Patent Application: US-44228295-A

Abstract:
the invention provides an isolated cdna sequence coding for murine interleukin 5 receptor , murine secretory interleukin 5 receptor , human interleukin 5 receptor , and human secretory interleukin 5 receptor and products including murine interleukin 5 receptor , murine secretory interleukin 5 receptor , and human interleukin 5 receptor which are produced using the isolated cdna sequence . these products may be useful for a therapeutic agent for autoimmune disorders and diseases with eosinophilia in which human il - 5 is believed to be involved .

Description:
the description which relates to murine il - 5r is indicated ( murine ) and which relates to human il - 5r is indicated ( human ). preparation of poly ( a ) + rna from mouse bone marrow cells ( murine ) in order to prepare the cdna coding for the il - 5r , mrnas are recovered from the mouse bone marrow cells having il - 5r . mouse bone marrow cells are obtainable by a long - term bone marrow cell culture in the presence of il - 5 ( growth factor 1 : 135 , 1989 ). a suitable source of cells may be a balb / c mouse bone marrow long - term culture cell line , y16 , which is early b cells and shows a strong response to il - 5 ( even at a concentration of 1 pg / ml of il - 5 ). rna is prepared from the cell according to the method described by okayama et al . ( methods in enzymology 154 : 3 1987 ). poly ( a ) + rna is recovered by fractionating the total rna with the affinity chromatography using an oligo ( dt ) cellulose column . the poly ( a ) + rna is reverse transcribed to cdna using random primers and reverse transcriptase ( gene 25 : 263 , 1983 ). the cdna larger than 1 . 0 kb is selected for cloning and inserted into the bstxi site of cdm 8 vector ( see fig2 a ) containing a cytomegalovirus promoter according to the method described by seed et al . ( proc . natl . acad . sci . u . s . a . 84 : 8573 , 1987 ). e . coli is transformed with the recombinant plasmid in order to provide cdna library expressible in mammals . cloning of il - 5r gene : transfection of cos7 cells using the dna of the transformant ( murine ) cos 7 cells ( green monkey kidney cells ) are transfected with the dna according to the deae dextran or protoplast fusion method . the cos7 transformant is screened using anti - il - 5r antibodies h7 and t21 according to the method described by seed et al . ( nature 329 : 840 , 1988 ). h7 and t21 antibodies and the cos7 suspension are incubated together . after incubation , the mixture is transferred to plates coated with goat anti - rat igg antibody ( h7 and t21 are rat igg antibodies ). then , plasmid dna is recovered from the cos7 cells immobilized on the bottom of the plate . the transformation - screening procedure described above is repeated several times . after screening , a group of the selected cos7 transformant is further screened by flow cytometry using fluorescein - conjugated h7 and t21 and the transformant containing il - 5r cdna is identified . the rough restriction maps of il - 5r cdna isolated above are shown in fig1 . pil - 5r . 8 is the cdna clone prepared first from the cdm 8 library . pil - 5r . 2 is obtained from the cdna library using the hindiii - psti fragment of pil - 5r . 8 as a probe according to the colony hybridization method . the nucleotide sequences of the cdna fragments of pil - 5r . 2 and pil - 5r . 8 are determined according to the method described by sanger et al ( proc . natl . acad . sci . u . s . a ., 74 : 5463 , 1977 ). the entire nucleotide sequence of the cdna fragment of pil - 5r . 8 and the deduced amino acid sequence are shown in seq id no . 15 . the nucleotide a of the initiation codon atg is numbered 303 and the amino acid methionine is numbered 1 . the cdna fragment of pil - 5r . 8 has 1808 nucleotides in length which codes for 415 amino acids . this polypeptide consists of 4 portions according to hydropathy plot ( of urfs and orfs , rusell f , doolittle , university science books , 1987 ): signal peptide ( see amino acids 1 - 17 of seq id no . 15 ), extracellular region , transmembrane region , and cytoplasmic region . the amino acids at positions 32 - 34 , 128 - 130 , 213 - 215 , 241 - 243 , 392 - 394 , and 412 - 414 of seq id no . 15 are presumably linked to n - linked oligosaccharide . pil - 5r . 2 lacks a transmembrane region ( fig1 and seq id no . 16 ) and therefore , il - 5r expressed by pil - 5r . 2 is a secretory type . as shown in seq id no . 16 , pil - 5r . 2 lacks the sequence between the nucleotide nos . 986 and 1164 the following two types of vectors , cdm8 and pcaggs , can be utilized as expression vectors for an isolated il - 5r cdna sequence ( see fig2 ). cdm 8 vector : the vector demonstrates an elevated dna expression in mammalian cells . the vector has two bstxi sites . the vector is digested with bstxi and both ends of cdna of interest are ligated to a bstxi linker . the cdna - linker complex is ligated to the bstxi digested vector . pcaggs vector : the vector contains a cmv enhancer upstream of the promoter of pags - 3 , which is a vector having a much stronger expression ability than that of cdm8 ( gene , 79 : 269 , 1989 ). the cdna insertion site of pcaggs is xhoi site substituted with ecori site in the exon of rabit β - globin gene region . the pcaggs vector demonstrate a higher level of dna product expression than pags - 3 . in the example of the invention , the pcaggs vector is used for expression test of il - 5r and the expressed murine il - 5r is tested by il - 5 binding test , il - 5 cross - linking test and immunoprecipitation test using monoclonal antibody , h7 . the murine il - 5r cdna encoding a secretory il - 5r is inserted into the xhoi ( ecori ) site of the pcaggs vector . cos7 ( green monkey kidney cell , atcc crl1651 ) is transfected with the recombinant plasmid and the resulting transformant is grown in a medium . the amino acid sequence of the peptide in the culture supernatant is determined . the n - terminal 20 amino acids thus determined are the same as those deduced from the nucleotide sequence of the murine il - 5r cdna . the cos7 culture supernatant containing soluble il - 5r inhibits the binding of il - 5 to il - 5r expressed on b cells or eosinophils . the cos7 transformant thus obtained is tested for the production of il - 5r capable of binding to il - 5 using 35 s - methionine and 35 s - cysteine labeled il - 5 ( j . immunol . 140 : 1175 , 1988 ; j . exp . med . 168 : 863 , 1988 ). binding of the labeled il - 5 is inhibited by the excess amount ( 100 - fold ) of the non - labelled il - 5 and thus the cdna clone pil - 5r . 8 is confirmed to code for il - 5r . cos7 cells are transfected with pil - 5r . 8 followed by cross - linking reaction and immunoprecipitation . cross - linking : il - 5r produced by the transformant is tested whether it is the same as those expressed by a il - 5 responsive early b cell , t88 - m , by the cross - linking test using 35 s - labeled il - 5 ( proc . natl . acad . sci . u . s . a ., 1989 , 86 : 2311 ). after electrophoresis , the band pattern on the gel indicates that the molecular weight comparable to il - 5 monomer is decreased ( about 22 , 000 ) under reduced condition . immunoprecipitation : the surface proteins of the transfected cells is 125 i - labeled and immunoprecipitated with anti - il - 5r antibodies , h7 ( int . immunol . 2 : 181 , 1990 ). il - 5r produced by the transformant are found to have a molecular weight of 60 , 000 . poly ( a ) + rna are prepared from il - 5 responsive cell lines such as y16 ( early - b cell ), bcl1 - b20 ( mouse b cell chronic leukemia lymphoma , in vitro line ); mouse myeloma cell , mopc104e , x5568 , l cell , il - 3 responsive cell line fdc - p1 derived from mouse bone marrow long - term cultured cell , and il - 2 responsive mouse t cell lines . 2 μg of each of poly ( a ) + rna are tested for the presence of il - 5r mrna by northern blot . northern blot is carried out using the hindiii - psti fragment of pil - 5r . 8 as a probe ( biochemistry 16 : 4743 , 1977 ). il - 5 responsive cell lines including y16 , bcl1 - b20 , mopc104e are found to express il - 5r mrna with the size of 5 - 5 . 