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WO1998038203A1 - Sodium-phosphate cotransporter in lithium therapy for the treatment of mental illness - Google Patents

Sodium-phosphate cotransporter in lithium therapy for the treatment of mental illness Download PDF

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Publication number
WO1998038203A1
WO1998038203A1 PCT/US1998/002875 US9802875W WO9838203A1 WO 1998038203 A1 WO1998038203 A1 WO 1998038203A1 US 9802875 W US9802875 W US 9802875W WO 9838203 A1 WO9838203 A1 WO 9838203A1
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Prior art keywords
lithium
seq
sodium
leu
val
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PCT/US1998/002875
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French (fr)
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Robert B. Gunn
Richard T. Timmer
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Emory University
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Priority to AU63263/98A priority Critical patent/AU6326398A/en
Publication of WO1998038203A1 publication Critical patent/WO1998038203A1/en
Priority to US10/314,790 priority patent/US20040002079A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • Na-PO 4 cotransport is the primary mechanism for the regulation of total body phosphate balance. It mediates both the gastrointestinal uptake and renal reabsorption of PO . Whereas 70% (high phosphate diet) to 85% (low phosphate diet) of dietary phosphate is absorbed by the GI tract the major control is in proximal reabsorption by the kidney of about 70% of the filtered load and in distal discretionary reabsorption of some fraction of the remainder. Na-PO 4 cotransport has been found in the plasma membrane of every mammalian cell examined.
  • phosphate metabolism includes most important biological molecules: nucleotides, DNA, RNA, glycolytic intermediates, phospholipids, and most proteins through regulatory or structural phosphorylations.
  • Na-PO 4 cotransport is essential for ATP synthesis.
  • Na-PO 4 cotransporters exist in the kidney, as two isoforms, type I and type II.
  • a related cotransporter is also present in liver where the protein has been partially purified, reconstituted in liposomes, and expressed in oocytes from liver mRNA. Its function in rat hepatocytes in primary culture is stimulated by insulin.
  • Na-PO cotransport activity has been extensively characterized in the duodenum/upper jejunum of many higher vertebrates. Neither the liver nor intestinal forms of the cotransporter have been cloned from mammalian sources. In contrast to mammals, teleosts appear to have an isoform of the Type II transporter present in the intestine.
  • BNPI brain-specific cDNA
  • the brain transporter is specific to the brain and mRNA transcripts are found in the neurons of the cerebral cortex, hippocampus, and cerebellum.
  • the neuronal transporter is also found in peripheral nerves and transports arsenate and Li + can substitute for Na + .
  • the erythrocyte form represents a third type of Na-PO 4 cotransporter, which applicants have discovered is also a retroviral receptor.
  • Retroviruses require specific cell-surface receptors for cell recognition and infection.
  • Two widely expressed mammalian retrovirus receptors PiT-1 (Glvr-1; Genbank L20859, U.S.Patent 5,414,076) and PiT-2 (Ram-1; Genbank L19931, U.S. Patent 5,550,221) have been cloned and shown to share 30% homology with Pho-4 + ', a phosphate uptake gene in Neurospora crassa and when these two mammalian genes are expressed in oocytes they induce sodium dependent phosphate cotransporter activity.
  • a murine cationic amino acid transporter has been shown to be a retrovirus receptor, thus, indicating there are at least two classes of transporters that are retrovirus receptors.
  • the two sodium phosphate cotransporters/retrovirus receptors (PiT-1 and PiT-2) are widely expressed in tissues and cells (thymus, marrow, lung, liver, heart, kidney, muscle and brain) and appear to be the ubiquitous housekeeping sodium-phosphate cotransporters that every cell requires in order to maintain the intracellular concentration of phosphate above electrochemical equilibrium.
  • the cotransporter/receptor isoforms in different species (human and mouse; rat and hamster) together with the differences in retroviral envelope proteins define the species specificity for susceptibility to infection by each retrovirus.
  • the ability to transfect cell lines from one species with the transporter/receptor isoform from another species and/or alter the envelope protein provides novel model systems and the means to design vectors that allow increased gene transfer into human hematopoietic progenitor cells and other cells.
  • a third cotransporter BNPI There are different isoforms of these three genes in different people.
  • cDNA and cRNA probes to PiT-1 or to PiT-2 and their mRNA products and antibodies to these proteins distinguish between individuals who are responders or non-responders to lithium treatment.
  • the sodium-phosphate cotransporter is the same cell membrane protein as the lithium-sodium countertransporter.
  • the present invention provides a readily performed diagnostic test to evaluate patient status, by measuring a combination of sodium, phophate or lithium flux in an in vitro membrane-based translation system.
  • Applicants have identified the gene product of PiT-1 as the lithium-sodium countertransporter across cell membranes.
  • the PiT-1 gene product is the erythrocyte isoform. Probes for this gene distinguish between responders and non-responders to lithium treatment.
  • Applicants have also identified the lithium-sodium countertransporter as the physiological mechanism for the extrusion of lithium from cells. It regulates the cell concentration of lithium. The activity of this transporter determines the therapeutic effect of lithium.
  • This invention provides a simple molecular biological test for the ability of cells to extrude lithium.
  • the only test to determine the activity of a lithium transporter is a laboratory measurement of lithium flux into or out of cells using chemical assays for lithium. See, e.g., Sarkadi, B. et al., J. Gen.Physiol.72 : 249 (1978).
  • the lithium-sodium countertransporter has significance for determining the responsiveness of humans with mental disorders to treatment with lithium salts. At present about half of patients treated with lithium do not improve. There are no techniques at present to diagnose whether a patient will be helped by lithium treatment, except by a time-consuming therapeutic trial.
  • the diagnostic test of the present invention allows genetic screening to predict whether a patient will respond to lithium transport. The test is also a screen for susceptibility to and extent of manic depressive illness. Further, the test is suitable to screen newborns in families with depression for their potential to develop the illness and whether they can respond to lithium treatment.
  • the sodium-phosphate cotransporter is identified as the same protein as the lithium-sodium countertransporter, and is suitable for diagnostic assays for mental illnesses susceptible to lithium therapy, including manic depression.
  • Various methods for evaluating the flux of lithium and other cations in appropriate cells are also disclosed, including reticulocytes.
  • Figure 1 is a schematic diagram of a cell 1 showing that the sodium-phosphate cotransporter is the same cell membrane protein as the sodium-lithium countertransporter.
  • the present invention relates to a purified DNA molecule coding for a lithium-sodium countertransporter. It also relates to a purified DNA molecule coding for an amino acid sequence selected from the group consisting of hPiT-1, hPiT-2, and hBNPI, said molecule useful for measuring lithium-sodium countertransport in human cells. Specifically, the present invention relates to a novel utility for the sequences identified as SEQ.ID.NO.: 1, SEQ.LD.NO.: 2, SEQ.LD.NO.: 3, SEQ.LD.NO.: 4, SEQ.LD.NO.: 5, and SEQ.LD.NO.: 6.
  • the human amphotrophic retrovirus receptor is useful as a lithium-sodium countertransporter, including the sequences identified as SEQ.LD.NO.: 1, SEQ.LD.NO.: 2, SEQ.LD.NO.: 3, SEQ.LD.NO.: 4, SEQ.LD.NO.: 5, and SEQ.LD.NO.: 6.
  • a first method of evaluating sensitivity to lithium therapy in manic depressive patients comprising the steps of
  • a second method of evaluating sensitivity to lithium therapy in manic depressive patients comprising the steps of (a) providing a sample of patient blood;
  • the first and second methods are drawn to sequences to any lithium-sodium countertransporter selected from the group consisting of hPiT-1, hPiT- 2, and hBNPI, said sequences identified as SEQ.LD.NO.: 1, SEQ.LD.NO.: 2, SEQ.LD.NO.: 3, SEQ.LD.NO.: 4, SEQ.LD.NO.: 5, and SEQ.LD.NO.: 6.
  • a third method of evaluating sensitivity to lithium therapy in manic depressive patients comprising the steps of
  • a fourth method of evaluating lithium-sodium countertransport in patients with mental illness comprising the steps of
  • Phosphorus is a major dietary element essential to most important biological molecules. Its absorption from the gut and reabsorption from the glomerular filtrate is by secondary active transport on a family of Na-PO 4 cotransporters.
  • the gene product responsible for this function in erythrocytes is pharmacologically distinct from the previously characterized renal brush border Na-PO cotransporter. Both the brain and peripheral nerve forms (PiT-1, PiT-2, BNPI) and the red blood cell form of the Na- PO 4 cotransporter can use Li + as a congener for Na + . Also, arsenate is transported by nerve membranes and probably by red blood cells. Therefore, these two tissues most likely have the same cotransporter/receptor.
  • the renal Na-PO 4 cotransporter presumably the apical isoform, has been cloned from several species. Applicants have identified the erythrocyte isoform as PiT-1.
  • FIG. 1 schematically shows a simplified diagram, with cell 1.
  • Lithium cation, Li + enters the cell as the anion LiCO 3 " , by the action of the AEl (Anion Exchange Protein, Band 3) countertransporter.
  • the lithium cation leaks into the cells by a minor unknown leak pathway.
  • Lt is pumped out by the sodium-phosphate cotransporter, which applicants have identified to be also the lithium-sodium countertransporter.
  • the hPiT-1 gene or other coding sequences for the lithium-sodium countertransporter are prepared for the expression systems and diagnostic assays of the present invention.
  • These polynucleotide sequences are prepared by ligation of other sequences, restriction endonuclease digestion, cloning, mutagenesis, organic synthesis, or combination thereof, in accordance with the principles and practice of constructing DNA sequences.
  • sequencing DNA e.g., verification of a construct at the end of a series of steps, dideoxy DNA sequencing is the preferred method.
  • Other DNA sequencing methods are well known.
  • Phosphoramidite chemistry in solid phase is the preferred method for the organic synthesis of oligodeoxynucleotides and polydeoxynucleotides. Many other organic synthetic methods are available and are readily adapted to the particular sequences of this invention by a person skilled in the art.
  • Amplification of DNA or cDNA is a common step in the detection of specific sequences in the diagnostic tests of the present invention. It is typically performed by the polymerase chain reaction (PCR). See, e.g., Mullins, K. et al., U.S. Pat. No.
  • the basic principle of PCR is the exponential replication of a DNA sequence by successive cycles of primer extension.
  • the extension products of one primer when hybridized to another primer, becomes a template for the synthesis of another nucleic acid molecule.
  • the primer template complexes act as substrate for DNA polymerase which, in performing its replication function, extends the primers.
  • the conventional enzyme for PCR applications is the thermostable DNA polymerase isolated from Thermus aquaticus, or Taq DNA polymerase. Numerous variations in the PCR protocol exist, and a particular procedure of choice in any given step in the constructions of this invention is readily performed by a skilled artisan.
  • primers for hPiT-1 are organically synthesized, based on its known sequence, and are hybridized to a sample of patient DNA. PCR in combination with reverse transcriptase, so-called RT-PCR, is then carried out to amplify the patient hPiT-1 genes. Subsequent analysis, e.g., by restriction fragment length polymorphism (RFLP), provides information on patient status.
  • RFLP restriction fragment length polymorphism
  • the source for mRNA is derived from enzymological manipulations, rather than isolation of naturally transcribed mRNA from, e.g., cell lines such as the erythroleukemic cell line K562.
  • Synthetic capped mRNA is preferably prepared by in vitro transcription of the appropriate linearized cDNA constructs containing the appropriate promoter for an RNA polymerase, e.g., T7 RNA polymerase. See, e.g., Fletcher, L. et al., J.Biol. Chem.265 : 19582 (1990), herein incorporated by reference for these purposes. Under these conditions, high yields of capped mRNA coding for hPiT-1, hPiT-2 or BNPI sequences are obtained, which migrate as a discrete band in gel electrophoresis.
  • the capped mRNA is then subjected to in vitro membrane-based translation, e.g., in Xenopus oocytes, microsomes or cultured cells, in an expression system designed to permit flux analysis of Na, PO 4 , and Li.
  • Preferred expression systems include Xenopus oocytes, and transfected HEK 293 cells.
  • Other suitable transfection systems include Dictyostelium discoideum cells, baculovirus-infected ceected Sf9 cells, and CHO cells. Selection of the appropriate cell system, as well as adjusting the experimental parameters to enhance translation, is readily determined within the skill of the art.
  • the gene for the countertransporter proteins such as the hPiT-1 gene, is also suitable for expression in an expression vector in a recombinant expression system.
  • the constructed sequence need not be the same as the original, or its complimentary sequence, but instead may be any sequence determined by the degeneracy of the DNA code. Conservative amino acid substitutions may also be employed, or other modifications, such as an amino terminal methionine.
  • a ribosome binding site active in the host expression system is Hgated to the 5' end of the chimeric coding sequence, giving a synthetic gene.
  • the resulting synthetic gene can be inserted into any one of a large variety of vectors for expression, by ligating to an appropriately linearized plasmid.
  • Expression in E. coli is suitable for expression of active lithium-sodium countertransporter protein, e.g., E. coli BL21.
  • a regulatable promoter is also suitable for the expression of these coding sequences, e.g., under the control of the E. coli lac promoter.
  • Other suitable regulatable promoters include trp, tac, recA, T7, lambda promoters.
  • the flux of a molecule is a measure of the number of molecules that cross the cell membrane per unit time and per unit of membrane (expressed either as area or number of cells or amount of cell protein).
  • the flux is measured by determining the appearance or disappearance (or both) of the molecule on one side of the membrane.
  • the amount on one side of the membrane is measured at different known times either by a chemical determination or by a radioactive determination if a tracer of the atoms or molecules is used.
  • Lithium, atomic number 3, atomic weight 6.9 Daltons has no radioactive isotopes of use for biological measurements. Chemical detemination must be used instead of a radioisotope.
  • the amount of lithium is most often determined by atomic absorption spectroscopy or emission spectroscopy.
  • the assay of lithium-sodium countertransport flux rate is made by the following steps:
  • the cells are mixed in a standard buffered solution containing sodium and lithium chloride solution, and subjected to repeated suspension, centrifugation, removal of supernatant fluid, and resuspension;
  • the cells in the standard solution are incubated in the presence of inhibitors of the Na, K, ATPase (e.g., ouabain at 10 "5 M) and in the presence of inhbitors of Anion Exchange protein (e.g., denitrostilbenedisulfonate at 2.5 x 10 "4 M), in suspension at body temperature;
  • inhibitors of the Na, K, ATPase e.g., ouabain at 10 "5 M
  • inhbitors of Anion Exchange protein e.g., denitrostilbenedisulfonate at 2.5 x 10 "4 M
  • RFLP restriction fragment length polymorphism
  • ASO allele-specific ohgonucleotides
  • Tagging or labeling the desired polynucleotide fragments can take various forms.
  • the radioisotope 32 P and other radioactive labels are not preferred because of laboratory safety and waste disposal requirements.
  • Alternative methods of labeling include chemical analogs, such as biotinylated analogs of TTP and UTP, which incorporate into the resulting DNA and RNA, respectively.
  • the biotin-labeled probe can be coupled to avidin, or streptavidin, and the complex detected by chemiluminescence, immunofluorescence, immunoperoxidase, immune colloidal gold techniques, or the like.
  • the biotin-labeled probe can also be detected with avidin conjugated to poly AP (calf intestinal alkaline phosphatase), assayed with the appropriate AP substrates.
  • Digoxigenin is a useful substitute for avidin in many applications, and it is readily detected with antibodies specific for digoxigenin.
  • Various combinations of such labels are readily carried out, e.g., a biotin-labeled probe detected with streptavidin conjugated to poly AP, or a biotin labeled probe detected with anti- biotin antibodies linked to AP, or other secondary labeling systems.
  • a biotin-labeled probe detected with streptavidin conjugated to poly AP or a biotin labeled probe detected with anti- biotin antibodies linked to AP, or other secondary labeling systems.
  • Monoclonal antibodies are the reagent of choice in the present invention, and a specifcally used to analyze patient cells for specific characteristics of the lithium-sodium countertransporter.
  • Monospecific antibodies to the lithium-sodium countertransporter are purified from mammalian antisera containing antibodies reactive against the lithium- sodium countertransporter or are prepared as monoclonal antibodies reactive with the lithium-sodium countertransporter using the technique of Kohler and Milstein, Nature 256: 495-497 (1975).
  • Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for the lithium-sodium countertransporter.
  • Homogenous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with the lithium-sodium countertransporter, as described above.
  • the lithium-sodium countertransporter specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats* horses and the like, with an appropriate concentration of the lithium-sodium countertransporter either with or without an immune adjuvant.
  • Preimmune serum is collected prior to the first immunization.
  • Each animal receives between about 0.1 mg and about 1000 mg of the lithium-sodium countertransporter associated with an acceptable immune adjuvant.
  • acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium parvum and tRNA.
  • the initial immunization consists of the lithium-sodium countertransporter in, preferably, Freund's complete adjuvant at multiple sites either subcutaneously (SC), intraperitoneally ( P) or both.
  • SC subcutaneously
  • P intraperitoneally
  • Each animal is bled at regular intervals, preferably weekly, to determine antibody titer.
  • the animals may or may not receive booster injections following the initial immunization. Those animals receiving booster injections are generally given an equal amount of the antigen in Freund's incomplete adjuvant by the same route.
  • Booster injections are given at about three week intervals until maximal titers are obtained.
  • the animals are bled, the serum collected, and aliquots are stored at about -20° C.
  • Monoclonal antibodies (mAb) reactive with the lithium-sodium countertransporter are prepared by immunizing inbred mice, preferably Balb/c, with the lithium-sodium countertransporter.
  • the mice are immunized by the LP or SC route with about 0.1 mg to about 10 mg, preferably about 1 mg, of the lithium-sodium countertransporter in about 0.5 ml buffer or saline incorporated in an equal volume of an acceptable adjuvant, as discussed above. Freund's complete adjuvant is preferred.
  • the mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks.
  • Immunized mice are given one or more booster immunizations of about 0.1 to about 10 mg of the lithium-sodium countertransporter in a buffer solution such as phosphate buffered saline by the intravenous (IN) route.
