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FIELD OF THE INVENTION
The present invention relates to human and mouse Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger (sodium ion-driven chloride/bicarbonate exchanger) proteins, which are a class of proteins involved in intracellular pH regulation. More specifically, the present invention relates to sodium ion-driven chloride/bicarbonate exchanger proteins, cells designed to express one of the proteins, which cells are of a species different from the origin of the one of the proteins expressed, DNAs encoding the proteins, antibodies to the proteins, and a method for selecting agonists/antagonists of the sodium ion-driven chloride/bicarbonate exchanger proteins.
BACKGROUND OF THE INVENTION
Regulation of intracellular pH (pH.sub.i) in response to various stimuli is a critical one among a number of cellular functions. A family of bicarbonate transporters is a major pH.sub.i regulator under physiological conditions in animal cells. Bicarbonate (HCO.sub.3 --) transporters are divided into four groups according to their functions [Boron, W. F. et al., J. Exp. Biol., 200:263-268(1997)]: Na.sup.+ -independent Cl--/HCO.sub.3 -- exchanger (alternatively called an anion exchanger, AE), Na.sup.+ --HCO.sub.3 -- cotransporter (NBC), K.sup.+ --HCO.sub.3 -- cotransporter, and Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger. Three AEs and three NBCs have been cloned and functionally characterized, but the molecular structure of the K.sup.+ --HCO.sub.3 -- cotransporter and the Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger have remained unknown.
A Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger was first discovered in invertebrate neurons and was later found in vertebrate neurons as well as non-neuronal cells, including brain, vascular endothelial cells, sperm, kidney and pancreatic .beta.-cells. Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger is an intracellular pH regulator that transports extracellular Na.sup.+ and HCO.sub.3 -- into the cells in exchange for intracellular Cl--, thereby playing an important role in cellular alkalinization.
In pancreatic .beta.-cells, glucose is the most important physiological regulator of insulin secretion. Glucose metabolism induces an increase in intracellular pH in the pancreatic cells. It has been shown that this glucose-induced pH.sub.i rise is evoked primarily by the action of Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger [Pace, C. S. et al., J. Membrane Biol., 73:39-43(1983)].
Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger is thus an important intracellular pH regulator in various cells, but its molecular basis is not known. Analysis of the molecular structure and function of Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger should be valuable not only for functional analysis of insulin secretion by pancreatic .beta.-cells but also for screening as well as for drug designing based on its molecular structure aimed at the development of therapeutics of diabetes mellitus.
On the above background, the present invention has as its objective to clone Na.sup.+ -driven Cl--/HCO.sub.3 -- exchangers, thereby obtaining their DNA for sequencing, providing cells of a different species expressing the DNAs, and determining the structure and function of the Na.sup.+ -driven Cl--/HCO.sub.3 -- exchangers.
SUMMARY OF THE INVENTION
Thus, the present invention provides a Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger protein comprising the amino acid sequence set forth as SEQ ID NO:2 or NO:4 in the Sequence Listing.
The present invention further provides a protein comprising an amino acid sequence having deletion, substitution, addition or insertion of one or more amino acids relative to the amino acid sequence set forth as SEQ ID NO:2 or NO:4 in the Sequence Listing and which, when expressed in a cell, functions as Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger.
The present invention further provides an above protein wherein the Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger, dependently upon both of extracellular bicarbonate and intracellular chloride ions, takes up extracellular sodium ion into the cell and transport intracellular sodium ion out of the cell.
The present invention further provides a cell in which one of the above proteins is expressed, wherein the cell is of a species different from the species of origin of the one of the proteins. Non-limiting examples of such cells of different species include Xenopus laevis oocytes and HEK293 cells. Expression of a Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger in such cells of different species may be achieved by transfection of a DNA encoding the Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger or by introduction of a cRNA corresponding to the Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger.
The present invention further provides antibodies to the above proteins. The antibodies may be monoclonal or polyclonal.
The present invention further provides a method for selection of agonists and antagonists of Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger, which method comprises bringing a cell of a different species expressing the protein into contact with a candidate compound, measuring the function of the Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger, comparing the result thus obtained with a result obtained by measuring the function of the sodium ion-driven chloride/bicarbonate exchanger of the cell which has not been brought into contact with the candidate compound, and thereby determining whether or not the candidate compound enhances or inhibits the function.
The present invention further provides a DNA comprising the nucleotide sequence set forth as SEQ ID NO:1 or NO:3 in the Sequence Listing, a DNA >2 comprising a nucleotide sequence consisting of nucleotides 67 through 3330 in the nucleotide sequence set forth as SEQ ID NO:1 in the Sequence Listing, and a DNA comprising a nucleotide sequence consisting of the nucleotides 83 through 3346 in the nucleotide sequence set forth as SEQ ID NO:3 in the Sequence Listing.
The present invention further provides a DNA comprising a nucleotide sequence having deletion, substitution, addition or insertion of one or more nucleotides relative to a DNA comprising a nucleotide sequence consisting of the nucleotides 67 through 3330 in the nucleotide sequence set forth as SEQ ID NO:1 in the Sequence Listing, and encoding:
(1) a protein comprising the amino acid sequence set forth as SEQ ID NO:2 in the Sequence Listing, or
(2) a protein comprising an amino acid sequence having deletion, substitution, addition or insertion of one or more amino acids relative to the amino acid sequence set forth as SEQ ID NO:2 in the Sequence Listing, which protein, when expressed in a cell, functions as Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger.
The present invention still further provides a DNA comprising a nucleotide sequence having deletion, substitution, addition or insertion of one or more nucleotides relative to a DNA comprising a nucleotide sequence consisting of the nucleotides 83 through 3346 in the nucleotide sequence set forth as SEQ ID NO:3 in the Sequence Listing, and encoding:
(1) a protein comprising the amino acid sequence set forth as SEQ ID NO:4 in the Sequence Listing, or
(2) a protein comprising an amino acid sequence having deletion, substitution, addition or insertion of one or more amino acids relative to the amino acid sequence set forth as SEQ ID NO:4 in the Sequence Listing, which protein, when expressed in a cell, functions as Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows RNA blot analysis of NCBE mRNA in rat tissues and hormone-secreting cell lines (a) and RT-PCR detection of NCBE mRNA from mouse pancreatic islets (b).
FIG. 2 shows a graph illustrating the effect of extracellular Na.sup.+ concentration on .sup.22 Na.sup.+ uptake.
FIG. 3 shows a graph illustrating the effect of extracellular HCO.sub.3 concentration on .sup.22 Na.sup.+ uptake.
FIG. 4 shows a graph illustrating the effect of intracellular Cl-- on .sup.36 Cl-- efflux.
FIG. 5 shows a graph illustrating the effect of DIDS on .sup.22 Na.sup.+ uptake.
FIG. 6 shows a graph illustrating the change in the intracellular pH in the presence and absence of 300 .mu.M DIDS, along with the change in the intracellular pH in control (non-transfected) cells.
FIG. 7 shows a graph illustrating the change observed in the intracellular pH when the environment is switched from a Na.sup.+ -free solution to a Na.sup.+ -containing solution, under a HCO.sub.3-- -free condition.
FIG. 8 shows a graph illustrating the change observed in the intracellular pH when the environment is switched from a Na.sup.+ -free solution to a Na.sup.+ -containing solution, under a Cl---free condition.
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, the cells of different species in which the protein of the present invention is expressed may be, for example, Xenopus laevis oocytes or HEK293 cells, and selected according to a given purpose from a variety of cells other than those from mouse or human. A conventional method well known in the art may be used for bringing about expression of a protein of the present invention in cells of species different from the species of origin of the protein.
In the present specification, the term "one or more" when used in the context of "an amino acid sequence having deletion, substitution, addition or insertion of one or more amino acids" means a number of one to ten in general, and preferably a number of one to a few (e.g., three or four).
Also in the present specification, the term "one or more" when used in the context of "a DNA comprising a nucleotide sequence having deletion, substitution, addition or insertion of one or more nucleotides" means a number of one to ten in general, and preferably a number of one to a few (e.g., three or four).
A variety of such mutant DNAs, as well as mutant proteins encoded by the DNAs, can be produced by means of recombinant DNA technology. First, mutations can be introduced into a cloned DNA fragment through any of different chemical or enzymatic processes. Mutant DNAs thus obtained are then sequenced for selection of particular mutants with intended merits. This method allows systematic preparation of different mutants regardless of their phenotypes. General methods for preparing mutant clones are as follows.
1. With the help of an oligonucleotide, substitution, deletion, insertion or addition of one or more nucleotides can be directly induced in a given DNA sequence. This method allows introduction of a number of mutations into a small region of a given DNA.
2. By using a relatively long oligonucleotide, a desired gene can be synthesized.
3. By means of region-specific mutagenesis, a desired mutation can be introduced into a large (1-3 kb) DNA region.
4. Linker-scanning mutagenesis of DNA is a method suitable to introduce a cluster point mutation into a relatively small (4-10 bp) DNA region.
5. PCR is also utilized as a method for directly introducing a mutation. [References: Current protocols in molecular biology. 3 vols., Edited by Ausubel F. M. et al., John Wiley & Sons, Inc., Current Protocols., Vol. 1, Chapter 8: Mutagenesis of cloned DNA, pages 8.0.1-8.5.10]
Also well known to those skilled in the art are methods for preparing plasmids or other vectors which can express a desired gene including different mutations obtained by the above methods. That is, by inserting a DNA comprising a desired gene into an expression vector DNA using a combination of restriction enzymes and a ligase, a recombinant plasmid is readily constructed which carries the desired gene. The recombinant plasmid thus obtained is then introduced into different cells to effect transfection, thereby producing transformed cells. A range of cells may be utilized, from prokaryotic cells, e.g. E. coli, to yeast, insect, plant and animal cells.
[Reference: Vectors essential data. Gacesa P. and Ramji D. P. 166 pages. BIOS Scientific Publishers Limited 1994., John Wiley & Sons in association with BIOS Scientific Publishers Ltd. Expression vectors, pages 9-12.]
Introduction of a recombinant plasmid into host cells may be carried out by calcium chloride method or by electroporation. Calcium chloride method is an efficient way for achieving transformation and it does not requires any apparatus specially designed for it. If still higher efficiency is needed, electroporation is recommended.
