDNA. A second region of homology between rat HCaRG at the 3'-end was found with the complementary sequence of a cDNA randomly picked from a cDNA library derived from 4 human multiple sclerosis lesions (Genbank access #N49076). Again, the cDNA coding sequence is incomplete and no function was attributed to the cDNA. Both are available royalty-free.
Example 4 - The human cDNA seauence Rat primers, derived from rat cDNA sequence of the HCaRG, were used for PCR amplfication on human genomic DNA and human parathyroid cDNAs. Some ' WO 97/49807 PGTICA97/0043' primer pairs were able to generate fragments that were cloned and sequenced. A
483bp cDNA fragment was found to be almost completely homologous to the rat sequence (Fig.4). Both the initiation codon and the EF-hand like motif are conserved.
Example 5 - In situ hybridization The expression of HCaRG was determined in WKY and SHR tissues by in situ hybridization. Labeled antisense probe hybridized to parathyroid glands of both WKY and SHR with a higher signal in the SHR gland (Figure 5). Expression was also detected in the medulla and zona fasciculate of the adrenal cortex (Figure 6)-. In the kidney, the labeling was almost exclusively in the renal cortex concentrated in tubular part contrasting with the absence of signal of the giomeruii (Figure 7). In all these organs, the signal was higher in the hypertensive animal.
Organs which showed only a very low level of expression were the liver and the heart ventricles. The sense probe was used as a negative control and showed very low signal in our conditions of hybridization demonstrating the specficity of the reaction.
Examale 6 - TPA. a Protein Kinase C AQOnist. Increases mRNA Levels We have initiated studies on the regulation of HCaRG. Hormonal signal transduction pathways are stimulated by different agonists in cultural parathyroid cells incubated in low or normal calcium medium. Our initiaai studies showed that TPA, a protein kinase C agonist, (Protein kinase C is the main target on intracellular calcium and is involved in the phosphoryiation regulation of many target proteins including, ionic channel, contractile proteins and hormonal receptors) increases the mRNA levels of HCaRG when cells are incubated in normal calcium medium. These data suggest that Protein kinase C could mediate, inside of the cell, these effects of extracellular caidum. Interestingly, the calcium sensor is linked to the protein kinase C pathway. Other hormonal systems are tested for their effects on HCaRG expression. These include glucocorticoids.
catecholamines, Vitamin D, parathormone, growth factors, cytokines. These tests define the mechanisms controlling HCaRG synthesis and delineate their anomalies in disease states.