  • Lymphocytes from antibody positive mice, preferably splenic lymphocytes, are obtained by removing spleens from immunized mice by standard procedures known in the art.
  • Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions which will allow the formation of stable hybridomas.
  • Fusion partners may include, but are not limited to: mouse myelomas P3/ ⁇ Sl/Ag 4-1; MPC-11; S-194 and Sp 2/0, with Sp 2/0 being preferred.
  • the antibody producing cells and myeloma cells are fused in polyethylene glycol, about 1000 mol. wt., at concentrations from about 30% to about 50%.
  • Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art.
  • DMEM Dulbecco's Modified Eagles Medium
  • Supernatant fluids are collected from growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay such as solid phase immunoradioassay (SPIRA) using the lithium-sodium countertransporter as the antigen.
  • SPIRA solid phase immunoradioassay
  • the culture fluids are also tested in the Ouchterlony precipitation assay to determine the isotype of the mAb.
  • Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press, 1973.
  • Monoclonal antibodies are produced in vivo by injection of pristane primed Balb/c mice, approximately 0.5 ml per mouse, with about 2x10 6 to about 6x10 6 hybridoma cells about 4 days after priming. Ascites fluid is collected at approximately 8-12 days after cell transfer and the monoclonal antibodies are purified by techniques known in the art.
  • In vitro production in anti-lithium-sodium countertransporter mAb is carried out by growing the hydridoma in DMEM containing about 2% fetal calf serum to obtain sufficient quantities of the specific mAb.
  • the mAb are purified by techniques known in the art.
  • Antibody titers of ascites or hybridoma culture fluids are determined by various serological or immunological assays which include, buy are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of the lithium-sodium countertransporter in body fluids or tissue and cell extracts.
  • Schizophrenic disorders are mental diseases with a tendency toward chronicity and are characterized by psychotic symptoms involving disturbances of thinking, feeling, and behavior.
  • Affective disorders also known as mood disorders, are psychopathologic states in which a disturbance of mood is either a primary determinant or constitutes the core manifestation.
  • a clinically useful division of affective disorders is bipolar (with periods of depression and elevation) and unipolar (depressions only) mood disturbances. Such bipolar mood disturbances are commonly known as manic depression.
  • Lithium usually given as a carbonate salt, attenuates bipolar mood swings, without affecting normal mood. It also appears to be useful in the treatment of aggressive personality disorders, which are typically classified outside of affective disorders. About 50% of bipolar patients respond to lithium therapy. Various clinical attributes are useful in assessing response to lithium, including the presence of manic episodes as the primary mood disorder, an episode frequency of less than about 2 years, as well as past or family history of lithium response. Applicants now provide another attribute to evaluate response to lithium, that is, lithium-sodium countertransport.
  • Lithium is commonly used to treat affective disorders.
  • the site of action of lithium is believed to be in the brain.
  • the steady state ratio of intracellular red blood cell lithium concentration to plasma lithium concentration during therapy shows great interindividual variation, although the lithium ratio is relatively constant for any one individual. Individual fluctuations of the lithium ratio have also been reported. The relative constancy of the ratio in an individual may be genetically determined.
  • the steady state lithium ratio across the red cell membrane is the result of three lithium transport processes: the Na,K,ATPase which is inhibited by ouabain and other cardiac glycos des, the anion exchange protein (AEl, band 3) and the lithium-sodium countertransport system.
  • the Na, K, ATPase pumps Li into the cell by substituting Li + at a normal K + binding site, but at therapeutic levels of Li + (l-2mM) and normal plasma Na and normal plasma K, the Na,K ATPase carries Li poorly ( «0.025 mmol/lit cell • h; «75 ⁇ mol/kg Hgb • h).
  • the anion exchanger is the principle mediator of the inward leak of Li as the ion pair LiCO 3 " . This transport is inhibited by stilbene disulfonates (SITS, DNDS, DLDS), phloretin and dipyridamol.
  • the lithium-sodium countertransporter normally pumps Li out of the cell against its electrochemical gradient so that [Li] ce ⁇ is lower than [Li] p ⁇ and [Li] ce ⁇ /[Li] p ⁇ , which lithium ratio is 0.2 to 0.8 in different individuals.
  • a higher steady ratio is expected in alkalosis (higher plasma HCO 3 " , CO 3 2" , and LiCO 3 ' ) due to increased AEl mediated leak into the cell, or when the lithium-sodium countertransport extrusion of lithium is slowed, either because cell Na + gradient is decreased or because the countertransporter is less effective.
  • the lithium-sodium countertransporter as the product of PiT-1 gene previously identified as retrovirus receptor and a NaPO 4 cotransporter.
  • the red cell NaPO 4 cotransporter transports Li instead of Na (i.e., LiPO 4 cotransport) and that is performs Na/Na exchange and lithium-sodium countertransport.
  • the lithium ratio has been implicated in the responsiveness of polar disease to lithium treatment, the development of essential hypertension (hypertension of unknown etiology), the'susceptibility of individuals to affective (bipolar) disorders, and the toxic side effects of Li therapy.
  • Lithium influxes, 32 PO influxes, sodium effluxes were measured in human red blood cells incubated in an isotonic media containing (mM): 150 (Na + Li + K)C1, 0.3 K 2 HPO 4 , 20 HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), 0.25 DNDS (4,4'-dinitro stilbene-2,2'-disulfonate to inhibit the Anion Exchange Protein Band3 pathway for phosphate transport), titrated to pH 7.64 with KOH at 20°C to give a pH of 7.40 at 37°C where the fluxes were performed.
  • hPiT-1 Human PiT-1 (hPiT-1) was cloned as the human isoform of the gibbon ape retrovirus receptor [Van Zeijl, M., et al (Proc. Nat. Acad. Sci. 91 : 1168 (1994)].
  • PCR primers were designed to amplify either the hPiT-1 or hPiT-2 isoforms.
  • the 1700 bp product was amplified by RT-PCR from total RNA isolated from K562 cells, and restriction analysis with Sphl identifies the product as being derived from hPiT-1 and not hPiT-2. This evidence, considered together with the kinetic evidence of preceeding Example, indicates that hPiT-1 is the sodium-phosphate cotransporter isoform present in both K562 cells and erythrocytes.
  • RT-PCR was carried out using 1 ⁇ g of total RNA isolated from K562 cells using the method of Chomczynski et al., Analytical Biochem. 162: 156 (1987).
  • the RT-PCR reaction was carried out in a single tube using recombinant Tth DNA- polymerase which is capable of reverse transcriptase activity under appropriate reaction condition.
  • the primers used in these experiments (FI and Rl) are based upon highly conserved regions between hPiTl and hPiT2 located in putative transmembrane domains in the N-terminal and C-terminal regions of the proteins.
  • the results from these experiments show that the products of Sphl digestion are 1000, 487 and 138 bp, which agree with the predicted sizes.
  • the gel patterns under the PiT-1 control after Sphl digestion are the same as for K562, after Sphl digestion; and both are different from those predicted for PiT-2 after Sphl digestion.
  • K562 cells have the human PiT-1
  • the template for expression of the Na-PO 4 cotransporter in the cell-free system or in Xenopus oocytes is capped mRNA prepared polymerase, prepared by in vitro transcription of linearized cDNA constructs containing the promoter for T7 RNA polymerase.
  • Samples of mRNAs are prepared as prepared as previously described in a 1 ml reaction mixture [Fletcher, L. et al., J. Biol. Chem.265; 19582(1990)]. These reaction conditions permit high yields of mRNA (-1000 mg or about 100-200 copies of RNA transcript per copy of template DNA) that are >80% capped and migrate as a single discrete band of the correct molecular weight on denaturing PAGE.
  • T7 RNA polymerase is purified as described by Davanloo, et al. Proc. Natl.Acad.Sci.Sl;,2035(l9S4).
  • the Na-PO cotransporter mRNA is expressed in a wheat germ cell-free system containing in vitro transcribed mRNA, dog pancreatic microsomes and signal recognition particle (SRP).
  • Cell-free translation is carried out in the presence of 34 mM [ 14 C]leucine (50 cpm/pmol), 50 mM each of the other 19 amino acids, unfractionated wheat germ tRNA and other components a previously described [Erickson, AH et al., Methods Enzymol. 96;38 (1983)].
  • the expression of the sodium-phosphate cotransporter is carried out in several heterologous expression systems, e.g. Dictyostelium discoideum cells, Xeonpus oocytes, HEK 293 cells, baculovirus-infected Sf9 cells, and CHO cells. Transfection, growth and selection of transformants are performed by well known techniques.
  • the expression of the Na-PO 4 cotransporter is assessed by immunoprecipitation with Na- PO 4 - specific antibodies of the protein from cells (Dictyostelium, HEK 293, etc.) grown in medium supplemented with 35 S-methionine.
  • the functional expression of the Na-PO transporter, both native and mutant forms, in transfected cells is monitored by determining the Na-dependent 32 Na-PO 4 flux , as described in an Example below. Negative controls include determining background levels of Na-dependent Na-PO 4 transport from cells transfected with the construct in the anti-sense orientation.
  • the two principal expression systems for heterologous expression of the erythrocyte Na- PO 4 cotransporter are injected Xenopus oocytes and transfected Dictostelium.
  • the cotransporter is expressed in another expression system such as HEK 293 or baculovirus-infected Sf9 cells.
  • stage V and IN oocytes are removed using standard anesthetic (0.17% 3-aminobenzoic acid) and surgical procedures.
  • the oocytes are placed in OR-2 medium and collagenase treated (2mg/ml) for 2.5 h.
  • oocytes are washed and defolliculated if needed by trituration and co-injected with 2.5 ng capped SEAP cR ⁇ A and 5-50 ng of capped transporter of cR ⁇ A (prepared as described above).
  • Capped SEAP cR ⁇ A prepared by in vitro transcription o ⁇ HIndlll linearized pGEM-SEAP.
  • the pGEM-SEAP construct contains the human placental alkaline phosphatase with a site-specific mutation at codon 489 to create a termination codon[Tate, S.S. et al., FASEB J. 4; 228 (1990)]. This stop codon results in a secreted form of alkaline phosphatase rather than a membrane anchored form.
  • cR ⁇ As are injected in a total volume of 50 nl using a ⁇ arishige injector. Following incubation overnight in Barth's medium, oocytes are sorted and placed in single wells of
  • alkaline phosphatase activity is measured by chromogenic assay.
  • the secreted alkaline phosphatase catalyzes the dephosporylation of nicotinamide adenine dinucleotide phosphate (NADP + ); the NAD formed then catalytically activates an NAD + -specific oxidation-reduction cycle driven by the enzymes alcohol dehydrogenase and diaphorase.
  • the chromophore formed is a violet colored formazan product of LNT-violet. Only those oocytes that express SEAP
  • Dictyostelium A second expression system for heterologous expression of the cloned cotransporter is Dictyostelium.
  • a significant advantage of the Dictyostelium expression system is that these cells are grown in suspension culture and are handled like red cells for flux measurements.
  • the principle expression vector e.g. pBS18 and its derivatives
  • Tn5 gene neomycin phosphotransferase II
  • the insert of interest is driven by the actin 8 promoter and the 2H3 transcriptional terminator.
  • the Tn5 gene permits selection of permanent transfectants in media containing G-418, to which the native slime mold is highly sensitive.
  • HEK-239 cells C. HEK-239 cells. Heterologus expression is also readily carried out with HEK- 293 cells, ATCC Accession No. CRL 1573.
  • HEK-293 (human embryonic kidney) cells were obtained from the American Type Culture Collection (ATCC) at passage 31. These cells were used to prepare seed stocks at passage 32. Cells were used until passage 45, after which fresh cultures were started from frozen passage 32 cells. The cells were grown in Minimal Essential Media (MEM) with Hank's salts and supplemented with L-glutamine and 5% fetal calf serum at 37°C in 5% CO 2 / 95% air. Transfection was carried out using standard calcium phosphate precipitation methods.
  • MEM Minimal Essential Media
  • FLUX MEASUREMENTS A. Flux measurements in cell-free expression system. The flux is measured by an adaptation of a rapid filtration method according to Macintyre, J.D. et al. Biochim. Biophys. Acta 644; 351 (1981). Briefly, microsomes are suspended, equilibrated and mixed in media containing either sodium or choline or N-methyl-alpha- glucamine as the dominant cation in a thermostatically controlled chamber. The flux is initiated by additional of 32 PO to the flux medium. Aliquots are removed at different times and filtered under vacuum using prewashed mixed cellulose-ester filters.
  • microsomes retained by the filter are rapidly washed with stopping solution containing 323 mM MgSO 4 (isotonic for microsomes).
  • a sample of flux suspension is used to measure total protein and specific activity.
  • the flux (pmol PO- ⁇ ig protein-min or ions/cotransporter molecule-min) is calculated from the slope of cpm/aliquot versus time.
  • Preloaded microsomes are used to verify the quantitative recovery of microsomes, the replication of sample counts, and the effectiveness of the wash in removing a extracellular marker, usually 14 C-PEG at
  • Oocytes Flux measurements in oocytes.
  • Oocytes are prepared and injected as described in Example above. Briefly, eight to ten oocytes are placed in individual wells of a 96- well culture plate in medium containing either Na or choline as the dominant cation. The flux is initiated by addition of 32 PO 4 to each well. As known times, oocytes are removed and washed three times in ice cold choline medium. The oocyte is then dissolved in 0.2 ml 10% SDS and the counted in a liquid scintillation counter. A sample of the 32 PO 4 incubation fluid is counted to calculate the extracellular specific activity. The influx (pmol/oocyte/hr) is calculated from the specific activity and the uptake. The difference between the flux in Na and choline media is the calculated
  • HL-5 medium contains 20 + 3 mM K. This is not a defined medium so the composition must be determined for each flux.
  • " -Cells are grown to a density of 1-3 x 10 6 /ml at 20°C in a shaking incubator. Approximately 10 7 cells are required for each data point on the influx curve. The cells are resuspended to 4 x 10 7 /ml in HL-5 in a thermostatted stirred chamber at a known pH. At time zero tracer (-0.6 ⁇ Ci/ml) is added and at known times thereafter samples (0.4 ml) are removed.
  • the samples are transferred to 7 ml of ice-cold stop solution (58 mM MgSO 4 ), immediately centrifuged for 30 seconds at 3000 x g in a rotor, the supernatant aspirated and discarded as radioactive waste.
  • the pellet of cells is resuspended thrice in 6 ml of stop solution, pelleted, and the supernatant aspirated.
  • To the drained pellets 1 ml of 0.1% DOC in 1 N NaOH is added for solubilization. Aliquots are counted for radioactivity or are assayed for protein.
  • the data are calculated as pmoles/mg protein and the data vs. time are fitted to a single exponential by nonlinear regression analysis and the inital slope (flux) and asymptote value constant (pmoles/mg protein) calculated.
  • HEK-293 Human embryonic kidney cells (HEK-293) are purchased from the American Type Culture Collection (ATCC, accession number CRL 1573) at passage 32 and grown in MEM and 5% fetal calf serum in a 5% CO /95% air 37°C incubator. They are maintained in T75 flasks and split weekly. At passage 45 decreased expression is observed, so frozen stocks at passage 34 are brought up.
  • the cells are transfected with cDNA harvested from bacteria and purified on an anion exchange column. At 24 hours the cells are plated onto 24-well plates at 3-5 x 10 5 cells/cm 2 and transport measured at 48-72 hr. The expression is low by 96 hr and absent at 5 and 10 days.
  • the medium is aspirated and washed once with 0.5 ml of a Na-free (143 mM N-methyl-D- glucamine Cl) HCO 3 -free MEM-like HEPES buffered medium for 3-5 min. Then it is preincubated at 37°C in room air for 30 minutes. The flux is initiated by adding 32 PO 4 or 22 Na containing media. Cells transfected with the vector only (e.g., pRBG4) are always treated and fluxed in parallel. 32 PO 4 influx in the absence of Na is always " measured. All fluxes are performed in duplicate.
  • a Na-free (143 mM N-methyl-D- glucamine Cl) HCO 3 -free MEM-like HEPES buffered medium for 3-5 min. Then it is preincubated at 37°C in room air for 30 minutes. The flux is initiated by adding 32 PO 4 or 22 Na containing media. Cells transfected with the vector only (e.g., pRBG4) are always
  • the plates are placed on a water thermostatted table for 5 minutes and the flux initiated by aspirating the preincubation medium from the last column of wells and adding the tracer solution at known times ( ⁇ 0.2 sec). This is done to successive columns of cells at approximately 30 minutes, 15 minutes, 10 minutes, 5 minutes, 2 minutes and 1 minute prior to terminating the influx simultaneously for all wells on the plate by 3 rapid ice cold washes (over 15 seconds total elapsed time) with (mM): 150 NaCl, 1.5 CaCl 2 , 1 MgCl 2 solution. Residual wash solution is aspirated and the cells in the dry wells are solubilized in 0.5 ml of 25 mM NaOH with 0.5% deoxycholate.
  • a 50 ⁇ l sample from each well is used to measure protein and 400 ⁇ l sample is counted in a liquid scintillation counter.
  • Quadruplicate 10 ⁇ l samples of the influx solution are counted contemporaneously with the flux samples for specific activity determination.
  • the pmol/ ⁇ g protein in each well is calculated and the slope of the linear least squares best fit to these values against sample times is the computed flux, according to Sarkadi, B. et al., J.Gen.Physiol.72 249 (1978).
  • 5 or 6 of the data points are used to calculate each flux.
  • Each condition is always measured in duplicate in both vector-only transfected and vector+insert transfected cells.
  • the hPiT-1 DNA was isolated, cloned and sequenced according to 5,414,076, herein incorporated by reference for this purpose. It is set forth as SEQUENCE LD NO.:l and SEQUENCE ID NO.:2.
  • the hPiT-2 DNA was isolated, cloned and sequenced according to 5,550,221, herein incorporated by reference for this purpose. It is set forth as SEQUENCE LD NO.:3 and SEQUENCE LD NO.:4.
  • BNPI DNA was isolated, cloned and sequenced according to Ni, B et al., J.Neurochem.66, 2227 (1996), herein incorporated by reference for this purpose. It is set forth as SEQUENCE LD NO.:5 and SEQUENCE ID NO.:6.
  • Polyclonal antibodies are prepared according to England, B.J. et al.,
  • hPiT-1 RESTRCITION LENGTH FRAGMENT POLYMORPHISM ANALYSIS A.
  • primers for hPiT-1 for example, CAGTTCAGTC AAGCCGTCAG ( SEQ ID NO : 7 ) and CCAGCCAACA GACACAACAG ( SEQ ID NO : 8 )
  • the hPiT-1 sequence is amplified by PCR and ASO, by the methods of Connor, B.J.et al., Proc.Natl.Acad.Sci. 80: 278 (1983), and Saiki, R.K. et al., Nature 324: 163
  • hPiT-2 sequence is amplified by PCR and ASO, by the methods of Connor, B.J.et al., Proc.Natl.Acad. Sci. 80: 278 (1983), and Saiki, R.K. et al., Nature 324: 163
  • BNPI sequence is amplified by PCR and ASO, by the methods of Connor, B.J.et al., Proc.Natl.Acad.Sci. 80: 278 (1983), and Saiki, R.K. et al, Nature 324: 163
  • CTCCGTGGGA GCCAATGATG TAGCAAATTC TTTTGGTACA GCTGTGGGCT CAGGTGTAGT 540
  • AGCACTTTCC AGAGCAAACT CACTGTTTAC AAGAACTCTT GGCCTTACGA AGTTTATAAC 180
  • CTCCGGGCAC TCCCAGTATT CTATGCTGCT ACCATAGCAA TCAATGTCTT TTCCATCATG 840
  • CTCCGCCGAA ACAACAGTTA CACCTGCTAC ACCGCAGCCA TTTGTGGGCT GCCAGTGCAC 1440
  • GGCTCCATCC CACACTGTTC ACCCAGGCTG CGGAGACTCA CCTTCCCGAG CTAACTTAAC 2400 TACTGTACAT AATAATATGT ATTAAACTGG TATCGTGGTG ATATAATGTG GTGCAGTTAC 2460
  • CCGCCGCATC ATGTCCACCA CCAACGTGCG CAAGTTGATG AACTGCGGAG GCTTCGGCAT 1620
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FEATURE
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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Abstract