[References: Current Protocols in Molecular Biology, 3 Vols. Edited by Ausbel F. M. et al., John Wiley & Sons, Inc., Current Protocols, Vol. 1, unit 1.8: Introduction of Plasmid DNA into Cells, pages 1.8.1-1.8.10]
There are known two types of transfection generally carried out on animal cell lines, i.e., a transient type and a stable and permanent type. In transient transfection, transformed cells are cultured for 1-4 days to allow transcription and replication of the introduced gene, and then the cells are harvested and their DNA analyzed. In many studies, alternatively, a stable transformant cell line is produced, in which the introduced gene is incorporated into the chromosomes. Examples of the method for transfection include calcium phosphate method, electroporation, and liposome fusion method. [Reference: Current protocols in molecular biology. 3 vols. Edited by Ausubel F. M. et al., John Wiley & Son, Inc., Current Protocols. Vol. 1, chapter 9: Introduction of DNA into mammalian cells, pages 9.0.1-9.17.3.]
Polyclonal and monoclonal antibodies to the Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger proteins of the present invention, or to their fragments or their analogues, are readily prepared using technologies well known in the art. Antibodies thus obtained may be used, for example, in immunohistochemistry of Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger protein expressed in cells of different species or for inhibition of its function by blocking the protein. Cells of different species in which the function of Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger is inhibited are used as a control in selection of agonists/antagonists of the protein.
A general method for preparing a monoclonal antibody in mg-scale directed to the Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger proteins of the present invention is as follows: Mice are inoculated with one of the antigen proteins to immunize. The spleen is removed from the mice exhibiting a sufficient antibody titer. The spleen cells are dissociated and B cells are selected and fused with myeloma cells of B cell origin to form hybridoma cells secreting the antibody. The monoclonal antibody secreted by the hybridoma cells is purified from the culture medium by using an affinity column, or by ion-exchange or gel filtration, etc. Polyclonal antibody of the present invention may also be prepared by a conventional method: using rabbits, horses, mice or guinea pigs as immunized animals, the antigen protein is inoculated along one of the schedules known in the art to immunize the animals, and then an immunoglobulin such as IgG is isolated from the collected serum.
[Reference: Current protocols in molecular biology, 3 vols. Edited by Ausubel F. M. et al., John Wiley & Sons, Inc., Current Protocols, Vol. 2, chapter 11: Immunology, pages 11.0.1-11.16.13.]
EXAMPLES
The present invention is described in further details with reference to examples. However, it is not intended that the present invention be limited to the examples.
To determine its structure and functional role, the present inventors cloned a Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger (designated NCBE) from cDNA library from MIN6, an insulin secreting mouse cell line. The primary structure, tissue distribution and functional characterization of Na.sup.+ -driven chloride (Cl--)/bicarbonate (HCO.sub.3 --) exchanger (NCBE) will be described below.
It was revealed that the mouse NCBE protein (SEQ ID NO:2) consists of 1,088 amino acids and has 65, 65 and 41% amino acid identity to the sodium bicarbonate cotransporter from human muscle, retina and kidney, respectively. The mouse NCBE has was found to have ten putative membrane spanning regions and the conserved 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-binding motif characteristic of anion exchangers and sodium bicarbonate cotransporters. NCBE mRNA is was shown to be expressed at high levels in the brain and in a mouse insulinoma cell line MIN6, and, though at low levels, also in pituitary, testis, kidney, and ileum. Through functional analysis of NCBE protein expressed in Xenopus laevis oocytes and HEK293 cells, it was demonstrated that the protein causes a rise in intracellular pH by transporting extracellular Na.sup.+ and HCO.sub.3 -- into cells in exchange for intracellular Cl--. Based on the findings, the present inventors concluded that the cloned NCBE is the Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger that regulates intracellular pH in native cells.
Then, to also identify a human NCBE, a partial sequence (2,746 bp) of the mouse Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger cDNA obtained above was first amplified by PCR. For this amplification, a DNA fragment having the sequence consisting of the nucleotides 250-270 of the sequence set forth as SEQ ID NO:1 in the Sequence Listing was used as a sense primer, and, as an antisense primer, a DNA fragment having a sequence complementary to the sequence consisting of the nucleotides 2976-2995 of the sequence set forth as SEQ ID NO:1 in the Sequence Listing. PCR conditions were as follows:
Initial denaturation:94.degree. C., 2 min
Amplification (20 cycles)
denaturation:94.degree. C., 15 sec
annealing:60.degree. C., 30 sec
extension:72.degree. C., 2 min
Final extension:72.degree. C., 7 min
The PCR product thus obtained was labeled with .sup.32 P-dCTP by nick translation and used to screen about 1 million phages from a human fetal brain cDNA library (Clontech). Four positive phage clones were obtained and their DNAs were digested with EcoRI. After agarose electrophoresis, corresponding bands were excised, and respective DNAs extracted to obtain inserts. Separately, pGEM7Z (Promega) was digested with EcoRI and treated with alkaline phosphatase. To this, the inserts obtained from the positive phages were ligated, respectively, for subcloning. The respective inserts were then sequenced on an autosequencer (ABI 310), and, based on the sequences thus obtained, the cDNA nucleotide sequence corresponding to human NCBE protein was determined (set forth as SEQ ID NO:3). According to the result, the sequence of human NCBE protein then was determined (set forth as SEQ ID NO:4 in the Sequence Listing).
The methods and results of the above experiments will be described below, focusing on the procedures followed and results obtained with mouse NCBE.
[Materials and Methods]
<cDNA Cloning>
A partial cDNA fragment of human kidney NBC cDNA [Burnham, C. E., et al., J. Biol. Chem., 272:19111-19114(1997)] amplified by PCR, using a human kidney cDNA as a template. The sense and antisense primers used in this were 5'-TTTGGAGAAAACCCCTGGT-3' (nt 2232-2250) (SEQ ID NO:5) and 5'-TGACATCATCCAGGAAGCTG-3' (nt 2912-2931) (SEQ ID NO:6). PCR was performed up to 40 cycles under the following conditions: denaturation at 94.degree. C. for 15 sec, annealing at 60.degree. C. for 30 sec, and extension at 72.degree. C. for 45 sec in a thermal cycler GeneAmp PCR system 9600 (PE Applied Biosystems, Foster, Calif.). The 700 bp-PCR product was subjected to screening of a MIN6 cDNA library [Inagaki, N., et al., Proc. Natl. Acad. Sci. USA, 91:2679-2683(1994)] as a probe under a low stringent condition previously described [Fukumoto, H. et al., Proc. Natl. Acad. Sci. USA, 85:5434-5438(1988)]. Positive clones were subcloned in pGEM-3Z vector (Promega, Madison, Wis.) and sequenced in both directions using ABI PRISM .TM. 377 DNA sequencer (PE Applied Biosystems).
<RNA blot analysis>
RNA blot analysis was performed using 10 .mu.g of total RNA from various tissues and cells. The RNAs were denatured with formaldehyde, electrophoresed on 1% agarose gel, and transferred onto a nylon membrane. The blots were probed with NCBE cDNA under a standard condition previously described [Wang, C -Z. et al., Biochem. Biophys. Res. Commun., 220: 196-202(1996)]. Before autoradiography, the blots were washed with 0.1.times.SSC and 0.1% SDS at room temperature for one hr and then at 50.degree. C. for another hour.
Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Total RNA was prepared from isolated mouse pancreatic islets with TRIZOL Reagent (Life Technologies, Inc., Rockvill, Md.). First-strand cDNA (10 ng) was generated using Superscript.TM. II reverse transcriptase (Life Technologies) with random primers. PCR was performed with Expand High Fidelity PCR System (Roch Diagnostics, Mannheim, Germany) using about 1 ng of template DNA in a 20 .mu.l reaction volume under a standard condition. The sense and antisense primers used were 5'-GTCATGTTAGACCAACAGGT-3' (nt 4283-4302) (SEQ ID NO:7) and 5'-GTTGTAATAGCGACACTC-3' (nt 4911-4928) (SEQ ID NO:8). The PCR product was resolved on 1% agarose gel and confirmed by DNA sequencing.
Functional Analysis of NCBE in Xenopus laevis oocytes
The coding sequence of NCBE in pSD5 was linearized by digestion with FspI and in vitro transcribed with SP6 RNA polymerase as previously described (Wang, C -Z. et al., Biochem. Biophys. Res. Commun., 220:196-202(1996)). Defolliculated oocytes were injected with NCBE cRNA (50 nl, 0.5 .mu.g/.mu.l) or water and incubated in 1.times.MBS medium (88 mM NaCl, 1 mM KCl, 0.8 mM MgCl.sub.2, 0.4 mM CaCl.sub.2, 0.3 mM Ca(NO.sub.3).sub.2, 2.4 mM NaHCO.sub.3 and 7.5 mM Tris, pH 7.4) for 3-5 days at 18.degree. C. before the studies. The oocytes were preincubated for one hr at 18.degree. C. in the standard solution (100 mM NaCl, 2 mM KCl, 1 mM MgCl.sub.2, 1 mM CaCl.sub.2, and 8 mM NaHCO.sub.3, pH 7.4).
For studies of dependency on extracellular Na.sup.+ concentration, the oocytes were then incubated in 1.4 ml of either 1, 10, 30 or 100 mM Na.sup.+ solution bubbled with 1.5% CO.sub.2, pH 7.4 with 0.074 MBq of .sup.22 Na+ (NEN.TM. Life Science Products, Boston, Mass.). In each solution, the Na.sup.+ in the standard solution was substituted with an equal molar amount of choline. A ten .mu.l aliquot was removed from the incubation solution for later determination of .sup.22 Na.sup.+ -specific activity. After 15 min, .sup.22 Na.sup.+ uptake was terminated by three washes with an ice-cold solution containing 1, 10, 30 or 100 mM Na.sup.+, pH 7.4, respectively, and the oocytes were then lyzed in 0.5 ml of 5% SDS and 4.5 ml of Aqueous Counting Scintillant (Amersham Pharmacia Biotech) was added. .sup.22 Na.sup.+ uptake was performed in either Cl---free 1, 10, 30 or 100 mM Na.sup.+ solution (pH 7.4). Extracellular Cl-- was substituted with an equal molar amount of gluconic acid, and extracellular Na.sup.+ was substituted with an equal molar amount of N-methyl-D-glucamine (NMG). The .sup.22 Na.sup.+ uptake for 15 min was also examined in the presence or absence of 300 .mu.M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, Sigma), an inhibitor of anion-transporters in the standard solution.