' WO 97/49807 PCT/CA97I00439 Example 7 - Chromosomal localization With the obtention of the cDNA coding for the HCaRG from human and rat and the putative full length open reading frame, our research includes genomic structure, search of genetic control elements. Our research relates to the pathophysiological regulation of its expression and to in vitro expression of a functional protein.
Southern blot analysis was performed on l0pg genomic DNA of SHR and BN.Ix rats with the following restriction enzymes BamH 1, Bgl II, EcoR f, Hind III, Kpn I and Pst I. The probe consisted of the '2P-labeled fragment of 860bp of HCaRG shown in Fig. 1. A clear RFLP genotyping for the B BN.Ix allele (12 kb) or S (SHR) allele (2.2 kb) was then detected with the Bgl II restriction enzyme (Fig.
8) in the 33 recombinant inbred strains. The strain distribution pattern of this RFLP was then analyzed by Pearson's correlation for segregation with 500 markers localized in the rat genetic map using the Map Manager program of Manly (version 2.6.5). The address of RATMAP is "http://www.ratrnap.gen.gu.se".
Example 8 - Pathoohvsiological regulation of expression of HCaRG
Northern blot and in situ hybridization experiments have shown that rat tissues which demonstrate a significant expression of HCaRG are the parathyroid gland, the medulla and inner cortical section of the adrenal gland, the cortical tubular segments of the kidney and the brain cortex and medulla. In most organs.
the expression was higher in SHR than in normotensive rat. The effect of dietary sodium and calcium is tested on HCaRG expression in these organs in salt sensitive and salt-resistant hypertensive rat strains with a protocol previously described in Chang et al, (Chang et al. 1990) and Tremblay et al. (Tremblay et al.
1991 ). These earlier reports have shown an increased in CPA activity by high sodium intake and normalization by high dietary calcium suggesting that this factor could be a biological marker of salt sensitivity in the population. We have recently detected the expression of HCaRG in human lymphocytes. This is a readily available source of human RNA and we have developed a semi-quantitative RT-PCR assay to quantify the mRNA levels of HCaRG in humans. Human samples are obtained from controls and patients with abnormal calcium metabolism such 'WO 97/d9807 PC'f/CA9710043~
as patients with cardiovascular diseases, osteoporosis, atheroscierosis and cancer. In addition, biopsies of cancer tissues are obtained. We have already detected the mRNA of HCaRG in colon cancer as well as in breast cancer. These studies use HCaRG as a biological marker of abnormal calcium metabolism in humans.
Example 9 - HCaRG expression in bacteria and antibody oreaaration Rat and human HCaRG are inserted into bacterial expression vectors in order to produce large amounts of HCaRG protein. For HCaRG, we use the pMAL-c2 (New England Biolabs) to generate a fusion protein of HCaRG following the maltose-binding protein. A blunt HCaRG cDNA obtained by PCR and starting at the initiator methionine is inserted in the Xmni site of pMAL-c2. This strategy places the HCaRG product next to the Factor Xa cleavage site of the fusion protein.
Because protein expression in E.coli varies according to the vector used and the nature of the protein expressed, we prepare other fusion proteins. The pGF,C-5X plasmid (Pharmacia) allows for the introduction of genes to produce giutathion S-transferase fusion proteins. The vector exists in thn~e frames and has extensive restriction insertion sites for easy insertion of foreign gene. For example, the cloned rat HCaRG is inserted in the EcoRl-Xhol sites to produce the fusion protein with HCaRG product localized after a Factor Xa cleavage site.
In both systems, the fusion proteins enable the rapid purification of the expressed protein through affinity chromatography. Crude bacterial extracts containing cytopiasmic proteins are analysed. According to the amount of protein syr>thesized, purfication steps are determined or crude extract is used directly. To generate antibodies by injection into rabbits, urea extracted aggregates, SDS-page purfied bands or protein extracts are used (Sambrook et al. 1989).
Exam~~le 10 - HCaRG exaression in mammalian cells Because bacteria are unable to post-translationally modify proteins as mammalian cells, a bacterial protein may be inactive. We express the HCaRG
protein in mammalian cells to circumvent this problem. Gene transfer techniques to COS7 cells are used routinely in the lab. The expression vector is pcDNAneol _17_ WO 9'f149~07 PCTICA97/00439 (Invitrogen) available in the lab. The cloned HCaRG is inserted as a Hind III-Bgl II
fragment in Hind III-BamHl sites in the vector to place the gene under the CMV
promoter. A plasmidic neo gene enables the selection of stable transformants.
High expression is selected by Northern blots and protein is purified when antibodies are available. Various biological activities however, are tested immediately on cells expressing HCaRG. The initial candidate activities are calcium channel function calmodulin-phosphodiesterase activator activity, cell proliferation, cell death and apoptosis.
Example 11 - Gene Therapy - The intracellular function of a protein can be also studied by inhibition of its expression by antisense molecules Recently, antisense oligonucleotides have been used extensively to inhibit expression of specfic genes (Stein et al. 1988a). Although, the exact mechanism of this inhibition is not known, evidence suggest that Rnase H-like activity degrades RNA oligonudeotide duplexes (Dash et al. 1989). While modified oiigonucieotides such as methylphosphonates diffuse freely across the cell membrane, unmodified and modfied oligonudeotides have been shown to be actively transported into living cells by binding to membrane receptors (Loke et al.
' 1989; Stein et al. 1988b). It is therefore possible to inhibit the expression of specific genes and their gene products by adding specfic antisense molecules to the culture medium. We explore the capacity of the oiigonudeotide antisense spanning the translation initiation site of HCaRG to inhibit PHF as welt as CPA
synthesis. Parathyroid cells or other cells expressing HCaRG, PHF or CPA are treated with antisense oligonucleotides. Cells are incubated in medium containing up to 100 uM antisense oligonucleotide. Lipofection helps to increase the percer>tage of uptake of oligonucleotides in certain cells. Fresh antisense molecules are added every 24 hrs. After 24 to 48 hrs cell culture medium is tested for the presence of PHF activity and intracellular CPA activity is assessed.
Non-sense and sense oiigonucleotides are used as control for determination of specifidty of the effect. Other parameters such as cyclic nucleotides and intracellular calcium levels are also measured since they may constitute an additional step to define the mechanism of action of HCaRG.
_18_ WO 97/49807 PGT/CA971~00439 METHODOLOGY
Cell culture Parathyroid cells (PTC) were isolated from SHR or WKY. Primary cultures were passaged in DMEM medium with 10% fetal calf serum. 48 hours before further studies, cells were maintained in F12 medium which contained low Ca concentration (0.5mM) or DMEM medium which contained normal Ca concentration (1.5mM).
RNA preparation Total RNA was prepared from PTC using standard guanidinium thiocyanate -phenol-chloroform method.
Rapid amplification of cDNA ends (RACE7.
From the putative amino acid sequence of parathyroid hypertensive factor, four mixtures of degenerate oligonucleotides were synthesized. A total of 144 different oligonudeotides were generated. From preliminary experiments of RACE
(described below) one mixture was selected. Four unique nucleotide sequences were selected as candidate primers from this mix, synthesized and used subsequently for 3'-RACE. In brief, for the 3'-RACE, total RNA from parathyroid cells was reverse-transcribed using a hybrid primer consisting of oligo(dT) (17 mer) extended by a unique 17-base oligonudeotide (adaptor). Amplification by potymerase chain reaction (PCR) was subsequent>)r performed using the adaptor primer, which bound to cDNA at its 3'-ends, and candidate primers mentioned above. For the 5'-RACE, reverse transcription was performed using an intemai primer derived from the sequence of the cDNA fragment generated by 3'-RACE.
A dA homopolymer was then appended to tail the first strand reaction products by using terminal transferase. Finally, PCR amplification was accomplished using the hybrid primer previously described and a second intemai primer upstream of the first one.
SHR parathyroid cDNA library Parathyroid glands were-removed from approximately 100 12 week old SHR rats and immediately frozen in liquid nitrogen. Glands were then added to a -1 &