The sodium-phosphate cotransporter existing on virtually every human cell is identified as the same protein as the lithium-sodium countertransporter, and is suitable for diagnostic assays for mental illnesses susceptible to lithium therapy, including manic depression.

Description

SODiIUM-PHOSPHATE COTRANSPORTER
JN LITHIUM THERAPY FOR THE TREATMENT OF
MENTAL ILLNESS
BACKGROUND OF THE INVENTION
The subject matter of this present invention was developed in part by one or more grants of the United States Government, NIH HL-28674 and NIH HL-08989. Na-PO4 cotransport is the primary mechanism for the regulation of total body phosphate balance. It mediates both the gastrointestinal uptake and renal reabsorption of PO . Whereas 70% (high phosphate diet) to 85% (low phosphate diet) of dietary phosphate is absorbed by the GI tract the major control is in proximal reabsorption by the kidney of about 70% of the filtered load and in distal discretionary reabsorption of some fraction of the remainder. Na-PO4 cotransport has been found in the plasma membrane of every mammalian cell examined. In cell membranes it is the principal mechanism for PO4 uptake against the usually negative membrane potential and makes the cytoplasmic and extracellular concentrations approximately equal (±3 -fold). Intracellularly, phosphate metabolism includes most important biological molecules: nucleotides, DNA, RNA, glycolytic intermediates, phospholipids, and most proteins through regulatory or structural phosphorylations. Within the mitochondrial membrane Na-PO4 cotransport is essential for ATP synthesis.
Na-PO4 cotransporters exist in the kidney, as two isoforms, type I and type II. A related cotransporter is also present in liver where the protein has been partially purified, reconstituted in liposomes, and expressed in oocytes from liver mRNA. Its function in rat hepatocytes in primary culture is stimulated by insulin. Na-PO cotransport activity has been extensively characterized in the duodenum/upper jejunum of many higher vertebrates. Neither the liver nor intestinal forms of the cotransporter have been cloned from mammalian sources. In contrast to mammals, teleosts appear to have an isoform of the Type II transporter present in the intestine. Alterations in intestinal P; reabsorption appear to be related to 1,25-dihydroxy-vitamin D3 status and/or dietary P; intake. Recently a brain-specific cDNA, designated BNPI, has been cloned that appears to encode a Na-PO cotransporter and is 32% identical to the rabbit renal cotransporter, NaPi-1. The brain transporter is specific to the brain and mRNA transcripts are found in the neurons of the cerebral cortex, hippocampus, and cerebellum. The neuronal transporter is also found in peripheral nerves and transports arsenate and Li+ can substitute for Na+ . Evidence suggests at least three, possibly four, distinct isoforms of the cotransporter, the renal types I and II, as well as the brain- specific form which may represent a special type. The erythrocyte form represents a third type of Na-PO4 cotransporter, which applicants have discovered is also a retroviral receptor.
Retroviruses require specific cell-surface receptors for cell recognition and infection. Two widely expressed mammalian retrovirus receptors PiT-1 (Glvr-1; Genbank L20859, U.S.Patent 5,414,076) and PiT-2 (Ram-1; Genbank L19931, U.S. Patent 5,550,221) have been cloned and shown to share 30% homology with Pho-4+', a phosphate uptake gene in Neurospora crassa and when these two mammalian genes are expressed in oocytes they induce sodium dependent phosphate cotransporter activity. Also, a murine cationic amino acid transporter has been shown to be a retrovirus receptor, thus, indicating there are at least two classes of transporters that are retrovirus receptors. The two sodium phosphate cotransporters/retrovirus receptors (PiT-1 and PiT-2) are widely expressed in tissues and cells (thymus, marrow, lung, liver, heart, kidney, muscle and brain) and appear to be the ubiquitous housekeeping sodium-phosphate cotransporters that every cell requires in order to maintain the intracellular concentration of phosphate above electrochemical equilibrium. Furthermore the cotransporter/receptor isoforms in different species (human and mouse; rat and hamster) together with the differences in retroviral envelope proteins define the species specificity for susceptibility to infection by each retrovirus. The ability to transfect cell lines from one species with the transporter/receptor isoform from another species and/or alter the envelope protein provides novel model systems and the means to design vectors that allow increased gene transfer into human hematopoietic progenitor cells and other cells. Two sodium-phosphate cotransporters, PiT-1 and PiT-2, are found in most cells. A third cotransporter BNPI There are different isoforms of these three genes in different people. cDNA and cRNA probes to PiT-1 or to PiT-2 and their mRNA products and antibodies to these proteins distinguish between individuals who are responders or non-responders to lithium treatment. Applicants have discovered that the sodium-phosphate cotransporter is the same cell membrane protein as the lithium-sodium countertransporter. This discover - has important implications for the diagnosis and therapy of patients in need of lithium for the treatment of manic depression. The present invention provides a readily performed diagnostic test to evaluate patient status, by measuring a combination of sodium, phophate or lithium flux in an in vitro membrane-based translation system. Applicants have identified the gene product of PiT-1 as the lithium-sodium countertransporter across cell membranes. The PiT-1 gene product is the erythrocyte isoform. Probes for this gene distinguish between responders and non-responders to lithium treatment. Applicants have also identified the lithium-sodium countertransporter as the physiological mechanism for the extrusion of lithium from cells. It regulates the cell concentration of lithium. The activity of this transporter determines the therapeutic effect of lithium. This invention provides a simple molecular biological test for the ability of cells to extrude lithium. Presently, the only test to determine the activity of a lithium transporter is a laboratory measurement of lithium flux into or out of cells using chemical assays for lithium. See, e.g., Sarkadi, B. et al., J. Gen.Physiol.72 : 249 (1978).
The lithium-sodium countertransporter has significance for determining the responsiveness of humans with mental disorders to treatment with lithium salts. At present about half of patients treated with lithium do not improve. There are no techniques at present to diagnose whether a patient will be helped by lithium treatment, except by a time-consuming therapeutic trial. The diagnostic test of the present invention allows genetic screening to predict whether a patient will respond to lithium transport. The test is also a screen for susceptibility to and extent of manic depressive illness. Further, the test is suitable to screen newborns in families with depression for their potential to develop the illness and whether they can respond to lithium treatment.
BRIEF DESCRIPTION OF THE INVENTION
The sodium-phosphate cotransporter is identified as the same protein as the lithium-sodium countertransporter, and is suitable for diagnostic assays for mental illnesses susceptible to lithium therapy, including manic depression. Various methods for evaluating the flux of lithium and other cations in appropriate cells are also disclosed, including reticulocytes.
DESCRIPTION OF THE FIGURES
Figure 1 is a schematic diagram of a cell 1 showing that the sodium-phosphate cotransporter is the same cell membrane protein as the sodium-lithium countertransporter.
DEFINITIONS AND ABBREVIATIONS
Pi Concentration of inorganic phosphate DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a purified DNA molecule coding for a lithium-sodium countertransporter. It also relates to a purified DNA molecule coding for an amino acid sequence selected from the group consisting of hPiT-1, hPiT-2, and hBNPI, said molecule useful for measuring lithium-sodium countertransport in human cells. Specifically, the present invention relates to a novel utility for the sequences identified as SEQ.ID.NO.: 1, SEQ.LD.NO.: 2, SEQ.LD.NO.: 3, SEQ.LD.NO.: 4, SEQ.LD.NO.: 5, and SEQ.LD.NO.: 6. In one embodiment of the present invention, applicants show that the human amphotrophic retrovirus receptor is useful as a lithium-sodium countertransporter, including the sequences identified as SEQ.LD.NO.: 1, SEQ.LD.NO.: 2, SEQ.LD.NO.: 3, SEQ.LD.NO.: 4, SEQ.LD.NO.: 5, and SEQ.LD.NO.: 6.
In another embodiment of the present invention, there is provided a first method of evaluating sensitivity to lithium therapy in manic depressive patients, comprising the steps of
(a) providing a sample of patient blood;
(b) extracting from the blood sample the patient's DNA;
(c) subjecting the DNA to hybridization with primers specific for any sequence coding for lithium-sodium countertransporter;
(d) polymerizing said sequences, to give polymerized sequences;
(e) amplifying said polymerized sequences, to give an amplified sample of patient sequences;
(f) digesting the amplified sample with one or more restriction endonucleases suitable for mapping sites on the DNA indicating susceptibility to lithium therapy.
In another embodiment of the present invention, there is provided a second method of evaluating sensitivity to lithium therapy in manic depressive patients, comprising the steps of (a) providing a sample of patient blood;
(b) extracting from the blood sample the patient's DNA;
(c) subjecting the DNA to hybridization with primers specific for any sequence coding for lithium-sodium countertransporter; (d) polymerizing said sequences, to give polymerized sequences;
(e) amplifying said polymerized sequences, to give an amplified sample of patient sequences;
(f) subjecting the amplified sample to in vitro membrane-based translation to give a translated sample within a cell; and (g) subjecting the translated sample to flux analysis of lithium, to evaluate sensitivity to lithium therapy in manic depressive patients.
Specifically, the first and second methods are drawn to sequences to any lithium-sodium countertransporter selected from the group consisting of hPiT-1, hPiT- 2, and hBNPI, said sequences identified as SEQ.LD.NO.: 1, SEQ.LD.NO.: 2, SEQ.LD.NO.: 3, SEQ.LD.NO.: 4, SEQ.LD.NO.: 5, and SEQ.LD.NO.: 6.
In another embodiment of the present invention, there is provided a third method of evaluating sensitivity to lithium therapy in manic depressive patients, comprising the steps of
(a) providing a sample of patient blood; (b) isolating the erythrocytes;
(c) subjecting the erythrocytes to flux analysis of lithium, to evaluate sensitivity to lithium therapy in manic depressive patients.
In another embodiment of the present invention, there is provided a fourth method of evaluating lithium-sodium countertransport in patients with mental illness, comprising the steps of
(a) providing a sample of patient blood;
(b) isolating the erythrocytes; (c) subjecting the erythrocytes to flux analysis of lithium, to evaluate lithium- sodium.
Phosphorus is a major dietary element essential to most important biological molecules. Its absorption from the gut and reabsorption from the glomerular filtrate is by secondary active transport on a family of Na-PO4 cotransporters. The gene product responsible for this function in erythrocytes is pharmacologically distinct from the previously characterized renal brush border Na-PO cotransporter. Both the brain and peripheral nerve forms (PiT-1, PiT-2, BNPI) and the red blood cell form of the Na- PO4 cotransporter can use Li+ as a congener for Na+. Also, arsenate is transported by nerve membranes and probably by red blood cells. Therefore, these two tissues most likely have the same cotransporter/receptor. The renal Na-PO4 cotransporter, presumably the apical isoform, has been cloned from several species. Applicants have identified the erythrocyte isoform as PiT-1.
Figure 1 schematically shows a simplified diagram, with cell 1. Lithium cation, Li+ enters the cell as the anion LiCO3 ", by the action of the AEl (Anion Exchange Protein, Band 3) countertransporter. Alternatively, the lithium cation leaks into the cells by a minor unknown leak pathway. Lt is pumped out by the sodium-phosphate cotransporter, which applicants have identified to be also the lithium-sodium countertransporter.
1. Manipulations of DNA for the Preparation of Expression Systems and Other
Purposes
Following well known and conventional practice, the hPiT-1 gene or other coding sequences for the lithium-sodium countertransporter are prepared for the expression systems and diagnostic assays of the present invention. These polynucleotide sequences are prepared by ligation of other sequences, restriction endonuclease digestion, cloning, mutagenesis, organic synthesis, or combination thereof, in accordance with the principles and practice of constructing DNA sequences. For sequencing DNA, e.g., verification of a construct at the end of a series of steps, dideoxy DNA sequencing is the preferred method. Other DNA sequencing methods are well known.
Many treatises on recombinant methods have been published, including J. Sambrook et al., Molecular Cloning: A Laboratory Manual 1989; L.G.Davis et al., Basic Methods in Molecular Biology Elsevier 1986; F.M. Ausubel, et al (eds.), Current Protocols in Molecular Biology, Wiley Interscience 1994 (loose-leaf). Such methods include plasmid purification, RNA isolation, Northern blots, Southern blots, Western blots, gel electrophoresis, cDNA library construction, DNA sequencing, amplification by the polymerase chain reaction, cell free translation of mRNAs, and ligation. Phosphoramidite chemistry in solid phase is the preferred method for the organic synthesis of oligodeoxynucleotides and polydeoxynucleotides. Many other organic synthetic methods are available and are readily adapted to the particular sequences of this invention by a person skilled in the art.
Amplification of DNA or cDNA is a common step in the detection of specific sequences in the diagnostic tests of the present invention. It is typically performed by the polymerase chain reaction (PCR). See, e.g., Mullins, K. et al., U.S. Pat. No.
4,800,159 and other published sources. The basic principle of PCR is the exponential replication of a DNA sequence by successive cycles of primer extension. The extension products of one primer, when hybridized to another primer, becomes a template for the synthesis of another nucleic acid molecule. The primer template complexes act as substrate for DNA polymerase which, in performing its replication function, extends the primers. The region in common with both primer extensions, upon denaturation, serves as template for a repeated primer extension. The conventional enzyme for PCR applications is the thermostable DNA polymerase isolated from Thermus aquaticus, or Taq DNA polymerase. Numerous variations in the PCR protocol exist, and a particular procedure of choice in any given step in the constructions of this invention is readily performed by a skilled artisan. For example, primers for hPiT-1 are organically synthesized, based on its known sequence, and are hybridized to a sample of patient DNA. PCR in combination with reverse transcriptase, so-called RT-PCR, is then carried out to amplify the patient hPiT-1 genes. Subsequent analysis, e.g., by restriction fragment length polymorphism (RFLP), provides information on patient status. 2. Translation of mRNA. Various techniques have been developed to synthesize or isolate large quantities of capped eukaryotic mRNAs, and are readily adaptable to mRNA coding for hPiT-1 and related sequences. Preferably the source for mRNA is derived from enzymological manipulations, rather than isolation of naturally transcribed mRNA from, e.g., cell lines such as the erythroleukemic cell line K562. Synthetic capped mRNA is preferably prepared by in vitro transcription of the appropriate linearized cDNA constructs containing the appropriate promoter for an RNA polymerase, e.g., T7 RNA polymerase. See, e.g., Fletcher, L. et al., J.Biol. Chem.265 : 19582 (1990), herein incorporated by reference for these purposes. Under these conditions, high yields of capped mRNA coding for hPiT-1, hPiT-2 or BNPI sequences are obtained, which migrate as a discrete band in gel electrophoresis.
