Patent Application: US-97787197-A

Abstract:
the present invention generally relates to a novel catalytic subunit of a lipid kinase designated p110δ . polynucleotides encoding p110δ and recombinant p110δ polypeptides are provided along with antibodies to p110δ , assays for identifying inhibitors of p110δ , and the like .

Description:
the present invention is illustrated by the following examples . example 1 describes the cloning and characterization of cdna encoding p110δ . p110δ was obtained by combining three separate cdna clones spanning the full length p110δ cdna . example 2 describes the expression and kinase activity of recombinant p110δ . example 3 describes the isolation of a mouse genomic p110δ clone . baculovirus expression of p110δ is described in example 4 . example 5 assesses the ability of recombinant p110δ to associate with p85 in transfected mammalian cells . the expression of p110δ in various human tissues is disclosed in example 6 . example 7 provides monoclonal antibodies specific for p110δ . example 8 describes experiments directed to chromosomal localization of p110δ . example 9 describes experiments related to the association of p110δ and growth factor receptors . example 10 discusses the use of transgenic animals which are engineered to include a disruption in the p110δ gene . degenerate oligonucleotide primers were designed for use in a pcr reaction based on sequences conserved in the catalytic domain of known pi 3 - kinases . the sense primer was gcagacggatccggigaygayhkiagrcarga ( seq id no : 3 ) encoding the sequence gddlrqd ( seq id no : 4 ), and the anti - sense primer was gcagacgaattcrwriccraartciryrtg ( seq id no : 5 ) encoding the amino acid sequence hidfgh ( seq id no : 6 ). bam hi and eco ri restriction sites are underlined . pcr reactions consisted of 100 ng of cdna template from human peripheral blood mononuclear cells ( pbmc ) activated for 18 hours with 10 ng / ml phorbol myristate and 250 ng / ml calcium ionophore ( sigma )!, 10 μg / ml oligonucleotide primers , 50 mm kcl , 10 mm tris hcl ( ph 8 . 4 ), 1 . 5 mm mgcl 2 , 200 mm dntps , and 1 u of taq polymerase in a final volume of 100 μl . reactions were performed using denaturation for 1 minute at 94 ° c ., annealing at 60 ° c . for 2 minutes and extension for at 72 ° c . for 1 minute for 3 cycles . the procedure was then repeated using 56 ° c . annealing temperature for 3 cycles , 52 ° c . annealing temperature for 3 cycles and 50 ° c . annealing temperature for 30 cycles . amplified products were gel purified , digested with bam hi and eco ri , and subcloned into the vector pbsskii + ( stratagene , la jolla , calif .) for sequencing . all dna for sequencing was prepared using the wizard miniprep dna purification system ( promega , madison , wis .). sequencing was performed on the applied biosystems model 373 automated sequencer . data bank searches were made using the blast program , and protein and dna alignments were made using the geneworks program ( intelligenetics inc . mountain view calif .). one clone contained a 399 bp insert that encoded a 133 amino acid open reading frame showing similarity to p110β . this clone was a partial clone of a new catalytic subunit of pi 3 - kinase designated p110δ . to identify a cdna encoding p110δ , specific oligonucleotide primers were designed based on the sequence of the 399 bp pcr product . the forward primer was catgctgaccctgcagatgat ( seq id no : 7 ) and the reverse primer was aacagctgcccactctctcgg ( seq id no : 8 ). these primers were used to screen a cdna library from human pbmc stimulated with pma and ionomycin ( as described above ) in the mammalian expression vector prc - cmv . successive rounds of pcr were performed initially on pools of 100 , 000 clones and subsequently on smaller pools until a single clone termed pbmc # 249 was isolated by colony hybridization using the pcr product labelled by random priming as a probe . this cdna was not full length . therefore to identify longer cdna clones the same approach was used to screen a cdna library from human macrophages ( also in the vector prccmv ). this led to the isolation of an additional cdna clone ( m # 928 ) which extended the cdna sequence by 1302 bp . the remaining 5 &# 39 ; end of the cdna encoding p110δ was obtained by 5 &# 39 ; race pcr ( clonetech , palo alto , calif .) two anti - sense gene - specific oligonucleotide primers were designed based on the 5 &# 39 ; end of cdna m # 928 for