Abstract:
The present invention provides novel ATP-sensitive potassium-channel proteins which are present ubiquitously in the living bodies of animals, and their genes.

Description:
This is a continuation of application Ser. No. 08/614,155 filed Mar. 12, 1996 now U.S. Pat. No. 5,919,692. 
    
    
     The present invention relates to proteins for novel ATP-sensitive potassium channels, huK ATP -1 and ruK ATP -1, that are expressed in various tissues of human and rat origins, and to genes encoding the same. The said proteins and genes can be used as diagnostic and therapeutic agents for potassium-channel related diseases such as diabetes, hypertension and endocrine insufficiencies. 
     BACKGROUND OF THE INVENTION 
     The etiology for diabetes is known to be mostly owing to disturbances of insulin secretion in the pancreatic β-cells. Consequently, elucidation of the molecular mechanism of insulin secretion is expected to play an important role in the clarification of causes for diabetes and the development of therapeutic agents against diabetes, but no detail has yet been made known on such molecular mechanism. 
     It has already been made clear that the ATP-sensitive potassium channel (K ATP ) being present on the cellular membrane plays a leading role in the cellular functions such as secretions and muscular contraction by coupling the state of metabolism in the cells with the membrane potential. 
     The K ATP  channel was first discovered in the cardiac muscle in 1983 [Noma, A., Nature 305:147 (1983)] and was thereafter confirmed to be present in tissues such as the pancreatic β-cell [Cook, D. L. et al., Nature 311: 271 (1984), Misler, S. et al., Proc. Natl. Acad. Sci. U.S.A. 83: 7119 (1986)], pituitary [Bernardi, H. et al., Proc. Natl. Acad. Sci. U.S.A., 90:1340 (1993)]. skeletal muscle [Spruce, A. E., et al., Nature, 316: 736 (1985)] and brain. 
     In addition, it has been suggested that there exists the molecular heterogeneity of such K ATP  channels [Ashcroft, F. M., Annu. Rev. Neurosci. 11: 97 (1988)]. 
     Particularly in the pancreatic β-cells, ATP produced by the metabolism of glucose brings about calcium ion inflow from the calcium channel by closing the K ATP  channel to cause depolarization, resulting in secretion of insulin. As is evident from this, the K ATP  channel plays a leading role in regulating the secretion of insulin. 
     The K ATP  channel belongs to a potassium channel family exhibiting electrophysiologically inward rectification, whereby the potassium channel family exhibiting inward rectification is classified into the four subfamilies, ROMK1, IRK1, GIRK1 and cK ATP -1, on the basis of the degree of amino acid sequence identity. 
     Nevertheless, there has not been clarified the molecular architecture for the K ATP  channel in the pancreatic β-cells. In addition, no information has been disclosed on the novel ATP-sensitive potassium channels (huK ATP -1 and ruK ATP -1) of the present invention for the detailed protein structure and the formation of complexes with other proteins, for example, the sulfonylurea binding protein. 
     SUMMARY OF THE INVENTION 
     In order to achieve the isolation, identification and functional analyses of a novel membrane channel, there are required the sophisticated techniques, such as molecular biological technique, cellular biological technique and electro-physiological technique. 
     Such being the case, the present inventors made ample and full use of such techniques to isolate human and rat genomes and cDNAs encoding the novel K ATP  channel (uK ATP -1) expressed in different tissues of mammalians and to identify their amino acid sequences (see FIGS. 1,  2 ,  3  and  4 ). The identified uK ATP -1 channel was expressed in the Xenopus oocyte system and mammalian cell lines. 
     Electrophysiological analysis demonstrated that uK ATP -1 is an ATP-sensitive potassium channel exhibiting inward rectification. The uK ATP -1 channel being expressed ubiquitously in tissues of mammalians inclusive of man and rats is involved in the maintenance of the membrane potential through the basal energy metabolism. 
     As is described in the above, the present invention relates to an ATP-sensitive potassium channel (uK ATP -1) which is ubiquitously present in mammalians, and encompasses the ATP-sensitive potassium channel proteins, identified DNA sequences encoding the same, plasmid having such sequences incorporated therein and furthermore recombinant cells (tranformants) having such plasmid transfected therein. In addition, this invention comprises the isolated UK ATP -1 proteins and recombinant proteins, their related materials such as agonists and antagonists, and drug designs inclusive of diagnostics and drugs for gene therapy. 
     DETAILED DESCRIPTION 
     huK ATP -1 of a human origin is composed of 424 amino acid residue (See FIG. 1 (SEQ ID NO: 1)) with a molecular weight of 47,965, while the one of a rat origin is likewise composed of 424 amino acid residue (see FIG. 4 (SEQ ID NO: 4)) with a molecular weight of 47,960. These two potassium channels exhibit 98% amino acid sequence identity, and such a marked homology leads us to the assumption that uK ATP -1 performs common, structurally and functionally basic actions in all mammalian cells. Among others, uK ATP -1 participates in the membrane potential and energy metabolism, suggesting that it could find application as a drug substance acting to prevent disturbances under unusual, extreme metabolic conditions inclusive of endocrine diseases, e.g. diabetes, starvation and ischemia. 