The capped mRNA is then subjected to in vitro membrane-based translation, e.g., in Xenopus oocytes, microsomes or cultured cells, in an expression system designed to permit flux analysis of Na, PO4, and Li. Preferred expression systems include Xenopus oocytes, and transfected HEK 293 cells. Other suitable transfection systems include Dictyostelium discoideum cells, baculovirus-infected ceected Sf9 cells, and CHO cells. Selection of the appropriate cell system, as well as adjusting the experimental parameters to enhance translation, is readily determined within the skill of the art.
3. Construction of expression vector.
The gene for the countertransporter proteins, such as the hPiT-1 gene, is also suitable for expression in an expression vector in a recombinant expression system. Of course, the constructed sequence need not be the same as the original, or its complimentary sequence, but instead may be any sequence determined by the degeneracy of the DNA code. Conservative amino acid substitutions may also be employed, or other modifications, such as an amino terminal methionine.
A ribosome binding site active in the host expression system is Hgated to the 5' end of the chimeric coding sequence, giving a synthetic gene. The resulting synthetic gene can be inserted into any one of a large variety of vectors for expression, by ligating to an appropriately linearized plasmid. Expression in E. coli is suitable for expression of active lithium-sodium countertransporter protein, e.g., E. coli BL21. A regulatable promoter is also suitable for the expression of these coding sequences, e.g., under the control of the E. coli lac promoter. Other suitable regulatable promoters include trp, tac, recA, T7, lambda promoters.
4. Diagnostic Assays to Measure Lithium-Sodium countertransport
The flux of a molecule is a measure of the number of molecules that cross the cell membrane per unit time and per unit of membrane (expressed either as area or number of cells or amount of cell protein). The flux is measured by determining the appearance or disappearance (or both) of the molecule on one side of the membrane. The amount on one side of the membrane is measured at different known times either by a chemical determination or by a radioactive determination if a tracer of the atoms or molecules is used. Lithium, atomic number 3, atomic weight 6.9 Daltons, has no radioactive isotopes of use for biological measurements. Chemical detemination must be used instead of a radioisotope. The amount of lithium is most often determined by atomic absorption spectroscopy or emission spectroscopy. The assay of lithium-sodium countertransport flux rate is made by the following steps:
1) a sample of whole blood, e.g. 10 ml, is taken from the patient by venipunture;
2) the cells are mixed in a standard buffered solution containing sodium and lithium chloride solution, and subjected to repeated suspension, centrifugation, removal of supernatant fluid, and resuspension;
3) the cells in the standard solution are incubated in the presence of inhibitors of the Na, K, ATPase (e.g., ouabain at 10"5 M) and in the presence of inhbitors of Anion Exchange protein (e.g., denitrostilbenedisulfonate at 2.5 x 10"4 M), in suspension at body temperature;
4) at given known times samples of cells are removed, cooled on ice to slow the further transport of lithium, then washed 3 times by centrifugation, aspirated to remove supernatant and resuspended in an ice cold lithium-free solution, to give washed cells; 5) the washed cells are lysed with lithium-free water;
6) aliquots of lysed cells are taken and diluted if necessary to measure hemaglobin [(van Kampen, E.J. et al., Clin.Chim.Acta 6:538 (1961)] and lithium by flame spectroscopy; and
7) the flux equals the change in lithium per g hemoglobin between samples from the same suspension, divided by the time between samples. 5. Genetic Screening Tests
A variety of methods exist for the evaluation and screening of human DNA sequences obtained as patient samples, for the purpose of patient evaluation. See generally, Caskey, C.T., Science 236: 1223 (1987); Bloch, W., Biochemistry 30:2735 (1991); Erlich, H.A et al., Science 252: 1643 (1991).
In the classic analysis of polynucleotide sequences by the technique of restriction fragment length polymorphism (RFLP), natural variations in DNA are detected by digestion of DNA, whether or not amplified, with a selected set of restriction endonucleases. The polymorphism need not overlap the site of etiological origin to be evaluated and tested, e.g., the PiT-1 gene, but instead may be a neighboring region linked thereto, e.g., linkage disequilibrium. In one modification of RFLP, a single base pair mutation of a DNA coding strand affects its digestion by a selected restriction endonuclease, and its presence is readily detected by the appropriate primers and PCR (polymerase chain reaction). These types of analytical methods are advantageous because there is no need for a hyribidization reaction of target to labeled probe.
In another technique, known as oligonucleotide complementarity, allele-specific ohgonucleotides (ASO) are synthesized for a variety of purposes. These ohgonucleotides are useful for either directly hybridizing to target DNA under specific stringency conditions, or for priming in vitro amplification by the polymerase chain reaction.
Tagging or labeling the desired polynucleotide fragments can take various forms. The radioisotope 32P and other radioactive labels are not preferred because of laboratory safety and waste disposal requirements. Alternative methods of labeling include chemical analogs, such as biotinylated analogs of TTP and UTP, which incorporate into the resulting DNA and RNA, respectively. The biotin-labeled probe can be coupled to avidin, or streptavidin, and the complex detected by chemiluminescence, immunofluorescence, immunoperoxidase, immune colloidal gold techniques, or the like. The biotin-labeled probe can also be detected with avidin conjugated to poly AP (calf intestinal alkaline phosphatase), assayed with the appropriate AP substrates. Digoxigenin is a useful substitute for avidin in many applications, and it is readily detected with antibodies specific for digoxigenin. Various combinations of such labels are readily carried out, e.g., a biotin-labeled probe detected with streptavidin conjugated to poly AP, or a biotin labeled probe detected with anti- biotin antibodies linked to AP, or other secondary labeling systems. 6. Preparation of antibodies specific for the lithium-sodium countertransporter protein, and allelic variants thereof. Monoclonal antibodies are the reagent of choice in the present invention, and a specifcally used to analyze patient cells for specific characteristics of the lithium-sodium countertransporter. Monospecific antibodies to the lithium-sodium countertransporter are purified from mammalian antisera containing antibodies reactive against the lithium- sodium countertransporter or are prepared as monoclonal antibodies reactive with the lithium-sodium countertransporter using the technique of Kohler and Milstein, Nature 256: 495-497 (1975). Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for the lithium-sodium countertransporter. Homogenous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with the lithium-sodium countertransporter, as described above. The lithium-sodium countertransporter specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats* horses and the like, with an appropriate concentration of the lithium-sodium countertransporter either with or without an immune adjuvant. Preimmune serum is collected prior to the first immunization. Each animal receives between about 0.1 mg and about 1000 mg of the lithium-sodium countertransporter associated with an acceptable immune adjuvant. Such acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium parvum and tRNA. The initial immunization consists of the lithium-sodium countertransporter in, preferably, Freund's complete adjuvant at multiple sites either subcutaneously (SC), intraperitoneally ( P) or both. Each animal is bled at regular intervals, preferably weekly, to determine antibody titer. The animals may or may not receive booster injections following the initial immunization. Those animals receiving booster injections are generally given an equal amount of the antigen in Freund's incomplete adjuvant by the same route. Booster injections are given at about three week intervals until maximal titers are obtained. At about 7 days after each booster immunization or about weekly after a single immunization, the animals are bled, the serum collected, and aliquots are stored at about -20° C.
Monoclonal antibodies (mAb) reactive with the lithium-sodium countertransporter are prepared by immunizing inbred mice, preferably Balb/c, with the lithium-sodium countertransporter. The mice are immunized by the LP or SC route with about 0.1 mg to about 10 mg, preferably about 1 mg, of the lithium-sodium countertransporter in about 0.5 ml buffer or saline incorporated in an equal volume of an acceptable adjuvant, as discussed above. Freund's complete adjuvant is preferred. The mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks. Immunized mice are given one or more booster immunizations of about 0.1 to about 10 mg of the lithium-sodium countertransporter in a buffer solution such as phosphate buffered saline by the intravenous (IN) route. Lymphocytes, from antibody positive mice, preferably splenic lymphocytes, are obtained by removing spleens from immunized mice by standard procedures known in the art. Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions which will allow the formation of stable hybridomas. Fusion partners may include, but are not limited to: mouse myelomas P3/ΝSl/Ag 4-1; MPC-11; S-194 and Sp 2/0, with Sp 2/0 being preferred. The antibody producing cells and myeloma cells are fused in polyethylene glycol, about 1000 mol. wt., at concentrations from about 30% to about 50%. Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art. Supernatant fluids are collected from growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay such as solid phase immunoradioassay (SPIRA) using the lithium-sodium countertransporter as the antigen. The culture fluids are also tested in the Ouchterlony precipitation assay to determine the isotype of the mAb. Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press, 1973.
Monoclonal antibodies are produced in vivo by injection of pristane primed Balb/c mice, approximately 0.5 ml per mouse, with about 2x106 to about 6x106 hybridoma cells about 4 days after priming. Ascites fluid is collected at approximately 8-12 days after cell transfer and the monoclonal antibodies are purified by techniques known in the art.
In vitro production in anti-lithium-sodium countertransporter mAb is carried out by growing the hydridoma in DMEM containing about 2% fetal calf serum to obtain sufficient quantities of the specific mAb. The mAb are purified by techniques known in the art.
Antibody titers of ascites or hybridoma culture fluids are determined by various serological or immunological assays which include, buy are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of the lithium-sodium countertransporter in body fluids or tissue and cell extracts.
It is readily apparent to those skilled in the art that the above described methods for producing monospecific antibodies may be utilized to produce antibodies specific polypeptide fragments of the lithium-sodium countertransporter, or full-length nascent lithium-sodium countertransporter polypeptide, or variants or alleles thereof.
7. Manic Depression and Other Affective Disorders
The classification of mental illness is fluid and subject to further adjustments and refinements. Two distinct types of mental illness are schizophrenic disorders and affective disorders. Schizophrenic disorders are mental diseases with a tendency toward chronicity and are characterized by psychotic symptoms involving disturbances of thinking, feeling, and behavior. Affective disorders, also known as mood disorders, are psychopathologic states in which a disturbance of mood is either a primary determinant or constitutes the core manifestation. A clinically useful division of affective disorders is bipolar (with periods of depression and elevation) and unipolar (depressions only) mood disturbances. Such bipolar mood disturbances are commonly known as manic depression.
Lithium, usually given as a carbonate salt, attenuates bipolar mood swings, without affecting normal mood. It also appears to be useful in the treatment of aggressive personality disorders, which are typically classified outside of affective disorders. About 50% of bipolar patients respond to lithium therapy. Various clinical attributes are useful in assessing response to lithium, including the presence of manic episodes as the primary mood disorder, an episode frequency of less than about 2 years, as well as past or family history of lithium response. Applicants now provide another attribute to evaluate response to lithium, that is, lithium-sodium countertransport.
8. Lithium Flux Mechanisms
Lithium is commonly used to treat affective disorders. The site of action of lithium is believed to be in the brain. The steady state ratio of intracellular red blood cell lithium concentration to plasma lithium concentration during therapy shows great interindividual variation, although the lithium ratio is relatively constant for any one individual. Individual fluctuations of the lithium ratio have also been reported. The relative constancy of the ratio in an individual may be genetically determined. The steady state lithium ratio across the red cell membrane is the result of three lithium transport processes: the Na,K,ATPase which is inhibited by ouabain and other cardiac glycos des, the anion exchange protein (AEl, band 3) and the lithium-sodium countertransport system. The Na, K, ATPase pumps Li into the cell by substituting Li+ at a normal K+ binding site, but at therapeutic levels of Li+ (l-2mM) and normal plasma Na and normal plasma K, the Na,K ATPase carries Li poorly («0.025 mmol/lit cell • h; «75 μmol/kg Hgb • h). In plasma like media with 24 mM bicarbonate, the anion exchanger is the principle mediator of the inward leak of Li as the ion pair LiCO3 ". This transport is inhibited by stilbene disulfonates (SITS, DNDS, DLDS), phloretin and dipyridamol. The lithium-sodium countertransporter normally pumps Li out of the cell against its electrochemical gradient so that [Li]ceιι is lower than [Li]pι and [Li]ceιι/[Li]pι, which lithium ratio is 0.2 to 0.8 in different individuals. A higher steady ratio is expected in alkalosis (higher plasma HCO3 ", CO3 2", and LiCO3 ') due to increased AEl mediated leak into the cell, or when the lithium-sodium countertransport extrusion of lithium is slowed, either because cell Na+ gradient is decreased or because the countertransporter is less effective.