race pcr reactions . the primary race primer was gggccacatgtagaggcagcgttccc ( seq id no : 9 ) and the nested race primer was ggcccaggcaatggggcagtccgcc ( seq id no : 10 ). marathon - race reactions were set up using marathon - ready cdna template from human leukocytes and the advantage core pcr reaction kit ( clonetech , palo alto , calif .) following the manufacturer &# 39 ; s protocol . touchdown pcr cycling conditions were modified to improve the specificity of the marathon - race pcr primary reaction as follows : denaturation at 94 ° c . for 2 minutes , followed by 5 cycles of denaturation at 94 ° c . for 30 seconds and annealing and extension at 72 ° c . for 3 minutes ; 5 cycles of denaturation at 94 ° c . for 30 seconds and annealing and extension at 70 ° c . for 3 minutes ; and 25 cycles of denaturation at 94 ° c . for 30 seconds and annealing and extension at 68 ° c . for 3 minutes . amplified products were used as templates in a nested pcr reaction using the previously described cycling parameters . the reamplified products were then analyzed by southern blotting using oligonucleotide probes specific for p110δ . probes ( 100 ng each ) were end - labelled with 32 p - γatp , and hybridized and washed under standard conditions ( frisch and sambrook ). the sequences of the two probes were gatgcggaacggctgctccaggg ( seq id no : 11 ) and ccagggaccacagggacacagag ( seq id no : 12 ). the specific 5 &# 39 ; race pcr products identified in this manner were gel purified and subcloned into the ta vector pcrii ( invitrogen , san diego , calif .) according to the manufacturer &# 39 ; s instructions . three independent clones were sequenced to ensure the veracity of the 5 &# 39 ; sequence . a full length cdna for p110δ was assembled from clones # 249 , m # 928 and the 5 &# 39 ; race pcr products . the 5 &# 39 ; race product was used as a template in pcr using the 5 &# 39 ; primer agttacggatccggcaccatg ( gactacaaggacgacgatgacaag ) ccccctggggtggactgccc ( seq id no : 13 ) and the 3 &# 39 ; primer ccacatgtagaggcagcgttcc ( seq id no : 14 ). the 5 &# 39 ; primer includes a bam hi site ( underlined ), and sequences that encode the flag peptide sequence dykddddk ( seq id no : 15 ) ( shown in parenthesis ) which is recognized by the m2 anti - flag monoclonal antibody ( kodak scientific imaging systems , new haven , conn .). the resulting pcr product was digested with bam hi and afl ii , and was ligated along with an afl ii / pvu i fragment derived from the clone m # 928 and a pvu ii / xba i fragment derived from pbmc clone # 249 into the bam hi / xba i sites of the mammalian expression vector pcdna3 ( invitrogen , san diego , calif .). the vector containing the flag - tagged composite p110δ cdna is designated pcdna3 : p110δflag . in the flag - tagged p110δ , the flag - tag is located immediately after the initiating methionine . a full - length composite cdna encoding p110δ is shown in seq id no : 1 . the sequence of p110δ includes an open reading frame of 3135 nucleotides which is predicted to encode a protein of approximately 114 kd . in addition , there are 197 bp of 5 &# 39 ; and 1894 bp of 3 &# 39 ; untranslated sequence . the sequence around the predicted initiating methionine is in good agreement with that required for optimal translational initiation kozak , m ., j . cell biol ., 115 : 887 - 992 ( 1991 )! and the presence of stop codons in the 5 &# 39 ; untranslated sequence is consistent with the isolation of the complete coding region of p110δ . comparison of the deduced amino acid sequence of p110δ ( seq id no : 2 ) with other pi 3 - kinases reveals that it is most closely related to p110β . similar to p110β , the catalytic domain of p110δ b is found in the c - terminus of the protein and is believed to be reside within amino acid residues 723 - 1044 of seq id no : 2 . an alignment of the predicted carboxyl terminal catalytic domains of the pi 3 - kinase family ( including p110δ residues 723 through 1044 of seq id no : 2 ) is shown in fig1 . table 1 shows the identity of p110δ to other members of the pi 3 - kinase family . p110δ is 72 % identical to p110β in this region but is less closely related to p110α ( 49 %) and p110γ ( 45 %). table 1 also shows that p110δ shows low identity to cpk / p170 and the yeast vps 34 protein , 31 and 32 % respectively . table 1______________________________________p110δ p110β p110α p110γ cpk / p170 vps34______________________________________p110δ -- 72 49 45 31 32p110β -- 49 48 37 31p110α -- 45 39 29p110γ -- 39 31cpk / p170 -- 28vps34 -- ______________________________________ dendrogram analysis revealed that p110β and p110δ form a distinct sub - branch of the pi 3 - kinase family . the distantly related atm gene and the catalytic subunit of dna dependent protein kinase have been included for comparison . it has been demonstrated that pi 3 - kinase is an important intermediate in the ras pathway hu et al . 