8 kb preparation of poly ( a ) + rna from human peripheral blood eosinophils ( human ) a dna sequence coding for human il - 5r is prepared from human peripheral blood eosinophils . eosinophils are isolated from peripheral blood of healthy volunteers and of a patient with eosinophilia by a density gradient centrifugation using ficoll ( migita , y ., et al . supra ). whole mrna is prepared from eosinophils according to the method described by okayama et al . ( ibid ). poly ( a ) + rna is recovered by fractionating the whole rna with the affinity chromatography using an oligo ( dt ) cellulose column . one of the poly ( a ) + rna preparation is derived from healthy volunteers and the other is derived from a patient with eosinophilia . the poly ( a ) + rna is reverse transcribed to cdna using random primers and reverse transcriptase as described above . the cdna of more than 1 . 0 kb fragments is selected for cloning . the cdna fragment derived from eosinophils of healthy volunteers ( helv - cdna ) is inserted into the bstxi site of vector pags - 3 ( miyazaki , et al ., 1989 , gene , 79 : 269 ) according to the method described by seed et al . ( ibid ). e . coli is then transformed with the recombinant plasmid ( helv - cdna library ). the cdna derived from eosinophils of patients with eosinophilia ( eosi - cdna ) is inserted into the ecori site of phage λgt10 using an ecori linker . e . coli is then infected with the recombinant phage ( eosi - cdna library ). the helv - cdna library is screened using the hindiii - psti fragment of pil - 5r . 8 . a positive clone is isolated and is designated as ph5r . 1 . ph5r . 1 lacks some of the nucleotide sequence of il - 5r . subsequently , the eosi - cdna library is screened using the nucleotide sequence of ph5r . 1 . two positive clones designated as hsil5r and hsil5r2 are isolated . fig6 shows restriction maps of the isolated il - 5r cdnas of hsil5r and hsil5r2 . the nucleotide sequence was determined according to the sanger &# 39 ; s method ( ibid ). hsil5r and hsil5r2 are membrane bound receptors . the cytoplasmic domain sequence of hsil5r2 is shorter than that of hsil5r . seq id no . 17 and no . 18 show the nucleotiode and deduced amino acid sequence of hsil5r ( 420 amino acids in length ) and hsil5r2 ( 396 amino acid in length ), respectively . the amino acid sequence is analyzed as described above . hsil5r and hsil5r2 consist of signal peptide region , extracellular region , transmembrane region , cytoplasmic region . the nucleotide sequence downstream of nucleotide position 1245 distinguishes hsil5r ( seq id no . 17 ) from hsil5r2 ( seq id no . 18 ). the amino acid sequence of hsil5r2 terminates after amino acid ile ( amino acid number 396 ) located immediately after the nucleotide position 1245 . human il - 5r cdna is inserted into a pcaggs vector , and cos 7 cells are transfected with the recombinant plasmid . λgt10 cdna clones , hsil5r and hsil5r2 are digested with ecori and the il - 5r cdna fragment is inserted into the ecori site of pcaggs . binding assay of transfectants with hsil5r or hsil5r2 to il - 5 ( human ) the il - 5r expression of the clones are tested using 35 s - methionine - and 35 s - cysteine - labeled murine il - 5 or 125 i - labeled human il - 5 . the human il - 5 is prepared as follows : the il - 5 cdna fragment is inserted into an expression vector derived from baculovirus . sf21 cells ( spodotera frugiperda ) are infected with the recombinant dna . the cells are cultured and the culture supernatant is tested for human il - 5 using anti - il - 5 monoclonal antibody , nc17 ( proc . natl . acad . sci . u . s . a . 84 : 4581 , 1987 ). the isolated human il - 5 is labeled with 125 i . binding assay is carried out as described for murine il - 5r . il - 5r produced by the positive clones is tested whether it is the same as those produced by eosinophils , by cross - linking test using 35 s - labeled murine il - 5 and 125 i - labeled human il - 5 as described above . poly ( a ) + rna may be prepared from human eosinophils , erythroleukemic cell line tf - 1 , eosinophilic leukemia cell line eol - 3 , atl - 2 adult t cell leukemia cell line atl - 2 , burkitt &# 39 ; s lymphoma cell line raji , and histiocytic lymphoma cell line u - 937 . 6 μg of each of poly ( a ) + rna is tested for the presence of il - 5r mrna using the entire sequence of hsil5r cdna as a probe . human eosinophils and tf - 1 cell line are found to express il - 5r mrna with the size of 1 . 4 kb and 5 . 3 kb . hsil5r cdna is inserted into the ecori site of bluescript sk (-). the construct is digested with sali and kpni . the sali - kpni digested fragment is then incubated with exonuclease iii so that the sequence coding for the cytoplasmic domain and transmembrane domain of human il - 5r can be removed . the digested fragment is blunted with mung bean exonuclease followed by a treatment with a klenow fragment and subjected to ligation ( gene 33 : 103 , 1985 ). after treatment , a clone is obtained which contains deletion from 3 &# 39 ; end to the nucleotide number 995 ( seq id no . 17 ), a site which corresponds to the starting point of deletion in the secretory murine il - 5r cdna . the deletion mutant is digested with ecori and bsshii . the resulting dna fragment is ligated to a linker containing a stop codon . after ligation , a dna - linker complex is inserted into an appropriate restriction site of any vector . alternatively , the hsil - 5r cdna fragment of the bluescript sk (-) construct is deleted from 3 &# 39 ; end to the nucleotide number 996 . as a result of frameshift , the construct contains two stop codons . the secretory human il - 5r construct thus obtained lacks dna sequences for a cytoplasmic domain and a transmembrane domain and codes for 333 amino acids . the secretory human il - 5r construct is introduced into host cells and the transfectant produces a secretory human il - 5r . an expression vector is selected according to host cells to be transfected . host cells include prokaryotes such as gram negative bacteria ( e . coli ) or gram positive bacteria ( bacillus ), yeast , and eukaryotic cell lines derived from insects and mammals . the following examples are described for murine secretory il - 5r and membrane type il - 5r . y16 ( 2 × 10 7 ) cells were placed in a 3 liter spinner culture bottle containing a medium ( rpmi 1640 , 4 % fcs , 5 × 10 - 5 m 2 - mercaptoethanol , 100 u / ml of penicillin , 100 μg / ml of streptomycin ) and 300 pg / ml of il - 5 . the bottle was sealed and incubated for a week . after incubation , about 5 × 10 9 cells were harvested . 1 × 10 9 cells were solubilized in 50 ml of 5 . 5m guanidium thiocyanate solution ( ph7 . 0 ) containing 25 mm sodium citrate , 0 . 5 % sodium laurylsulcosine , and 0 . 2m 2 - mercaptoethanol according to the method described by okayama et al . ( supra ). the cell lysate was layered onto cesium trifluoroacetic acid solution ( density : 1 . 5 g / ml ) containing 0 . 1m edta / ph7 . 0 . the mixture was centrifuged at 15 ° c ., at 125 , 000 g , for 24 hours . after centrifugation , the rna pellet was dissolved in distilled water containing 10 mm tris - hcl / ph7 . 5 and 1 mm edta . the rna solution was loaded onto an oligo ( dt ) cellulose column and the pass - through was loaded onto the column again ( molecular cloning , 1989 , chapter 7 , p26 , cold spring harbor labolatory press ). the oligo ( dt ) bounded fraction was eluted and 30 μg of poly ( a ) + rna was recovered . 30 μg of the poly ( a ) + rna thus obtained was used to synthesize cdna using a cdna synthesis kit ( brl , bethesda , md .) according to the method described by seed ( supra ). the cdm8 vector ( see fig2 a ) was digested with bstxi . after digestion , an approximately 4100 bp fragment was purified by a potassium acetate density gradient centrifugation . the cdna was ligated to a bstxi linker and a cdna - linker complex containing cdna having a size of 1 , 000 bp or more was selected by a potassium acetate density gradient centrifugation . the fractionated fragments were subjected to ligation with the purified cdm8 vector . e . coli mc1061 / p3 was transformed with the construct and about 2 million transformants were obtained as a cdna library . cos7 ( 5 × 10 5 ) cells were placed in each of 100 plates ( 6 cm ). the following day , the cos7 cell was transfected with 2 μg of the plasmid dna ( per plate ) prepared from the cdna library according to the deae - dextran method . on day 3 , the cos7 cells were removed from the plates and incubated with antibodies , h7 and t21 . the cos7 cell was screened for the presence of the h7 and t21 antigens using goat anti - rat igg antibodies ( panning technique ). after screening , plasmid dnas were prepared from the h7 and t21 antigen positive cos7 cells . then , e . coli mc1061 / p3 was transformed with the plasmid dnas . fresh cos7 cells were fused with the transformants according to the protoplast fusion method . the cos7 cells were screened for the presence of the h7 and t21 antigens according to the panning technique . after four cycles of the procedure described above , fresh cos7 cells were transformed and the transformant was screened by the panning technique using goat anti - rat igg antibody f ( ab &# 39 ;) 2 fragment . this transformation - screening procedure was repeated two times in order to eliminate the contamination of fc recepter genes . after screening , 50 independent colonies were selected and the plasmid dna was prepared . fresh cos7 cells were then transfected with the plasmid dna and the transformants were tested for the presence of the h7 and t21 antigens . one of the transformants was found to be antigen positive and designated as pil - 5r . 