     For example, the inflow and outflow of calcium ions caused by the opening and closing of UK ATP -1 during the onset of ischemia is closely connected with ischemic disturbances. In other words, there is a possibility that the agonists and antagonists for the opening and closing of uK ATP -1 would constitute a suppressory agent against ischemic disturbances. 
     From the comparative studies of huK ATP -1 and ruK ATP -1 with other potassium channels for the amino acid sequence, it was confirmed that uK ATP -1 of the present invention belongs to a novel family of the inward rectifier potassium channels; the central region of the uK ATP -1 protein showed incresed homology with other inward rectifier potassium channels. A hydropathy plot indicated the presence of two hydrophobic regions, which are composed of two transmembrane regions characteristic of the inward rectifier potassium channels and one pore region [Nicholas, C. G., Trends Pharmacol. Sci., 14: 320 (1993), Jan, L. Y. and Jan, Y. N., Nature, 371: 119 (1994)]. 
     With reference to ruK ATP -1 (Inagaki, N. et al., J. B. C., 270: 5691 (1995)], it was reported that in the second intracellular region, there are two potential cAMP-dependent protein kinase phosphorylation sites (Thr-234 and Ser-385) and seven potential protein kinase C dependent phosphorylation sites (Ser-224, Thr-345, Ser-354, Ser-379, Ser-385, Ser-391 and Ser-397), while there are one (Thr-63) and four potential casein kinase II dependent phosphorylation sites (Thr-234, Ser-281, Thr-329 and Ser-354) in the first and second intracellular regions, respectively, with no N-linked glycosylation site being present in the intracellular regions. The same findings were obtained with huK ATP -1 [Inagaki, N., et al., in press (1995)]. 
     Then, the present inventors identified the nucleotide sequences and entire amino acid sequences of huK ATP - 1  and ruK ATP -1, thus enabling not only proteins themselves of huK ATP -1 and ruK ATP -1 but also their mutants to be synthesized in large quantities by expressing the DNAs encoding huK ATP -1 and ruK ATP -1 and their mutants in bacteria or animal cells with use of the known genetic engineering techniques. It is furthermore added that huK ATP - 1  and its fragments are useful for the hybridization diagnosis of depleted huK ATP -1 DNA, with the mutants of huK ATP -1 being of use in the studies on the sugar metabolism in cells, particularly insulin-dependent and independent diabetes. 
     The DNAs of novel huK ATP -1 and ruK ATP -1 according to the present invention were identified based on a cDNA library and genome library. The DNA encoding huK ATP -1 shows a length of about 9.7 kb, being composed of three exons and is present on the chromosome at 12p11.23. The chromosomal DNA can be obtained by probing a genome DNA library with use of cDNAs for uK ATP -1 and its fragment, as well. The isolated uK ATP -1 DNA can easily be subjected to nucleotide depletion, insertion or replacement by the known techniques to prepare its mutants. 
     By employing the known techniques, it is easy to link nucleotide sequences encoding other proteins or synthetic polypeptides to uK ATP -1 or its variants at the 5′ and 3′ ends to thereby prepare fusion proteins, or derivatives thereof. 
     For example, a fusion protein is prepared as a precursor protein and undergoes cleavage in vivo or in vitro to thereby perform functions; such fusion protein provides target-tissue and membrane orientation in addition to its proper function. In such a case, the fusion proteins contain sugar-chain binding amino acids, and can be modified to derivatives having tissue orientation or physiological activities activated by adding new sugar chains. 
     In order to produce uK ATP -1, its mutants or their derivatives, the corresponding coding DNA is incorporated into a reproducible plasmid, and host cells being transformed with such plasmid are incubated. The host cells include bacteria, yeasts and animal cells. 
     Prokaryotes such as bacteria are suited for the cloning of deoxyribonucleotides. For example, pER 322 plasmid derived from  E. coli  contains a gene resistant to ampicillin or tetracycline and can provide a practical means of identifying the transformed cells. Furthermore, the microbial plasmids contain a promoter which can be used to express their proteins themselves. In addition to prokaryotes, eukaryotes such as yeasts can work well, with a plasmid YRp7 being utilizable especially in allowing the expression in yeasts of the species Saccharomyces [Stinchomb et al., Nature, 282: 39 (1979)]. 
     Animal cells are also used as a host, and particularly the incubation of vertebra cells is employable easily and constitutes a conventional means [Krause and Paterson, Tissue Culture, Academic Press (1973)]. As the cell lines, there are mentioned AtT-20, Hela cells, Chinese hamster ovary (CHO), COSM6, COS-7 and the like. The promoters of Polyomavirus, Adenovirus 2, Cytomegalovirus and Simian virus 40 are used to control the function of expression plasmid in such cell lines, wherein pCMV is a plasmid which finds widened application in the expression systems of animal cells [Thomsen et al., PNAS, 81: 659 (1984)]. 