For the study of dependency on extracellular HCO.sub.3 -- concentration, Na.sup.+ uptake experiments were performed in 1, 3, 10 or 30 mM HCO.sub.3 -- solutions bubbled with 1.5% CO.sub.2 at 18.degree. C., pH 7.4, including 0.074 MBq of .sup.22 Na.sup.+. The solutions contained 2 mM KCl, 1 mM MgCl.sub.2 and 1 mM CaCl.sub.2, pH 7.4, and further 107 mM NaCl and 1 mM NaHCO.sub.3 for 1 mM HCO.sub.3 -- solution, 105 mM NaCl and 3 mM NaHCO.sub.3 for 3 mM HCO.sub.3 -- solution, 98 mM NaCl and 10 mM NaHCO.sub.3 for 10 mM HCO.sub.3 -- solution, and 78 mM NaCl and 30 mM NaHCO.sub.3 for 30 mM HCO.sub.3 -- solution.
For .sup.36 Cl-- efflux experiment, the oocytes were preincubated for one hour in the Cl---free solution for depletion of intracellular Cl--, or Cl-- containing standard solution. The oocytes were incubated in 0.074 MBq of .sup.36 Cl---containing solution (NEN.TM. Life Science Products) at 18.degree. C. for one hour bubbling with 1.5% CO.sub.2. The oocytes were rapidly washed three times with the corresponding, respective solutions and then transferred into 1.5 ml of each a Cl---free solution bubbled with 1.5% CO.sub.2, pH 7.4. A 10-.mu.l aliquot was removed from the incubation solution for later determination of .sup.36 Cl-- specific activity. .sup.36 Cl-- activities in the solution were measured at 0, 5, 15, 25 and 35 min. The oocytes were treated as described above for the measurement of the remaining intracellular .sup.36 Cl--. Portions of the medium from respective time points were counted and the values were summed to determine flux. .sup.36 C-- efflux was presented as a percent relative to the total cellular .sup.36 Cl-- released. .sup.22 Na.sup.+ and .sup.36 Cl-- activities were measured with beta scintillation counter (Aloka, Japan).
<Functional Analysis of NCBE in HEK293 Cells>
HEK293 cells were plated at a density of 3.times.10.sup.5 cells per 3.5 cm-diameter dish containing a coverslip, and cultured in Dulbecco's modified Eagle's medium (DMEM, high glucose) supplemented with 10% fetal bovine serum, streptomycin (60.5 .mu.g/ml), and penicillin (100 .mu.g/ml) at 37.degree. C. under a humidified condition of 95% air and 5% CO.sub.2. Cells were transfected with 1 .mu.g of the full-length NCBE cDNA in the pcDNA3.1 vector (Invitrogen, Groningen, The Netherlands) using Lipofectamine, Lipofectamine Plus, and Opti-MEM I reagents (Life Technologies, Gaithersburg, Md.) according to the manufacturer's instructions. The cells were studied 48-72 hours after transfection. Changes in intracellular pH were monitored using 2',7'-bis-(2-carboxyethyl)-5-(6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM, Molecular Probe, Eugene, Oreg.) (Burnham, C. E., et al., J. Biol. Chem., 272:19111-19114(1997)). HEK293 cells were loaded with 1 .mu.M BCECF-AM for one hour and monitored for changes in intracellular pH by dual-excitation wavelength method with a computerized image processor (490 nm/450 nm; 520-560 nm emission) (Argus-50; Hamamatsu Photonics, Hamamatsu, Japan). .DELTA.pH.sub.i was determined as the difference between the intracellular pH before and 10 min after switching to the test solution. The pH.sub.i calibration curve was generated using KCl/nigericin technique (Thomas, J. A. et al., Biochemistry 18:2210-2218(1979)). In all the experiments, the cells were first acidified by NH.sub.4.sup.+ -prepulse with 40 mM NH.sub.4 Cl-containing solution for 5 min before switching to the Na.sup.+ -containing respective test solutions (Burnham, C. E., et al., J. Biol. Chem., 272:19111-19114(1997)).
To estimate Na.sup.+ -dependency of the intracellular pH (.DELTA.pH.sup.i) recovery from intracellular acidification, a Na.sup.+ -free solution (115 mM tetramethylammonium chloride (TMA-Cl), 25 mM KHCO.sub.3, 0.8 mM K.sub.2 HPO.sub.4, 0.2 mM KH.sub.2 PO.sub.4, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2, 10 mM HEPES) and a Na.sup.+ -containing solution (TMA-Cl and KHCO.sub.3 in the Na.sup.+ -free solution were replaced with 90 mM NaCl, 25 mM KCl, and 25 mM NaHCO.sub.3) were used.
To test for HCO---dependency, a HCO.sub.3 ---free, Na.sup.+ -free solution (115 mM TMA-Cl, 0.8 mM K.sub.2 HPO.sub.4, 0.2 mM KH.sub.2 PO.sub.4, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2, 10 mM HEPES) and a HCO.sub.3 ---free, Na.sup.+ -containing solution (in which TMA-Cl in the HCO.sub.3 ---free, Na.sup.+ -free solution was replaced with 90 mM NaCl and 25 mM KCl) were used.
To determine Cl---dependency, a Cl---free, Na.sup.+ -free solution (25 mM KHCO.sub.3, 0.8 mM K.sub.2 HPO.sub.4, 0.2 mM KH.sub.2 PO.sub.4, 10 mM HEPES, 115 mM NMG-gluconate) and a Cl---free, Na.sup.+ -containing solution (in which NMG-gluconate was replaced with 115 mM sodium gluconate) were used and the results were compared with each other.
All the solutions were bubbled with 95% O.sub.2 and 5% CO.sub.2, and their pH adjusted to 7.4. The osmolarity of each solution was adjusted with sucrose. The assays were carried out at 37.degree. C.
<Statistical Analysis>
The results were expressed as means .+-. SE. Statistical significance between experiments was determined by Student's t test.
[Results and Discussion]
NCBE is structurally related to Na.sup.+ --HCO.sub.3 -- transporters.
As described above, the cDNA encoding Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger (NCBE) was cloned from a MIN6 cDNA by screening it using a partial human kidney Na.sup.+ --HCO.sub.3 -- cotransporter (NBC) cDNA as a probe. The thus determined nucleotide sequence (NCBE) is set forth as SEQ ID NO:1 in the Sequence Listing. The composite 5,385-bp nucleotide sequence contains an open reading frame, which follows an in-frame termination signal upstream of the "ATG" and encodes a protein of 1,088 amino acids set forth as SEQ ID NO:1 having a predicted molecular weight of 122 kDa. A hydrophobicity analysis indicates that the amino acid sequence has putative membrane spanning segments (TM1 to TM10) at the following positions, respectively.
TM 1: amino acids 479.about.499
TM2: amino acids 514.about.534
TM3: amino acids 564.about.584
TM4: amino acids 693.about.713
TM5: amino acids 733.about.753
TM6: amino acids 780.about.800
TM7: amino acids 826.about.846
TM8: amino acids 882.about.901
TM9: amino acids 905.about.924
TM10: amino acids 972.about.992
In the amino acid sequence, there are three potential N-linked glycosylation sites in the extracellular loops between the third (TM3) and fourth (TM4) spanning region (Asn-647, Asn-657 and Asn-667). Putative DIDS-binding motif is at amino acids 815-818.
Comparison of amino acid sequence between NCBE and other NBCs showed that NCBE has 65%, 65% and 41% amino acid identity to human muscle NBC [Pushkin, A. et al., J. Biol. Chem., 274:16569-16575(1999)], human retina NBC [Ishibashi, K. et al., Biochem. Biophys. Res. Commun., 24:535-538(1998)], and human kidney NBC [Burnham, C. E., et al., J. Biol. Chem., 272:19111-19114(1997)], respectively. This indicates that NCBE represents a novel bicarbonate transporter. The amino acid sequences in the putative transmembrane regions and DIDS-binding motif Lys Leu Lys Lys (residue 815-818) are well conserved in NCBE, while those in the intracellular amino- and carboxyl-terminal regions and in the large extracellular loop between the third and the fourth membrane spanning regions are rather divergent.
NCBE is expressed at high levels in the brain and insulin-secreting clonal pancreatic .beta.-cells.
RNA blot analysis revealed a 5.5 kb NCBE mRNA is expressed at high levels in brain and the insulin secreting cell line MIN6 cells and expressed at low levels in pituitary, testis, kidney, and ileum (FIG. 1, a). RT-PCR analysis shows that NCBE is also expressed in pancreatic islets (FIG. 1, b).
In the figure, "a" represents the result of the RNA blot analysis of NCBE mRNA in rat tissues and hormone-secreting cell lines. The size of hybridized transcripts is indicated. "b" represents the results of RT-PCR detection of NCBE mRNA in mouse pancreatic islets. DNA length markers and RT-PCR products are shown in lanes 1 and 2, respectively.
NCBE is a Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger that regulates intracellular pH (pH.sub.i).
The present inventors examined the functional properties of NCBE using Xenopus laevis oocyte system. .sup.22 Na.sup.+ uptake and .sup.36 Cl-- efflux were measured 3-5 days after injection of the cRNAs or water (control). Bubbling with 1.5% CO.sub.2 to acidify the oocytes, the present inventors first examined the effect of extracellular Na.sup.+ concentration on .sup.22 Na.sup.+ uptake. The results are shown in FIG. 3.
FIG. 3 illustrates the relation between .sup.22 Na.sup.+ uptake (nmol/oocyte/hour) and extracellular Na.sup.+ concentration. In the figure, .box-solid. and .circle-solid. indicate the results obtained with the cells injected with NCBE cRNA, and .quadrature. and .largecircle. the results obtained with the cells injected with water. .box-solid. and .quadrature. indicate the results obtained using Cl---containing extracellular solutions, and .circle-solid. and .largecircle. indicate the results obtained using Cl---free extracellular solutions. The respective data represent the mean .+-.SE (standard error) for 7 to 16 oocytes from two independent experiments. * and .dagger. (p<0.05) indicate the presence/absence of statistical significance in the difference from water-injected cells and from incubation in Cl---free extracellular solutions, respectively, with 10, 30 or 100 mM Na.sup.+.