8 . the cdna library prepared from y16 as described above was screened for the presence of il - 5r cdna using the fragment inserted in pil - 5r . 8 as a probe according to the colony hybridization method ( molecular cloning , 1989 , chapter 1 , p90 , cold spring harbor laboratory press ): the hindiii - psti fragment was prepared from pil - 5r . 8 and radiolabeled with α - 32 p - dctp according to the random primer method . the transformants of the cdna library were grown on a solid lb agarose medium ( approximately 10 , 000 colonies per 10 cm plate ) overnight . the colonies were transferred to a nitrocellulose membrane and the dnas on the membrane were hybridized to the radiolabeled profe . positive colonies were identified through autoradiography . one of the transformants was isolated and designated as pil - 5r . 2 . the cdna fragment of pil - 5r . 8 was digested with xbai and inserted into a m13mp19 vector . the construct was digested with bamhi and kpni . the bamhi - kpni digested fragment was then digested with exonuclease iii : the fragment was digested up to ten minutes with stopping digestion every minute . the digested fragment was blunted with mungbean exonuclease followed by a treatment with a klenow fragment and subjected to ligation ( gene 33 : 103 , 1985 ). e . coli jm109 was transformed with the constructs to produce different sizes of the m13 deletion mutants . single - stranded dnas were prepared from the mutants ( methods in enzymology 101 : 58 , 1983 ) and the nucleotide sequence was determined using the m 13 primer , 5 &# 39 ;- gttttcccagtcacgac - 3 &# 39 ; according to the sanger &# 39 ; s method . single - stranded dna was also prepared from the m13 mutant containing the cdna fragment of a reversed orientation and the nucleotide sequence was determined as described above . the nucleotide sequence thus obtained from the m13 mutant containing the cdna fragment of a right orientation was found to be complementary to the one of m13 mutant containing the cdna fragment of a reversed orientation . seq id no . 15 shows the complete nucleotide sequence of pil - 5r . 8 . the first 17 amino acids are believed to be the signal peptide ( nucleic . acids . res . 14 : 4683 , 1986 ) and the amino acids from amino acid position 340 to amino acid position 361 are considered to be the transmembrane region according to the hydropathy plot . amino acids 32 - 34 , 128 - 130 , 213 - 215 , 241 - 243 , 392 - 394 , and 412 - 414 appear to be sites of n - linked oligosaccharide addition . the previously estimated molecular weight ( 45 , 284 ) of il - 5r differs from the real molecular weight ( about 60 , 000 ) of il - 5r produced by the cos7 cells transfected with pil - 5 . 8 . the difference of the weight may be due to the addition of n - linked oligosacharide . nucleotide position 1467 represents the beginning of sequence of pil - 5r . 2 the nucleotide sequence of pil - 5r . 2 shown in seq id no . 16 was determined using primers ( 17 - mers ) synthesized based on the nucleotide sequence of pil .- 5r . 8 , a t7 primer ( 5 &# 39 ;- atggaaattaatacg - 3 &# 39 ;), and a primer for the 3 &# 39 ; end of cdm8 ( 5 &# 39 ;- tgcaggcgcagaactgg - 3 &# 39 ;) according to the sanger &# 39 ; s method . the pil - 5r . 2 is a frame shift mutant , resulting in translation termination to give 4 peptides . the polypeptide encoded by pil - 5r . 2 is a secretory il - 5r which is likely to act on b cells or eosinophils in the process of differentiation . pil - 5r . 8 ( cdm8 vector ) was digested with xhoi and the il - 5r cdna fragment was inserted into the xhoi site of pcaggs vector ( see fig2 b ) whose ecori site had been replaced with a xhoi site . the new construct was designated as pcaggs . 5r . 8 . e . coli was transformed with the construct and the transformant was designated as e . coli 5r . 8 . e . coli 5r . 8 was deposited with fermentation research institute , agency of industrial science and technology , ministry of international trade and industry of 1 - 3 , higashi 1 - chome , tsukuba - shi , ibaraki - ken , 305 japan and was assigned the accession number ferm bp 3085 . cos7 cells were transfected with pil - 5r . 8 or pcaggs . 5r . 8 and the cells were harvested two days later . 2 - 10 × 10 4 cells were incubated with different concentrations of 35 s - labeled il - 5 ( 2 . 5 × 10 8 cpm / μg ) in the presence or absence of 100 - fold excess of non - labeled il - 5 at 37 ° c . for 10 minutes . after incubation , the number of il - 5 binding per cell was counted and the dissociation constant was calculated . fig3 ( a ), ( b ), ( c ) shows the scatchard plot analysis ( ann n . y . acad . sci , 51 : 660 , 1949 ) of 35 s - labeled il - 5 binding to il - 5r expressed on transfectants and y16 cells . the inset shows the direct binding data (□: total binding , ▪: non - specific binding ). fig3 ( a ) shows the results when cos7 cells were transfected with pil - 5r . 8 : the dissociation constant was 2 nm and the number of the il - 5 binding was 12 , 000 / cell . fig3 ( b ) shows the results when cos7 cells were transfected with pcaggs . 5r . 8 : the dissociation constant was 9 . 6 nm and the number of the il - 5 binding was 880 , 000 / cell . fig3 ( c ) shows the results when y16 cells were also tested for the il - 5 binding . a high affinity il - 5r and a low affinity il - 5r were found in the y16 cells . the high affinity il - 5r has the number of il - 5 binding of 1 , 200 / cell with the dissociation constant ( k d ) of 20 pm . the low affinity il - 5r has the number of il - 5 binding of 22 , 000 / cell with the dissociation constant ( k d ) of 5 . 1 nm . these results suggest that the inserted il - 5r cdna encodes a low affinity il - 5 recepter . because cos7 cells transfected with pcaggs . 5r . 8 were found to express il - 5r at a higher level than those transfected with pil - 5r . 8 , pcaggs . 5r . 8 was used for the following experiments . cos7 cells ( 1 × 10 5 ) were transfected with pcaggs or pcaggs5r . 8 and the transformants were incubated with 4 nm 35 s - labeled il - 5 in the presence or absence of 100 - fold excess of non - labeled il - 5 as follows : a pcaggs transformant without non - labeled il - 5 ( lane 1 ), a pcaggs transformant with non - labeled il - 5 ( lane 2 ), a pcaggs5r . 8 transformant without non - labeled il - 5 ( lane 3 , 5 ), a pcaggs5r . 8 transformant with non - labeled il - 5 ( lane 4 , 6 ). the mixture was incubated at 37 ° c . for 10 minutes . cells were washed extensively and then disuccinimidyl tartarate ( dst )( piece chemical , rockford , ill .) was added to the cell suspension . the cell suspension was incubated at 4 ° c . for 30 minutes and then 1 % triton x - 100 was added to the suspension to disrupt the cells . the disrupted cell suspension was loaded on a 7 . 5 % sds - polyacrylamide gel in the reducing ( lane 5 , 6 ) or non - reducing ( lane 1 - 4 ) conditions . after electrophoresis , the gel was analyzed with bio - analyzer 100 ( fuji film ). the results are shown in fig4 . a band of approximately 90 - 100 kd in size was found which could be a low affinity il - 5r previously reported by mita , et al ., in proc . natl . acad . sci . u . s . a . 86 : 2311 , 1989 . in contrast , the molecular weight of the band in the reducing condition was about 75 kd ( lane 5 in fig4 ). the difference was due to the dissociation of monomeric 35 s - labeled il - 5 ( mw : 22 , 000 ) from the il - 5 - il - 5r complex , because biologically active il - 5 binds to its receptor as a disulfide - linked dimer . the surfaces of the pcaggs . 5r . 8 transfected cos7 ( 5 × 10 6 ) cells were labeled with 125 i using iodobeads ( pierce chemical , rockford , ill .). the cell was disrupted and h7 antibody was added to the cell lysate . protein g - sepharose ( pharmacia , piscataway , n . j .) was added to the mixture and the mixture was incubated at 4 ° c . for 12 hours . the proteins adsorbed on the sepharose was loaded on the sds - page . after electrophoresis under a reducing condition , the gel was analyzed with bio - analyzer 100 . the band ( mw : about 60 kd ) was found only in the lane where the sample was prepared from the cell transfected with pcaggs . 