     The DNA sequences for the channel protein and huK ATP -1 and ruK ATP -1 according to the present invention begin with the initiation codon “ATG”. In cases where the recombinant cells are used to synthesize such protein, there is no need to add ATG to the desired DNA, thus making the manipulation easy. When uK ATP -1 is expressed in a prokaryote transformed with  E. coli , consequently, there is generally synthesized a protein of the amino acid sequence beginning with Met. The N-terminated met of the resultant protein may be eliminated according to the purpose of application. 
     In cases in which uK ATP -1 is synthesized in recombinant animal cells, similarly, proteins having Met contained or eliminated at the N-terminal are bio-synthesized, and both are useful for individually intended application purposes. 
     uK ATP -1 and its fragments can be administered to animals for their immunization to thereby produce antibodies. Also, immunization of animals permits a monoclonal antibody to be produced from cells secreting the desired antibody. 
     It has become easy to prepare uK ATP -1 in large quantities, thus providing better understanding of the same at the molecular level. Accordingly, the production of uK ATP -1 and its mutants or analogs raises the possibility to develop diagnostics or therapeutics for the channel-protein related diseases. 
     In particular, such proteins can be utilized in the procedures of investigating into a substance suited for diagnostics and therapeutics, or a substance that exerts agonistic or antagonistic action on uK ATP -1. For example, a testing procedure with animal cells can be conducted by injecting cRNA for uK ATP -1 into cells to conduct expression, followed by addition of sulfonylurea to study their interactions [Kayano, T. et al., J. Biol. Chem., 265: 13276 (1990), Example 4]. 
     Additionally, the pertinent information has been obtained on the DNA sequence of uK ATP -1, facilitating DNA or RNA encoding their fractional sequences to be prepared. Such relatively short DNA sequences possess the capability to hybridize with the gene to be selected, and can find application as a probe, which probe is effective for detection of cDNAs in different tissues. 
     The probe as prepared with use of uK ATP -1 can be utilized to produce nucleic acids capable of hybridization from a variety of organisms and their tissues. The resultant nucleic acids may be the same type as uK ATP -1 or its isoform and include nucleic acids encoding the novel proteins. 
     The prepared probe is utilizable in the gene diagnosis of potassium-channel related diseases; investigation can be conducted into patients&#39; nucleotide sequences hybridized with the probe capable of detecting the disease genes. 
     The blocker and opener agents for the potassium channel have heretofore been used as therapeutics against diabetes and hypertension. uK ATP -1 and its mutants, their derivatives and monoclonal antibodies to them, when processed into pharmaceutical preparations, can be administered to patients to thereby alleviate through neutralization the adverse effects brought about by an excess of such blocker or opener agents administered clinically. When uK ATP -1 itself shows functional insufficiency, such pharmaceutical preparations can be administered to thereby make up for such deficient functions of uK ATP -1. 
     The present invention comprises the preparation of drugs for gene therapy being applicable in the essential treatment method. The nucleotide sequences for uK ATP -1 or its mutants and their derivatives can be incorporated into plasmid or stem cells, which are then given patients to open up the possibility of finding application as a drug for gene therapy. 
     Below described are the examples to illustrate the present invention in more detail, while referring to the appended drawings. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 (SEQ ID NO:1) is an illustration of the amino acid sequences corresponding to the base sequences as shown in FIGS. 2 (SEQ ID NO: 2) and  3  (SEQ ID NO: 3). 
     FIG. 2 (SEQ ID NO:2) is an illustration of the base sequence of uK ATP -1 of a human origin as obtained in Example 5. 
     FIG. 3 (SEQ ID NO: 3) is an illustration of the amino acid sequence corresponding to FIGS. 5 and 6. 
     FIG. 4 (SEQ ID NO: 4) is an illustration of the base sequence of ruK ATP -1 of a rat origin. 
     FIG. 5A shows the results of electrophysiological analysis of ruK ATP -1 with use of Xenopus oocytes. The oocytes injected with cRNA of rUK ATP -1 exhibited inward rectification under conditions of 45 mM [K + ] concentrated extracellular fluid, which rectification was however blocked with 300 μM of Ba 2+  added to the extracellular fluid. The control, which comprised injection of water, was observed to produce negligible slight inward electric current alone. 
     FIG. 5B is a plot of potassium-concentration dependent electric current versus voltage, leading to the confirmation that the uK ATP -1 evidently is an inward rectifier potassium channel. 
     FIG. 5C is a plot of reversible voltage versus a logarithm of extracellular K +  concentration in the oocyte injected with cRNA for ruK ATP -1, indicating the dependency of the reversible voltage on the extracellular K +  concentration. 
     FIG. 6 is a single-channel analysis of HEK 239 transformed cells having uK ATP -1 expressed the rein, wherein A represents recordings of single-channel current and B is a current-voltage relationship, demonstrating the presence of a K +  current showing inward rectification. 
    