There is evidence that the differences in the steady state ratio are principally due to differences in the activity of the Na/Li exchanger (lithium sodium countertrnsporter). For example, there is a correlation between the Li influx on the lithium-sodium countertransporter (which is reversible and will run backward given a reversed Na gradient) and the steady state ratio. Also, the steady state Na ratio does not correlate with the steady state Li ratio in different donor cells after 24 hr in vitro. Thus the "tightness" of the Na/Li coupling varies among individuals.
Applicants have identified the lithium-sodium countertransporter as the product of PiT-1 gene previously identified as retrovirus receptor and a NaPO4 cotransporter. Applicants have shown that the red cell NaPO4 cotransporter transports Li instead of Na (i.e., LiPO4 cotransport) and that is performs Na/Na exchange and lithium-sodium countertransport. The lithium ratio has been implicated in the responsiveness of polar disease to lithium treatment, the development of essential hypertension (hypertension of unknown etiology), the'susceptibility of individuals to affective (bipolar) disorders, and the toxic side effects of Li therapy.
EXAMPLE 1
KLNETIC EVIDENCE THAT THE SODIUM-PHOSPHATE COTRANSPORTER IS THE MAJOR MOLECULAR MECHANISM FOR Na-Li EXCHANGE LN HUMAN RED BLOOD CELLS. Lithium influxes, 32PO influxes, sodium effluxes were measured in human red blood cells incubated in an isotonic media containing (mM): 150 (Na + Li + K)C1, 0.3 K2HPO4, 20 HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), 0.25 DNDS (4,4'-dinitro stilbene-2,2'-disulfonate to inhibit the Anion Exchange Protein Band3 pathway for phosphate transport), titrated to pH 7.64 with KOH at 20°C to give a pH of 7.40 at 37°C where the fluxes were performed. First, external lithium (in the absence of sodium) activated phosphate influx. Lithium activation of phosphate influx was increased by 1, 7.5 and 75 mM external sodium. Second, external lithium stimulated Na efflux in the presence of 10"5 M ouabain (an inhibitor of the Na-K pump) and was further stimulated by external phosphate. These results indicate that the majority of sodium efflux is on the sodium-phosphate cotransporter. Third, external phosphate concentrations slightly inhibited lithium influxes at low (0.1 -0.3 mM) phosphate concentrations. Fourth, arsenate inhibited sodium-phosphate cotransport in red blood cells with a Ki of 5.2 mM, more than 10 fold greater than in HEK-293 cells, which have a renal ype II sodium-phosphate cotransporter. The collective results indicate that a mechanism for Na-Li exchange on the sodium-phosphate cotransporter. Phosphate is likely to be an important regulator of lithium transport and its therapeutic effects. EXAMPLE 2
KINETIC CHARACTERIZATION OF SODIUM-PHOSPHATE COTRANSPORTER LN THE ERYTHROLEUKEMIC CELL LINE K562: LDENTLFICATION OF THE ERYTHROCYTE SODLUM PHOSPHATE COTRANSPORTER AS hPiT- 1.
Na-dependent 32PO4 influx into the erythroleukemic line of K562 cells was measured. The 32PO4 influx was linear with time of over 30 minutes and was activated over 100-fold by 140 mM Na compared to isomolar substitution by 140 mM N-methyl- D-glucamine. The activation of 32PO4 influx by extracellular phosphate (pH 7.4 at 37° C) was hyperbolic with a Km P04 = 0.36mM and Vmax = 4500 nmol PO^g protein - min in 140 mM Na+. The
Figure imgf000021_0001
40 mM when PO40 was 0.3 mM. There was no activation of phosphate influx by Rb, K, or Cs. However, 140 mM Li activated phosphate influx to 18.7% of that realized in the presence of 140 mM Na. The Ki value for arsenate inhibition of Na-dependent 32PO4 influx K562 cells was 2.6 mM. These and other kinetic characteristics of sodium-phosphate cotransport in K562 cells are identical to those previously described for human erythrocytes. See Shoemaker et. al., J. Gen. Physiol, 92:449 (1988).
EXAMPLE 3
MOLECULAR LDENTLFICATION OF THE SODLUM-PHOSPHATE COTRANSPORTER LN ERYTHROLEUKEMIC CELL LINE K562 AND
ERYTHROCYTES AS HPIT-1.
Human PiT-1 (hPiT-1) was cloned as the human isoform of the gibbon ape retrovirus receptor [Van Zeijl, M., et al (Proc. Nat. Acad. Sci. 91 : 1168 (1994)]. PCR primers were designed to amplify either the hPiT-1 or hPiT-2 isoforms. The 1700 bp product was amplified by RT-PCR from total RNA isolated from K562 cells, and restriction analysis with Sphl identifies the product as being derived from hPiT-1 and not hPiT-2. This evidence, considered together with the kinetic evidence of preceeding Example, indicates that hPiT-1 is the sodium-phosphate cotransporter isoform present in both K562 cells and erythrocytes.
RT-PCR was carried out using 1 μ g of total RNA isolated from K562 cells using the method of Chomczynski et al., Analytical Biochem. 162: 156 (1987). The RT-PCR reaction was carried out in a single tube using recombinant Tth DNA- polymerase which is capable of reverse transcriptase activity under appropriate reaction condition. The primers used in these experiments (FI and Rl) are based upon highly conserved regions between hPiTl and hPiT2 located in putative transmembrane domains in the N-terminal and C-terminal regions of the proteins. The results from these experiments show that the products of Sphl digestion are 1000, 487 and 138 bp, which agree with the predicted sizes. The gel patterns under the PiT-1 control after Sphl digestion are the same as for K562, after Sphl digestion; and both are different from those predicted for PiT-2 after Sphl digestion. Thus K562 cells have the human PiT-1 isoform.
EXAMPLE 4
SYNTHESIS OF mRNA FOR CELL-FREE TRANSLATION AND XENOPUS OOCYTE-INJECTION EXPERIMENTS.
The template for expression of the Na-PO4 cotransporter in the cell-free system or in Xenopus oocytes is capped mRNA prepared polymerase, prepared by in vitro transcription of linearized cDNA constructs containing the promoter for T7 RNA polymerase. Samples of mRNAs are prepared as prepared as previously described in a 1 ml reaction mixture [Fletcher, L. et al., J. Biol. Chem.265; 19582(1990)]. These reaction conditions permit high yields of mRNA (-1000 mg or about 100-200 copies of RNA transcript per copy of template DNA) that are >80% capped and migrate as a single discrete band of the correct molecular weight on denaturing PAGE. T7 RNA polymerase is purified as described by Davanloo, et al. Proc. Natl.Acad.Sci.Sl;,2035(l9S4). EXAMPLE 5
CELL-FREE EXPRESSION OF NA-PO4 COTRANSPORTER
The Na-PO cotransporter mRNA is expressed in a wheat germ cell-free system containing in vitro transcribed mRNA, dog pancreatic microsomes and signal recognition particle (SRP). Cell-free translation is carried out in the presence of 34 mM [14C]leucine (50 cpm/pmol), 50 mM each of the other 19 amino acids, unfractionated wheat germ tRNA and other components a previously described [Erickson, AH et al., Methods Enzymol. 96;38 (1983)].
Assuming each Na-PO4 cotransporter polypeptide contains~75 leucine (hPiT-1 has 71), synthesis under these conditions yields Na-PO cotransporter with a specific activity of -3750 cpm/pmol. Wheat germ extract is prepared as described by Lax, S.R. et Methods Enzymol. 118;109 (1986), and dog pancreatic microsomes and SRP are prepared according to Walter, P. et al., Methods Enzymol.9&, 84-93, & 682- 691(1983). Under the appropriate conditions the wheat germ system provides excellent activity, e.g. 5 5 mol β-globin polypeptide is synthesized per mol β-globin mRNA template per hour. These data indicate that a 1 ml reaction containing 100 pmol mRNA synthesizes up to 50 mg Na-PO cotransporter protein (or nearly 500 pmol) per hour. The synthesis of Na-PO cotransporter is monitored by SDS-PAGE and fluorography to determine that the correct molecular weight polypeptide is produced. Translocation and glycosylation is assessed by endogycosidase H and endoglycosidase F treatment. Treated and untreated samples are analyzed by SDS- PAGE.
EXAMPLE 6 HETEROLOGOUS EXPRES SION OF THE NA-PO4 COTRANSPORTER
The expression of the the sodium-phosphate cotransporter is carried out in several heterologous expression systems, e.g. Dictyostelium discoideum cells, Xeonpus oocytes, HEK 293 cells, baculovirus-infected Sf9 cells, and CHO cells. Transfection, growth and selection of transformants are performed by well known techniques. The expression of the Na-PO4 cotransporter is assessed by immunoprecipitation with Na- PO4- specific antibodies of the protein from cells (Dictyostelium, HEK 293, etc.) grown in medium supplemented with 35S-methionine. The functional expression of the Na-PO transporter, both native and mutant forms, in transfected cells is monitored by determining the Na-dependent 32Na-PO4 flux , as described in an Example below. Negative controls include determining background levels of Na-dependent Na-PO4 transport from cells transfected with the construct in the anti-sense orientation. The two principal expression systems for heterologous expression of the erythrocyte Na- PO4 cotransporter are injected Xenopus oocytes and transfected Dictostelium. Alternatively or additionally, the cotransporter is expressed in another expression system such as HEK 293 or baculovirus-infected Sf9 cells.
A. Injected Xenopus oocytes. Stage V and IN oocytes are removed using standard anesthetic (0.17% 3-aminobenzoic acid) and surgical procedures. The oocytes are placed in OR-2 medium and collagenase treated (2mg/ml) for 2.5 h.
Individual oocytes are washed and defolliculated if needed by trituration and co-injected with 2.5 ng capped SEAP cRΝA and 5-50 ng of capped transporter of cRΝA (prepared as described above). Capped SEAP cRΝA prepared by in vitro transcription oϊHIndlll linearized pGEM-SEAP. The pGEM-SEAP construct contains the human placental alkaline phosphatase with a site-specific mutation at codon 489 to create a termination codon[Tate, S.S. et al., FASEB J. 4; 228 (1990)]. This stop codon results in a secreted form of alkaline phosphatase rather than a membrane anchored form. cRΝAs are injected in a total volume of 50 nl using a Νarishige injector. Following incubation overnight in Barth's medium, oocytes are sorted and placed in single wells of a
96 well plate containing 200 ul Barth's medium. Five hours after the oocytes are placed into individual wells, 50 ul of medium is removed for SEAP activity assay. Alkaline phosphatase activity is measured by chromogenic assay. The secreted alkaline phosphatase catalyzes the dephosporylation of nicotinamide adenine dinucleotide phosphate (NADP+); the NAD formed then catalytically activates an NAD+-specific oxidation-reduction cycle driven by the enzymes alcohol dehydrogenase and diaphorase. The chromophore formed is a violet colored formazan product of LNT-violet. Only those oocytes that express SEAP
(10 units activity/50 ul at 29 h post-injection) are used in the flux measurements. There is substantial correlation between the level of SEAP activity detected at 29 h post-injection and the level of 36C1 flux in oocytes co- injected with SEAP and human AEl or the level of Na-activated 32PO4 influx in oocytes injected with SEAP and mRNA for PiT- 1 , PiT-2, or BNPI. Those oocytes that are positive for SEAP expression are incubated for an additional 1- 5 days with daily changes in Barth's medium before influx assays are carried out.
B. Dictyostelium. A second expression system for heterologous expression of the cloned cotransporter is Dictyostelium. A significant advantage of the Dictyostelium expression system is that these cells are grown in suspension culture and are handled like red cells for flux measurements. The principle expression vector (e.g. pBS18 and its derivatives) is based upon selection using the Tn5 gene (neomycin phosphotransferase II) driven by the actin 6 promoter.
The insert of interest is driven by the actin 8 promoter and the 2H3 transcriptional terminator. The Tn5 gene permits selection of permanent transfectants in media containing G-418, to which the native slime mold is highly sensitive.
C. HEK-239 cells. Heterologus expression is also readily carried out with HEK- 293 cells, ATCC Accession No. CRL 1573. HEK-293 (human embryonic kidney) cells were obtained from the American Type Culture Collection (ATCC) at passage 31. These cells were used to prepare seed stocks at passage 32. Cells were used until passage 45, after which fresh cultures were started from frozen passage 32 cells. The cells were grown in Minimal Essential Media (MEM) with Hank's salts and supplemented with L-glutamine and 5% fetal calf serum at 37°C in 5% CO2 / 95% air. Transfection was carried out using standard calcium phosphate precipitation methods. Specifically, five days before the transfection, cells were plated in T75 flasks (75 cm2) at 2.5 x 104/ cm2. On the day of the transfection, the cell density was usually 2-3 x 105 / cm2. The cells were washed and fresh media (20 mL) was placed in each culture flask. A 1.0 mL suspension containing the calcium phosphate - DNA precipitate from 40 μg of plasmid DNA was added drop-wise with mixing to the media overlaying the cells. The cells were returned to the incubator for 4 h, then 2mL of 18% (v/v) glycerol was added to the media ("glycerol shocked"), and the cells incubated for an additional two minutes at room temperature. The media was then quickly aspirated from the flask, the cells washed one time with
25 mL Dulbecco's phosphate-buffered saline, fresh media added to the cells (25 mL) and the cells were incubated overnight. The next morning the cells were trypsinized by standard methods, resuspended to a final density of 1.5 - 1.7 x 105 and 1.0 mL of the cell suspension was used to replate the cells in 24-well plates (16 mm diameter wells) at a density of 8.0 x 104 cells/cm2 (1.6 x 105 cells
/ well). Flux measurements were carried out at 48 ± 6 hr post transfection.
EXAMPLE 7
FLUX MEASUREMENTS A. Flux measurements in cell-free expression system. The flux is measured by an adaptation of a rapid filtration method according to Macintyre, J.D. et al. Biochim. Biophys. Acta 644; 351 (1981). Briefly, microsomes are suspended, equilibrated and mixed in media containing either sodium or choline or N-methyl-alpha- glucamine as the dominant cation in a thermostatically controlled chamber. The flux is initiated by additional of 32PO to the flux medium. Aliquots are removed at different times and filtered under vacuum using prewashed mixed cellulose-ester filters. The microsomes retained by the filter are rapidly washed with stopping solution containing 323 mM MgSO4 (isotonic for microsomes). A sample of flux suspension is used to measure total protein and specific activity. The flux (pmol PO-Λig protein-min or ions/cotransporter molecule-min) is calculated from the slope of cpm/aliquot versus time. Preloaded microsomes are used to verify the quantitative recovery of microsomes, the replication of sample counts, and the effectiveness of the wash in removing a extracellular marker, usually 14C-PEG at
0°C. The probable 32PO4 influxes into microsomes are calculated assuming a single copy of the Na-PO cotransporter in each 0.05 um microsome, and using kinetic data from erythrocytes, assuming that there are 450 copies of the cotransporter per red blood cell. These calculations indicate that the half-times will be >10 h and are therefore measured by this technique (ty2 > 5 sec).