As shown in FIG. 2, the increase in .sup.22 Na.sup.+ uptake was dependent on extracellular Na.sup.+ concentrations, with a linear pattern observed in NCBE cRNA-injected oocytes over the physiological range of Na.sup.+ concentrations. The water-injected oocytes showed no increase in .sup.22 Na.sup.+ uptake. Comparison of Na.sup.+ uptake between the results obtained with Cl---containing and Cl---free solutions showed significantly higher Na.sup.+ uptake in the presence of extracellular Cl-- than the in the absence of extracellular Cl-- (FIG. 2). These results indicate that NCBE transports extracellular Na.sup.+ into the cells and that extracellular Cl-- participates in acceleration of the NCBE's activity.
The present inventors, then, examined the effect of extracellular bicarbonate ion on .sup.22 Na.sup.+ uptake. The results are shown in FIG. 3. The respective data represent the mean.+-.SE (standard error) for 11 to 16 oocytes from two independent experiments. * (p<0.05) indicates comparison with water-injected cells. As evident from the figure, increased extracellular bicarbonate ion significantly boosted Na.sup.+ uptake in a concentration-dependent manner in the NCBE cRNA-injected oocytes, while the water-injected oocytes did not show any such change in Na.sup.+ uptake. These results indicate that extracellular bicarbonate ion is necessary in transporting Na.sup.+ into the cells.
To determine whether Cl-- is transported into or out of the cells by NCBE, the present inventors examined .sup.36 Cl-- efflux from Xenopus laevis oocytes. As .sup.36 Cl-- influx was not detected in water-injected oocytes, analysis was made only for .sup.36 Cl-- efflux from NCBE cRNA-injected oocytes. The rate (%) of .sup.36 Cl-- efflux from NCBE cRNA-injected oocytes was measured from 0 to 35 min under the intracellular Cl---depleted condition by preincubation with a Cl---free solution and under the intracellular Cl-- non-depleted condition by preincubation with Cl---containing solution. The results are shown in FIG. 4. In the figure, .circle-solid. indicates the results obtained with cells under the intracellular Cl-- non-depleted condition (preincubation in the Cl---containing solution), and .tangle-solidup. indicates the results obtained with cells under the intracellular Cl---depleted condition (preincubation in the Cl---free solution). The data represent the mean .+-.SE (standard error) for 16 to 17 oocytes from three independent experiments. * (p<0.05) indicates comparison with intracellular Cl---depleted cells, at 5, 15, 25, and 35 min.
Comparison made among results of .sup.36 Cl-- efflux under the different conditions indicates that NCBE transports intracellular Cl-- out of the cells. Taken together, these results demonstrate that NCBE exchanges extracellular Na.sup.+ and bicarbonate ion with intracellular Cl--.
The present inventors also examined the effect of DIDS, an inhibitor of anion-transporter, on .sup.22 Na.sup.+ uptake. Expression was assessed in the absence or presence of 0.3 mM DIDS. The results are shown in FIG. 5. The data represent the mean .+-.SE (standard error) for 21 to 22 oocytes from three independent experiments. * (p<0.05) indicates comparison with cRNA+DIDS.
While the .sup.22 Na.sup.+ uptake in NCBE cRNA-injected oocytes was 31.4.+-.2.1 nmol/oocyte/hour (n=21) in the absence of DIDS, it was 6.0.+-.0.7 nmol/oocyte/hour (n=14) in the presence of 300 .mu.M DIDS. In water-injected oocytes, the uptake was 1.6.+-.0.3 (n=22) and 2.1.+-.0.4 (n=19) nmol/oocytes/hour in the absence and presence of DIDS, respectively. Thus, DIDS was shown to partially inhibit .sup.22 Na.sup.+ uptake by NCBE (FIG. 5).
To clarify the role of NCBE in the regulation of intracellular pH, changes in intracellular pH were measured under various conditions using HEK293 cells transiently transfected with NCBE. All the experiments were performed under conditions where the intracellular pH was acidified with NH.sub.4 +prepulse. To determine whether the change in the intracellular pH is dependent on extracellular Na.sup.+, the environment of the cells was switched from a Na.sup.+ -free solution to a Na.sup.+ -containing solution. The results are shown in FIG. 6. FIG. 6 is a graph illustrates a trace of control (non-transfected) cells and NCBE-transfected cells with or without 300 .mu.M DIDS. The environment of the cells was switched from a Na.sup.+ -free solution to a Na.sup.+ -containing solution.
As shown in the figure, a rapid recovery of intracellular pH (.DELTA.pH.sub.i) was observed only in the NCBE-transfected cells in the presence of 1 mM 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), a specific inhibitor of Na.sup.+ /H+exchanger (.DELTA.pH.sub.i was 0.239.+-.0.028 (n=97) in the NCBE-transfected cells and 0.003.+-.0.015 (n=70) in the control. p<0.05) (FIG. 6). This recovery in intracellular pH was partially inhibited by 300 .mu.M DIDS (.DELTA.pH.sub.i was 0.023.+-.0.042 (n=89). p<0.05).
To determine whether this change in intracellular pH is bicarbonate ion-dependent, the environment of the NCBE-transfected cells was switched from a HCO.sub.3 ---free, Na.sup.+ -free solution to a HCO.sub.3 ---free but Na.sup.+ -containing solution, in the presence of 1 mM EIPA. However, as shown in FIG. 7, no recovery of intracellular pH was detected (.DELTA.pH.sub.i was 0.002.+-.0.014 (n=71)).
Finally, an examination for Cl-- dependency was also made by the present inventors. NCBE-transfected cells were kept in a Cl---free solution (under an intracellular Cl---depletion condition) throughout the experiments. Under this condition, the environment of the cells was switched from a Na.sup.+ -free solution to a Na.sup.+ -containing solution. In the presence of 1 mM EIPA, as shown in FIG. 8, no recovery of intracellular pH was detected [.DELTA.pH, was 0.067.+-.0.012 (n=95)].
These results indicate that recovery of intracellular pH from intracellular acidification is detected only where extracellular Na.sup.+ and HCO.sub.3 -- and intracellular Cl-- are present.
The studies of the function of NCBE heterologously expressed in Xenopus laevis oocytes and HEK293 cells show that NCBE allows intracellular pH to recover from acute intracellular acidification, by transporting extracellular Na.sup.+ and HCO.sub.3 -- in exchange for intracellular Cl-- (FIGS. 3 and 4). NCBE is functionally distinct from so far reported anion exchangers and Na.sup.+ --HCO.sub.3 -- cotransporters. This is because: 1) NCBE, expressed in Xenopus laevis, exhibited a Na.sup.+ uptake increase dependent on intracellular Cl--, 2) it shows the ability of exporting Cl-- out of the cells, and, furthermore, 3) the NCBE, expressed in HEK239 cells, elevates intracellular pH in a manner dependent upon extracellular Na.sup.+ and HCO.sub.3 --, and intracellular Cl--. These properties are similar to those of Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger described in native cells. The cloned NCBE, therefore, is concluded to be a Na.sup.+ -driven Cl--/HCO.sub.3 -- exchanger.
Possible physiological relevance of NCBE. That NCBE mRNA is expressed in insulin secreting cell line MIN6 and pancreatic islets implies its physiological relevance. It has been shown that glucose-induced insulin secretion is accompanied by a rise in intracellular pH in pancreatic .beta.-cells. While several intracellular pH regulators have been suggested to be present in pancreatic .beta.-cells, their molecular basis has not been known so far. NCBE is the first intracellular pH-regulating exchanger whose primary structure and functional properties have been determined. NCBE most likely contributes to the process for recovery of intracellular pH in pancreatic .beta.-cells that have been acidified by glucose metabolism. NCBE mRNA occurs also in the testis, although its expression level is low. It has been shown that intracellular pH regulates many functions in sperm including sperm capacitation. As sperm capacitation results in the increase in intracellular pH, which requires functional Na.sup.+, Cl-- and HCO.sub.3 ---dependent acid-efflux pathway, NCBE could participate in the process of sperm capacitation. NCBE mRNA is also expressed at high levels in the brain. Though physiological studies suggests that NCBE is present in hippocampal neurons and astrocytes, its physiological significance of such cells remains unknown at present.