    
     EXAMPLE 1 
     cDNA Cloning of a Novel Inward Rectifier Potassium Channel (ruK ATP -1) 
     A cDNA fragment of GIRK, rat G protein regulating, inward rectifier potassium channel, was amplified by the polymerase chain reaction (PCR) method. Using a  32 P-labeled rat GIRK cDNA fragment as a probe, search was made into a cDNA library made from rat islets of Langerhans in the vector of λgt22. The isolated ruK ATP -1 cDNA was cut into suitable DNA fragments, and after subcloning into M13mp18 or mp19, base sequencing was performed by the chain terminator method (see FIGS.  5  and  6 ). 
     EXAMPLE 2 
     Expression in Xenopus Laevis Oocytes and Electrophysiological Analysis 
     A 20 ng quantity of cRNA synthesized in vitro from plasmid pGEM11Z containing a full-length ruK ATP -1 cDNA with the RNA polymerase after being linearized through treatment with a restriction enzyme Not1 was injected into Xenopus oocytes, followed by electrophysiological analysis 2 or 3 days later. (see FIG.  7 ). As is illustrated in FIGS. 7A and 7B, there was observed a K +  electric current showing weak inward rectification. The K +  electric current was suppressed by adding Ba 2+  in the exracellular fluid. 
     EXAMPLE 3 
     Single-channel Analysis of HEK 239 Cells Having ruK ATP -1 Expressed 
     HEK 239 cells were cultured in minimum essential Eagle&#39;s medium supplemented with 10% of horse serum. The expression plasmid (pCMV6b) carrying a full-length ruK ATP -1 coding cDNA was transfected into HEK 239 cells with use of Lipofectamine to prepare transformed HEK 293 cells. The transformed cells produced in this. manner were subjected to single channel analysis, with the results being shown in FIGS. 8A and 8B. 
     As is evident in FIGS. 8A and 8B, the outward electric current flowing through the channel was suppressed by the intracellular Mg 2+ , revealing that uK ATP -1 is an inward rectifier K +  channel; uK ATP -1 exhibited a single-channel conductance of ca. 70 pS. FIG. 7 illustrates effects of ATP on the uK ATP -1 channel activity as observed in the inside-out mode. When 1 μM of ATP was added inside the cellular membrane, the channel was open but closed completely upon addition 1 mA of ATP. The results indicate that uK ATP -1 is an ATP-regulated K ATP  channel. 
     EXAMPLE 4 
     RNA Blotting Analysis 
     A 20 μg portion of RNA extracted individually from various tissues and cell lines as well as 10 μg of RNA extracted from the pituitary and thyroid glands were denatured with formaldehyde and electrophoresed on 1% agarose gel, followed by transferring onto a Nylon membrane. Using  32 P labeled ruK ATP -1 cDNA as a probe, hybridization was carried out, with the expression of uK ATP -1 mRNA being observed in almost all tissues. 
     EXAMPLE 5 
     Cloning of cDNA and Gene of uK ATP -1 of a Human Origin 
     In order to isolate cDNA encoding uK ATP -1 of a human origin, search was effected into a human lung cDNA library using  32 P labeled ruK ATP -1 cDNA of a rat origin as a probe. The resultant clone was subjected to sub-cloning into M13mp18, M13mp19 and pGEM3Z, followed by base sequencing by the chain terminator method. 
     