WO 97/49807 PCT/CA97/~00439 guanidium thiocyanate solution and homogenized in this solution. mRNA was obtained by phenol-chloroform extraction, ethanol precipitation and separation on an oligo(dT) column. mRNA was stored in ethanol at -80°C until used.
The cDNA library was constructed using the above mRNA as a template and the ZAP-cDNA' synthesis kit (Stratagene; La Jolla, CA). A summary of the protocol is as follows: mRNA was reverse transcribed using Moloney-Murine leukemia virus reverse transcriptase. Second strand synthesis was then carried out using DNA poiymerase in the presence of RNAse H. The cDNA was then extracted using phenol: chloroform, precipitated with sodium acetate, washed with 80% ethanol and resuspended in sterile water. Following this step the cDNA
termini were blunted by incubation with Klenow fragment and dNTPs in a ligase buffer. cDNA was again precipitated and washed. Following this wash, EcoRl adaptors were ligated on using T4 ligase and the ends kinased using T4 poiynucieotide kinase. This mixture was then digested with Xho 1 to release adaptors and residual linker-primer from the 3' end of the cDNA. The resultant mixture was then separated on a Sephacryl S-400 column. Eluted cDNA was precipitated with 100% cold ethanol and resuspended in sterile water.
Following the above process, cDNA was ligated into the Uni-XAP XR vector using T4 DNA ligase, thus forming the cDNA library. The resultant library was packaged into Gigapack 11 Gold packaging extract. The packaged product was plated onto XLI-Blue MRF' cells and recombinant numbers determined. The library was then amplified by mixing the packaging mixture with host bacteria (XU-Blue MRF' cells). The library was stored at -80°C until screened.
For screening, phage were plated onto bacterial host plates (Xt_I-Blue MRF7 and incubated overnight. After chilling at 4°C for 2 hours a nitrocellulose fitter was overlaid for 2 minutes. The ftlter was then denatured in 1.5M
NaCU0.5M
NaOH and neutralized in 1.5M NaCI with 0.5M Tris-CI (pH 8.0). The fitter was then rinsed and the DNA crosslinked to the filter with UV light. Filters were then pre-hybridized and hybridized using standard techniques with a digoxigenin-dUTP
labeled probe.