# SEQUENCE LISTING
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catgcagtca aaaactaggc ttgtattaaa tgctttagag atatttgaag ag
#ttttgtgg 4100
ggcttttcat tttaaatctt taccagaaat atgctactga gtttctctcc ca
#ttgacaag 4160
ggttgcttcc cgaataagcc tatgacatac atacttacgg aatgccacat gg
#tgcaacat 4220
tgtacatttg atgccagccc tggcagctgt tctgctgacc atggtcatgt gc
#tgctaagt 4280
ttggttccta tcatgttgtc atgttagacc aacaggtctc caactgtatt tt
#gttttttt 4340
tgcaaagctc ttttccacat tttaactaaa tgcatgttgt ggaaaaatag tc
#tttgaaat 4400
aaaatttcag attttgttag aaaaggttat gtaaatactt cagtccatat ga
#aacagttc 4460
aactttattg aaacaggaag gagattatgg atttttgagt attactaaat at
#aaatttca 4520
tttaattttc aataaatgtg ctttaataca aaacaaaata tcataggggt ct
#tagttcct 4580
aaaaaagtat caatgattaa caaccttata atctttcaat gtccaggttt ag
#aaaaattc 4640
agagccttct gggttttata aattacatgt actctgtgta aatacacata at
#tagaaaaa 4700
tcctctttgc ttttaagcta atgaagacga gagacaacag agcctacata ac
#cttaatat 4760
tctgatatct tgaacaaaaa atttcctcag aatcctttca ggagccattt tt
#ttaatgag 4820
atatgagcca aaattgtgag aagaattttc agttcgtaaa gtctgtattt at
#aaatggta 4880
aagaaaaatg caaaattctt ttccaaatgt gctacctttg tgatagttgt aa
#tagcgaca 4940
ctctctctaa acattctcgc tgtctatgac ttagcaggcc aatccccaaa gc
#actctcct 5000
ggtgtctcta gagtgtcatg tctgttctgt tgaaatgacc agtgagtgac ac
#ttcacatg 5060
atcactggtt taaacaggca atcagcctat gaaattctgt atttctgaat at
#ttttatag 5120
taattttgtt cttgtgtgaa ttttaatgct atctctatct taatcttaat at
#tttgaaat 5180
cacataaaat ataagaaaat gtagtattct atatttactc taatttcaga tt
#cctggtca 5240
aaattactga atatcttgaa tgtaatttat tgcaatgttt aagtactgtg ta
#aatgtgac 5300
aggatattgt gtttttcaaa actaagaaat gttatgtgga aataaatatt ta
#tcctaaaa 5360
aaaaaaaaaa aaaaaaaaaa aaaaa
#
# 5385
<210> SEQ ID NO 2
<211> LENGTH: 1088
<212> TYPE: PRT
<213> ORGANISM: Mus musculus
<400> SEQUENCE: 2
Met Glu Ile Lys Asp Gln Gly Ala Gln Met Gl
#u Pro Leu Leu Pro Thr
1 5
# 10
# 15
Arg Asn Asp Glu Glu Ala Val Val Asp Arg Gl
#y Gly Thr Arg Ser Ile
20
# 25
# 30
Leu Lys Thr His Phe Glu Lys Glu Asp Leu Gl
#u Gly His Arg Thr Leu
35
# 40
# 45
Phe Ile Gly Val His Val Pro Leu Gly Gly Ar
#g Lys Ser His Arg Arg
50
# 55
# 60
His Arg His Arg Gly His Lys His Arg Lys Ar
#g Asp Arg Glu Arg Asp
65
#70
#75
#80
Ser Gly Leu Glu Asp Gly Arg Glu Ser Pro Se
#r Phe Asp Thr Pro Ser
85
# 90
# 95
Gln Arg Val Gln Phe Ile Leu Gly Thr Glu As
#p Asp Asp Glu Glu His
100
# 105
# 110
Leu Pro His Asp Leu Phe Thr Glu Leu Asp Gl
#u Ile Cys Trp Arg Glu
115
# 120
# 125
Gly Glu Asp Ala Glu Trp Arg Glu Thr Ala Ar
#g Trp Leu Lys Phe Glu
130
# 135
# 140
Glu Asp Val Glu Asp Gly Gly Glu Arg Trp Se
#r Lys Pro Tyr Val Ala
145 1
#50 1
#55 1
#60
Thr Leu Ser Leu His Ser Leu Phe Glu Leu Ar
#g Ser Cys Ile Leu Asn
165
# 170
# 175
Gly Thr Val Leu Leu Asp Met His Ala Asn Th
#r Ile Glu Glu Ile Ala
180
# 185
# 190
Asp Met Val Leu Asp Gln Gln Val Ser Ser Gl
#y Gln Leu Asn Glu Asp
195
# 200
# 205
Val Arg His Arg Val His Glu Ala Leu Met Ly
#s Gln His His His Gln
210
# 215
# 220
Asn Gln Lys Lys Leu Ala Asn Arg Ile Pro Il
#e Val Arg Ser Leu Ala
225 2
#30 2
#35 2
#40
Asp Ile Gly Lys Lys Gln Ser Glu Pro Asn Se
#r Met Asp Lys Asn Ala
245
# 250
# 255
Gly Gln Val Val Ser Pro Gln Ser Ala Pro Al
#a Cys Ala Glu Asn Lys
260
# 265
# 270
Asn Asp Val Ser Arg Glu Asn Ser Thr Val As
#p Phe Ser Lys Val Asp
275
# 280
# 285
Leu His Phe Met Lys Lys Ile Pro Pro Gly Al
#a Glu Ala Ser Asn Ile
290
# 295
# 300
Leu Val Gly Glu Leu Glu Phe Leu Asp Arg Al
#a Val Val Ala Phe Val
305 3
#10 3
#15 3
#20
Arg Leu Ser Pro Ala Val Leu Leu Gln Gly Le
#u Ala Glu Val Pro Ile
325
# 330
# 335
Pro Ser Arg Phe Leu Phe Ile Leu Leu Gly Pr
#o Leu Gly Lys Gly Gln
340
# 345
# 350
Gln Tyr His Glu Ile Gly Arg Ser Ile Ala Th
#r Leu Met Thr Asp Glu
355
# 360
# 365
Val Phe His Asp Val Ala Tyr Lys Ala Lys As
#p Arg Asn Asp Leu Val
370
# 375
# 380
Ser Gly Ile Asp Glu Phe Leu Asp Gln Val Th
#r Val Leu Pro Pro Gly
385 3
#90 3
#95 4
#00
Glu Trp Asp Pro Ser Ile Arg Ile Glu Pro Pr
#o Lys Asn Val Pro Ser
405
# 410
# 415
Gln Glu Lys Arg Lys Ile Pro Ala Val Pro As
#n Gly Thr Ala Ala His
420
# 425
# 430
Gly Glu Ala Glu Pro His Gly Gly His Ser Gl
#y Pro Glu Leu Gln Arg
435
# 440
# 445
Thr Gly Arg Ile Phe Gly Gly Leu Met Leu As
#p Ile Lys Arg Lys Ala
450
# 455
# 460
Pro Phe Phe Trp Ser Asp Phe Arg Asp Ala Ph
#e Ser Leu Gln Cys Leu
465 4
#70 4
#75 4
#80
Ala Ser Phe Leu Phe Leu Tyr Cys Ala Cys Me
#t Ser Pro Val Ile Thr
485
# 490
# 495
Phe Gly Gly Leu Leu Gly Glu Ala Thr Glu Gl
#y Arg Ile Ser Ala Ile
500
# 505
# 510
Glu Ser Leu Phe Gly Ala Ser Met Thr Gly Il
#e Ala Tyr Ser Leu Phe
515
# 520
# 525
Gly Gly Gln Pro Leu Thr Ile Leu Gly Ser Th
#r Gly Pro Val Leu Val
530
# 535
# 540
Phe Glu Lys Ile Leu Phe Lys Phe Cys Lys Gl
#u Tyr Gly Leu Ser Tyr
545 5
#50 5
#55 5
#60
Leu Ser Leu Arg Ala Ser Ile Gly Leu Trp Th
#r Ala Thr Leu Cys Ile
565
# 570
# 575
Ile Leu Val Ala Thr Asp Ala Ser Ser Leu Va
#l Cys Tyr Ile Thr Arg
580
# 585
# 590
Phe Thr Glu Glu Ala Phe Ala Ser Leu Ile Cy
#s Ile Ile Phe Ile Tyr
595
# 600
# 605
Glu Ala Leu Glu Lys Leu Phe Glu Leu Ser Gl
#u Thr Tyr Pro Ile Asn
610
# 615
# 620
Met His Asn Asp Leu Glu Leu Leu Thr Gln Ty
#r Ser Cys Asn Cys Met
625 6
#30 6
#35 6
#40
Glu Pro His Ser Pro Ser Asn Asp Thr Leu Ly
#s Glu Trp Arg Glu Ser
645
# 650
# 655
Asn Leu Ser Ala Ser Asp Ile Ile Trp Gly As
#n Leu Thr Val Ser Glu
660
# 665
# 670
Cys Arg Ser Leu His Gly Glu Tyr Val Gly Ar
#g Ala Cys Gly His Gly
675
# 680
# 685
His Pro Tyr Val Pro Asp Val Leu Phe Trp Se
#r Val Ile Leu Phe Phe
690
# 695
# 700
Ser Thr Val Thr Met Ser Ala Thr Leu Lys Gl
#n Phe Lys Thr Ser Arg
705 7
#10 7
#15 7
#20
Tyr Phe Pro Thr Lys Val Arg Ser Ile Val Se
#r Asp Phe Ala Val Phe
725
# 730
# 735
Leu Thr Ile Leu Cys Met Val Leu Ile Asp Ty
#r Ala Ile Gly Ile Pro
740
# 745
# 750
Ser Pro Lys Leu Gln Val Pro Ser Val Phe Ly
#s Pro Thr Ile Tyr Asp
755
# 760
# 765
Arg Gly Trp Phe Val Thr Pro Leu Gly Pro As
#n Pro Trp Trp Thr Ile
770
# 775
# 780
Ile Ala Ala Ile Ile Pro Ala Leu Leu Cys Th
#r Ile Leu Ile Phe Met
785 7
#90 7
#95 8
#00
Asp Gln Gln Ile Thr Ala Val Ile Ile Asn Ar
#g Lys Glu His Lys Leu
805
# 810
# 815
Lys Lys Gly Cys Gly Tyr His Leu Asp Leu Le
#u Met Val Ala Val Met
820
# 825
# 830
Leu Gly Val Cys Ser Ile Met Gly Leu Pro Tr
#p Phe Val Ala Ala Thr
835
# 840
# 845
Val Leu Ser Ile Thr His Val Asn Ser Leu Ly
#s Leu Glu Ser Glu Cys
850
# 855
# 860
Ser Ala Pro Gly Glu Gln Pro Lys Phe Leu Gl
#y Ile Arg Glu Gln Arg
865 8