       
         
           
             4 
           
           
             
               424 amino acids 
               amino acid 
               single 
               linear 
             
              1
Met Leu Ala Arg Lys Ser Ile Ile Pro Glu Glu Tyr Val Leu Ala Arg
                5                   10                  15
Ile Ala Ala Glu Asn Leu Arg Lys Pro Arg Ile Arg Asp Arg Leu Pro
            20                  25                  30
Lys Ala Arg Phe Ile Ala Lys Ser Gly Ala Cys Asn Leu Ala His Lys
        35                  40                  45
Asn Ile Arg Glu Gln Gly Arg Phe Leu Gln Asp Ile Phe Thr Thr Leu
    50                  55                  60
Val Asp Leu Lys Trp Arg His Thr Leu Val Ile Phe Thr Met Ser Phe
65                  70                  75                  80
Leu Cys Ser Trp Leu Leu Phe Ala Ile Met Trp Trp Leu Val Ala Phe
                85                  90                  95
Ala His Gly Asp Ile Tyr Ala Tyr Met Glu Lys Ser Gly Met Glu Lys
            100                 105                 110
Ser Gly Leu Glu Ser Thr Val Cys Val Thr Asn Val Arg Ser Phe Thr
        115                 120                 125
Ser Ala Phe Leu Phe Ser Ile Gln Val Gln Val Thr Ile Gly Phe Gly
    130                 135                 140
Gly Arg Met Met Thr Glu Glu Cys Pro Leu Ala Ile Thr Val Leu Ile
145                 150                 155                 160
Leu Gln Asn Ile Val Gly Leu Ile Ile Asn Ala Val Met Leu Gly Cys
                165                 170                 175
Ile Phe Met Lys Thr Ala Gln Ala His Arg Arg Ala Glu Thr Leu Ile
            180                 185                 190
Phe Ser Arg His Ala Val Ile Ala Val Arg Asn Gly Lys Leu Cys Phe
        195                 200                 205
Met Phe Arg Val Gly Asp Leu Arg Lys Ser Met Ile Ile Ser Ala Ser
    210                 215                 220
Val Arg Ile Gln Val Val Lys Lys Thr Thr Thr Pro Glu Gly Glu Val
225                 230                 235                 240
Val Pro Ile His Gln Leu Asp Ile Pro Val Asp Asn Pro Ile Glu Ser
                245                 250                 255
Asn Asn Ile Phe Leu Val Ala Pro Leu Ile Ile Cys His Val Ile Asp
            260                 265                 270
Lys Arg Ser Pro Leu Tyr Asp Ile Ser Ala Thr Asp Leu Ala Asn Gln
        275                 280                 285
Asp Leu Glu Val Ile Val Ile Leu Glu Gly Val Val Glu Thr Thr Gly
    290                 295                 300
Ile Thr Thr Gln Ala Arg Thr Ser Tyr Ile Ala Glu Glu Ile Gln Trp
305                 310                 315                 320
Gly His Arg Phe Val Ser Ile Val Thr Glu Glu Glu Gly Val Tyr Ser
                325                 330                 335
Val Asp Tyr Ser Lys Phe Gly Asn Thr Val Lys Val Ala Ala Pro Arg
            340                 345                 350
Cys Ser Ala Arg Glu Leu Asp Glu Lys Pro Ser Ile Leu Ile Gln Thr
        355                 360                 365
Leu Gln Lys Ser Glu Leu Ser His Gln Asn Ser Leu Arg Lys Arg Asn
    370                 375                 380
Ser Met Arg Arg Asn Asn Ser Met Arg Arg Asn Asn Ser Ile Arg Arg
385                 390                 395                 400
Asn Asn Ser Ser Leu Met Val Pro Lys Val Gln Phe Met Thr Pro Glu
                405                 410                 415
Gly Asn Gln Asn Thr Ser Glu Ser
            420
 