Flux measurements in oocytes. Oocytes are prepared and injected as described in Example above. Briefly, eight to ten oocytes are placed in individual wells of a 96- well culture plate in medium containing either Na or choline as the dominant cation. The flux is initiated by addition of 32PO4 to each well. As known times, oocytes are removed and washed three times in ice cold choline medium. The oocyte is then dissolved in 0.2 ml 10% SDS and the counted in a liquid scintillation counter. A sample of the 32PO4 incubation fluid is counted to calculate the extracellular specific activity. The influx (pmol/oocyte/hr) is calculated from the specific activity and the uptake. The difference between the flux in Na and choline media is the calculated
Na-dependent phosphate influx. C. Flux measurements in Dictyostelium discoideum. HL-5 medium contains 20 + 3 mM K. This is not a defined medium so the composition must be determined for each flux." -Cells are grown to a density of 1-3 x 106/ml at 20°C in a shaking incubator. Approximately 107 cells are required for each data point on the influx curve. The cells are resuspended to 4 x 107/ml in HL-5 in a thermostatted stirred chamber at a known pH. At time zero tracer (-0.6 μCi/ml) is added and at known times thereafter samples (0.4 ml) are removed. The samples are transferred to 7 ml of ice-cold stop solution (58 mM MgSO4), immediately centrifuged for 30 seconds at 3000 x g in a rotor, the supernatant aspirated and discarded as radioactive waste. The pellet of cells is resuspended thrice in 6 ml of stop solution, pelleted, and the supernatant aspirated. To the drained pellets 1 ml of 0.1% DOC in 1 N NaOH is added for solubilization. Aliquots are counted for radioactivity or are assayed for protein. The data are calculated as pmoles/mg protein and the data vs. time are fitted to a single exponential by nonlinear regression analysis and the inital slope (flux) and asymptote value constant (pmoles/mg protein) calculated.
D. Flux measurements in cultured mammaliam cells. Human embryonic kidney cells (HEK-293) are purchased from the American Type Culture Collection (ATCC, accession number CRL 1573) at passage 32 and grown in MEM and 5% fetal calf serum in a 5% CO /95% air 37°C incubator. They are maintained in T75 flasks and split weekly. At passage 45 decreased expression is observed, so frozen stocks at passage 34 are brought up. The cells are transfected with cDNA harvested from bacteria and purified on an anion exchange column. At 24 hours the cells are plated onto 24-well plates at 3-5 x 105 cells/cm2 and transport measured at 48-72 hr. The expression is low by 96 hr and absent at 5 and 10 days. Usually the medium is aspirated and washed once with 0.5 ml of a Na-free (143 mM N-methyl-D- glucamine Cl) HCO3-free MEM-like HEPES buffered medium for 3-5 min. Then it is preincubated at 37°C in room air for 30 minutes. The flux is initiated by adding 32PO4 or 22Na containing media. Cells transfected with the vector only (e.g., pRBG4) are always treated and fluxed in parallel. 32PO4 influx in the absence of Na is always "measured. All fluxes are performed in duplicate. The plates are placed on a water thermostatted table for 5 minutes and the flux initiated by aspirating the preincubation medium from the last column of wells and adding the tracer solution at known times (± 0.2 sec). This is done to successive columns of cells at approximately 30 minutes, 15 minutes, 10 minutes, 5 minutes, 2 minutes and 1 minute prior to terminating the influx simultaneously for all wells on the plate by 3 rapid ice cold washes (over 15 seconds total elapsed time) with (mM): 150 NaCl, 1.5 CaCl2, 1 MgCl2 solution. Residual wash solution is aspirated and the cells in the dry wells are solubilized in 0.5 ml of 25 mM NaOH with 0.5% deoxycholate. A 50 μl sample from each well is used to measure protein and 400 μl sample is counted in a liquid scintillation counter. Quadruplicate 10 μl samples of the influx solution are counted contemporaneously with the flux samples for specific activity determination. The pmol/μg protein in each well is calculated and the slope of the linear least squares best fit to these values against sample times is the computed flux, according to Sarkadi, B. et al., J.Gen.Physiol.72 249 (1978). Usually 5 or 6 of the data points are used to calculate each flux. Each condition is always measured in duplicate in both vector-only transfected and vector+insert transfected cells.
EXAMPLE 8
ISOLATION ,CLONTNG, and SEQUENCING of LLITHLUM-SODLUM
COUNTERTRANSPORTERS.
The hPiT-1 DNA was isolated, cloned and sequenced according to 5,414,076, herein incorporated by reference for this purpose. It is set forth as SEQUENCE LD NO.:l and SEQUENCE ID NO.:2. The hPiT-2 DNA was isolated, cloned and sequenced according to 5,550,221, herein incorporated by reference for this purpose. It is set forth as SEQUENCE LD NO.:3 and SEQUENCE LD NO.:4.
The BNPI DNA was isolated, cloned and sequenced according to Ni, B et al., J.Neurochem.66, 2227 (1996), herein incorporated by reference for this purpose. It is set forth as SEQUENCE LD NO.:5 and SEQUENCE ID NO.:6.
EXAMPLE 9
PREPARATION OF ANTIBODIES SPECIFIC FOR THE ERYTHROCYTE NA-PO4 COTRANSPORTER.
Polyclonal antibodies are prepared according to England, B.J. et al.,
Biochim.Biophys.Acta 623: 171 (1980), and Tirnmer, RT. et al., J.Biol.Chem. 268:
24863 (1993). Monoclonal antibodies are prepared according to Kohler, G. et al.,
Nature 256: 495 (1975).
EXAMPLE 10
RESTRCITION LENGTH FRAGMENT POLYMORPHISM ANALYSIS A. Using primers for hPiT-1, for example, CAGTTCAGTC AAGCCGTCAG ( SEQ ID NO : 7 ) and CCAGCCAACA GACACAACAG ( SEQ ID NO : 8 ) , the hPiT-1 sequence is amplified by PCR and ASO, by the methods of Connor, B.J.et al., Proc.Natl.Acad.Sci. 80: 278 (1983), and Saiki, R.K. et al., Nature 324: 163
(1986). Subsequent digestion with Taql, Pvul, Mbόl, and Sacl restriction endouncleases is performed.
B. Using primers for hPiT-2, for example,
ACAACGAGAC GGTGGAGACT (SEQ ID NO: 9) and TGCGGTGTAG CAGGTGTAAC (SEQ ID NO: 10), the hPiT-2 sequence is amplified by PCR and ASO, by the methods of Connor, B.J.et al., Proc.Natl.Acad. Sci. 80: 278 (1983), and Saiki, R.K. et al., Nature 324: 163
(1986). Subsequent digestion with Taql, Pvul, Mbol, and Sacl restriction endouncleases is performed.
C. Using primers for BNPI, for example,
CCTCGCCGCT ACATTATCGC (SEQ ID NO: 11) and
CGAAGCCTCC GCAGTTCATC (SEQ ID NO: 12), the BNPI sequence is amplified by PCR and ASO, by the methods of Connor, B.J.et al., Proc.Natl.Acad.Sci. 80: 278 (1983), and Saiki, R.K. et al, Nature 324: 163
(1986). Subsequent digestion with Taql, Pvul, Mbol, and Sacl restriction endouncleases is performed.
While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the usual variations, adaptations, modifications or deletions as come within the scope of the following claims and its equivalents.
SEQUENCE LISTING
(1) GENERAL" INFORMATION:
(1) APPLICANT: EMORY UNIVERSITY Gunn, Robert B. Timmer, Richard T.
(11) TITLE OF INVENTION: SODIUM-PHOSPHATE COTRANSPORTER IN LITHIUM THERAPY FOR THE TREATMENT OF MENTAL ILLNESS
(111) NUMBER OF SEQUENCES: 12
(Iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Kilpatrick Stockton LLP
(B) STREET: Suite 2800, 1100 Peachtree Street NE
(C) CITY: Atlanta
(D) STATE: GA
(E) COUNTRY: USA
(F) ZIP: 30309-4530
(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 #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US98/
(B) FILING DATE: ll-FEB-1998
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA
(A) APPLICATION NUMBER: US 60/039,462
(B) FILING DATE: 27-FEB-1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Meredith, Roy D.
(B) REGISTRATION NUMBER: 30,777
(C) REFERENCE/DOCKET NUMBER: EMU153PCT (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (404) 815-6500
(B) TELEFAX: (404) 815-6555
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 679 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(viii) POSITION IN GENOME:
(B) MAP POSITION: 2qll-ql4
(ix) FEATURE:
(A) NAME/KEY: hPIT-1
(B) LOCATION: 1..679
(D) OTHER INFORMATION: /label= Receptorl
/note= "/organisπ=Homosapiens/eel l_line=HL60 /tissue_lib=lambda HGR6, 7, and 16; Clontech #1020b/map=2qll-ql4"
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: US 5,414,076 P (x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: O'Hara, B., et al . , "Characterization of Human Gene Conferring Sensitivity to Infection by Gibbon Ape Leukemia Virus," Cell Growth Differ. 1(3):119-127 (1990) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
Met Ala Thr Leu He Thr Ser Thr Thr Ala Ala Thr Ala Ala Ser Gly 1 -. 5 10 15
Pro Leu Val Asp Tyr Leu Trp Met Leu He Leu Gly Phe He He Ala 20 25 30
Phe Val Leu Ala Phe Ser Val Gly Ala Asn Asp Val Ala Asn Ser Phe 35 40 45
Gly Thr Ala Val Gly Ser Gly Val Val Thr Leu Lys Gin Ala Cys He 50 55 60
Leu Ala Ser He Phe Glu Thr Val Gly Ser Val Leu Leu Gly Ala Lys 65 70 75 80
Val Ser Glu Thr He Arg Lys Gly Leu He Asp Val Glu Met Tyr Asn 85 90 95
Ser Thr Gin Gly Leu Leu Met Ala Gly Ser Val Ser Ala Met Phe Gly 100 105 110
Ser Ala Val Trp Gin Leu Val Ala Ser Phe Leu Lys Leu Pro He Ser 115 120 125
Gly Thr His Cys He Val Gly Ala Thr He Gly Phe Ser Leu Val Ala 130 135 140
Lys Gly Gin Glu Gly Val Lys Trp Ser Glu Leu He Lys He Val Met 145 150 155 160
Ser Trp Phe Val Ser Pro Leu Leu Ser Gly He Met Ser Gly He Leu 165 170 175
Phe Phe Leu Val Arg Ala Phe He Leu His Lys Ala Asp Pro Val Pro 180 185 190
Asn Gly Leu Arg Ala Leu Pro Val Phe Tyr Ala Cys Thr Val Gly He 195 200 205
Asn Leu Phe Ser He Met Tyr Thr Gly Ala Pro Leu Leu Gly Phe Asp 210 215 220
Lys Leu Pro Leu Trp Gly Thr He Leu He Ser Val Gly Cys Ala Val 225 230 235 240 Phe Cys Ala Leu He Val Trp Phe Phe Val Cys Pro Arg Met Lys Arg 245 250 255
Lys He Glu Arg Glu He Lys Cys Ser Pro Ser Glu Ser Pro Leu Met 260 265 270
Glu Lys Lys Asn Ser Leu Lys Glu Asp His Glu Glu Thr Lys Leu Ser 275 280 285
Val Gly Asp He Glu Asn Lys His Pro Val Ser Glu Val Gly Pro Ala 290 295 300
Thr Val Pro Leu Gin Ala Val Val Glu Glu Arg Thr Val Ser Phe Lys 305 310 315 320
Leu Gly Asp Leu Glu Glu Ala Pro Glu Arg Glu Arg Leu Pro Ser Val 325 330 335
Asp Leu Lys Glu Glu Thr Ser He Asp Ser Thr Val Asn Gly Ala Val 340 345 350
Gin Leu Pro Asn Gly Asn Leu Val Gin Phe Ser Gin Ala Val Ser Asn 355 360 365
Gin He Asn Ser Ser Gly His Ser Gin Tyr His Thr Val His Lys Asp 370 375 380
Ser Gly Leu Tyr Lys Glu Leu Leu His Lys Leu His Leu Ala Lys Val 385 390 395 400
Gly Asp Cys Met Gly Asp Ser Gly Asp Lys Pro Leu Arg Arg Asn Asn 405 410 415
Ser Tyr Thr Ser Tyr Thr Met Ala He Cys Gly Met Pro Leu Asp Ser 420 425 430
Phe Arg Ala Lys Glu Gly Glu Gin Lys Gly Glu Glu Met Glu Lys Leu 435 440 445
Thr Trp Pro Asn Ala Asp Ser Lys Lys Arg He Arg Met Asp Ser Tyr 450 455 460
Thr Ser Tyr Cys Asn Ala Val Ser Asp Leu His Ser Ala Ser Glu He 465 470 475 480 Asp Met Ser Val Lys Ala Ala Met Gly Leu Gly Asp Arg Lys Gly Ser 485 490 495
Asn Gly Ser Leu Glu Glu. Trp Tyr Asp Gin Asp Lys Pro Glu Val Ser 500 505 510
Leu Leu Phe Gin Phe Leu Gin He Leu Thr Ala Cys Phe Gly Ser Phe 515 520 525
Ala His Gly Gly Asn Asp Val Ser Asn Ala He Gly Pro Leu Val Ala 530 535 540
Leu Tyr Leu Val Tyr Asp Thr Gly Asp Val Ser Ser Lys Val Ala Thr 545 550 555 560
Pro He Trp Leu Leu Leu Tyr Gly Gly Val Gly He Cys Val Gly Leu 565 570 575
Trp Val Trp Gly Arg Arg Val He Gin Thr Met Gly Lys Asp Leu Thr 580 585 590
Pro He Thr Pro Ser Ser Gly Phe Ser He Glu Leu Ala Ser Ala Leu 595 600 605
Thr Val Val He Ala Ser Asn He Gly Leu Pro He Ser Thr Thr His 610 615 620
Cys Lys Val Gly Ser Val Val Ser Val Gly Trp Leu Arg Ser Lys Lys 625 630 635 640
Ala Val Asp Trp Arg Leu Phe Arg Asn He Phe Met Ala Trp Phe Val 645 650 655
Thr Val Pro He Ser Gly Val He Ser Ala Ala He Met Ala He Phe 660 665 670
Arg Tyr Val He Leu Arg Met 675
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3220 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: mRNA (iii) "HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: hPit-1
(B) LOCATION: 1..3220
(D) OTHER INFORMATION: /product= "Leukemia Virus Receptor 1"
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: US 5,414,076 P
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
GAGCTGTCCC CGGTGCCGCC GACCCGGGCC GTGCCGTGTG CCCGTGGCTC CAGCCGCTGC 60
CGCCTCGATC TCCTCGTCTC CCGCTCCGCC CTCCCTTTTC CCTGGATGAA CTTGCGTCCT 120
TTCTCTTCTC CGCCATGGAA TTCTGCTCCG TGCTTTTAGC CCTCCTGAGC CAAAGAAACC 180
CCAGACAACA GATGCCCATA CGCAGCGTAT AGCAGTAACT CCCCAGCTCG GTTTCTGTGC 240
CGTAGTTTAC AGTATTTAAT TTTATATAAT ATATATTATT TATTATAGCA TTTTTGATAC 300
CTCATATTCT GTTTACACAT CTTGAAAGGC GCTCAGTAGT TCTCTTACTA AACAACCACT 360
ACTCCAGAGA ATGGCAACGC TGATTACCAG TACTACAGCT GCTACCGCCG CTTCTGGTCC 420
TTTGGTGGAC TACCTATGGA TGCTCATCCT GGGCTTCATT ATTGCATTTG TCTTGGCATT 480
CTCCGTGGGA GCCAATGATG TAGCAAATTC TTTTGGTACA GCTGTGGGCT CAGGTGTAGT 540
GACCCTGAAG CAAGCCTGCA TCCTAGCTAG CATCTTTGAA ACAGTGGGCT CTGTCTTACT 600
GGGGGCCAAA GTGAGCGAAA CCATCCGGAA GGGCTTGATT GACGTGGAGA TGTACAACTC 660
GACTCAAGGG CTACTGATGG CCGGCTCAGT CAGTGCTATG TTTGGTTCTG CTGTGTGGCA 720
ACTCGTGGCT TCGTTTTTGA AGCTCCCTAT TTCTGGAACC CATTGTATTG TTGGTGCAAC 780
TAπGGπTC TCCCTCGTGG CAAAGGGGCA GGAGGGTGTC AAGTGGTCTG AACTGATAAA 840
AATTGTGATG TCTTGGπCG TGTCCCCACT GCTTTCTGGA ATTATGTCTG GAATTTTATT 900 CTTCCTGGTT CGTGCATTCA TCCTCCATAA GGCAGATCCA GTTCCTAATG GTTTGCGAGC 960
TTTGCCAGTT-TTCTATGCCT GCACAGTTGG AATAAACCTC TTTTCCATCA TGTATACTGG 1020
AGCACCGTTG CTGGGCTTTG ACAAACTTCC TCTGTGGGGT ACCATCCTCA TCTCGGTGGG 1080
ATGTGCAGTT TTCTGTGCCC TTATCGTCTG GTTCTTTGTA TGTCCCAGGA TGAAGAGAAA 1140
AATTGAACGA GAAATAAAGT GTAGTCCTTC TGAAAGCCCC TTAATGGAAA AAAAGAATAG 1200
CTTGAAAGAA GACCATGAAG AAACAAAGTT GTCTGTTGGT GATATTGAAA ACAAGCATCC 1260
TGTTTCTGAG GTAGGGCCTG CCACTGTGCC CCTCCAGGCT GTGGTGGAGG AGAGAACAGT 1320 CTCATTCAAA CTTGGAGATT TGGAGGAAGC TCCAGAGAGA GAGAGGCTTC CCAGCGTGGA 1380
CTTGAAAGAG GAAACCAGCA TAGATAGCAC CGTGAATGGT GCAGTGCAGT TGCCTAATGG 1440 GAACCTTGTC CAGTTCAGTC AAGCCGTCAG CAACCAAATA AACTCCAGTG GCCACTCCCA 1500
GTATCACACC GTGCATAAGG ATTCCGGCCT GTACAAAGAG CTACTCCATA AATTACATCT 1560
TGCCAAGGTG GGAGATTGCA TGGGAGACTC CGGTGACAAA CCCTTAAGGC GCAATAATAG 1620
CTATACTTCC TATACCATGG CAATATGTGG CATGCCTCTG GATTCAπCC GTGCCAAAGA 1680
AGGTGAACAG AAGGGCGAAG AAATGGAGAA GCTGACATGG CCTAATGCAG ACTCCAAGAA 1740 GCGAATTCGA ATGGACAGTT ACACCAGTTA CTGCAATGCT GTGTCTGACC TTCACTCAGC 1800
ATCTGAGATA GACATGAGTG TCAAGGCAGC GATGGGTCTA GGTGACAGAA AAGGAAGTAA 1860 TGGCTCTCTA GAAGAATGGT ATGACCAGGA TAAGCCTGAA GTCTCTCTCC TCTTCCAGTT 1920
CCTGCAGATC CTTACAGCCT GCTTTGGGTC ATTCGCCCAT GGTGGCAATG ACGTAAGCAA 1980
TGCCATTGGG CCTCTGGTTG CTπATATTT GGπTATGAC ACAGGAGATG TTTCTTCAAA 2040 AGTGGCAACA CCAATATGGC πCTACTCTA TGGTGGTGTT GGTATCTGTG TTGGTCTGTG 2100
GGTTTGGGGA AGAAGAGTTA TCCAGACCAT GGGGAAGGAT CTGACACCGA TCACACCCTC 2160
TAGTGGCπC AGTATTGAAC TGGCATCTGC CCTCACTGTG GTGATTGCAT CAAATATTGG 2220 CCTTCCCATC AGTACAACAC ATTGTAAAGT GGGCTCTGTT GTGTCTGTTG GCTGGCTCCG 2280
GTCCAAGAAG GCTGTTGACT GGCGTCTCTT TCGTAACATT TTTATGGCCT GGTTTGTCAC 2340
AGTCCCCATT TCTGGAGTTA TCAGTGCTGC CATCATGGCA ATCTTCAGAT ATGTCATCCT 2400 CAGAATGTGA AGCTGTTTGA GATTAAAATT TGTGTCAATG TTTGGGACCA TCTTAGGTAT 2460
TCCTGCTCCC CTGAAGAATG ATTACAGTGT TAACAGAAGA CTGACAAGAG TCTTTTTATT 2520
TGGGAGCAGA GGAGGGAAGT GTTACTTGTG CTATAACTGC TTTTGTGCTA AATATGAATT 2580
GTCTCAAAAT TAGCTGTGTA AAATAGCCCG GGTTCCACTG GCTCCTGCTG AGGTCCCCTT 2640
TCCTTCTGGG CTGTGAATTC CTGTACATAT TTCTCTACTT TTTGTATCAG GCTTCAATTC 2700
CATTATGTTT TAATGTTGTC TCTGAAGATG ACTTGTGATT TTTTTTTCTT TTTTTTAAAC 2760
CATGAAGAGC CGTTTGACAG AGCATGCTCT GCGTTGTTGG TTTCACCAGC TTCTGCCCTC 2820
ACATGCACAG GGATTTAACA ACAAAAATAT AACTACAACT TCCCTTGTAG TCTCTTATAT 2880
AAGTAGAGTC CTTGGTACTC TGCCCTCCTG TCAGTAGTGG CAGGATCTAT TGGCATATTC 2940
GGGAGCTTCT TAGAGGGATG AGGTTCTTTG AACACAGTGA AAAπTAAAT TAGTAACTTT 3000
TTTGCAAGCA GTπAπGAC TGTTATTGCT AAGAAGAAGT AAGAAAGAAA AAGCCTGTTG 3060 GCAATCTTGG TTATTTCTTT AAGATTTCTG GCAGTGTGGG ATGGATGAAT GAAGTGGAAT 3120
GTGAACπTG GGCAAGπAA ATGGGACAGC CTTCCATGTT CATTTGTCTA CCTCTTAACT 3180
GAATAAAAAA GCCTACAGTT TTTAGAAAAA ACCCGAATTC 3220
INFORMATION FOR SEQ ID NO: 3:
(i) .SEQUENCE CHARACTERISTICS:
* (A) LENGTH: 652 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: placenta