#70 8
#75 8
#80
Val Thr Gly Leu Met Ile Phe Ile Leu Met Gl
#y Ser Ser Val Phe Met
885
# 890
# 895
Thr Ser Ile Leu Lys Phe Ile Pro Met Pro Va
#l Leu Tyr Gly Val Phe
900
# 905
# 910
Leu Tyr Met Gly Ala Ser Ser Leu Lys Gly Il
#e Gln Leu Phe Asp Arg
915
# 920
# 925
Ile Lys Leu Phe Trp Met Pro Ala Lys His Gl
#n Pro Asp Phe Ile Tyr
930
# 935
# 940
Leu Arg His Val Pro Leu Arg Lys Val His Le
#u Phe Thr Val Ile Gln
945 9
#50 9
#55 9
#60
Met Ser Cys Leu Gly Leu Leu Trp Ile Ile Ly
#s Val Ser Arg Ala Ala
965
# 970
# 975
Ile Val Phe Pro Met Met Val Leu Ala Leu Va
#l Phe Val Arg Lys Leu
980
# 985
# 990
Met Asp Phe Leu Phe Thr Lys Arg Glu Leu Se
#r Trp Leu Asp Asp Leu
995
# 1000
# 1005
Met Pro Glu Ser Lys Lys Lys Lys Leu Glu As
#p Ala Glu Lys Glu Glu
1010
# 1015
# 1020
Glu Gln Ser Met Leu Ala Met Glu Asp Glu Gl
#y Thr Val Gln Leu Pro
1025 1030
# 1035
# 1040
Leu Glu Gly His Tyr Arg Asp Asp Pro Ser Va
#l Ile Asn Ile Ser Asp
1045
# 1050
# 1055
Glu Met Ser Lys Thr Ala Met Trp Gly Asn Le
#u Leu Val Thr Ala Asp
1060
# 1065
# 1070
Asn Ser Lys Glu Lys Glu Ser Arg Phe Pro Se
#r Lys Ser Ser Pro Ser
1075
# 1080
# 1085
<210> SEQ ID NO 3
<211> LENGTH: 4138
<212> TYPE: DNA
<213> ORGANISM: Homo sapience
<400> SEQUENCE: 3
taagcagagc gagtgccggg ctgagtgtaa gacactgaag acactgcaga gc
#aaggtgct 60
tattccagag gcgttacaaa ac atg gag att aaa gac cag
# gga gcc caa atg 112
# Met Glu Ile Lys Asp Gln G
#ly Ala Gln Met
# 1
# 5
# 10
gag ccg ctg ctg cct acg aga aat gat gaa ga
#a gca gtt gtg gat aga 160
Glu Pro Leu Leu Pro Thr Arg Asn Asp Glu Gl
#u Ala Val Val Asp Arg
15
# 20
# 25
ggt gga act cgt tct att ctc aaa aca cac tt
#t gag aaa gaa gat tta 208
Gly Gly Thr Arg Ser Ile Leu Lys Thr His Ph
#e Glu Lys Glu Asp Leu
30
# 35
# 40
gaa ggt cat cga aca cta ttt att gga gta ca
#t gtg ccc ttg gga gga 256
Glu Gly His Arg Thr Leu Phe Ile Gly Val Hi
#s Val Pro Leu Gly Gly
45
# 50
# 55
aga aaa agc cat cga cgt cac agg cat cgt gg
#t cat aaa cac aga aag 304
Arg Lys Ser His Arg Arg His Arg His Arg Gl
#y His Lys His Arg Lys
60
# 65
# 70
aga gac aga gaa aga gat tca gga tta gag ga
#t gga agg gag tca cct 352
Arg Asp Arg Glu Arg Asp Ser Gly Leu Glu As
#p Gly Arg Glu Ser Pro
75
#80
#85
#90
tct ttt gac acc cca tca cag agg gta cag tt
#t att ctt gga acc gag 400
Ser Phe Asp Thr Pro Ser Gln Arg Val Gln Ph
#e Ile Leu Gly Thr Glu
95
# 100
# 105
gat gat gac gag gaa cac att cct cat gac ct
#t ttc aca gaa ctg gat 448
Asp Asp Asp Glu Glu His Ile Pro His Asp Le
#u Phe Thr Glu Leu Asp
110
# 115
# 120
gag att tgt tgg cgt gaa ggt gag gac gct ga
#g tgg cga gaa aca gcc 496
Glu Ile Cys Trp Arg Glu Gly Glu Asp Ala Gl
#u Trp Arg Glu Thr Ala
125
# 130
# 135
agg tgg ttg aag ttt gaa gaa gat gtg gaa ga
#t gga gga gaa agg tgg 544
Arg Trp Leu Lys Phe Glu Glu Asp Val Glu As
#p Gly Gly Glu Arg Trp
140
# 145
# 150
agc aag cct tat gtg gct act ctt tca ttg ca
#c agc ttg ttt gaa ttg 592
Ser Lys Pro Tyr Val Ala Thr Leu Ser Leu Hi
#s Ser Leu Phe Glu Leu
155 1
#60 1
#65 1
#70
aga agt tgt att ctg aat gga act gtg ttg ct
#g gac atg cat gcc aac 640
Arg Ser Cys Ile Leu Asn Gly Thr Val Leu Le
#u Asp Met His Ala Asn
175
# 180
# 185
act tta gaa gaa att gca gat atg gtt ctt ga
#c caa caa gtg agc tca 688
Thr Leu Glu Glu Ile Ala Asp Met Val Leu As
#p Gln Gln Val Ser Ser
190
# 195
# 200
ggt cag ctg aat gaa gat gta cgc cat agg gt
#c cat gag gca ttg atg 736
Gly Gln Leu Asn Glu Asp Val Arg His Arg Va
#l His Glu Ala Leu Met
205
# 210
# 215
aaa cag cat cat cat cag aat cag aaa aaa ct
#c acc aac agg att ccc 784
Lys Gln His His His Gln Asn Gln Lys Lys Le
#u Thr Asn Arg Ile Pro
220
# 225
# 230
att gtt cgt tcc ttt gct gat att ggc aag aa
#a cag tca gaa cca aat 832
Ile Val Arg Ser Phe Ala Asp Ile Gly Lys Ly
#s Gln Ser Glu Pro Asn
235 2
#40 2
#45 2
#50
tcc atg gac aaa aat gca ggt cag gtt gtt tc
#t cct cag tct gct cca 880
Ser Met Asp Lys Asn Ala Gly Gln Val Val Se
#r Pro Gln Ser Ala Pro
255
# 260
# 265
gcc tgt gtt gaa aat aaa aat gat gtt agc ag
#a gaa aac agc act gtt 928
Ala Cys Val Glu Asn Lys Asn Asp Val Ser Ar
#g Glu Asn Ser Thr Val
270
# 275
# 280
gac ttt agc aag gtt gat ctg cat ttt atg aa
#a aag att cct cca ggt 976
Asp Phe Ser Lys Val Asp Leu His Phe Met Ly
#s Lys Ile Pro Pro Gly
285
# 290
# 295
gct gaa gca tcg aac atc tta ctg gga gaa ct
#g gag ttc ttg gat cga 1024
Ala Glu Ala Ser Asn Ile Leu Leu Gly Glu Le
#u Glu Phe Leu Asp Arg
300
# 305
# 310
aca gta gtt gcg ttt gtc agg ttg tct cca gc
#t gta ttg ctt caa gga 1072
Thr Val Val Ala Phe Val Arg Leu Ser Pro Al
#a Val Leu Leu Gln Gly
315 3
#20 3
#25 3
#30
ctg gct gaa gtc cca atc cca acc aga ttt tt
#g ttc att ctt ctg gga 1120
Leu Ala Glu Val Pro Ile Pro Thr Arg Phe Le
#u Phe Ile Leu Leu Gly
335
# 340
# 345
ccc ctg gga aag ggt caa cag tac cat gag at
#t ggc aga tca att gca 1168
Pro Leu Gly Lys Gly Gln Gln Tyr His Glu Il
#e Gly Arg Ser Ile Ala
350
# 355
# 360
acc cta atg aca gat gag gta ttt cat gat gt
#t gcc tat aaa gct aaa 1216
Thr Leu Met Thr Asp Glu Val Phe His Asp Va
#l Ala Tyr Lys Ala Lys
365
# 370
# 375
gat cgt aat gac ttg gta tca gga att gat ga
#g ttt ctg gat cag gtt 1264
Asp Arg Asn Asp Leu Val Ser Gly Ile Asp Gl
#u Phe Leu Asp Gln Val
380
# 385
# 390
act gtt ctc cct cct gga gaa tgg gat cca ag
#c att cga ata gag cct 1312
Thr Val Leu Pro Pro Gly Glu Trp Asp Pro Se
#r Ile Arg Ile Glu Pro
395 4
#00 4
#05 4
#10
ccc aaa aat gtt cct tcc cag gag aag agg aa
#g att cct gct gta cca 1360
Pro Lys Asn Val Pro Ser Gln Glu Lys Arg Ly
#s Ile Pro Ala Val Pro
415
# 420
# 425
aat gga aca gca gct cat ggg gaa gca gag cc
#c cac gga gga cat agt 1408
Asn Gly Thr Ala Ala His Gly Glu Ala Glu Pr
#o His Gly Gly His Ser
430
# 435
# 440
gga cct gaa ctc cag cga act gga agg att tt
#t ggg gga ctt att tta 1456
Gly Pro Glu Leu Gln Arg Thr Gly Arg Ile Ph
#e Gly Gly Leu Ile Leu
445
# 450
# 455
gat atc aaa aga aaa gct cca tac ttc tgg ag
#t gac ttc aga gat gct 1504
Asp Ile Lys Arg Lys Ala Pro Tyr Phe Trp Se
#r Asp Phe Arg Asp Ala
460
# 465
# 470
ttc agc ctg cag tgc tta gca tct ttt cta tt
#t ctc tac tgc gcg tgt 1552
Phe Ser Leu Gln Cys Leu Ala Ser Phe Leu Ph
#e Leu Tyr Cys Ala Cys
475 4
#80 4
#85 4
#90
atg tct cct gtc atc acg ttt gga gga ctg ct
#g gga gaa gca act gaa 1600
Met Ser Pro Val Ile Thr Phe Gly Gly Leu Le
#u Gly Glu Ala Thr Glu
495
# 500
# 505
ggg cgt ata agt gca att gaa tct ctc ttt gg
#a gca tcc atg acc ggg 1648
Gly Arg Ile Ser Ala Ile Glu Ser Leu Phe Gl
#y Ala Ser Met Thr Gly
510
# 515
# 520
ata gcc tat tct ctc ttt ggt gga cag cct ct