           
           
             
               1275 base pairs 
               nucleic acid 
               single 
               linear 
             
              2
ATGTTGGCCA GAAAGAGTAT CATCCCGGAG GAGTATGTGC TGGCGCGCAT CGCCGCAGAG     60
AACCTGCGCA AGCCGCGCAT CCGAGACCGC CTCCCCAAAG CCCGCTTCAT CGCCAAGAGC    120
GGGGCCTGCA ACCTGGCGCA TAAGAACATC CGTGAGCAAG GACGCTTTCT ACAGGACATC    180
TTCACCACCT TGGTGGACCT GAAATGGCGC CACACGCTGG TCATCTTTAC CATGTCCTTC    240
CTCTGCAGCT GGCTGCTCTT CGCTATCATG TGGTGGCTGG TGGCCTTTGC CCATGGGGAC    300
ATCTATGCTT ACATGGAGAA AAGTGGAATG GAGAAAAGTG GTTTGGAGTC CACTGTGTGT    360
GTGACTAATG TCAGGTCTTT CACTTCTGCT TTTCTCTTCT CCATTGAAGT TCAAGTTACC    420
ATTGGGTTTG GAGGGAGGAT GATGACAGAG GAATGCCCTT TGGCCATCAC GGTTTTGATT    480
CTCCAGAATA TTGTGGGTTT GATCATCAAT GCAGTCATGT TAGGCTGCAT TTTCATGAAA    540
ACAGCTCAGG CTCACAGAAG GGCAGAAACT TTGATTTTCA GCCGCCATGC TGTGATTGCC    600
GTCCGAAATG GCAAGCTGTG CTTCATGTTC CGAGTGGGTG ACCTGAGGAA AAGCATGATC    660
ATTAGTGCCT CTGTGCGCAT CCAGGTGGTC AAGAAAACAA CTACACCTGA AGGGGAGGTG    720
GTTCCTATTC ACCAACTGGA CATTCCTGTT GATAACCCAA TCGAGAGCAA TAACATTTTT    780
CTGGTGGCCC CTTTGATCAT CTGCCACGTG ATTGACAAGC GCAGTCCCCT GTATGACATC    840
TCAGCAACTG ACCTGGCCAA CCAAGACTTG GAGGTCATAG TTATTCTGGA AGGAGTGGTT    900
GAAACTACTG GCATCACCAC ACAAGCACGA ACCTCCTACA TTGCTGAGGA CATCCAATGG    960
GGCCACCGCT TTGTGTCCAT TGTGACTGAG GAAGAAGGAG TGTATTCTGT GGATTACTCC   1020
AAATTTGGCA ACACTGTTAA AGTAGCTGCT CCACGGTGCA GTGCCCGAGA GCTGGATGAG   1080
AAACCTTCCA TCCTTATTCA GACCCTCCAA AAGAGTGAAC TGTCTCATCA AAATTCTCTG   1140
AGGAAGCGCA ACTCCATGAG AAGAAACAAT TCCATGAGGA GGAACAATTC TATCCGAAGG   1200
AACAATTCTT CCCTCATGGT ACCAAAGGTG CAATTTATGA CTCCAGAAGG AAATCAAAAC   1260
ACATCGGAAT CATGA                                                    1275
 