wo 9»49so~ rcr~ca9»ooa.'.
Northern blot hybridization:
lONg of total RNA from parathyroid cells or tissues was denatured at 68°C
and separated on a denaturing formamide -1 % agarose gel. The gel was transferred onto nitrocellulose by vacuum transfer with 20XSSC. The membrane was exposed to UV light to fix RNA, and pre-hybridized in a solution containing SSPE. SDS, denhardt's and dextran sulfate for at least 4 hours. Hybridization was performed overnight in the same buffer containing 32P labeled probes generated from cDNA clones) by PCR or random labeling method.
Subcloning PCR fragments from RACE experiments were isolated from agarose gel and extracted by the phenol-chloroform method. pSP72 piasmid, used as vector was digested at the Smal site and blunted PCR fragment were inserted using T4 DNA ligase. Transformed OHSa bacteria were grown and colonies were selected by PCR.
Sequencing Double-stranded sequencing of the Boned cDNA inserts was carried out using Sequenase Version 2.0 (USB). 5Ng of template was denatured, annealed with T7 or SP6 primers, labelled with S35-1 dATP by extension using the chain termination method of Sanger.
In situ hybridization Tissues from SHR and WKY rats were rinsed in phosphate buffer, fixed in 4% paraformaidehyde and imbedded in paraffin. 3-5 um sections were cut and mounted on microscope slides pretreated with aminopropylthiethoxysiiane.
Slides were dried at 37°C then at 60°C for 10 minutes prior to use.
The probe was 3'-RACE 300bp fragment which had been subcloned into the BAM H1 site of a pSP72 vector. Briefly, the plasmid DNA was purified and linearized with Hindlll and EcoRl digestion followed by phenol-chloroform extraction. After gel confirmation, the DNA was transcribed using T7 or Sp6 polymerases to create sense and antisense riboprobes. The probes were labeled _21 _ with digoxigenin-UTP using a tailing reaction. Probes were validated by dot-blot hybridization using template DNA.
Prehybridization of slides occurred after de-waxing the slides in xyiene followed by progressive ethanol-water hydration (95% 50%). The slides were rinsed in phosphate buffered saline and proteinase K added (20pg/mi for 20 min at room temperature). Following this, the slides were rinsed in a glycine buffer.
PBS
and then dehydrated in ethanol. Actual pre-hybridization was with 50%
formamide, 0.2% SDS, 0.1 % Sarcosyl, 5X SSC and 2% blocking reagent {Boehringer Mannheim Biochemica) for 1 hour at 60°C.
Hybridization was carried out by adding the probe (200 nglmi) at a volume of 50NI section. The slides were incubated at 60°C overnight in a chamber humidified with 4X SSC and 50% formamide. During hybridization, a coversfip was in place over the tissue sections. Following hybridization, the coverslip was removed and the sections rinsed in 4X SSC then washed in 4X SSC for 15 minutes followed by washing in 2X SSC for 15 minutes, all at room temperature.
Finally, the sections were washed with 0.1 % SSC for 30 minutes at 60°C.
Hybridization was detected by color reaction. Sections were washed with Buffers 1 & 2 (Boehringer Mannheim Biochemica; D1G Luminescent Detection Kit).
The sections were then incubated with anti-DIG alkaline phophatase antibody 1:500 in buffer 2 for 40 minutes. The sections were then washed in buffer 1, twice, for 15 minutes each and then in buffer 3 for 2 minutes. Incubation in the color solution (NBTIx-phos) was then canied out for 45 minutes, the slides washed in distilled water and dry-mounted with Geltol.
The present invention has been described in detail and with particular reference to the preferred embodiments; however, it wilt be understood by one having ordinary skill in the art that changes can be made thereto without departing from the spirit and scope thereof.

WO 97149807 PCTICA971'0043°
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each ind'rviduai publication.
patent or patent application was specfically and individually indicated to be incorporated by reference in its entirety.

WO 97/49807 PGTlCA97100a3~
References Brown, E. M. 1994. Homeostatic mechanisms regulating extraceilular and intracellular calcium metabolism. p. 15-54. In J.P. Bllezikian, R. Marcus, and M.A.
Levine. (ed.), The parathyroids: Basic and clinical concepts. Raven Press, New York.
Change, E., J. Kunes, P. Hamet, and J. Tremblay. 1990. Increase of calmodulin activator in hypertension. Am J Hypertens 3:210S-215S.
Cohn, D.V., B.H. Fasciotto, J.X. Zhang and S.U. Gorr. 1994. Chemistry and biology of chromogranin A (secretory protein-I) of the parathyroid and other endocrine glands. p. 107-119. In J.P. Bilezikian, R. Marcus, and MA. Levine.
(ed), The parathyroids: Basic and clincial concepts. Raven Press, New York.
Dash, P., I. 1. Lotan, M. Knapp, E.R. Kandel, and P. Goeiet. 1989. Proc Natl Acad Sci USA 84;7896-281.
Huang, S.L., Y.I. Wen, D.B. Kupranycz, S.C. Pang, G. Schiager, P. Hamet, and J. Tremblay. 1988. Abnormality of caimodulin in hypertension: evidence of the presence of an activator. J. Clin invest 82:276-281.
Lewanczuk, R. Z. and P.K. T. Pang. 1989. tn vivo potentiation of vasopressors by spontaneously hypertensive rat plasma: correlation with blood pressure and calcium uptake. Clin Exp Hypertens (A) 11:1471-1485.
Loke, S:L., C.A. Stein, X.H. Zhang, K. Mori, M. Nakanishi, C. Subasinghe, J.S.
Cohen, and L.M. Neckers. 1989. Characterization of oligonucieotide transport into living cells. Pro Natl Acad Sci USA 86:3474-3478.
Pang, P.K. T. and RZ Lewanczuk, 1989. Parathyroid origin of a new circulating hypertens'rve factor in spontaneously hypertensive rats. Am J. Hypertens 2:898-902.
Sambrook, J., E.F. Fritsch and T. Maniatis. 1989. Plasmid vectors, p.1.1-1.110.
In J. Sambrook, E. F. Fritsch, and T. Maniatis (ed.), Molecular cloning: A
laboratory manual. Cold Spring harbor Laboratory Press, New York.