#t acc ata tta ggc agt 1696
Ile Ala Tyr Ser Leu Phe Gly Gly Gln Pro Le
#u Thr Ile Leu Gly Ser
525
# 530
# 535
aca gga cca gtt ttg gtg ttt gaa aag att tt
#g ttt aaa ttt tgc aaa 1744
Thr Gly Pro Val Leu Val Phe Glu Lys Ile Le
#u Phe Lys Phe Cys Lys
540
# 545
# 550
gaa tat ggg ctg tca tac cta tct tta aga gc
#t agc att gga ctt tgg 1792
Glu Tyr Gly Leu Ser Tyr Leu Ser Leu Arg Al
#a Ser Ile Gly Leu Trp
555 5
#60 5
#65 5
#70
act gca act cta tgt atc ata ctt gtg gcc ac
#a gat gct agt tcc ctt 1840
Thr Ala Thr Leu Cys Ile Ile Leu Val Ala Th
#r Asp Ala Ser Ser Leu
575
# 580
# 585
gtc tgc tac atc act cgg ttt act gaa gaa gc
#t ttt gct tcc ctg att 1888
Val Cys Tyr Ile Thr Arg Phe Thr Glu Glu Al
#a Phe Ala Ser Leu Ile
590
# 595
# 600
tgc atc att ttc att tat gag gcc ctg gag aa
#g ttg ttt gaa ctc agt 1936
Cys Ile Ile Phe Ile Tyr Glu Ala Leu Glu Ly
#s Leu Phe Glu Leu Ser
605
# 610
# 615
gaa gca tat cca atc aac atg cat aat gat ct
#g gaa ctg ctg aca caa 1984
Glu Ala Tyr Pro Ile Asn Met His Asn Asp Le
#u Glu Leu Leu Thr Gln
620
# 625
# 630
tac tcg tgt aac tgt gtg gaa ccg cat aat cc
#c agc aat ggc aca ttg 2032
Tyr Ser Cys Asn Cys Val Glu Pro His Asn Pr
#o Ser Asn Gly Thr Leu
635 6
#40 6
#45 6
#50
aag gaa tgg agg gaa tcc aat att tct gcc tc
#t gac ata att tgg gag 2080
Lys Glu Trp Arg Glu Ser Asn Ile Ser Ala Se
#r Asp Ile Ile Trp Glu
655
# 660
# 665
aac cta act gtg tca gaa tgc aaa tca ttg ca
#t gga gag tat gtt gga 2128
Asn Leu Thr Val Ser Glu Cys Lys Ser Leu Hi
#s Gly Glu Tyr Val Gly
670
# 675
# 680
cgg gcc tgt ggc cat gat cac cca tat gtt cc
#a gat gtt cta ttt tgg 2176
Arg Ala Cys Gly His Asp His Pro Tyr Val Pr
#o Asp Val Leu Phe Trp
685
# 690
# 695
tct gtg atc ctg ttc ttt tcc aca gtt act ct
#g tca gcc acc ctg aag 2224
Ser Val Ile Leu Phe Phe Ser Thr Val Thr Le
#u Ser Ala Thr Leu Lys
700
# 705
# 710
cag ttc aag act agc aga tat ttt cca acc aa
#g gtt cga tcc ata gtg 2272
Gln Phe Lys Thr Ser Arg Tyr Phe Pro Thr Ly
#s Val Arg Ser Ile Val
715 7
#20 7
#25 7
#30
agt gac ttt gct gtc ttt ctt aca att ctg tg
#t atg gtt tta att gac 2320
Ser Asp Phe Ala Val Phe Leu Thr Ile Leu Cy
#s Met Val Leu Ile Asp
735
# 740
# 745
tat gcc att ggg atc cca tct cca aaa cta ca
#a gta cca agt gtt ttc 2368
Tyr Ala Ile Gly Ile Pro Ser Pro Lys Leu Gl
#n Val Pro Ser Val Phe
750
# 755
# 760
aag ccc act aga gat gat cgt ggc tgg ttt gt
#t acg cct tta ggt cca 2416
Lys Pro Thr Arg Asp Asp Arg Gly Trp Phe Va
#l Thr Pro Leu Gly Pro
765
# 770
# 775
aac cca tgg tgg aca gta ata gct gct ata at
#t cca gct ctg ctt tgt 2464
Asn Pro Trp Trp Thr Val Ile Ala Ala Ile Il
#e Pro Ala Leu Leu Cys
780
# 785
# 790
act att cta att ttc atg gac caa cag att ac
#a gct gtc atc atc aac 2512
Thr Ile Leu Ile Phe Met Asp Gln Gln Ile Th
#r Ala Val Ile Ile Asn
795 8
#00 8
#05 8
#10
agg aaa gag cat aag cta aag aaa ggt tgt gg
#g tac cat ctg gac cta 2560
Arg Lys Glu His Lys Leu Lys Lys Gly Cys Gl
#y Tyr His Leu Asp Leu
815
# 820
# 825
tta atg gtg gct gtc atg ctc ggt gta tgc tc
#c atc atg ggc ctg cca 2608
Leu Met Val Ala Val Met Leu Gly Val Cys Se
#r Ile Met Gly Leu Pro
830
# 835
# 840
tgg ttt gtg gct gcc aca gtc ctc tcc atc ac
#t cat gtc aat agc cta 2656
Trp Phe Val Ala Ala Thr Val Leu Ser Ile Th
#r His Val Asn Ser Leu
845
# 850
# 855
aaa ctg gaa tca gaa tgc tca gct cca gga ga
#a caa ccc aaa ttt ctc 2704
Lys Leu Glu Ser Glu Cys Ser Ala Pro Gly Gl
#u Gln Pro Lys Phe Leu
860
# 865
# 870
ggc att cgg gag caa agg gtt act ggg ctt at
#g att ttt att ctt atg 2752
Gly Ile Arg Glu Gln Arg Val Thr Gly Leu Me
#t Ile Phe Ile Leu Met
875 8
#80 8
#85 8
#90
ggt tca tca gtc ttt atg acc agt att ctg aa
#g ttt att ccc atg cca 2800
Gly Ser Ser Val Phe Met Thr Ser Ile Leu Ly
#s Phe Ile Pro Met Pro
895
# 900
# 905
gtg cta tat gga gtg ttt ctt tat atg ggt gc
#t tca tct cta aag gga 2848
Val Leu Tyr Gly Val Phe Leu Tyr Met Gly Al
#a Ser Ser Leu Lys Gly
910
# 915
# 920
att cag ttc ttt gat agg ata aag ctc ttc tg
#g atg ccg gca aaa cat 2896
Ile Gln Phe Phe Asp Arg Ile Lys Leu Phe Tr
#p Met Pro Ala Lys His
925
# 930
# 935
caa cca gat ttt ata tac cta agg cac gta cc
#g ctt cga aaa gtg cat 2944
Gln Pro Asp Phe Ile Tyr Leu Arg His Val Pr
#o Leu Arg Lys Val His
940
# 945
# 950
ctc ttc aca att att cag atg agt tgc ctt gg
#c ctt ttg tgg ata ata 2992
Leu Phe Thr Ile Ile Gln Met Ser Cys Leu Gl
#y Leu Leu Trp Ile Ile
955 9
#60 9
#65 9
#70
aaa gtt tca aga gct gct att gtc tct ccc at
#g atg gtg tta tcc ctg 3040
Lys Val Ser Arg Ala Ala Ile Val Ser Pro Me
#t Met Val Leu Ser Leu
975
# 980
# 985
gtt ttt gta aga aag ttg atg gac ttg ttg tt
#c acg aaa cgg gaa ctc 3088
Val Phe Val Arg Lys Leu Met Asp Leu Leu Ph
#e Thr Lys Arg Glu Leu
990
# 995
# 1000
tgc tgg ttg gat gat ttg atg cct gag agt aa
#g aaa aag aaa ctg gaa 3136
Cys Trp Leu Asp Asp Leu Met Pro Glu Ser Ly
#s Lys Lys Lys Leu Glu
1005
# 1010
# 1015
tat gct gaa aaa gaa gaa gaa caa tgt gtg ct
#a cct atg gaa gat gag 3184
Tyr Ala Glu Lys Glu Glu Glu Gln Cys Val Le
#u Pro Met Glu Asp Glu
1020
# 1025
# 1030
ggc aca gta caa ctc cca ttg gaa ggg cac ta
#t aga gat gat cca tct 3232
Gly Thr Val Gln Leu Pro Leu Glu Gly His Ty
#r Arg Asp Asp Pro Ser
1035 1040
# 1045
# 1050
gtg atc aat ata tct gat gaa atg tca aag ac
#t gcc ttg tgg agg aac 3280
Val Ile Asn Ile Ser Asp Glu Met Ser Lys Th
#r Ala Leu Trp Arg Asn
1055
# 1060
# 1065
ctt ctg att act gcc gat aac tca aaa gat aa
#g gag tca agc ttt cct 3328
Leu Leu Ile Thr Ala Asp Asn Ser Lys Asp Ly
#s Glu Ser Ser Phe Pro
1070
# 1075
# 1080
tcc aaa agc tcc cct tcc taa tcactctaga agctgattc
#c ccaaagcatt 3379
Ser Lys Ser Ser Pro Ser
1085
gaaagccgaa aagagaagaa agctgactca gggatagttg ttgacaggga ga
#cttgtcta 3439
tgactcgatc ttcaatttat tttttacata tatatgagaa gagtgtcaca at
#tattaata 3499
aaactgcttg gatcatgtat ggtaaattct gtccctcaac ccaaatccac tt
#tcatacgg 3559
taagtagggc aaaacttgtt tcatttcggt gttaaaattt cggagcagga ga
#catccctg 3619
tgagcagaaa caatagccaa tgcagaatct gtgtgttcct tgctgaacgt aa
#gacatttg 3679
taaactggat tctgattgtc agttttatga gagcaatagc ttccttaaag ag
#ataagtca 3739
tatacaccta gtttgtattc tcatacttta gagacctgaa gacgcctgat aa
#tttcattc 3799
aggagaattt ttgaaaggta gtcaaacttc tttttagttt ttatagctta gc
#attagtga 3859
cttatttcaa aagacccaaa tcaaaaagtt agtttgaaag cattttttaa ta
#attgtatt 3919
tatgcatttg gctactgtaa gttttgctcc atggaataat gatgtgatag ca
#aaaatgaa 3979
taagactatg aataagttcc tacatgaagg ttaatgtcag tggtgaaaaa tc
#ttattatg 4039
ctccaatata ctgccagcat gctgagtata cttggatcat aaaaaactgt tt
#catttttc 4099
ttatttattt tatgcatagg aatattcatt ccggaattc