5r . 8 ( fig5 ). the il - 5r cdna fragment obtained by xhoi digestion of pil - 5r . 2 was inserted into pcaggs vector by the similar method as in the case of pil - 5r . 8 and the construct was designated as pcaggs . 5r . 2 . e . coli was transformed with the pcaggs . 5r . 2 and the transformant was designated as e . coli 5r . 2 . e . coli 5r . 2 was deposited with fermentation research institute , agency of industrial science and technology , ministry of international trade and industry of 1 - 3 , higashi 1 - chome , tsukuba - shi , ibaraki - ken , 305 japan and was assigned the accession number ferm bp 3084 . cos7 cells were transfected with pcaggs . 5r . 2 dna according to the deae dextran method and was cultured in serum free medium ( iscove &# 39 ; s dmem ) for two days . the culture supernatant was concentrated and the concentrate was electrophoresed on sds - page . a band ( mw : approximately 50 , 000 ) was found in the lane on which the culture supernatant of pcaggs . 5r . 2 transfected cos7 was loaded , while no band was found in the lane on which the culture supernatant of the pcaggs vector alone was loaded . the culture supernatant of the pcaggs . 5r . 2 transfected cos7 was loaded onto a column filled with h7 antibodies bound glycosylhard - gel ( seikagaku kogyo , tokyo ). the column was washed with 2 mm hepfs solution containing 0 . 1 % chaps and then h7 bound fractions were eluted out with 350 mm acetic acid . the fractions were lyophilized and then solubilized in a sample buffer for sds - page . the mixture was electrophoresed according to the method described by laemmli in nature 227 : 680 , 1970 . the protein on the gel was transferred to a polyvinylidene difluoride membrane ( millipore , bedford , mass .) according to the electroblotting method . the band corresponding to a molecular weight of about 50 , 000 was cut out of the membrane and analyzed with a gas phase sequencer 447a ( with hplc system , applied biosystem co .). the amino acid sequence of the n terminus of secretory - il - 5r was as follows : asp - leu - leu - asn - his - lys - lys - phe - leu - leu - leu - pro - pro - val - x - phe - thr - ile - lys - ala . this amino acid sequence was found to be the same one ( amino acid number 18 - 37 ) deduced from the nucleotide sequence of pil - 5r . 8 , membrane bound il - 5r cdna clone . the amino acid sequence ( amino acid number 1 - 17 ) is believed to be a signal peptide . x ( amino acid number 15 ) may be asn , which is deduced from the nucleotide sequence of cdna , and to which a n - linked oligosaccharide is believed to bind . eosinophils were obtained from 28 liter of peripheral blood of healthy volunteers and 50 ml of peripheral blood of a patient with eosinophilia . after removing erythrocytes , fractions containing eosinophils ( 1 . 09 g / ml ) were collected from each sample by a density gradient centrifugation using ficoll . the fraction contained 50 % eosinophils and the number of eosinophis was 2 . 8 × 10 9 healthy volunteers ( helv )! and 2 . 0 × 10 9 eosinophilia ( eosi )!. 5 μg of poly ( a ) + rna was recovered from each cell source as described above . 5 μg of each poly ( a ) + rna thus obtained was used to synthesize cdna ( helv - cdna , eosi - cdna ) as described above . the helv - cdna was ligated to a bstxi linker and a fragment of helv - cdna - linker complex having a size of 1 , 000 bp or more was selected . the fragment was then inserted into a bstxi digested pags - 3 vector . e . coli mc1061 was transformed with the recombinant plasmid and about one million transformants were obtained ( helv - cdna library ). the eosi - cdna was ligated to a ecori linker and fragments of eosi - cdna - linker complex having a size of 1 , 000 bp or more were selected . the fragments were inserted into a ecori digested λgt10 vector . e . coli c600hfl was infected with the recombinant phage and 1 . 6 million independent plaques were obtained ( eosi - cdna library ). screening of helv - and eosi - cdna libraries according to the colony - hybridization method the helv - cdna library was screened according to the colony hybridization method . one million colonies of the helv - cdna library were grown on a solid medium and the colonies were transferred to 100 sheets of nitrocellulose membranes ( 8 cm in diameter ). after dna fixation , the membrane was placed in a bag containing 10 × denhardt &# 39 ; s solution , 6 × ssc ( 0 . 9m nacl , 0 . 09m sodium citrate ), 100 μg / ml of heat - denatured salmon sperm dna . the 32 p - labeled , 1 . 2 kb hindiii - psti fragment of pil - 5r . 8 was added to the bag and hybridization was carried out at 65 ° c . for 24 hours under less stringent conditions . the membrane was washed at 45 ° c . in a solution containing 1 × ssc and 0 . 1 % sds . after washing , a x - ray film was overlayed on the membrane for autoradiography as described above . a positive clone was obtained and was designated as ph5r . 1 . however , the cdna fragment of ph5r . 1 was found to contain only 1 . 0 kb , which was not an right size for il - 5r . subsequently , the eosi - cdna library was screened using the xhoi digested , 1 . 0 kb fragment of ph5r . 1 as a probe according to the protocol of colony / plaque screen . approximately one million clones of the eosi - cdna library was grown on a solid medium and the plaques were transferred to nylon membranes ( 13 cm in diameter , colony / plaque screen , dupont - nen , boston , mass .). hybridization was carried out at 65 ° c . for 24 hours in a solution containing 1 % sds , 1m nacl , 10 % dextran sulfate , 100 μg / ml of heat - denatured salmon sperm dna . after hybridization , the membrane was washed at 65 ° c . for an hour in a solution containing 2 × ssc and 1 % sds . two positive clones containing about 2 kb cdna fragment were obtained and designated as hsil5r and hsil5r2 . e . coli was transformed with hsil5r or hsil5r2 and the transformants were designated as e . coli hsil5r or e . coli hsil5r2 , respectively . the transformants were deposited with fermentation research institute , agency of industrial science and technology , ministry of international trade and industry of 1 - 3 , higashi 1 - chome , tsukuba - shi , ibaraki - ken , 305 japan and were assigned the accession number as follows : hils5r and hsil5r2 were digested with ecori , and the ecori digested il - 5r fragment was inserted into the ecori site of bluescript ks (-) vector ( stratagene , la jolla , calif .). the nucleotide sequence was determined according to the sanger &# 39 ; s method . the sequence was determined in both 5 &# 39 ; and 3 &# 39 ; direction . initial primers were synthesized according to the sequence of the 5 &# 39 ; upstream of the il - 5r cdna fragment ( t3 primer ) and of the 3 &# 39 ; downstream of the il - 5r cdna fragment ( t7 primer ). after the 5 &# 39 ; and 3 &# 39 ; end sequences were determined , subsequent primers were synthesized according the sequence analyzed by the dna sequencing . the nucleotide sequence thus determined was found to be complementary . seq id no . 17 shows the nucleotide and the corresponding amino acid sequence of hsil5r . the first 20 amino acids are hypothetically a signal peptide and amino acids 345 to 365 are believed to be a transmembrane region according to hydropathy plot . these assumption are based on the same model as those of mouse . amino acids 35 - 37 , 131 - 133 , 137 - 139 , 142 - 144 , 216 - 218 , and 244 - 246 seem to be the sites of n - linked oligosaccharide addition . the estimated molecular weight ( 45 , 556 ) of il - 5r from cdna clone differs from the real molecular weight ( about 60 , 000 ) of il - 5r produced by the transformed cos7 cell . the difference of the weight may be due to the n - linked oligosaccharide . the nucleotide sequence downstream of nucleotide position 1245 distinguishes hsil5r ( seq id no . 17 ) from hsil5r2 ( seq id no . 18 ). seq id no . 18 shows the nucleotide and the corresponding amino acid sequence of hsil5r2 . the amino acid sequence of hsil5r2 terminates at ile ( amino acid number 396 ), while hsil5r contains additional 24 amino acids following ser at amino acid no . 