(ix) FEATURE:
(A) NAME/KEY: hPiT-2
(B) LOCATION: 1..652
(D) OTHER INFORMATION: /label= Receptor2
/note= "/organism=homo sapiens /sex=male /tissue_type=placenta tissue_lib=Stratagene #936203"
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: van Zeijl, M., et al . , "A Human Amphotropic Retrovirus Receptor is a Second Member of the Gibbon Ape Leukemia Virus Receptor Family," Proc. Natl. Acad. Sci., U.S.A., 91 (3) : 1168-1172 (1994) (x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER:' US 5,550,221 P
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Met Al a Met Asp Gl u Tyr Leu Trp Met Val H e Leu Gly Phe He He 1 5 10 15
Al a Phe He Leu Al a Phe Ser Val Gly Al a Asn Asp Val Al a Asn Ser 20 25 30
Phe Gly Thr Al a Val Gly Ser Gly Val Val Thr Leu Arg Gi n Al a Cys He Leu Ala Ser He Phe Glu Thr Thr Gly Ser Val Leu Leu Gly Ala 50 55 60
Lys Val Gly Glu Thr He Arg Lys Gly He He Asp Val Asn Leu Tyr 65 70 75 80
Asn Glu Thr Val Glu Thr Leu Met Ala Gly Glu Val Ser Ala Met Val 85 90 95
Gly Ser Ala Val Trp Gin Leu He Ala Ser Phe Leu Arg Leu Pro He 100 105 110
Ser Gly Thr His Cys He Val Gly Ser Thr He Gly Phe Ser Leu Val 115 120 125
Ala He Gly Thr Lys Gly Val Gin Trp Met Glu Leu Val Lys He Val 130 135 140
Ala Ser Trp Phe He Ser Pro Leu Leu Ser Gly Phe Met Ser Gly Leu 145 150 155 160
Leu Phe Val Leu He Arg He Phe He Leu Lys Lys Glu Asp Pro Val 165 170 175
Pro Asn Gly Leu Arg Ala Leu Pro Val Phe Tyr Ala Ala Thr He Ala 180 185 190
He Asn Val Phe Ser He Met Tyr Thr Gly Ala Pro Val Leu Gly Leu 195 200 205
Val Leu Pro Met Trp Ala He Ala Leu He Ser Phe Gly Val Ala Leu 210 215 220
Leu Phe Ala Phe Phe Val Trp Leu Phe Val Cys Pro Trp Met Arg Arg 225 230 235 240
Lys He Thr Gly Lys Leu Gin Lys Glu Gly Ala Leu Ser Arg Val Ser 245 250 255
Asp Glu Ser Leu Ser Lys Val Gin Glu Ala Glu Ser Pro Val Phe Lys 260 265 270
Glu Leu Pro Gly Ala Lys Ala Asn Asp Asp Ser Thr He Pro Leu Thr 275 280 285 Gly Ala Ala Gly Glu Thr Leu Gly Thr Ser Glu Gly Thr Ser Ala Gly 290 295 300
Ser His Pro Arg Ala Ala Tyr Gly Arg Ala Leu Ser Met Thr His Gly 305 310 315 320
Ser Val Lys Ser Pro He Ser Asn Gly Thr Phe Gly Phe Asp Gly His 325 330 335
Thr Arg Ser Asp Gly His Val Tyr His Thr Val His Lys Asp Ser Gly 340 345 350
Leu Tyr Lys Asp Leu Leu His Lys He His He Asp Arg Gly Pro Glu 355 360 365
Glu Lys Pro Ala Gin Glu Ser Asn Tyr Arg Leu Leu Arg Arg Asn Asn 370 375 380
Ser Tyr Thr Cys Tyr Thr Ala Ala He Cys Gly Leu Pro Val His Ala 385 390 395 400
Thr Phe Arg Ala Ala Asp Ser Ser Ala Pro Glu Asp Ser Glu Lys Leu 405 410 415
Val Gly Asp Thr Val Ser Tyr Ser Lys Lys Arg Leu Arg Tyr Asp Ser 420 425 430
Tyr Ser Ser Tyr Cys Asn Ala Val Ala Glu Ala Glu He Glu Ala Glu 435 440 445
Glu Gly Gly Val Glu Met Lys Leu Ala Ser Glu Leu Ala Asp Pro Asp 450 455 460
Gin Pro Arg Glu Asp Pro Ala Glu Glu Glu Lys Glu Glu Lys Asp Ala 465 470 475 480
Pro Glu Val His Leu Leu Phe His Phe Leu Gin Val Leu Thr Ala Cys 485 490 495
Phe Gly Ser Phe Ala His Gly Gly Asn Asp Val Ser Asn Ala He Gly 500 505 510
Pro Leu Val Ala Leu Trp Leu He Tyr Lys Gin Gly Gly Val Thr Gin 515 520 525 Glu Ala Ala Thr Pro Val Trp Leu Leu Phe Tyr Gly Gly Val Gly He 530 535 540
Cys Thr Gly Leu Trp Val Trp Gly Arg Arg Val He Gin Thr Met Gly 545 550 555 560
Lys Asp Leu Thr Pro He Thr Pro Ser Ser Gly Phe Thr He Glu Leu 565 570 575
Ala Ser Ala Phe Thr Val Val He Ala Ser Asn He Gly Leu Pro Val 580 585 590
Ser Thr Thr His Cys Lys Val Gly Ser Val Val Ala Val Gly Trp He 595 600 605
Arg Ser Arg Lys Ala Val Asp Trp Arg Leu Phe Arg Asn He Phe Val 610 615 620
Ala Trp Phe Val Thr Val Pro Val Ala Gly Leu Phe Ser Ala Ala Val 625 630 635 640
Met Ala Leu Leu Met Tyr Gly He Leu Pro Tyr Val 645 650
(2) INFORMATION FOR SEQ ID NO : 4 :
(i) SEQUENCE CHARACTERISTICS :-
(A) LENGTH: 3175 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: mRNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: placenta
(ix) FEATURE:
(A) NAME/KEY: hPiT-2
(B) LOCATION: 1..3175
(D) OTHER INFORMATION: /product= "Leukemia virus receptor 2" /label= Receptor2 (x) PUBLICATION INFORMATION:'
(H) DOCUMENT NUMBER: van Zeijl, M., et al . , "A Human Amphotropic Retrovirus Receptor is a Second Member of the Gibbon Ape Leukemia Virus Receptor Family," Proc. Natl. Acad. Sci., U.S.A., 91(3) :1168-1172 (1994)
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: US 5,550,221 P
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CAGATCGGGA AGAAAAATAT GGAATGTGTT TTACCGCTGA CTGAACACAA CCAAATGAAC 60
TGTCCTGACA GTAGTTTGCA AACCAGCAGC TAGCAGTTTG TCCAGCCTCT AACATTGTCC 120
AGCACTTTCC AGAGCAAACT CACTGTTTAC AAGAACTCTT GGCCTTACGA AGTTTATAAC 180
CTCAAGCTTT GTTTATTTAA AATATTCCTG CAAAAGAAAA GTACCCGGCA CCCACTTTCC 240
AAAATGGCCA TGGATGAGTA TTTGTGGATG GTCATTTTGG GTTTCATCAT AGCTTTCATC 300
TTGGCCTTTT CTGTTGGTGC AAACGATGTT GCCAACTCCT TTGGTACAGC CGTGGGCTCT 360
GGTGTGGTGA CCTTGAGGCA GGCATGCATT TTAGCTTCAA TATTTGAAAC CACCGGCTCC 420
GTGTTACTAG GCGCCAAAGT AGGAGAAACC ATTCGCAAAG GTATCATTGA CGTGAACCTG 480
TACAACGAGA CGGTGGAGAC TCTCATGGCT GGGGAAGTTA GTGCCATGGT TGGTTCCGCT 540
GTGTGGCAGC TGATTGCTTC CTTCCTGAGG CTTCCAATCT CAGGAACGCA CTGCATTGTG 600
GGTTCTACTA TAGGATTCTC ACTGGTCGCA ATCGGTACCA AAGGTGTGCA GTGGATGGAG 660
CTTGTCAAGA TTGTTGCTTC TTGGTTTATA TCTCCACTGT TGTCTGGTTT CATGTCTGGC 720
CTGCTGTTTG TACTCATCAG AATTTTCATC TTAAAAAAGG AAGACCCTGT TCCCAATGGC 780
CTCCGGGCAC TCCCAGTATT CTATGCTGCT ACCATAGCAA TCAATGTCTT TTCCATCATG 840
TACACAGGAG CACCAGTGCT CGGCCTTGTT CTCCCCATGT GGGCCATAGC CCTCAπTCC 900
TTTGGTGTCG CCCTCCTGTT CGCTTTTTTT GTGTGGCTCT TCGTGTGTCC GTGGATGCGG 960
AGGAAAATAA CAGGCAAATT ACAAAAAGAA GGTGCTTTAT CACGAGTATC TGACGAAAGC 1020 CTCAGTAAGG TTCAGGAAGC AGAGTCCCCA GTATTTAAAG AGCTACCAGG TGCCAAGGCT 1080
AATGATGACA GCACCATCCC GCTCACGGGA GCAGCAGGGG AGACACTGGG GACCTCGGAA 1140 GGCACTTCTG CGGGCAGCCA CCCTCGGGCT GCATACGGAA GAGCACTGTC CATGACCCAT 1200
GGCTCTGTGA AATCGCCCAT CTCCAACGGC ACCTTCGGCT TCGACGGCCA CACCAGGAGC 1260
GACGGTCATG TGTACCACAC CGTGCACAAA GACTCGGGGC TCTACAAAGA TCTGCTGCAC 1320
AAAATCCACA TCGACAGGGG CCCCGAGGAG AAGCCAGCCC AGGAAAGCAA CTACCGGCTG 1380
CTCCGCCGAA ACAACAGTTA CACCTGCTAC ACCGCAGCCA TTTGTGGGCT GCCAGTGCAC 1440
GCCACCTTTC GAGCTGCGGA CTCATCGGCC CCAGAGGACA GTGAGAAGCT GGTGGGCGAC 1500
ACCGTGTCCT ACTCCAAGAA GAGGCTGCGC TACGACAGCT ACTCGAGCTA CTGTAACGCG 1560
GTGGCAGAGG CGGAGATCGA GGCGGAGGAG GGCGGCGTGG AGATGAAGCT GGCGTCGGAG 1620 CTGGCCGACC CTGACCAGCC GCGAGAGGAC CCTGCAGAGG AGGAGAAGGA GGAGAAGGAC 1680
GCACCCGAGG πCACCTCCT GTTCCATπC CTGCAGGTCC TCACCGCCTG TTTCGGGTCC 1740
TTTGCTCACG GCGGCAATGA CGTGAGTAAT GCCATCGGTC CCCTGGTAGC CTTGTGGCTG 1800
ATTTACAAAC AAGGCGGGGT AACGCAAGAA GCAGCTACAC CCGTCTGGCT GCTGTHTAT 1860
GGAGGAGTTG GAATCTGCAC AGGCCTCTGG GTCTGGGGGA GAAGAGTGAT CCAGACCATG 1920
GGGAAGGACC TCACTCCCAT CACGCCGTCC AGCGGCTTCA CGATCGAGCT GGCCTCAGCC 1980
TTCACAGTGG TGATCGCCTC CAACATCGGG CTTCCAGTCA GCACCACGCA CTGTAAGGTG 2040
GGCTCGGTGG TGGCCGTGGG CTGGATCCGC TCCCGCAAGG CTGTGGACTG GCGCCTCTTT 2100 CGGAACATCT TCGTGGCCTG GTTCGTGACC GTCCCTGTGG CTGGGCTGTT CAGCGCTGCT 2160 GTCATGGCTC TTCTCATGTA TGGGATCCTT CCATATGTGT GATTTGTCTT CπCCAGCTG 2220
CAAACAGCTA AAGGGATGGT CTGGTGTTGG CGTGTGGGAG ACATGTGTGC TCGTGCCGCA 2280
CATACACATC CTGGCCGTGC ACGGCTCTCT CATGACCAGC TCTCTGCCTC CCTTCCAGGA 2340
GGCTCCATCC CACACTGTTC ACCCAGGCTG CGGAGACTCA CCTTCCCGAG CTAACTTAAC 2400 TACTGTACAT AATAATATGT ATTAAACTGG TATCGTGGTG ATATAATGTG GTGCAGTTAC 2460
TTATATATTA'AATATCTATT GTATCCATAG AATAGGCAGC ATTATTTCAA ACATATTCAA 2520
GTTGGGAGTG GAGATCATTG CCTAGAAGTC AATATTCAAT AAATCTTGTA CATAACTATT 2580
TCGATGGCAA ATGTTAAGCC TTCTAAAAGG AAAGTGTAGA TTGGAAAATG ATTTTTTTTC 2640
CAAATGATGT TTTTGCCTTC TAATATACTG TAAGGTAATG AGCTTCAGAA CAGGCAACCT 2700
GACCCTGCAG AGGTCGCGTG CTGTGGGATG ACAGCGGGAC GGGAGCTCAC AAGTGCTTTC 2760 ACTGAAGATT TGTTCATATA CTGTGTATTG ATTGTTGTGT AATATATCAT CATTGCTTTT 2820
GTAAATACGT AAAACTGTAA TTTTTTAATG GTGTGCTTCC CTTATACTTT TTGATCAGAG 2880
AATTTTGGAA AGTACCAAAG AAGCAGGGGA ATCATTGGCC AGTGTTACGT TTTCACATTG 2940
TCTGTCTCCC ACCCTCACTG ATCACGCCTG CCCCAGAGCA GTGTGTGGCG GTGACACCGT 3000
CACCCAGCAT GCGCCACGCC GTCGTCCCAC CAGCAGTGCC ACCGCCACCA CACCCCAGAT 3060
CCCACCCACC TTGCAGTGGC TTTCTTGTCA TCAGAGTAGA GAATGCACAG GTGTTGGTGA 3120
GGGCGTGTGG CTGAGCACTA CATGTCAAGT CAGAGTCAGT TTCTATCCAA TTCTC 3175
(2) INFORMATION FOR SEQ ID NO: 5:
(i) -SEQUENCE CHARACTERISTICS:
(A) LENGTH: 560 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(ix) FEATURE:
(A) NAME/KEY: hBNPI
(B) LOCATION: 1..560
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: Ni, B., et al . ,
J. Neuroche ., 66:2227 (1996)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Met Glu Phe Arg Gin Glu Glu Phe Arg Lys Leu Ala Gly Arg Ala Leu 1 5 10 15
Gly Lys Leu His Arg Leu Leu Glu Lys Arg Gin Glu Gly Ala Glu Thr 20 25 30
Val Glu Leu Ser Ala Asp Gly Arg Pro Val Thr Thr Gin Thr Arg Asp 35 40. 45
Pro Pro Val Val Asp Cys Thr Cys Phe Gly Leu Pro Arg Arg Tyr He 50 55 60
He Ala He Met Ser Gly Leu Gly Phe Cys He Ser Phe Gly He Arg 65 70 75 80
Cys Asn Leu Gly Val Ala He Val Ser Met Val Asn Asn Ser Thr Thr 85 90 95
His Arg Gly Gly His Val Val Val Gin Lys Ala Gin Phe Ser Trp Asp 100 105 110 Pro Glu Thr Val Gly Leu He His Gly Ser Phe Phe Trp Gly Tyr He 115 120 125
Val Thr Gin He Pro Gly Gly Phe He Cys Gin Lys Phe Ala Ala Asn 130 ,135 140
Arg Val Phe Gly Phe Ala He Val Ala Thr Ser Thr Leu Asn Met Leu 145 150 155 160
He Pro Ser Ala Ala Arg Val His Tyr Gly Cys Val He Phe Val Arg 165 170 175
He Leu Gin Gly Leu Val Glu Gly Val Thr Tyr Pro Ala Cys His Gly 180 185 190
He Trp Ser Lys Trp Ala Pro Pro Leu Glu Arg Ser Arg Leu Ala Thr 195 200 205
Thr Ala Phe Cys Gly Ser Tyr Ala Gly Ala Val Val Ala Met Pro Leu 210 215 220
Ala Gly Val Leu Val Gin Tyr Ser Gly Trp Ser Ser Val Phe Tyr Val 225 230 235 240
Tyr Gly Ser Phe Gly He Phe Trp Tyr Leu Phe Trp Leu Leu Val Ser 245 250 255
Tyr Glu Ser Pro Ala Leu His Pro Ser He Ser Glu Glu Glu Arg Lys 260 265 270
Tyr He Glu Asp Ala He Gly Glu Ser Ala Lys Leu Met Asn Pro Leu 275 280 285
Thr Lys Phe Ser Thr Pro Trp Arg Arg Phe Phe Thr Ser Met Pro Val 290 295 300
Tyr Ala He He Val Ala Asn Phe Cys Arg Ser Trp Thr Phe Tyr Leu 305 310 315 320
Leu Leu He Ser Gin Pro Asp Tyr Phe Glu Glu Val Phe Gly Phe Glu 325 330 335
He Ser Lys Val Gly Leu Val Ser Ala Leu Pro His Leu Val Met Thr 340 345 350 He He Val Pro He Gly Gly Gin He Ala Asp Phe Leu Arg Ser Arg 355 360 365
Arg He Met Ser Thr Thr Asn Val Arg Lys Leu Met Asn Cys Gly Gly 370 .375 380
Phe Gly Met Glu Ala Thr Leu Leu Leu Val Val Gly Tyr Ser His Ser 385 390 395 400
Lys Gly Val Ala He Ser Phe Leu Val Leu Ala Val Gly Phe Ser Gly 405 410 415
Phe Ala He Ser Gly Phe Asn Val Asn His Leu Asp He Ala Pro Arg 420 425 430
Tyr Ala Ser He Leu Met Gly He Ser Asn Gly Val Gly Thr Leu Ser 435 440 445
Gly Met Val Cys Pro He He Val Gly Ala Met Thr Lys His Lys Thr 450 455 460
Arg Glu Glu Trp Gin Tyr Val Phe Leu He Ala Ser Leu Val His Tyr 465 470 475 480
Gly Gly Val He Phe Tyr Gly Val Phe Ala Ser Gly Glu Lys Gin Pro 485 490 495
Trp Ala Glu Pro Glu Glu Met Ser Glu Glu Lys Cys Gly Phe Val Gly 500 505 510
His Asp Gin Leu Ala Gly Ser Asp Asp Ser Glu Met Glu Asp Glu Ala 515 520 525
Glu Pro Pro Gly Ala Pro Pro Ala Pro Pro Pro Ser Tyr Gly Ala Thr 530 535 540
His Ser Thr Phe Gin Pro Pro Arg Pro Pro Pro Pro Val Arg Asp Tyr 545 550 555 560
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2716 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: hBNPI
(B) LOCATION: 1..2716
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: Ni, B., et al . ,
J. Neuroche ., 66:2227 (1996)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
CGATAAGCTT GATATCGAAT TCCGGACTCT TGCTCGGGCG CCTTAACCCG GCGTTCGGTT 60
CATCCCGCAG CGCCAGTTCT GCTTACCAAA AGTGGCCCAC TAGGCACTCG CATTCCACGC 120
CCGGCTCCAC GCCAGCGAGC CGGGCTTCTT ACCCATTTAA AGTTTGAGAA TAGGTTGAGA 180
TCGTπCGGC CCCAAGACCT CTAATCAπC GCTTTACCGG ATAAAACTGC GTGGCGGGGG 240
TGCGTCGGGT CTGCGAGAGC GCCAGCTATC CTGAGGGAAA CTTCGGAGGG AACCAGCTAC 300
TAGATGGπC GATTAGTCTT TCGCCCCTAT ACCCAGGTCG GACGACCGAT TTGCACGTCA 360
GGACCGCTAC GGACCTCCAC CAGAGTTTCC TCTGGCTTCG CCCTGCCCAG GCGATCGGCG 420
GGGGGGACCC GCGGGGTGAC CGGCGGCAGG AGCCGCCACC ATGGAGTTCC GCCAGGAGGA 480
GTπCGGAAG CTAGCGGGTC GTGCTCTCGG GAAGCTGCAC CGCCTTCTGG AGAAGCGGCA 540
GGAAGGCGCG GAGACGGTGG AGCTGAGTGC GGATGGGCGC CCGGTGACCA CGCAGACCCG 600
GGACCCGCCG GTGGTGGACT GCACCTGCTT CGGCCTCCCT CGCCGCTACA TTATCGCCAT 660
CATGAGTGGT CTGGGCTTCT GCATCAGCTT TGGCATCCGC TGCAACCTGG GCGTGGCCAT 720
CGTCTCCATG GTCAATAACA GCACGACCCA CCGCGGGGGC CACGTGGTGG TGCAGAAAGC 780
CCAGTTCAGC TGGGATCCAG AGACTGTCGG CCTCATACAC GGCTCCTTTT TCTGGGGCTA 840
CATTGTCACT CAGATTCCAG GAGGATTTAT CTGTCAAAAA TTTGCAGCCA ACAGAGTTTT 900 CGGCTTTGCT ATTGTGGCAA CATCCACTCT AAACATGCTG ATCCCCTCAG CTGCCCGCGT 960
CCACTATGGC TGTGTCATCT TCGTGAGGAT CCTGCAGGGG TTGGTAGAGG GGGTCACATA 1020
CCCCGCCTGC CATGGGATCT GGAGCAAATG GGCCCCACCC TTAGAACGGA GTCGCCTGGC 1080
GACGACAGCC TTTTGTGGTT CCTATGCTGG GGCGGTGGTC GCGATGCCCC TCGCCGGGGT 1140
CCTTGTGCAG TACTCAGGAT GGAGCTCTGT TTTCTACGTC TACGGCAGCT TCGGGATCTT 1200
CTGGTACCTG TTCTGGCTGC TCGTCTCCTA CGAGTCCCCC GCGCTGCACC CCAGCATCTC 1260
GGAGGAGGAG CGCAAGTACA TCGAGGACGC CATCGGAGAG AGCGCGAAAC TCATGAACCC 1320
CCTCACGAAG TTTAGCACTC CCTGGCGGCG CTTCπCACG TCTATGCCAG TCTATGCCAT 1380
CATCGTGGCC AACTTCTGCC GCAGCTGGAC GTTCTACCTG CTGCTCATCT CCCAGCCCGA 1440
CTACTTCGAA GAAGTGπCG GCTTCGAGAT CAGCAAGGTA GGCCTGGTGT CCGCGCTGCC 1500
CCACCTGGTC ATGACCATCA TCGTGCCCAT CGGCGGCCAG ATCGCGGACT TCCTGCGGAG 1560
CCGCCGCATC ATGTCCACCA CCAACGTGCG CAAGTTGATG AACTGCGGAG GCTTCGGCAT 1620
GGAAGCCACG CTGCTGπGG TGGTCGGCTA CTCGCACTCC AAGGGCGTGG CCATCTCCTT 1680
CCTGGTCCTA GCCGTGGGCT TCAGCGGCTT CGCCATCTCT GGGTTCAACG TGAACCACCT 1740
GGACATAGCC CCGCGCTACG CCAGCATCCT CATGGGCATC TCCAACGGCG TGGGCACACT 1800
GTCGGGCATG GTGTGCCCCA TCATCGTGGG GGCCATGACT AAGCACAAGA CTCGGGAGGA 1860
GTGGCAGTAC GTGTTCCTAA TTGCCTCCCT GGTGCACTAT GGAGGTGTCA TCTTCTACGG 1920
GGTCTTTGCT TCTGGAGAGA AGCAGCCGTG GGCAGAGCCT GAGGAGATGA GCGAGGAGAA 1980
GTGTGGCTTC GTTGGCCATG ACCAGCTGGC TGGCAGTGAC GACAGCGAAA TGGAGGATGA 2040
GGCTGAGCCC CCGGGGGCAC CCCCTGCACC CCCGCCCTCC TATGGGGCCA CACACAGCAC 2100
ATTTCAGCCC CCCAGGCCCC CACCCCCTGT CCGGGACTAC TGACCATGTG CCTCCCACTG 2160
AATGGCAGTT TCCAGGACCT CCATTCCACT CATCTCTGGC CTGAGTGACA GTGTCAAGGA 2220
ACCCTGCTCC TCTCTGTCCT GCCTCAGGCC TAAGAAGCAC TCTCCCTTGT TCCCAGTGCT 2280 GTCAAATCCT CTTTCCTTCC CAATTGCCTC TCAGGGGTAG TGAAGCTGCA GACTGACAGT 2340
TTCAAGGATA" CCCAAATTCC CCTAAAGGTT CCCTCTCCAC CCGTTCTGCC TCAGTGGTTT 2400 CAAATCTCTC CTTTCAGGGC TTTATTTGAA TGGACAGTTC GACCTCTTAC TCTCTCTTGT 2460
GGTTTTGAGG CACCCACACC CCCCGCTTTC CTTTATCTCC AGGGACTCTC AGGCTAACCT 2520 TTGAGATCAC TCAGCTCCCA TCTCCTTTCA GAAAAATTCA AGGTCCTCCT CTAGAAGTTT 2580
CAAATCTCTC CCAACTCTGT TCTGCATCTT CCAGATTGGT TTAACCAATT ACTCGTCCCC 2640
GCCATTCCAG GGATTGATTC TCACCAGCGT TTCTGATGGA AAATGGCGGG AATTCCTGCA 2700
GCCCGGGGGA TCCACT 2716
(2) INFORMATION FOR SEQ ID NO: 7:
(i) -SEQUENCE CHARACTERISTICS: "(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: primer for hPiT-1
(B) LOCATION: 1..20
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CAGTTCAGTC AAGCCGTCAG 20
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: complementary strand primer for hPiT-1
(B) LOCATION: 1..20
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 : CCAGCCAACA GACACAACAG 20
(2) INFORMATION FOR SEQ ID NO: 9:
;i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: primer for hPiT-1
(B) LOCATION: 1..20
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ACAACGAGAC GGTGGAGACT 20
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: complementary strand primer for hPiT-2
(B) LOCATION: 1..20
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: TGCGGTGTAG CAGGTGTAAC 20
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:
(A) NAME/KEY: primer for hBNPI -. (B) LOCATION: 1..20
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
CCTCGCCGCT ACATTATCGC 20
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: complementary strand primer for hBNPI
(B) LOCATION: 1..20
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CGAAGCCTCC GCAGTTCATC 20