           
           
             
               424 amino acids 
               amino acid 
               single 
               linear 
             
              3
Met Leu Ala Arg Lys Ser Ile Ile Pro Glu Glu Tyr Val Leu Ala Arg
                5                   10                  15
Ile Ala Ala Glu Asn Leu Arg Lys Pro Arg Ile Arg Asp Arg Leu Pro
            20                  25                  30
Lys Ala Arg Phe Ile Ala Lys Ser Gly Ala Cys Asn Leu Ala His Lys
        35                  40                  45
Asn Ile Arg Glu Gln Gly Arg Phe Leu Gln Asp Ile Phe Thr Thr Leu
    50                  55                  60
Val Asp Leu Lys Trp Arg His Thr Leu Val Ile Phe Thr Met Ser Phe
65                  70                  75                  80
Leu Cys Ser Trp Leu Leu Phe Ala Ile Met Trp Trp Leu Val Ala Phe
                85                  90                  95
Ala His Gly Asp Ile Tyr Ala Tyr Met Glu Lys Gly Ile Thr Glu Lys
            100                 105                 110
Ser Gly Leu Glu Ser Ala Val Cys Val Thr Asn Val Arg Ser Phe Thr
        115                 120                 125
Ser Ala Phe Leu Phe Ser Ile Glu Val Gln Val Thr Ile Gly Phe Gly
    130                 135                 140
Gly Arg Met Met Thr Glu Glu Cys Pro Leu Ala Ile Thr Val Leu Ile
145                 150                 155                 160
Leu Gln Asn Ile Val Gly Leu Ile Ile Asn Ala Val Met Leu Gly Cys
                165                 170                 175
Ile Phe Met Lys Thr Ala Gln Ala His Arg Arg Ala Glu Thr Leu Ile
            180                 185                 190
Phe Ser Arg His Ala Val Ile Ala Val Arg Asn Gly Lys Leu Cys Phe
        195                 200                 205
Met Phe Arg Val Gly Asp Leu Arg Lys Ser Met Ile Ile Ser Ala Ser
    210                 215                 220
Val Arg Ile Gln Val Val Lys Lys Thr Thr Thr Pro Glu Gly Glu Val
225                 230                 235                 240
Val Pro Ile His Gln Gln Asp Ile Pro Val Asp Asn Pro Ile Glu Ser
                245                 250                 255
Asn Asn Ile Phe Leu Val Ala Pro Leu Ile Ile Cys His Val Ile Asp
            260                 265                 270
Lys Arg Ser Pro Leu Tyr Asp Ile Ser Ala Thr Asp Leu Val Asn Gln
        275                 280                 285
Asp Leu Glu Val Ile Val Ile Leu Glu Gly Val Val Glu Thr Thr Gly
    290                 295                 300
Ile Thr Thr Gln Ala Arg Thr Ser Tyr Ile Ala Glu Glu Ile Gln Trp
305                 310                 315                 320
Gly His Arg Phe Val Ser Ile Val Thr Glu Glu Glu Gly Val Tyr Ser
                325                 330                 335
Val Asp Tyr Ser Lys Phe Gly Asn Thr Val Arg Val Ala Ala Pro Arg
            340                 345                 350
Cys Ser Ala Arg Glu Leu Asp Glu Lys Pro Ser Ile Leu Ile Gln Thr
        355                 360                 365
Leu Gln Lys Ser Glu Leu Ser His Gln Asn Ser Leu Arg Lys Arg Asn
    370                 375                 380
Ser Met Arg Arg Asn Asn Ser Met Arg Arg Ser Asn Ser Ile Arg Arg
385                 390                 395                 400
Asn Asn Ser Ser Leu Met Val Pro Lys Val Gln Phe Met Thr Pro Glu
                405                 410                 415
Gly Asn Gln Cys Pro Ser Glu Ser
            420
 