1993 ; rodriguez - viciana et al ., embo journal , 15 : 2442 - 2451 ( 1996 )!. a constitutively active form of pi 3 - kinase has been shown to increase transcription of the c - fos gene , activate the protein kinase raf , and stimulate oocyte maturation hu et al ., 1995 !. the effects of pi 3 - kinase in these systems can be blocked by co - expression of a dominant negative form of ras indicating that pi 3 - kinase acts upstream of ras . additional studies have shown that ras can physically interact with pi 3 - kinase in vitro and stimulate its kinase activity rodriguez - viciana et al ., 1996 !. thus pi 3 - kinase can either act as an effector of ras - dependent signalling or be directly activated by interaction with ras . a specific region at the amino terminus of the p110 subunits termed the ras regulatory domain is responsible for this interaction rodriguez - viciana et al . 1996 !. comparison of the sequence of p110δ with other p110 subunits indicates that this region is also conserved in p110δ including a lysine residue which has been shown to be essential for physical association with ras ( rodriguez - viciana et al ., 1996 ). thus p110δ is also likely to interact with the ras pathway . fig2 presents an alignment of the proposed ras binding sites of four p110 subunits including p110δ residues 141 through 310 of seq id no : 2 . the flag - tagged p110δ was expressed by transfecting pcdna3 : p110δflag into cos cells using deae dextran . three days after transfection , expression of p110δ was determined by immunoprecipitations and western blotting using the m2 monoclonal antibody ( kodak scientific imaging systems ) according to the manufacturer &# 39 ; s instructions . pi 3 - kinase activity was determined as described hu et al ., mol . cell . biol ., 13 : 7677 - 7688 ( 1993 )!. to determine the pi 3 - kinase activity of p110δ b , 5μl of immunoprecipitated p110δ was mixed with 1 μl of pi / egta and incubated at room temperature for 10 minutes pi / egta is 10 mg / ml pi ( sigma ) in chcl 3 , which has been dried under a vacuum , resuspended in 20 mg / ml dmso in the presence or absence of various concentrations of the pi3 kinase inhibitor wortmannin and diluted 1 : 10 in 5 mm egta ! and added to 1 μl 10 × hm buffer ( 200 mm hepes ph7 . 2 , 50 mm mncl 2 ), 0 . 5 μl γ 32 patp ( 10 mci / ml - 300 ci / mmol ), 1 μl 100 μm atp , and 1 . 5 μl h 2 o and incubated at 30 ° c . for 15 minutes . the reactions were terminated by addition of 100 μl 1m hcl . lipids were extracted with 200 μl chcl 3 / meoh ( 1 : 1 ) by vortexing for 1 minute followed by centrifugation at 16 , 000 × g for 2 minutes at room temperature . the lipids were further extracted with 80 μl 1m hcl / meoh ( 1 : 1 ) by vortexing for 1 minute , followed by centrifugation at 16 , 000 × g for 2 minutes at room temperature . the lipids were dried under vacuum , resuspended in 10 μl chcl 3 / meoh ( 1 : 1 ) and spotted 2 cm from the bottom of a dry silica gel 60 chromatography plate ( vwr ) that had been pre - impregnated with 1 % k 2 c 2 o 4 in h 2 o . 250 μg of crude phosphoinositides ( sigma ) were spotted as markers . the products were resolved by chromatography for 2 hours in chcl 3 / meoh / 4n nh 4 oh ( 9 : 7 : 2 ), allowed to dry and placed in an iodine vapor tank for 5 minutes in order to visualize the crude standards . the position of the standards was marked with a pencil and the plate was autoradiographed . phosphorylated lipids were generated in the kinase assays . the major product was phosphatidyl inositol phosphate ( pip ). furthermore , the generation of these phosphorylated lipids was inhibited in a dose dependent manner by wortmannin ( approximately 50 % of the activity was inhibited at 100 nm wortmannin ) demonstrating that p110δ is a functional pi3 kinase . a mouse genomic clone encoding p110δ was isolated as described below . a mouse 129 svev lambda genomic library ( stratagene , la jolla , calif .) was screened using a fragment of the human cdna clone for p110δ ( corresponding to amino acids 739 to 1044 of seq id no . : 2 ) labelled to high specific activity (. sup .