~WO 97/d9807 PCTICA97/00439 Stein, C.A, and J.S. Cohen. 1988a. Oligodeoxynucleotides as inhibitors of gene expression: a review. Cancer 48:2659-2668.
Stein, C.A., K. Mori, S.L. Loke, C. Subasinghe, K. Shinozuka, J.S. Cohen, and L.M. Meckers. 1988b. Gene 72:333-341.
T~emblay, J., E. Chang, J. Kunes, and P. Hamet, 1991. Cyclic nucleotides and calmodulin-phosphodiesterase activator: potential biochemical markers of salt sensitivity. Clin Exp. Hypertens (A) 13:735-743.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: TREMBLAY, :JOHANNE
HAMfsT, PAVEL
LEWAI~TCZUK, RICHARD
GOSSARD, FRANCIS
(ii) TITLE OF INVENTION: A NOVEL HYPERTENSION RELATED CALCIUM
REGULATED Gf;NE (HCaRG) ( iii ) NUMBER OF SEQUf:NCES : 4 (iv) CORRESPONDENCE ADDRESS
(A) ADDRESSEE: GOUDREAU GAGE DUBUC
(B) STREET: 39:00 800 PLACE VICTORIA,P.O.BOX 242 (C) CITY: MONTREAL
(D) STATE: QUEBEC
( E ) COUNTRY : C'.ANADA
(F) ZIP: H4Z 1.E9 (v) COMPUTER READAESLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentl:n Release #1.0, Version #1.30 (vi) CURRENT APPLICP,TION DATA:
(A) APPLICATION NUMBER: CA 2,256,123 (B) FILING DATE: 31-L~EC-1998 (C) CLASSIFICP.TION:
(viii) ATTORNEY/AGENT INFORMF,TION:
(A) NAME: BRITT, KATHfERINE
(C) REFERENCE/DOCKET NUMBER: KB/12725.8 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 514-39'7-7419 (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: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:

(A) ORGANISM: Rattus rattus (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 132..803 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GCACGAGCCA CAGCCAGCTA CC'GCGGCTAG GTTCCTCCAG GTGCAGAGGG CGGTAAAGGC 60 TTGGTTTGTA TTTGTAATGC AF.CTGTGG7.'T AGGACCTTCT CTTCGGACTG GTCAAGAAAC 120 Met Ser Ala. Leu Gl.y Ala Ala Ala Pro Tyr Leu His His Pro Ala Asp Ser His Ser Gly Arch Val Ser Phe Leu Gly Ser Gln Pro 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 AGC ACC TTC AGA AAG TTG TTG AAp. 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 .~1AA 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 'rCA 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 ~~AG 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 Gln Glu Glu Leu Gln Glu Leu Gly Ile Pro Gln Asp Leu Ile Gly Asp 'CTG GCC AGT TTG GCA TTT GGG AGT CAA CGC CCT CTT CTC GAC TCT GTA 554 :~eu Ala Ser Leu Ala Phe Gly Ser Gln Arg Pro Leu Leu Asp Ser Val i~la Gln Gln Gln Gly Ser Ser Leu Pro His Val Ser Tyr Phe Arg Trp (~GG GTG GAC GTG GCC ATC 'TCA ACC AGC GCT CAG TCC CGC TCC CTG CAA 650 i~rg Val Asp Val Ala Ile Ser Thr Ser Ala Gln Ser Arg Ser Leu Gln (.CG AGT GTT CTC ATG CAG ~~TG AAG CTC ACA GAT GGA TCT GCA CAC CGC 698 _ ~~r,..~_....~_......._...~.._.._.._ ~ _.~._..__ _ .... ~, r. ___ __. r _ Pro Ser Val Leu Met Gln Leu Lys Leu Thr Asp Gly Ser Ala His Arg TTC GAG GTG CCC ATA GCC AAA TT7.' CAG GAG CTG CGG TAC AGT GTA GCC 746 Phe Glu Val Pro Ile Ala Lys Phe Gln Glu Leu Arg Tyr Ser Val Ala Leu Val Leu Lys Glu Met Ala Glu Leu Glu Lys Lys Cys Glu Arg Lys CTG CAG GAC TGACTGAACC C'TGGTACTGT GGGTGCTGAA GCTGGTACCA 843 Leu Gln Asp GAACACAGCC CCCCACTGGT GA.TGAGCCC'.A ACTCCATTGA GGTCCTGCAT GTGAGAACGT 903 ATTTTAAGTG AAAAGACAGC GGGACTTTC.'A GGTTTTGTTT TATGAGTCAA CAGCTGGGCA 963 GGGTGGCACA GTTTATAATC TC'AGCCCTTG GAAGTCTGAG GCTGGAGAAT GGGAAGTGTA 1023 AGCTGGGCCT GGCTTTCATA GT'GAGGCTC'A GTGTCGAATT AAAGAGGTAA AGCAACTATT 1083 (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 3er His Ser Gly Arg Val Ser Phe Leu Gly Ser Gln Pro Ser Pro Glu 'dal Thr Ala Val Ala Gln Leu Leu Lys Asp Leu Asp Arg Ser Thr Phe ~~rg Lys Leu Leu Lys Leu Val Val Gly Ala Leu His Gly Lys Asp Cys i~rg Glu Ala Val Glu Gln Leu Gly Ala Ser Ala Asn Leu Ser Glu Glu i~rg Leu Ala Val Leu Leu .Ala Gly Thr His Thr Leu Leu Gln Gln Ala 7~eu Arg Leu Pro Pro Ala ,Ser Leu Lys Pro Asp Ala Phe Gln Glu Glu i Leu Gln Glu Leu Gly Ile Pro Gln Asp Leu Ile Gly Asp Leu Ala Ser 115 1.20 125 Leu Ala Phe Gly Ser Gln Arg Pro Leu Leu Asp Ser Val Ala Gln Gln Gln Gly Ser Ser Leu Pro His Val. Ser Tyr Phe Arg Trp Arg Val Asp Val Ala Ile Ser Thr Ser Ala Gln Ser Arg Ser Leu Gln Pro Ser Val Leu Met Gln Leu Lys Leu Thr Asp Gly Ser Ala His Arg Phe Glu Val Pro Ile Ala Lys Phe Gln Glu Leu Arg Tyr Ser Val Ala Leu Val Leu Lys Glu Met Ala Glu Leu Glu Lys Lys Cys Glu Arg Lys Leu Gln Asp (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1355 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 295..966 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