#
# 4138
<210> SEQ ID NO 4
<211> LENGTH: 1088
<212> TYPE: PRT
<213> ORGANISM: Homo sapience
<400> SEQUENCE: 4
Met Glu Ile Lys Asp Gln Gly Ala Gln Met Gl
#u Pro Leu Leu Pro Thr
1 5
# 10
# 15
Arg Asn Asp Glu Glu Ala Val Val Asp Arg Gl
#y Gly Thr Arg Ser Ile
20
# 25
# 30
Leu Lys Thr His Phe Glu Lys Glu Asp Leu Gl
#u Gly His Arg Thr Leu
35
# 40
# 45
Phe Ile Gly Val His Val Pro Leu Gly Gly Ar
#g Lys Ser His Arg Arg
50
# 55
# 60
His Arg His Arg Gly His Lys His Arg Lys Ar
#g Asp Arg Glu Arg Asp
65
#70
#75
# 80
Ser Gly Leu Glu Asp Gly Arg Glu Ser Pro Se
#r Phe Asp Thr Pro Ser
85
# 90
# 95
Gln Arg Val Gln Phe Ile Leu Gly Thr Glu As
#p Asp Asp Glu Glu His
100
# 105
# 110
Ile Pro His Asp Leu Phe Thr Glu Leu Asp Gl
#u Ile Cys Trp Arg Glu
115
# 120
# 125
Gly Glu Asp Ala Glu Trp Arg Glu Thr Ala Ar
#g Trp Leu Lys Phe Glu
130
# 135
# 140
Glu Asp Val Glu Asp Gly Gly Glu Arg Trp Se
#r Lys Pro Tyr Val Ala
145 1
#50 1
#55 1
#60
Thr Leu Ser Leu His Ser Leu Phe Glu Leu Ar
#g Ser Cys Ile Leu Asn
165
# 170
# 175
Gly Thr Val Leu Leu Asp Met His Ala Asn Th
#r Leu Glu Glu Ile Ala
180
# 185
# 190
Asp Met Val Leu Asp Gln Gln Val Ser Ser Gl
#y Gln Leu Asn Glu Asp
195
# 200
# 205
Val Arg His Arg Val His Glu Ala Leu Met Ly
#s Gln His His His Gln
210
# 215
# 220
Asn Gln Lys Lys Leu Thr Asn Arg Ile Pro Il
#e Val Arg Ser Phe Ala
225 2
#30 2
#35 2
#40
Asp Ile Gly Lys Lys Gln Ser Glu Pro Asn Se
#r Met Asp Lys Asn Ala
245
# 250
# 255
Gly Gln Val Val Ser Pro Gln Ser Ala Pro Al
#a Cys Val Glu Asn Lys
260
# 265
# 270
Asn Asp Val Ser Arg Glu Asn Ser Thr Val As
#p Phe Ser Lys Val Asp
275
# 280
# 285
Leu His Phe Met Lys Lys Ile Pro Pro Gly Al
#a Glu Ala Ser Asn Ile
290
# 295
# 300
Leu Leu Gly Glu Leu Glu Phe Leu Asp Arg Th
#r Val Val Ala Phe Val
305 3
#10 3
#15 3
#20
Arg Leu Ser Pro Ala Val Leu Leu Gln Gly Le
#u Ala Glu Val Pro Ile
325
# 330
# 335
Pro Thr Arg Phe Leu Phe Ile Leu Leu Gly Pr
#o Leu Gly Lys Gly Gln
340
# 345
# 350
Gln Tyr His Glu Ile Gly Arg Ser Ile Ala Th
#r Leu Met Thr Asp Glu
355
# 360
# 365
Val Phe His Asp Val Ala Tyr Lys Ala Lys As
#p Arg Asn Asp Leu Val
370
# 375
# 380
Ser Gly Ile Asp Glu Phe Leu Asp Gln Val Th
#r Val Leu Pro Pro Gly
385 3
#90 3
#95 4
#00
Glu Trp Asp Pro Ser Ile Arg Ile Glu Pro Pr
#o Lys Asn Val Pro Ser
405
# 410
# 415
Gln Glu Lys Arg Lys Ile Pro Ala Val Pro As
#n Gly Thr Ala Ala His
420
# 425
# 430
Gly Glu Ala Glu Pro His Gly Gly His Ser Gl
#y Pro Glu Leu Gln Arg
435
# 440
# 445
Thr Gly Arg Ile Phe Gly Gly Leu Ile Leu As
#p Ile Lys Arg Lys Ala
450
# 455
# 460
Pro Tyr Phe Trp Ser Asp Phe Arg Asp Ala Ph
#e Ser Leu Gln Cys Leu
465 4
#70 4
#75 4
#80
Ala Ser Phe Leu Phe Leu Tyr Cys Ala Cys Me
#t Ser Pro Val Ile Thr
485
# 490
# 495
Phe Gly Gly Leu Leu Gly Glu Ala Thr Glu Gl
#y Arg Ile Ser Ala Ile
500
# 505
# 510
Glu Ser Leu Phe Gly Ala Ser Met Thr Gly Il
#e Ala Tyr Ser Leu Phe
515
# 520
# 525
Gly Gly Gln Pro Leu Thr Ile Leu Gly Ser Th
#r Gly Pro Val Leu Val
530
# 535
# 540
Phe Glu Lys Ile Leu Phe Lys Phe Cys Lys Gl
#u Tyr Gly Leu Ser Tyr
545 5
#50 5
#55 5
#60
Leu Ser Leu Arg Ala Ser Ile Gly Leu Trp Th
#r Ala Thr Leu Cys Ile
565
# 570
# 575
Ile Leu Val Ala Thr Asp Ala Ser Ser Leu Va
#l Cys Tyr Ile Thr Arg
580
# 585
# 590
Phe Thr Glu Glu Ala Phe Ala Ser Leu Ile Cy
#s Ile Ile Phe Ile Tyr
595
# 600
# 605
Glu Ala Leu Glu Lys Leu Phe Glu Leu Ser Gl
#u Ala Tyr Pro Ile Asn
610
# 615
# 620
Met His Asn Asp Leu Glu Leu Leu Thr Gln Ty
#r Ser Cys Asn Cys Val
625 6
#30 6
#35 6
#40
Glu Pro His Asn Pro Ser Asn Gly Thr Leu Ly
#s Glu Trp Arg Glu Ser
645
# 650
# 655
Asn Ile Ser Ala Ser Asp Ile Ile Trp Glu As
#n Leu Thr Val Ser Glu
660
# 665
# 670
Cys Lys Ser Leu His Gly Glu Tyr Val Gly Ar
#g Ala Cys Gly His Asp
675
# 680
# 685
His Pro Tyr Val Pro Asp Val Leu Phe Trp Se
#r Val Ile Leu Phe Phe
690
# 695
# 700
Ser Thr Val Thr Leu Ser Ala Thr Leu Lys Gl
#n Phe Lys Thr Ser Arg
705 7
#10 7
#15 7
#20
Tyr Phe Pro Thr Lys Val Arg Ser Ile Val Se
#r Asp Phe Ala Val Phe
725
# 730
# 735
Leu Thr Ile Leu Cys Met Val Leu Ile Asp Ty
#r Ala Ile Gly Ile Pro
740
# 745
# 750
Ser Pro Lys Leu Gln Val Pro Ser Val Phe Ly
#s Pro Thr Arg Asp Asp
755
# 760
# 765
Arg Gly Trp Phe Val Thr Pro Leu Gly Pro As
#n Pro Trp Trp Thr Val
770
# 775
# 780
Ile Ala Ala Ile Ile Pro Ala Leu Leu Cys Th
#r Ile Leu Ile Phe Met
785 7
#90 7
#95 8
#00
Asp Gln Gln Ile Thr Ala Val Ile Ile Asn Ar
#g Lys Glu His Lys Leu
805
# 810
# 815
Lys Lys Gly Cys Gly Tyr His Leu Asp Leu Le
#u Met Val Ala Val Met
820
# 825
# 830
Leu Gly Val Cys Ser Ile Met Gly Leu Pro Tr
#p Phe Val Ala Ala Thr
835
# 840
# 845
Val Leu Ser Ile Thr His Val Asn Ser Leu Ly
#s Leu Glu Ser Glu Cys
850
# 855
# 860
Ser Ala Pro Gly Glu Gln Pro Lys Phe Leu Gl
#y Ile Arg Glu Gln Arg
865 8
#70 8
#75 8
#80
Val Thr Gly Leu Met Ile Phe Ile Leu Met Gl
#y Ser Ser Val Phe Met
885
# 890
# 895
Thr Ser Ile Leu Lys Phe Ile Pro Met Pro Va
#l Leu Tyr Gly Val Phe
900
# 905
# 910
Leu Tyr Met Gly Ala Ser Ser Leu Lys Gly Il
#e Gln Phe Phe Asp Arg
915
# 920
# 925
Ile Lys Leu Phe Trp Met Pro Ala Lys His Gl
#n Pro Asp Phe Ile Tyr
930
# 935
# 940
Leu Arg His Val Pro Leu Arg Lys Val His Le
#u Phe Thr Ile Ile Gln
945 9
#50 9
#55 9
#60
Met Ser Cys Leu Gly Leu Leu Trp Ile Ile Ly
#s Val Ser Arg Ala Ala
965
# 970
# 975
Ile Val Ser Pro Met Met Val Leu Ser Leu Va
#l Phe Val Arg Lys Leu
980
# 985
# 990
Met Asp Leu Leu Phe Thr Lys Arg Glu Leu Cy
#s Trp Leu Asp Asp Leu
995
# 1000
# 1005
Met Pro Glu Ser Lys Lys Lys Lys Leu Glu Ty
#r Ala Glu Lys Glu Glu
1010
# 1015
# 1020
Glu Gln Cys Val Leu Pro Met Glu Asp Glu Gl
#y Thr Val Gln Leu Pro
1025 1030
# 1035
# 1040
Leu Glu Gly His Tyr Arg Asp Asp Pro Ser Va
#l Ile Asn Ile Ser Asp
1045
# 1050
# 1055
Glu Met Ser Lys Thr Ala Leu Trp Arg Asn Le
#u Leu Ile Thr Ala Asp
1060
# 1065
# 1070
Asn Ser Lys Asp Lys Glu Ser Ser Phe Pro Se
#r Lys Ser Ser Pro Ser
1075
# 1080
# 1085
<210> SEQ ID NO 5
<211> LENGTH: 19
<212> TYPE: DNA
<213> ORGANISM: Homo sapience
<400> SEQUENCE: 5
tttggagaaa acccctggt
#
#
# 19
<210> SEQ ID NO 6
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Homo sapience
<400> SEQUENCE: 6
tgacatcatc caggaagctg
#
#
# 20
<210> SEQ ID NO 7
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Homo sapience
<400> SEQUENCE: 7
gtcatgttag accaacaggt
#
#
# 20
<210> SEQ ID NO 8
<211> LENGTH: 18
<212> TYPE: DNA
<213> ORGANISM: Homo sapience
<400> SEQUENCE: 8
gttgtaatag cgacactc
#
#
# 18
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