396 . the amino acid sequences of hsil5r and hsil5r2 are identical from met ( amino acid number 1 ) to gly ( amino acid number 395 ) except for an amino acid at position 129 where the amino acid is val on the sequence of hsil5 and ile on the sequence of hsil5r2 . the bluescript ks (-) recombinant was digested with ecori . the restriction fragments containing il - 5r of hsil5r and hsil5r2 were inserted into pcaggs . the resulting constructs were designated as pcaggs . hsil - 5r and pcaggs . hsil5r2 . cos7 cells were transfected with these recombinant dnas and the transformed cells were tested for their chemical characteristics using 35 s - labeled murine il - 5 or 125 i - labeled human il - 5 ( 2 × 10 6 cpm / μg ) according to the cross - linking method . binding of 125 i - labeled human il - 5 to il - 5r expressed on the cos7 cell ( pcaggs . hsil5r transformant ) was shown in the inset of fig7 a , and the results analyzed by scatchard plot was shown in fig7 a . binding of 125 i - labeled human il - 5 to il - 5r expressed on cos7 cell ( pcaggs . hsil5r2 transformant ) was shown in the inset of fig7 b , and the results analyzed by scatchard plot was shown in fig7 b . binding of 35 s - labeled murine il - 5 to il - 5r expressed on the cos7 cells ( pcaggs . hsil5r transformant ) was shown in the inset of fig7 c , and the results analyzed by scatchard plot was shown in fig7 c . binding of 35 s - labeled murine il - 5 to il - 5r expressed on cos7 cell ( pcaggs . hsil5r2 transformant ) was shown in the inset of fig7 d , and the results analyzed by scatchard plot were shown in fig7 d . a high affinity il - 5r with the dissociation constant ( k d ) of less than 100 pm was not detectable by 125 i - labeled human il - 5 because of poor specific radioactivity . to calculate the dissociation constant of a high affinity il - 5r , we used 35 s - labeled mouse il - 5 which has high specific radioactivity and is not denatured . the dissociation constant of the pcaggs . hsil5r transfected cos7 cells were about 590 pm when radiolabeled human il - 5 was used , while the dissociation constant of the same pcaggs . hs il5r transfected cos7 cells were about 250 pm when radiolabeled mouse il - 5 was used . the dissociation constant of the pcaggs . hsil5r2 transfected cos7 cells were about 410 pm with radiolabeled human il - 5 , while the dissociation constant of the same pcaggs . hsil5r2 transfected cos7 cells were about 355 pm when radiolabeled mouse il - 5 was used . these results are comparable to the dissociation constant ( 170 - 330 pm ) of eosinophils from healthy adult peripheral blood that we reported previously . the data of the previous report were calculated by scatchard analysis of binding assays using 35 s - labeled mouse il - 5 . the dissociation constant thus determined was higher than that of mouse low affinity il - 5r and fell into the average value of normal human eosinophils . taken altogether , the isolated il - 5r cdna fragment was expressed on the surface of the cos7 cells and the il - 5r expressed on the cell surface are responsible for the binding of human il - 5 . fig8 shows inhibitory effects of cytokines on the binding of il - 5 to il - 5r . il - 5r expressed on the cos7 transformants specifically binds to human and mouse il - 5 but not to human il - 2 , human il - 3 , human il - 4 , human il - 6 , human gm - csf or human g - csf . the cos7 transformant ( 1 × 10 5 cells ) carrying pcaggs . hsil5r or pcaggs . hsil5r2 and either 5 . 5 nm 35 s - labeled mouse il - 5 or 1 nm 125 i - labeled human il - 5 were mixed in the presence or absence of 250 - fold excess of non - labeled il - 5 . after one hour incubation at 4 ° c ., 1 mm bis ( sulfosuccinimidyl ) suberate ( pierce chemical co ., rockford , ill .) was added to the mixture . the mixture was further incubated at 4 ° c . for 30 minutes . after the incubation , binding was analyzed as described above . in fig9 cos7 cells transfected with a pcaggs vector alone or pcaggshsil5r were incubated with 35 s - labeled murine il - 5 ( a ; lane 1 , 2 , 3 ) or 125 i - labeled human il - 5 ( b ; lane 4 , 5 , 6 ). cos7 cells transfected with a pcaggs . hsil5r were incubated with 35 s - labeled murine il - 5 in the presence ( lane 3 ) or absence ( lane 2 ) of 250 - fold excess of non - labeled il - 5 , or were incubated with 125 i - labeled human il - 5 in the presence ( lane 6 ) or absence ( lane 5 ) of 250 - fold excess amount of non - labeled il - 5 . there were two bands corresponding to about 105 kd ( lane 2 ) and 86 kd ( lane 5 ). since murine il - 5 is 45 kd and human il - 5 is 31 kd , the molecular weight of human il - 5r could be estimated to be 55 , 000 - 60 , 000 . this molecular weight of human il - 5r is almost the same as that of il - 5r expressed on eosinophils as we reported previously ( cellular immunology , 133 ; 484 - 469 ). in the presence of a 250 - fold excess of non - labeled il - 5 , no band was found ( lanes 3 , 6 in fig9 ). same experiment was carried out using pcaggs . hsil5r2 and the results were very similar to that described above . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 18 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 1245 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 1 : atggtgcctgtgttactaattcttgtgggagctttggcaacactgcaagctgacttactt60aatcacaaaaagtttttacttctaccacctgtcaattttaccattaaagccactggatta120gctcaagttcttttacactgggacccaaatcctgaccaagagcaaaggcatgttgatcta180gagtatcacgtgaaaataaatgccccacaagaagacgaatatgataccagaaagactgaa240agcaaatgtgtgaccccccttcatgaaggctttgcagctagcgtgaggaccattctgaag300agcagccatacaactctggccagcagttgggtttctgctgaactcaaagctccaccagga360tctcctggaacctcggttacgaatttaacttgtaccacacacactgttgtaagtagccac420acccacttaaggccataccaagtgtcccttcgttgcacctggcttgttgggaaggatgcc480cctgaggacacacagtatttcctatactacaggtttggtgttttgactgaaaaatgccaa540gaatacagcagagatgcactgaacagaaatactgcatgctggtttcccaggacatttatc600aacagcaaagggtttgaacagcttgctgtgcacattaatggctcaagcaagcgtgctgca660atcaagccctttgatcagctgttcagtccacttgccattgaccaagtgaatcctccaagg720aatgtcacagtggaaattgaaagcaattctctctatatacagtgggagaaaccactttct780gcctttccagatcattgctttaactatgagctgaaaatttacaacacaaaaaatggtcac840attcagaaggaaaaactgatcgccaataagttcatctcaaaaattgatgatgtttctaca900tattccattcaagtgagagcagctgtgagctcaccttgcagaatgccaggaaggtggggc960gagtggagtcaacctatttatgtgggaaaggaaaggaagtccttggtagaatggcatctc1020attgtgctcccaacagctgcctgcttcgtcttgttaatcttctcactcatctgcagagtg1080tgtcatttatggaccaggttgtttccaccggttccggccccaaagagtaacatcaaagat1140ctccctgtggttactgaatatgagaaaccttcgaatgaaaccaaaattgaagttgtacat1200tgtgtggaagaggttggatttgaagtcatgggaaattccacgttt1245 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 1808 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 2 : gaaataattggtaaacacagaaaatgtttcaatagaaaaaagaggaaacagaacactgtg60tagccctgttatcagcagagacagagctaacgctggggataccaaactagaagaagctca120ctggacaggtcccggtatgcagttctatttttgttgatggctctgtatctaatgtgttca180tttgtaccaaggatctaaccagggtcttccagagtctgagcaagcttctcccactgagct240acatcacagccccctgtttattggaagaagaaatacttacacctttccagtattcggcta300ccatggtgcctgtgttactaattcttgtgggagctttggcaacactgcaagctgacttac360ttaatcacaaaaagtttttacttctaccacctgtcaattttaccattaaagccactggat420tagctcaagttcttttacactgggacccaaatcctgaccaagagcaaaggcatgttgatc480tagagtatcacgtgaaaataaatgccccacaagaagacgaatatgataccagaaagactg540aaagcaaatgtgtgaccccccttcatgaaggctttgcagctagcgtgaggaccattctga600agagcagccatacaactctggccagcagttgggtttctgctgaactcaaagctccaccag660gatctcctggaacctcggttacgaatttaacttgtaccacacacactgttgtaagtagcc720acacccacttaaggccataccaagtgtcccttcgttgcacctggcttgttgggaaggatg780cccctgaggacacacagtatttcctatactacaggtttggtgttttgactgaaaaatgcc840aagaatacagcagagatgcactgaacagaaatactgcatgctggtttcccaggacattta900tcaacagcaaagggtttgaacagcttgctgtgcacattaatggctcaagcaagcgtgctg960caatcaagccctttgatcagctgttcagtccacttgccattgaccaagtgaatcctccaa1020ggaatgtcacagtggaaattgaaagcaattctctctatatacagtgggagaaaccacttt1080ctgcctttccagatcattgctttaactatgagctgaaaatttacaacacaaaaaatggtc1140acattcagaaggaaaaactgatcgccaataagttcatctcaaaaattgatgatgtttcta1200catattccattcaagtgagagcagctgtgagctcaccttgcagaatgccaggaaggtggg1260gcgagtggagtcaacctatttatgtgggaaaggaaaggaagtccttggtagaatggcatc1320tcattgtgctcccaacagctgcctgcttcgtcttgttaatcttctcactcatctgcagag1380tgtgtcatttatggaccaggttgtttccaccggttccggccccaaagagtaacatcaaag1440atctccctgtggttactgaatatgagaaaccttcgaatgaaaccaaaattgaagttgtac1500attgtgtggaagaggttggatttgaagtcatgggaaattccacgttttgatggcattttg1560ccattctgaaatgaactcatacaggactccgtgataagagcaaggactgctatttcttgg1620caaggaggtatttcaaatgaacactcagagccaggcggtggtagagctcgcctttaatac1680cagcacctgggatgcacagacgggaggatttctgagttcgaggccagcttggtctataaa1740gtgagttccaggacagccagagctacacagagaaaccctgtctcgaaaaaacaaacaaac1800aaacaaac1808 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 996 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 3 : atggtgcctgtgttactaattcttgtgggagctttggcaacactgcaagctgacttactt60aatcacaaaaagtttttacttctaccacctgtcaattttaccattaaagccactggatta120gctcaagttcttttacactgggacccaaatcctgaccaagagcaaaggcatgttgatcta180gagtatcacgtgaaaataaatgccccacaagaagacgaatatgataccagaaagactgaa240agcaaatgtgtgaccccccttcatgaaggctttgcagctagcgtgaggaccattctgaag300agcagccatacaactctggccagcagttgggtttctgctgaactcaaagctccaccagga360tctcctggaacctcggttacgaatttaacttgtaccacacacactgttgtaagtagccac420acccacttaaggccataccaagtgtcccttcgttgcacctggcttgttgggaaggatgcc480cctgaggacacacagtatttcctatactacaggtttggtgttttgactgaaaaatgccaa540gaatacagcagagatgcactgaacagaaatactgcatgctggtttcccaggacatttatc600aacagcaaagggtttgaacagcttgctgtgcacattaatggctcaagcaagcgtgctgca660atcaagccctttgatcagctgttcagtccacttgccattgaccaagtgaatcctccaagg720aatgtcacagtggaaattgaaagcaattctctctatatacagtgggagaaaccactttct780gcctttccagatcattgctttaactatgagctgaaaatttacaacacaaaaaatggtcac840attcagaaggaaaaactgatcgccaataagttcatctcaaaaattgatgatgtttctaca900tattccattcaagtgagagcagctgtgagctcaccttgcagaatgccaggaaggtggggc960gagtggagtcaacctatttatgtggaaaccttcgaa996 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 1355 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 4 : tattcggctaccatggtgcctgtgttactaattcttgtgggagctttggcaacactgcaa60gctgacttacttaatcacaaaaagtttttacttctaccacctgtcaattttaccattaaa120gccactggattagctcaagttcttttacactgggacccaaatcctgaccaagagcaaagg180catgttgatctagagtatcacgtgaaaataaatgccccacaagaagacgaatatgatacc240agaaagactgaaagcaaatgtgtgaccccccttcatgaaggctttgcagctagcgtgagg300accattctgaagagcagccatacaactctggccagcagttgggtttctgctgaactcaaa360gctccaccaggatctcctggaacctcggttacgaatttaacttgtaccacacacactgtt420gtaagtagccacacccacttaaggccataccaagtgtcccttcgttgcacctggcttgtt480gggaaggatgcccctgaggacacacagtatttcctatactacaggtttggtgttttgact540gaaaaatgccaagaatacagcagagatgcactgaacagaaatactgcatgctggtttccc600aggacatttatcaacagcaaagggtttgaacagcttgctgtgcacattaatggctcaagc660aagcgtgctgcaatcaagccctttgatcagctgttcagtccacttgccattgaccaagtg720aatcctccaaggaatgtcacagtggaaattgaaagcaattctctctatatacagtgggag780aaaccactttctgcctttccagatcattgctttaactatgagctgaaaatttacaacaca840aaaaatggtcacattcagaaggaaaaactgatcgccaataagttcatctcaaaaattgat900gatgtttctacatattccattcaagtgagagcagctgtgagctcaccttgcagaatgcca960ggaaggtggggcgagtggagtcaacctatttatgtggaaaccttcgaatgaaaccaaaat1020tgaagttgtacattgtgtggaagaggttggatttgaagtcatgggaaattccacgttttg1080atggcattttgccattctgaaatgaactcatacaggactccgtgataagagcaaggactg1140ctatttcttggcaaggaggtatttcaaatgaacactcagagccaggcggtggtagagctc1200gcctttaataccagcacctgggatgcacagacgggaggatttctgagttcgaggccagct1260tggtctataaagtgagttccaggacagccagagctacacagagaaaccctgtctcgaaaa1320aacaaacaaacaaacaaacaaacaaaaatgaacac1355 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 415 amino acids ( b ) type : amino acid ( c ) strandedness : unknown ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 5 : metvalprovalleuleuileleuvalglyalaleualathrleugln151015alaaspleuleuasnhislyslyspheleuleuleuproprovalasn202530phethrilelysalathrglyleualaglnvalleuleuhistrpasp354045proasnproaspglngluglnarghisvalaspleuglutyrhisval505560lysileasnalaproglngluaspglutyraspthrarglysthrglu65707580serlyscysvalthrproleuhisgluglyphealaalaservalarg859095thrileleulysserserhisthrthrleualasersertrpvalser100105110alagluleulysalaproproglyserproglythrservalthrasn115120125leuthrcysthrthrhisthrvalvalserserhisthrhisleuarg130135140protyrglnvalserleuargcysthrtrpleuvalglylysaspala145150155160progluaspthrglntyrpheleutyrtyrargpheglyvalleuthr165170175glulyscysglnglutyrserargaspalaleuasnargasnthrala180185190cystrppheproargthrpheileasnserlysglyphegluglnleu195200205alavalhisileasnglyserserlysargalaalailelysprophe210215220aspglnleupheserproleualaileaspglnvalasnproproarg225230235240asnvalthrvalgluilegluserasnserleutyrileglntrpglu245250255lysproleuseralapheproasphiscyspheasntyrgluleulys260265270iletyrasnthrlysasnglyhisileglnlysglulysleuileala275280285asnlyspheileserlysileaspaspvalserthrtyrserilegln290295300valargalaalavalserserprocysargmetproglyargtrpgly305310315320glutrpserglnproiletyrvalglylysgluarglysserleuval325330335glutrphisleuilevalleuprothralaalacysphevalleuleu340345350ilepheserleuilecysargvalcyshisleutrpthrargleuphe355360365proprovalproalaprolysserasnilelysaspleuprovalval370375380thrglutyrglulysproserasngluthrlysilegluvalvalhis385390395400cysvalglugluvalglyphegluvalmetglyasnserthrphe405410415 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 398 amino acids ( b ) type : amino acid ( c ) strandedness : unknown ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 6 : aspleuleuasnhislyslyspheleuleuleuproprovalasnphe151015thrilelysalathrglyleualaglnvalleuleuhistrpasppro202530asnproaspglngluglnarghisvalaspleuglutyrhisvallys354045ileasnalaproglngluaspglutyraspthrarglysthrgluser505560lyscysvalthrproleuhisgluglyphealaalaservalargthr65707580ileleulysserserhisthrthrleualasersertrpvalserala859095gluleulysalaproproglyserproglythrservalthrasnleu100105110thrcysthrthrhisthrvalvalserserhisthrhisleuargpro115120125tyrglnvalserleuargcysthrtrpleuvalglylysaspalapro130135140gluaspthrglntyrpheleutyrtyrargpheglyvalleuthrglu145150155160lyscysglnglutyrserargaspalaleuasnargasnthralacys165170175trppheproargthrpheileasnserlysglyphegluglnleuala180185190valhisileasnglyserserlysargalaalailelyspropheasp195200205glnleupheserproleualaileaspglnvalasnproproargasn210215220valthrvalgluilegluserasnserleutyrileglntrpglulys225230235240proleuseralapheproasphiscyspheasntyrgluleulysile245250255tyrasnthrlysasnglyhisileglnlysglulysleuilealaasn260265270lyspheileserlysileaspaspvalserthrtyrserileglnval275280285argalaalavalserserprocysargmetproglyargtrpglyglu290295300trpserglnproiletyrvalglylysgluarglysserleuvalglu305310315320trphisleuilevalleuprothralaalacysphevalleuleuile325330335pheserleuilecysargvalcyshisleutrpthrargleuphepro340345350provalproalaprolysserasnilelysaspleuprovalvalthr355360365glutyrglulysproserasngluthrlysilegluvalvalhiscys370375380valglugluvalglyphegluvalmetglyasnserthrphe385390395 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 332 amino acids ( b ) type : amino acid ( c ) strandedness : unknown ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 7 : metvalprovalleuleuileleuvalglyalaleualathrleugln151015alaaspleuleuasnhislyslyspheleuleuleuproprovalasn202530phethrilelysalathrglyleualaglnvalleuleuhistrpasp354045proasnproaspglngluglnarghisvalaspleuglutyrhisval505560lysileasnalaproglngluaspglutyraspthrarglysthrglu65707580serlyscysvalthrproleuhisgluglyphealaalaservalarg859095thrileleulysserserhisthrthrleualasersertrpvalser100105110alagluleulysalaproproglyserproglythrservalthrasn115120125leuthrcysthrthrhisthrvalvalserserhisthrhisleuarg130135140protyrglnvalserleuargcysthrtrpleuvalglylysaspala145150155160progluaspthrglntyrpheleutyrtyrargpheglyvalleuthr165170175glulyscysglnglutyrserargaspalaleuasnargasnthrala180185190cystrppheproargthrpheileasnserlysglyphegluglnleu195200205alavalhisileasnglyserserlysargalaalailelysprophe210215220aspglnleupheserproleualaileaspglnvalasnproproarg225230235240asnvalthrvalgluilegluserasnserleutyrileglntrpglu245250255lysproleuseralapheproasphiscyspheasntyrgluleulys260265270iletyrasnthrlysasnglyhisileglnlysglulysleuileala275280285asnlyspheileserlysileaspaspvalserthrtyrserilegln290295300valargalaalavalserserprocysargmetproglyargtrpgly305310315320glutrpserglnproiletyrvalgluthrpheglu325330 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 315 amino acids ( b ) type : amino acid ( c ) strandedness : unknown ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 8 : aspleuleuasnhislyslyspheleuleuleuproprovalasnphe151015thrilelysalathrglyleualaglnvalleuleuhistrpasppro202530asnproaspglngluglnarghisvalaspleuglutyrhisvallys354045ileasnalaproglngluaspglutyraspthrarglysthrgluser505560lyscysvalthrproleuhisgluglyphealaalaservalargthr65707580ileleulysserserhisthrthrleualasersertrpvalserala859095gluleulysalaproproglyserproglythrservalthrasnleu100105110thrcysthrthrhisthrvalvalserserhisthrhisleuargpro115120125tyrglnvalserleuargcysthrtrpleuvalglylysaspalapro130135140gluaspthrglntyrpheleutyrtyrargpheglyvalleuthrglu145150155160lyscysglnglutyrserargaspalaleuasnargasnthralacys165170175trppheproargthrpheileasnserlysglyphegluglnleuala180185190valhisileasnglyserserlysargalaalailelyspropheasp195200205glnleupheserproleualaileaspglnvalasnproproargasn210215220valthrvalgluilegluserasnserleutyrileglntrpglulys225230235240proleuseralapheproasphiscyspheasntyrgluleulysile245250255tyrasnthrlysasnglyhisileglnlysglulysleuilealaasn260265270lyspheileserlysileaspaspvalserthrtyrserileglnval275280285argalaalavalserserprocysargmetproglyargtrpglyglu290295300trpserglnproiletyrvalgluthrpheglu305310315 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 1260 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 9 : atgatcatcgtggcgcatgtattactcatccttttgggggccactgagatactgcaagct60gacttacttcctgatgaaaagatttcacttctcccacctgtcaatttcaccattaaagtt120actggtttggctcaagttcttttacaatggaaaccaaatcctgatcaagagcaaaggaat180gttaatctagaatatcaagtgaaaataaacgctccaaaagaagatgactatgaaaccaga240atcactgaaagcaaatgtgtaaccatcctccacaaaggcttttcagcaagtgtgcggacc300atcctgcagaacgaccactcactactggccagcagctgggcttctgctgaacttcatgcc360ccaccagggtctcctggaacctcagttgtgaatttaacttgcaccacaaacactacagaa420gacaattattcacgtttaaggtcataccaagtttcccttcactgcacctggcttgttggc480acagatgcccctgaggacacgcagtattttctctactataggtatggctcttggactgaa540gaatgccaagaatacagcaaagacacactggggagaaatatcgcatgctggtttcccagg600acttttatcctcagcaaagggcgtgactggcttgcggtgcttgttaacggctccagcaag660cactctgctatcaggccctttgatcagctgtttgcccttcacgccattgatcaaataaat720cctccactgaatgtcacagcagagattgaaggaactcgtctctctatccaatgggagaaa780ccagtgtctgcttttccaatccattgctttgattatgaagtaaaaatacacaatacaagg840aatggatatttgcagatagaaaaattgatgaccaatgcattcatctcaataattgatgat900ctttctaagtacgatgttcaagtgagagcagcagtgagctccatgtgcagagaggcaggg960ctctggagtgagtggagccaacctatttatgtgggaaatgatgaacacaagcccttgaga1020gagtggtttgtcattgtgattatggcaaccatctgcttcatcttgttaattctctcgctt1080atctgtaaaatatgtcatttatggatcaagttgtttccaccaattccagcaccaaaaagt1140aatatcaaagatctctttgtaaccactaactatgagaaagctgggtccagtgagacggaa1200attgaagtcatctgttatatagagaagcctggagttgagaccctggaggattctgtgttt1260 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 2006 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 10 : cggtcctcgccatcttctgttgagtactggtcggaacaagaggatcgtctgtagacagga60tatgatcatcgtggcgcatgtattactcatccttttgggggccactgagatactgcaagc120tgacttacttcctgatgaaaagatttcacttctcccacctgtcaatttcaccattaaagt180tactggtttggctcaagttcttttacaatggaaaccaaatcctgatcaagagcaaaggaa240tgttaatctagaatatcaagtgaaaataaacgctccaaaagaagatgactatgaaaccag300aatcactgaaagcaaatgtgtaaccatcctccacaaaggcttttcagcaagtgtgcggac360catcctgcagaacgaccactcactactggccagcagctgggcttctgctgaacttcatgc420cccaccagggtctcctggaacctcagttgtgaatttaacttgcaccacaaacactacaga480agacaattattcacgtttaaggtcataccaagtttcccttcactgcacctggcttgttgg540cacagatgcccctgaggacacgcagtattttctctactataggtatggctcttggactga600agaatgccaagaatacagcaaagacacactggggagaaatatcgcatgctggtttcccag660gacttttatcctcagcaaagggcgtgactggcttgcggtgcttgttaacggctccagcaa720gcactctgctatcaggccctttgatcagctgtttgcccttcacgccattgatcaaataaa780tcctccactgaatgtcacagcagagattgaaggaactcgtctctctatccaatgggagaa840accagtgtctgcttttccaatccattgctttgattatgaagtaaaaatacacaatacaag900gaatggatatttgcagatagaaaaattgatgaccaatgcattcatctcaataattgatga960tctttctaagtacgatgttcaagtgagagcagcagtgagctccatgtgcagagaggcagg1020gctctggagtgagtggagccaacctatttatgtgggaaatgatgaacacaagcccttgag1080agagtggtttgtcattgtgattatggcaaccatctgcttcatcttgttaattctctcgct1140tatctgtaaaatatgtcatttatggatcaagttgtttccaccaattccagcaccaaaaag1200taatatcaaagatctctttgtaaccactaactatgagaaagctgggtccagtgagacgga1260aattgaagtcatctgttatatagagaagcctggagttgagaccctggaggattctgtgtt1320ttgactgtcactttggcatcctctgatgaactcacacatgcctcagtgcctcagtgaaaa1380gaacagggatgctggctcttggctaagaggtgttcagaatttaggcaacactcaatttac1440ctgcgaagcaatacacccagacacaccagtcttgtatctcttaaaagtatggatgcttca1500tccaaatcgcctcacctacagcagggaagttgactcatccaagcattttgccatgttttt1560tctccccatgccgtacagggtagcacctcctcacctgccaatctttgcaatttgcttgac1620tcacctcagacttttcattcacaacagacagcttttaaggctaacgtccagctgtattta1680cttctggctgtgcccgtttggctgtttaagctgccaattgtagcactcagctaccatctg1740aggaagaaagcattttgcatcagcctggagtgaatcatgaacttggattcaagactgtct1800tttctatagcaagtgagagccacaaattcctcacccccctacattctagaatgatctttt1860tctaggtagattgtgtatgtgtgtgtatgagagagagagagagagagagagagagagaga1920gagaaattatctcaagctccagaggcctgatccaggatacatcatttgaaaccaactaat1980ttaaaagcataatagagctaatatat2006 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 1188 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 11 : atgatcatcgtggcgcatgtattactcatccttttgggggccactgagatactgcaagct60gacttacttcctgatgaaaagatttcacttctcccacctgtcaatttcaccattaaagtt120actggtttggctcaagttcttttacaatggaaaccaaatcctgatcaagagcaaaggaat180gttaatctagaatatcaagtgaaaataaacgctccaaaagaagatgactatgaaaccaga240atcactgaaagcaaatgtgtaaccatcctccacaaaggcttttcagcaagtgtgcggacc300atcctgcagaacgaccactcactactggccagcagctgggcttctgctgaacttcatgcc360ccaccagggtctcctggaacctcaattgtgaatttaacttgcaccacaaacactacagaa420gacaattattcacgtttaaggtcataccaagtttcccttcactgcacctggcttgttggc480acagatgcccctgaggacacgcagtattttctctactataggtatggctcttggactgaa540gaatgccaagaatacagcaaagacacactggggagaaatatcgcatgctggtttcccagg600acttttatcctcagcaaagggcgtgactggcttgcggtgcttgttaacggctccagcaag660cactctgctatcaggccctttgatcagctgtttgcccttcacgccattgatcaaataaat720cctccactgaatgtcacagcagagattgaaggaactcgtctctctatccaatgggagaaa780ccagtgtctgcttttccaatccattgctttgattatgaagtaaaaatacacaatacaagg840aatggatatttgcagatagaaaaattgatgaccaatgcattcatctcaataattgatgat900ctttctaagtacgatgttcaagtgagagcagcagtgagctccatgtgcagagaggcaggg960ctctggagtgagtggagccaacctatttatgtgggaaatgatgaacacaagcccttgaga1020gagtggtttgtcattgtgattatggcaaccatctgcttcatcttgttaattctctcgctt1080atctgtaaaatatgtcatttatggatcaagttgtttccaccaattccagcaccaaaaagt1140aatatcaaagatctctttgtaaccactaactatgagaaagctggaatt1188 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 2024 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 12 : tagatgctggggttgcagccacgagcatagacacgacagacacggtcctcgccatcttct60gttgagtactggtcggaacaagaggatcgtctgtagacaggatatgatcatcgtggcgca120tgtattactcatccttttgggggccactgagatactgcaagctgacttacttcctgatga180aaagatttcacttctcccacctgtcaatttcaccattaaagttactggtttggctcaagt240tcttttacaatggaaaccaaatcctgatcaagagcaaaggaatgttaatctagaatatca300agtgaaaataaacgctccaaaagaagatgactatgaaaccagaatcactgaaagcaaatg360tgtaaccatcctccacaaaggcttttcagcaagtgtgcggaccatcctgcagaacgacca420ctcactactggccagcagctgggcttctgctgaacttcatgccccaccagggtctcctgg480aacctcaattgtgaatttaacttgcaccacaaacactacagaagacaattattcacgttt540aaggtcataccaagtttcccttcactgcacctggcttgttggcacagatgcccctgagga600cacgcagtattttctctactataggtatggctcttggactgaagaatgccaagaatacag660caaagacacactggggagaaatatcgcatgctggtttcccaggacttttatcctcagcaa720agggcgtgactggcttgcggtgcttgttaacggctccagcaagcactctgctatcaggcc780ctttgatcagctgtttgcccttcacgccattgatcaaataaatcctccactgaatgtcac840agcagagattgaaggaactcgtctctctatccaatgggagaaaccagtgtctgcttttcc900aatccattgctttgattatgaagtaaaaatacacaatacaaggaatggatatttgcagat960agaaaaattgatgaccaatgcattcatctcaataattgatgatctttctaagtacgatgt1020tcaagtgagagcagcagtgagctccatgtgcagagaggcagggctctggagtgagtggag1080ccaacctatttatgtgggaaatgatgaacacaagcccttgagagagtggtttgtcattgt1140gattatggcaaccatctgcttcatcttgttaattctctcgcttatctgtaaaatatgtca1200tttatggatcaagttgtttccaccaattccagcaccaaaaagtaatatcaaagatctctt1260tgtaaccactaactatgagaaagctggaatttaaattcaagcatgttttaacttttggtt1320taaggtacttgggtgtacctggcagtgttgtaagctctttacattaattaattaactctc1380taggtactgttatcttcattttataaacaaggcagctgaagttgagagaaataagtaacc1440tgtcctaggtcacacaattaggaaatgacagatctggcagtctatttccaggcagtctat1500ttccacgaggtcatgagtgcgaaagagggactaggggaagaatgattaactccagggagc1560tgacttttctagtgtgcttacctgttttgcatctctcaaggatgtgccatgaagctgtag1620ccaggtggaattgtaccacagccctgacatgaacacctgatggcagctgctgggttggag1680cctagacaaaaacatgaagaaccatggctgctgcctgagcccatcgtgctgtaattatag1740aaaaccttctaagggaagaatatgctgatatttttcagataagtaccccttttataaaaa1800tcctccaagttagccctcgattttccatgtaaggaaacagaggctttgagataatgtctg1860tctcctaagggacaaagccaggacttgatcctgtcttaaaaatgcaaaatgtagtacttc1920ttccatcaaaggtagacatgcactaagggacaggttttggcttggtatcagaatacattt1980ttaaaagctgtgtaagaattgaacgggctgtactagggggtata2024 ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 420 amino acids ( b ) type : amino acid ( c ) strandedness : unknown ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 13 : metileilevalalahisvalleuleuileleuleuglyalathrglu151015ileleuglnalaaspleuleuproaspglulysileserleuleupro202530provalasnphethrilelysvalthrglyleualaglnvalleuleu354045glntrplysproasnproaspglngluglnargasnvalasnleuglu505560tyrglnvallysileasnalaprolysgluaspasptyrgluthrarg65707580ilethrgluserlyscysvalthrileleuhislysglypheserala859095servalargthrileleuglnasnasphisserleuleualaserser100105110trpalaseralagluleuhisalaproproglyserproglythrser115120125valvalasnleuthrcysthrthrasnthrthrgluaspasntyrser130135140argleuargsertyrglnvalserleuhiscysthrtrpleuvalgly145150155160thraspalaprogluaspthrglntyrpheleutyrtyrargtyrgly165170175sertrpthrgluglucysglnglutyrserlysaspthrleuglyarg180185190asnilealacystrppheproargthrpheileleuserlysglyarg195200205asptrpleualavalleuvalasnglyserserlyshisseralaile210215220argpropheaspglnleuphealaleuhisalaileaspglnileasn225230235240proproleuasnvalthralagluilegluglythrargleuserile245250255glntrpglulysprovalseralapheproilehiscyspheasptyr260265270gluvallysilehisasnthrargasnglytyrleuglnileglulys275280285leumetthrasnalapheileserileileaspaspleuserlystyr290295300aspvalglnvalargalaalavalsersermetcysargglualagly305310315320leutrpserglutrpserglnproiletyrvalglyasnaspgluhis325330335lysproleuargglutrpphevalilevalilemetalathrilecys340345350pheileleuleuileleuserleuilecyslysilecyshisleutrp355360365ilelysleupheproproileproalaprolysserasnilelysasp370375380leuphevalthrthrasntyrglulysalaglysersergluthrglu385390395400ilegluvalilecystyrileglulysproglyvalgluthrleuglu405410415aspservalphe420 ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 