Claims

WHAT IS CLAIMED IS:
1. A purified DNA molecule coding for a lithium-sodium countertransporter.
2. A purified DNA molecule coding for an amino acid sequence selected from the group consisting of hPiT-1, hPiT-2, and hBNPI, said molecule useful for measuring lithium-sodium countertransport in human cells.
3. The purified DNA molecule of claims 1 or 2, wherein the nucleotide sequence is SEQ.ID.NO.: 2.
4. The purified DNA molecule of claims 1 or 2, said DNA encoding for the amino acid sequence of SEQ.ID.NO.: 1.
5. The purified DNA molecule of claims 1 or 2, wherein the nucleotide sequence is SEQ.ID.NO.: 4.
6. The purified DNA molecule of claims 1 or 2, said DNA encoding for the amino acid sequence of SEQ.ID.NO.: 3.
7. The purified DNA molecule of claims 1 or 2, wherein the nucleotide sequence is SEQ.ID.NO.: 6.
8. The purified DNA molecule of claims 1 or 2, said DNA encoding for the amino acid sequence of SEQ.ID.NO. : 5.
9. A human amphotrophic retrovirus receptor useful as a lithium-sodium countertransporter.
10. The receptor of claim 9, wherein its nucleotide sequence is SEQ.ID.NO. 2.
11. The receptor of claim 9, wherein its amino acid sequence is SEQ.ID.NO. : 1.
12. The receptor of claim 9, wherein its nucleotide sequence is SEQ.ID.NO. 4.
13. The receptor of claim 9, wherein its amino acid sequence is SEQ.ID.NO.: 3.
14. The receptor of claim 9, wherein its nucleotide sequence is SEQ.ID.NO. 6.
15. The receptor of claim 9, wherein its amino acid sequence is SEQ.ID.NO.: 5.
16. A method of evaluating sensitivity to lithium therapy in manic depressive patients, comprising the steps of
(a) providing a sample of patient blood;
(b) extracting from the blood sample the patient's DNA;
(c) subjecting the DNA to hybridization with primers specific for any sequence coding for lithium-sodium countertransporter; (d) polymerizing said sequences, to give polymerized sequences;
(e) amplifying said polymerized sequences, to give an amplified sample of patient sequences;
(f) digesting the amplified sample with one or more restriction endonucleases suitable for mapping sites on the DNA indicating susceptibility to lithium therapy.
17. A method of evaluating sensitivity to lithium therapy in manic depressive patients, comprising the steps of
(a) providing a sample of patient blood; (b) extracting from the blood sample the patient's DNA; (c) subjecting the DNA to hybridization with. primers specific for any sequence coding for lithium-sodium countertransporter;
(d) polymerizing said sequences, to give polymerized sequences;
(e) amplifying said polymerized sequences, to give an amplified sample of patient sequences;
(f) subjecting the amplified sample to in vitro membrane-based translation to give a translated sample within a cell; and
(g) subjecting the translated sample to flux analysis of lithium, to evaluate sensitivity to lithium therapy in manic depressive patients.
18. The method of claims 16 or 17, wherein the sequence coding for the lithium- sodium countertransporter is selected from the group consisting of hPiT-1, hPiT-2, and hBNPI.
19. The method of claims 16 or 17, wherein the sequence is the nucleotide sequence coding for the lithium-sodium countertransporter selected from the group consisting of SEQ.ID.NO. :2, SEQ.ID.NO. :4,. and SEQ.ID.NO. :6.
20. The method of claims 16 or 17, wherein the sequence is the amino acid sequence for the lithium-sodium countertransporter is selected from the group consisting of SEQ.ID.NO. :1, SEQ.ID.NO.:3,. SEQ.ID.NO.:5.
21. A method of evaluating sensitivity to lithium therapy in manic depressive patients, comprising the steps of (a) providing a sample of patient blood;
(b) isolating the erythrocytes;
(c) subjecting the erythrocytes to flux analysis of lithium, to evaluate sensitivity to lithium therapy in manic depressive patients.
22. A method of evaluating lithium-sodium countertransport in patients with mental illness, comprising the steps of (a) providing a sample of patient blood;
(b) isolating the erythrocytes;
(c) subjecting the erythrocytes to flux analysis of lithium, to evaluate lithium- sodium countertransport.
PCT/US1998/002875 1997-02-27 1998-02-11 Sodium-phosphate cotransporter in lithium therapy for the treatment of mental illness WO1998038203A1 (en)

Priority Applications (2)

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AU63263/98A AU6326398A (en) 1997-02-27 1998-02-11 Sodium-phosphate cotransporter in lithium therapy for the treatment of mental illness
US10/314,790 US20040002079A1 (en) 1998-02-11 2002-12-09 Sodium-phosphate cotransporter in lithium therapy for the treatment of mental illness

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US60/039,462 1997-02-27

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10147006A1 (en) * 2001-09-24 2003-04-17 Gruenenthal Gmbh Identifying agents for treatment and diagnosis of diseases, e.g. depression or viral infections, from binding to inorganic phosphate transporters, also new agents

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5288678A (en) * 1990-10-19 1994-02-22 Beckman Instruments, Inc. Indirect potentiometric method and diluent for analysis of lithium
US5414076A (en) * 1989-08-24 1995-05-09 American Cyanamid Company DNA encoding gibbon ape leukemia virus receptor
US5550221A (en) * 1993-04-16 1996-08-27 American Cyanamid Company Nucleic acid molecule encoding the amphotropic virus receptor
US5618918A (en) * 1995-04-27 1997-04-08 Eli Lilly And Company Human brain sodium dependent inorganic phosphate cotransporter

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5414076A (en) * 1989-08-24 1995-05-09 American Cyanamid Company DNA encoding gibbon ape leukemia virus receptor
US5288678A (en) * 1990-10-19 1994-02-22 Beckman Instruments, Inc. Indirect potentiometric method and diluent for analysis of lithium
US5550221A (en) * 1993-04-16 1996-08-27 American Cyanamid Company Nucleic acid molecule encoding the amphotropic virus receptor
US5633348A (en) * 1993-04-16 1997-05-27 American Cyanamid Company Amphotropic virus receptor
US5618918A (en) * 1995-04-27 1997-04-08 Eli Lilly And Company Human brain sodium dependent inorganic phosphate cotransporter
US5618677A (en) * 1995-04-27 1997-04-08 Eli Lilly And Company Human brain sodium dependent inorganic phosphate cotransporter assay
US5686266A (en) * 1995-04-27 1997-11-11 Eli Lilly And Company Human brain sodium dependent inorganic phosphate cotransproter and related nucleic acid compounds

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
JOHANN S. V., ET AL.: "A HUMAN AMPHOTROPIC RETROVIRUS RECEPTOR IS A SECOND MEMBER OF THE GIBBON APE LEUKEMIA VIRUS RECEPTOR FAMILY.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 91., 1 February 1994 (1994-02-01), US, pages 1168 - 1172., XP002910069, ISSN: 0027-8424, DOI: 10.1073/pnas.91.3.1168 *
KLINGER H. P., ET AL.: "CHARACTERIZATION OF A HUMAN GENE CONFERRING SENSITIVITY TO INFECTION BY GIBBON APE LEUKEMIA VIRUS.", CELL GROWTH AND DIFFERENTIATION., THE ASSOCIATION, PHILADELPHIA, PA, US, vol. 01., 1 January 1990 (1990-01-01), US, pages 119 - 127., XP002910068, ISSN: 1044-9523 *
MILLER D. G., ET AL.: "CLONING OF THE CELLULAR RECEPTOR FOR AMPHOTROPIC MURINE RETROVIRUSES REVEALS HOMOLOGY TO THAT FOR GIBBON APE LEUKEMIA VIRUS", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 91., 1 January 1994 (1994-01-01), US, pages 78 - 82., XP002910070, ISSN: 0027-8424, DOI: 10.1073/pnas.91.1.78 *
NADI N. S., ET AL.: "CLONING AND EXPRESSION OF A CDNA ENCODING A BRAIN-SPECIFIC NA+-DEPENDENT INORGANIC PHOSPHATE COTRANSPORTER.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 91., 1 June 1994 (1994-06-01), US, pages 5607 - 5611., XP002910071, ISSN: 0027-8424, DOI: 10.1073/pnas.91.12.5607 *
WILSON C. A., ET AL.: "PROPERTIES OF A UNIQUE FORM OF THE MURINE AMPHOTROPIC LEUKEMIA VIRUS RECEPTOR EXPRESSED ON HAMSTER CELLS.", JOURNAL OF VIROLOGY., THE AMERICAN SOCIETY FOR MICROBIOLOGY., US, vol. 68., no. 12., 1 December 1994 (1994-12-01), US, pages 7697 - 7703., XP002910072, ISSN: 0022-538X *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10147006A1 (en) * 2001-09-24 2003-04-17 Gruenenthal Gmbh Identifying agents for treatment and diagnosis of diseases, e.g. depression or viral infections, from binding to inorganic phosphate transporters, also new agents

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