c;GGCAGGCAG TTGAGGTGGA TT.AAACCAAA CCCAGCTACG CAAAATCTTA GCATACTCCT 60 ~~AATTACCCA CATAGGATGA AT.AATAGCAG TTCTACCGTA CAACCCCGGA GGCGCAGACC 120 GGTTTCCCCG CCGCCCACCC GG.ACGCCGAC GAAAGCCAGC GAGCTCCTCA GCCTCAGGCA 240 'CCTGCATCTG GGACCGACCT CC'rGGGCTGG CTGATCAAAG AGGAAGCAGC AGCA ATG 297 Met '.CCT GCT GTG GGG GCT GCA .ACT CCA TAC CTG CAT CAT CCT GGT GAT AGT 345 Ser Ala Val Gly Ala Ala Thr Pro Tyr Leu His His Pro Gly Asp Ser CAC AGT GGC CGA GTG AGT TTC TTC: GGG GCC CAG CTT CCT CCA GAG GTG 393 His Ser Gly Arg Val Ser Phe Lieu Gly Ala Gln Leu Pro Pro Glu Val Ala Ala Met Ala Arg Leu Leu Gly Asp Leu Asp Arg Ser Thr Phe Arg AAG TTG CTG AAG TTT GTG GTC AGC' AGC CTG CAG GGG GAG GAC TGC CGA 489 Lys Leu Leu Lys Phe Val Val Ser Ser Leu Gln Gly Glu Asp Cys Arg GAC GGT GTG CAG CGT CTT GGG GTC' AGC GCC AAC CTG CCG GAG GAG CAG 537 Asp Gly Val Gln Arg Leu Gly Val Ser Ala Asn Leu Pro Glu Glu Gln ~TG 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 ~GT 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 ~~AG 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 'Jal Phe Gly Ser Gln Arg Pro Leu Leu Asp Ser Val Ala Gln Gln Gln Gly Ala Trp Leu Pro His Val Ala Asp Phe Arg Trp Arg Val Asp Val ~~la Ile Ser Thr Ser Ala Leu Ala Arg Ser Leu Gln Pro Ser Val Leu ~~TG CAG CTG AAG CTT TCA GAT GGG TCA GCA TAC CGC TTT GAG GTC CCC 873 Idet Gln Leu Lys Leu Ser Asp Gly Ser Ala Tyr Arg Phe Glu Val Pro ~~CA GCC AAG TTC CAG GAG CTG CGG TAC AGC GTG GCC CTG GTC CTA AAG 921 'Chr Ala Lys Phe Gln Glu Leu Arg Tyr Ser Val Ala Leu Val Leu Lys GAG ATG GCA GAT CTG GAG .AAG AGG TGT GAG CGC AGA CTG CAG GAC 966 Glu Met Ala Asp Leu Glu Lys Arg Cys Glu Arg Arg Leu Gln Asp :?10 215 220 '.CGACCCCTCA CTTGACCAGT CCCATTCAGA TCCGGCTTGG ACAGGCACCT GAGATGGTGC 1026 (:AAAGTGCAG CTGACTCTTC CC.ACGACAGC CCTGGCCTTC CCATCAGGCA GGCTCTTCAG 1086 i TTTTCTTTTT GGAAGTAAAG Cp,GCTAAAFvC ATGTTTCTAT AGGTGAGTGT TGGACCTTCA 1206 CACCTCCCCT TCCCTGTACA TT'TGTCTTT'G GTGCTGGACG TGGCCATGTG AGGCCAGGTT 1266 GAGGCCCTTT GTAGACAACA TA.CAGTTGC',T CAGCCTGGCC CCATGTAGCC AGGTGCTTTT 1326 GTAGATCTTG TGTTTCAGGC AG'~GGCCCGG 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 (xi) SEQUENCE DESCRIPTION': SEQ ID N0:4:
lHet Ser Ala Val Gly Ala Ala Thr Pro Tyr Leu His His Pro Gly Asp Ser His Ser Gly Arg Val Ser Phe Leu Gly Ala Gln Leu Pro Pro Glu 'Val Ala Ala Met Ala Arg Leu Leu Gly Asp Leu Asp Arg Ser Thr Phe ;Arg Lys Leu Leu Lys Phe Val Val Ser Ser Leu Gln Gly Glu Asp Cys ;Arg Asp Gly Val Gln Arg Leu Gly Val Ser Ala Asn Leu Pro Glu Glu Gln Leu Gly Ala Leu Leu Ala Gly Met His Thr Leu Leu Gln Gln Ala :~eu Arg Leu Pro Pro Thr Ser Leu Lys Pro Asp Thr Phe Arg Asp Gln :~eu Gln Glu Leu Cys Ile Pro Gln Asp Leu Val Gly Asp Leu Ala Ser 'lal Val Phe Gly Ser Gln Arg Pro Leu Leu Asp Ser Val Ala Gln Gln c;ln Gly Ala Trp Leu Pro His Val Ala Asp Phe Arg Trp Arg Val Asp :L45 150 155 160 'lal Ala Ile Ser Thr Ser .Ala Leu Ala Arg Ser Leu Gln Pro Ser Val 7~eu Met Gln Leu Lys Leu Ser Asp Gly Ser Ala Tyr Arg Phe Glu Val _____........ __ ...... ..... _ _. ....~._~..._..~.._..._...
_.....__._._.._.w~...__.,~___.._... . ._...~._.~,..~ ....._. .... _......
..____.._..... .T...__......._._..~..~.._ Pro Thr Ala Lys Phe Gln Glu Leu Arg Tyr Ser Val Ala Leu Val Leu Lys Glu Met Ala Asp Leu Glu Lye, Arg Cys Glu Arg Arg Leu Gln Asp ___.__ ._.~.v~_._____._ ~~_.___...._..r..~~p..~.~,~...~._.....w__..___. _..__-_._.__.. _.. _.I _