396 amino acids ( b ) type : amino acid ( c ) strandedness : unknown ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 14 : metileilevalalahisvalleuleuileleuleuglyalathrglu151015ileleuglnalaaspleuleuproaspglulysileserleuleupro202530provalasnphethrilelysvalthrglyleualaglnvalleuleu354045glntrplysproasnproaspglngluglnargasnvalasnleuglu505560tyrglnvallysileasnalaprolysgluaspasptyrgluthrarg65707580ilethrgluserlyscysvalthrileleuhislysglypheserala859095servalargthrileleuglnasnasphisserleuleualaserser100105110trpalaseralagluleuhisalaproproglyserproglythrser115120125ilevalasnleuthrcysthrthrasnthrthrgluaspasntyrser130135140argleuargsertyrglnvalserleuhiscysthrtrpleuvalgly145150155160thraspalaprogluaspthrglntyrpheleutyrtyrargtyrgly165170175sertrpthrgluglucysglnglutyrserlysaspthrleuglyarg180185190asnilealacystrppheproargthrpheileleuserlysglyarg195200205asptrpleualavalleuvalasnglyserserlyshisseralaile210215220argpropheaspglnleuphealaleuhisalaileaspglnileasn225230235240proproleuasnvalthralagluilegluglythrargleuserile245250255glntrpglulysprovalseralapheproilehiscyspheasptyr260265270gluvallysilehisasnthrargasnglytyrleuglnileglulys275280285leumetthrasnalapheileserileileaspaspleuserlystyr290295300aspvalglnvalargalaalavalsersermetcysargglualagly305310315320leutrpserglutrpserglnproiletyrvalglyasnaspgluhis325330335lysproleuargglutrpphevalilevalilemetalathrilecys340345350pheileleuleuileleuserleuilecyslysilecyshisleutrp355360365ilelysleupheproproileproalaprolysserasnilelysasp370375380leuphevalthrthrasntyrglulysalaglyile385390395 ( 2 ) information for seq id no : 15 :( i ) sequence characteristics :( a ) length : 1808 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : cds ( b ) location : 303 .. 1547 ( xi ) sequence description : seq id no : 15 : gaaataattggtaaacacagaaaatgtttcaatagaaaaaagaggaaacagaacactgtg60tagccctgttatcagcagagacagagctaacgctggggataccaaactagaagaagctca120ctggacaggtcccggtatgcagttctatttttgttgatggctctgtatctaatgtgttca180tttgtaccaaggatctaaccagggtcttccagagtctgagcaagcttctcccactgagct240acatcacagccccctgtttattggaagaagaaatacttacacctttccagtattcggcta300ccatggtgcctgtgttactaattcttgtgggagctttggcaacactg347metvalprovalleuleuileleuvalglyalaleualathrleu151015caagctgacttacttaatcacaaaaagtttttacttctaccacctgtc395glnalaaspleuleuasnhislyslyspheleuleuleuproproval202530aattttaccattaaagccactggattagctcaagttcttttacactgg443asnphethrilelysalathrglyleualaglnvalleuleuhistrp354045gacccaaatcctgaccaagagcaaaggcatgttgatctagagtatcac491aspproasnproaspglngluglnarghisvalaspleuglutyrhis505560gtgaaaataaatgccccacaagaagacgaatatgataccagaaagact539vallysileasnalaproglngluaspglutyraspthrarglysthr657075gaaagcaaatgtgtgaccccccttcatgaaggctttgcagctagcgtg587gluserlyscysvalthrproleuhisgluglyphealaalaserval80859095aggaccattctgaagagcagccatacaactctggccagcagttgggtt635argthrileleulysserserhisthrthrleualasersertrpval100105110tctgctgaactcaaagctccaccaggatctcctggaacctcggttacg683seralagluleulysalaproproglyserproglythrservalthr115120125aatttaacttgtaccacacacactgttgtaagtagccacacccactta731asnleuthrcysthrthrhisthrvalvalserserhisthrhisleu130135140aggccataccaagtgtcccttcgttgcacctggcttgttgggaaggat779argprotyrglnvalserleuargcysthrtrpleuvalglylysasp145150155gcccctgaggacacacagtatttcctatactacaggtttggtgttttg827alaprogluaspthrglntyrpheleutyrtyrargpheglyvalleu160165170175actgaaaaatgccaagaatacagcagagatgcactgaacagaaatact875thrglulyscysglnglutyrserargaspalaleuasnargasnthr180185190gcatgctggtttcccaggacatttatcaacagcaaagggtttgaacag923alacystrppheproargthrpheileasnserlysglypheglugln195200205cttgctgtgcacattaatggctcaagcaagcgtgctgcaatcaagccc971leualavalhisileasnglyserserlysargalaalailelyspro210215220tttgatcagctgttcagtccacttgccattgaccaagtgaatcctcca1019pheaspglnleupheserproleualaileaspglnvalasnpropro225230235aggaatgtcacagtggaaattgaaagcaattctctctatatacagtgg1067argasnvalthrvalgluilegluserasnserleutyrileglntrp240245250255gagaaaccactttctgcctttccagatcattgctttaactatgagctg1115glulysproleuseralapheproasphiscyspheasntyrgluleu260265270aaaatttacaacacaaaaaatggtcacattcagaaggaaaaactgatc1163lysiletyrasnthrlysasnglyhisileglnlysglulysleuile275280285gccaataagttcatctcaaaaattgatgatgtttctacatattccatt1211alaasnlyspheileserlysileaspaspvalserthrtyrserile290295300caagtgagagcagctgtgagctcaccttgcagaatgccaggaaggtgg1259glnvalargalaalavalserserprocysargmetproglyargtrp305310315ggcgagtggagtcaacctatttatgtgggaaaggaaaggaagtccttg1307glyglutrpserglnproiletyrvalglylysgluarglysserleu320325330335gtagaatggcatctcattgtgctcccaacagctgcctgcttcgtcttg1355valglutrphisleuilevalleuprothralaalacysphevalleu340345350ttaatcttctcactcatctgcagagtgtgtcatttatggaccaggttg1403leuilepheserleuilecysargvalcyshisleutrpthrargleu355360365tttccaccggttccggccccaaagagtaacatcaaagatctccctgtg1451pheproprovalproalaprolysserasnilelysaspleuproval370375380gttactgaatatgagaaaccttcgaatgaaaccaaaattgaagttgta1499valthrglutyrglulysproserasngluthrlysilegluvalval385390395cattgtgtggaagaggttggatttgaagtcatgggaaattccacgttt1547hiscysvalglugluvalglyphegluvalmetglyasnserthrphe400405410415tgatggcattttgccattctgaaatgaactcatacaggactccgtgataagagcaaggac1607tgctatttcttggcaaggaggtatttcaaatgaacactcagagccaggcggtggtagagc1667tcgcctttaataccagcacctgggatgcacagacgggaggatttctgagttcgaggccag1727cttggtctataaagtgagttccaggacagccagagctacacagagaaaccctgtctcgaa1787aaaacaaacaaacaaacaaac1808 ( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 1355 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( ix ) feature :( a ) name / key : cds ( b ) location : 13 .. 1008 ( xi ) sequence description : seq id no : 16 : tattcggctaccatggtgcctgtgttactaattcttgtgggagctttg48metvalprovalleuleuileleuvalglyalaleu1510gcaacactgcaagctgacttacttaatcacaaaaagtttttacttcta96alathrleuglnalaaspleuleuasnhislyslyspheleuleuleu152025ccacctgtcaattttaccattaaagccactggattagctcaagttctt144proprovalasnphethrilelysalathrglyleualaglnvalleu303540ttacactgggacccaaatcctgaccaagagcaaaggcatgttgatcta192leuhistrpaspproasnproaspglngluglnarghisvalaspleu45505560gagtatcacgtgaaaataaatgccccacaagaagacgaatatgatacc240glutyrhisvallysileasnalaproglngluaspglutyraspthr657075agaaagactgaaagcaaatgtgtgaccccccttcatgaaggctttgca288arglysthrgluserlyscysvalthrproleuhisgluglypheala808590gctagcgtgaggaccattctgaagagcagccatacaactctggccagc336alaservalargthrileleulysserserhisthrthrleualaser95100105agttgggtttctgctgaactcaaagctccaccaggatctcctggaacc384sertrpvalseralagluleulysalaproproglyserproglythr110115120tcggttacgaatttaacttgtaccacacacactgttgtaagtagccac432servalthrasnleuthrcysthrthrhisthrvalvalserserhis125130135140acccacttaaggccataccaagtgtcccttcgttgcacctggcttgtt480thrhisleuargprotyrglnvalserleuargcysthrtrpleuval145150155gggaaggatgcccctgaggacacacagtatttcctatactacaggttt528glylysaspalaprogluaspthrglntyrpheleutyrtyrargphe160165170ggtgttttgactgaaaaatgccaagaatacagcagagatgcactgaac576glyvalleuthrglulyscysglnglutyrserargaspalaleuasn175180185agaaatactgcatgctggtttcccaggacatttatcaacagcaaaggg624argasnthralacystrppheproargthrpheileasnserlysgly190195200tttgaacagcttgctgtgcacattaatggctcaagcaagcgtgctgca672phegluglnleualavalhisileasnglyserserlysargalaala205210215220atcaagccctttgatcagctgttcagtccacttgccattgaccaagtg720ilelyspropheaspglnleupheserproleualaileaspglnval225230235aatcctccaaggaatgtcacagtggaaattgaaagcaattctctctat768asnproproargasnvalthrvalgluilegluserasnserleutyr240245250atacagtgggagaaaccactttctgcctttccagatcattgctttaac816ileglntrpglulysproleuseralapheproasphiscyspheasn255260265tatgagctgaaaatttacaacacaaaaaatggtcacattcagaaggaa864tyrgluleulysiletyrasnthrlysasnglyhisileglnlysglu270275280aaactgatcgccaataagttcatctcaaaaattgatgatgtttctaca912lysleuilealaasnlyspheileserlysileaspaspvalserthr285290295300tattccattcaagtgagagcagctgtgagctcaccttgcagaatgcca960tyrserileglnvalargalaalavalserserprocysargmetpro305310315ggaaggtggggcgagtggagtcaacctatttatgtggaaaccttcgaa1008glyargtrpglyglutrpserglnproiletyrvalgluthrpheglu320325330tgaaaccaaaattgaagttgtacattgtgtggaagaggttggatttgaagtcatgggaaa1068ttccacgttttgatggcattttgccattctgaaatgaactcatacaggactccgtgataa1128gagcaaggactgctatttcttggcaaggaggtatttcaaatgaacactcagagccaggcg1188gtggtagagctcgcctttaataccagcacctgggatgcacagacgggaggatttctgagt1248tcgaggccagcttggtctataaagtgagttccaggacagccagagctacacagagaaacc1308ctgtctcgaaaaaacaaacaaacaaacaaacaaacaaaaatgaacac1355 ( 2 ) information for seq id no : 17 :( i ) sequence characteristics :( a ) length : 2006 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( ix ) feature :( a ) name / key : cds ( b ) location : 62 .. 1324 ( xi ) sequence description : seq id no : 17 : 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( 2 ) information for seq id no : 18 :( i ) sequence characteristics :( a ) length : 2024 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( ix ) feature :( a ) name / key : cds ( b ) location : 104 .. 1291 ( xi ) sequence description : seq id no : 18 : 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