           
           
             
               1275 base pairs 
               nucleic acid 
               single 
               linear 
             
              4
ATGCTGGCCA GGAAGAGCAT CATCCCGGAG GAGTATGTGC TGGCCCGCAT CGCGGCGGAG     60
AACCTGCGCA AACCGCGCAT CCGCGACCGC CTCCCCAAAG CCCGCTTCAT CGCCAAGAGC    120
GGAGCCTGCA ACCTGGCTCA CAAGAACATC CGAGAGCAAG GTCGCTTCCT GCAGGACATC    180
TTCACCACCT TGGTAGACCT GAAGTGGCGT CACACGCTGG TCATCTTCAC CATGTCCTTC    240
CTCTGCAGCT GGCTGCTCTT CGCTATCATG TGGTGGCTGG TGGCCTTCGC CCACGGGGAC    300
ATCTATGCTT ACATGGAGAA AGGCATCACG GAGAAGAGTG GCCTGGAGTC TGCCGTCTGT    360
GTGACCAATG TCAGGTCATT CACTTCTGCG TTTCTCTTCT CCATCGAGGT TCAAGTGACC    420
ATTGGGTTTG GAGGGAGAAT GATGACTGAG GAGTGCCCTC TGGCCATCAC GGTTTTGATT    480
CTGCAGAACA TTGTGGGTCT GATCATCAAC GCGGTCATGT TGGGCTGCAT CTTCATGAAG    540
ACGGCCCAGG CCCACAGAAG GGCAGAGACG CTGATTTTCA GCCGCCATGC TGTAATTGCG    600
GTCCGTAATG GCAAGCTGTG CTTCATGTTC CGGGTGGGTG ACCTGAGGAA AAGCATGATC    660
ATTAGCGCCT CGGTGCGCAT CCAGGTGGTC AAGAAAACCA CGACGCCAGA AGGAGAGGTG    720
GTGCCTATTC ACCAGCAGGA CATCCCTGTG GATAATCCCA TCGAGAGCAA TAACATCTTC    780
CTAGTGGCCC CTTTGATCAT CTGCCATGTG ATTGATAAGC GTAGCCCCCT GTACGATATC    840
TCAGCCACTG ACCTTGTCAA CCAAGACCTG GAGGTCATAG TGATTCTCGA GGGCGTGGTG    900
GAAACCACGG GCATCACCAC GCAAGCGCGG ACCTCCTACA TTGCAGAGGA GATCCAGTGG    960
GGACACCGCT TCGTGTCGAT TGTGACTGAG GAGGAGGGAG TGTACTCTGT GGACTATTCT   1020
AAATTTGGTA ATACTGTGAG ACTGGCGGCG CCAAGATGCA GTGCCCGGGA GCTGGACGAG   1080
AAACCTTCCA TCTTGATTCA GACCCTCCAA AAGAGTGAAC TGTCGCACCA GAATTCTCTG   1140
AGGAAGCGCA ACTCTATGAG AAGAAACAAC TCCATGAGGA GGAGCAACTC CATCCGGAGG   1200
AATAACTCTT CCCTCATGGT GCCCAAGGTG CAATTCATGA CTCCAGAAGG AAACCAGTGC   1260
CCATCAGAAT CATGA                                                    1275