Claims (30)

1. A nucleic acid molecule isolated from parathyroid of a mammal and whose expression is regulated by extracellular calcium concentration.

2. The isolated nucleic acid molecule of claim 1, wherein the mammal is a rat, and having the sequence set out in Figure 3.

3. The isolated nucleic acid molecule of claim 1, wherein the mammal is a human, and having the sequence set out in Figure 4.

4. The isolated nucleic acid molecule of claim 1, wherein the mammal is a rat, and having a homology of 60% or greater to all or part of the sequence set out in Figure 3.

5. The isolated nucleic acid molecule of claim 1, wherein the mammal is a human, and having a homology of 60% or greater to all or part of the sequence set out in Figure 4.

6. The isolated nucleic acid molecule of claim 1, wherein the mammal is a rat, and having a homology of 60% or greater to all or part of the translated portion of the sequence set out in Figure 3.

7. The isolated nucleic acid molecule of claim 1, wherein the mammal is a human, and having a homology of 60% or greater to all or part of the translated portion of the sequence set out in Figure 4.

8. A purified and isolated protein encoded by the nucleic acid molecule of claim 1.

9. A mimetic of the purified and isolated protein of claim 8.

10. A protein having a homology of 60% or greater to the protein of claim 8.

11. A pharmaceutical composition, comprising at least a portion of the protein of claim 8 and a pharmaceutically acceptable carrier, auxiliary or excipient.

12. A composition as claimed in claim 11, wherein the composition contains at least a portion of the amino acid sequence encoded by the nucleic acid molecule of a human as set out in Figure 4.

13. A composition as claimed in claim 11, wherein the composition contains at least a portion of the amino acid sequence encoded by the nucleic acid molecule of a rat as set out in Figure 4.

14. A pharmaceutical composition for use in gene therapy, comprising the nucleic acid molecule of claim 1, or a portion thereof, in a pharmaceutically acceptable vehicle.

15. The pharmaceutical composition of claim 14, wherein the molecule comprises a sense or an antisense sequence to the nucleic acid sequence of a human as set out in Figure 4.

16. A kit for the treatment or detection of a disease, disorder or abnormal physical state, comprising all or part of the nucleic acid molecule of claim 1,

17. A kit for the treatment or detection of a disease, disorder or abnormal physical state, comprising all or part of the protein of claim 8.

18. A kit for the treatment or detection of a disease, disorder or abnormal physical state, comprising an antibody to the protein of claim 8.

19. A kit for the treatment or detection of a disease, disorder or abnormal physical state, comprising all or part of the mimetic of claim 9.

20. The kit of claim 16, wherein the disorder is seceded from a group consisting of hypertension, hyperthyroidism, stroke, osteoporosis, osteopetrosis, cancer, heart failure, insulin dependent and independent diabetes, arteriosclerosis, gastrointestinal disease, inflammatory bowel disease and asthma.

21. A use of the pharmaceutical composition as claimed in claim 11, to treat a patient suffering from a disease, disorder or abnormal physical state related to the abnormal modulation of calcium.

22. A use of the protein of claim 8 to screen for inhibitors to such protein.

23. A method of treating a patient suffering from a condition caused by the abnormal modulation of calcium comprising administering an effective amount of a composition as claimed in claim 11 to the patient.

24. A method of using gene therapy to treat a patient suffering from a condition caused by the abnormal modulation of calcium comprising administering an effective amount of a composition as claimed in claim 14 to the patient.

25. The method of claim 24 wherein the composition includes a nucleic acid sequence which is a sense or an antisense sequence to the sequence of a human as set out in Figure 4.

26. A method for assaying for abnormal calcium levels in a patient which comprises (a) reacting a sample of the patient with a nucleic acid molecule of claim 1, or a portion thereof, under conditions where the sample and the molecule, or a portion thereof, are capable of forming a complex; (b) assaying for complexes, free molecule, or a portion thereof; and (c) comparing with a control.

27. A method for assaying for abnormal calcium levels in a patient which comprises (a) reacting a sample of the patient with a protein of claim 8 or a portion or a mimetic thereof, or with an antibody to said protein, under conditions where the sample and the protein, or a portion or a mimetic thereof, or an antibody thereto, are capable of forming a complex; (b) assaying for complexes, free protein, or a portion or a mimetic thereof, or an antibody thereto, and (c) comparing with a control.

28. A method for screening for efficacy of products for use in modulating (enhancing or inhibiting) abnormal calcium levels, the assay includes (a) reacting a sample of a patient with a protein of this invention, or a portion or a mimetic thereof, or an antibody thereto, under conditions where the sample and the protein, or a portion or a mimetic thereof, or an antibody thereto, are capable of forming a complex; (b) assaying for complexes, free protein, or a portion or a mimetic thereof: or an antibody thereto, and (c) comparing with a control.

29. A method for screening for efficacy of a product for use in modulating (enhancing or inhibiting) abnormal calcium levels, the assay includes (a) reacting the product with a protein of this invention, or a portion or a mimetic thereof, or an antibody thereto, under conditions where the product and the protein, or a portion or a mimetic thereof, or an antibody thereto, are capable of forming a complex; (b) assaying for complexes, free protein, or a portion or a mimetic thereof; or an antibody thereto, and (c) comparing with a control.

30. A method for screening for efficacy of a product for use in modulating (enhancing or inhibiting) abnormal calcium levels, the assay includes (a) reacting the product with a nucleic acid molecule of this invention, or a portion thereof, under conditions where the product and the molecule, or a portion thereof, are capable of forming a complex; (b) assaying for complexes, free molecule, or a portion thereof; and (c) comparing with a control.