˜ 1 × 10 9 dpm / ug dna ) by random priming using the random primed dna labelling kit ( boehringer mannheim ). hybridization was performed for sixteen hours at 42 ° c . in buffer containing 50 % formamide , 5 × ssc , 5 × denhardts , 0 . 05m na phosphate , and 100 ug / ml salmon sperm dna . filters were washed in 0 . 2 × ssc / 0 . 1 % sds at 50 ° c . a single clone was isolated . purified phage dna was digested with not i and inserts were subcloned into the vector pbsskii + ( stratagene , la jolla , calif .) for sequencing . this clone was approximately 16 kb and included the entire catalytic region of p110δ . recombinant p110δ may be expressed in sf9 insect cells using a baculovirus expression system . as discussed in example 1 , flag - tagged p110δ encoding sequences are useful in expressing the kinases of this invention . upon expression in insect cells , a monoclonal antibody that recognizes the flag tag ( eastman kodak , rochester , n . y .) is used to purify large quantities of the flag - pik - related kinase fusion protein . infected insect cells are incubated for 48 hours and lysed in lysis buffer ( 25 mm 2 - glycerolphosphate , 50 mm sodium phosphate ph 7 . 2 , 0 . 5 % triton - x 100 , 2 mm edta , 2 mm egta , 25 mm sodium fluoride , 100 μm sodium vanadate , 1 mm pmsf , 1 μg / ml leupeptin , 1 μg / ml pepstatin , 1 mm benzamidine , and 2 mm dtd ). expressed flag fusion proteins are purified over a column containing anti - flag antibody m2 affinity resin ( eastman kodak ). the column is washed with 20 column volumes of lysis buffer , then 5 column volumes of 0 . 5m lithium chloride , 50 mm tris ph 7 . 6 , 1 mm dtt , and then eluted either with 0 . 1m glycine ph 3 . 0 followed by immediate neutralization or by competitive elution with the flag peptide . for histidine tagged proteins , ni - nta agarose ( qiagen ) is used for protein purification . plasmids for expression of p85 and p110δ in the baculovirus expression sytstem were prepared as follows . the plasmid pcdna3 : p85 dna as described in example 5 was digested with bamhi and ecori and the 2 . 5 kb flag - p85 band containing the entire p85 coding region with the flag tag was gel purified and inserted in bamhi - ecori site of pfastbac dual ( gibco brl ). the ligation mixture was transformed into e . coli xl - 1 blue ( stratagene ) and plated on ampicillin containing plate . a clone was purified that carries the pfastbac - dual - p85 plasmid . the pfastbac - dual - p85 plasmid was transformed into e . coli dh10 bac cells and white colonies were selected on plates containing kanomycin , gentamycin , tetracyoline , x - gel and iptg . one white colony was restreaked on a similar plate for repurification . recombinant p85 - bacmid dna was purified from this clone . the plasmid pcdna3 : p110δ containing the entire p110δ coding region with the flag tag was digested with bamhi and xbai , gel purified and inserted into the bamhi - xbai site of pfastbac htb ( gibco brl ) such that the coding region of flag - tagged p110δ was in frame with the coding sequences of the histidine - tag present in the vector . the ligation mixture was then transformed into e . coli xl - 1 blue ( stratagene ). a clone carrying pfast - bac htb p110δ was isolated and the plasmid dna was isolated and the plasmid dna was purified . p110δ - bacmid dna was prepared by transforming e . coli dh10 bac cells as described for p85 - bacmid . to prepare virus stocks , the p85 - bacmid and the p110δ - bacmid dnas were separately transfected into sf - 9 cells according to the gibco brl suggested protocol . forty - eight hours after transfection , the sf9 cell pellet and baculovirus produced by the transfected cells were harvested . the virus was stored at 4 ° c . in grace &# 39 ; s complete media containing 10 % fbs , pennicillin - streptomycin , and gentamicin . this viral prep was used to make a high titer ( p2 ) virus stock . the p2 virus stock was used to infect a 50 ml culture of sf9 cells . the cells were collected 48 hours after infection and centrifuged at low speed to pellet the cells without lysis . the cell pellet was stored at - 20 ° c . for 24 hours before lysis . the cells were lysed in 5 ml of lysis buffer ( 50 mm tris , ph 8 . 0 ; 500 mm nacl ; 1 % np40 ; 100 μm pmsf ). expression of p85 and p110δ was confirmed by immunoblot using the m2 antibody anti - flag as a probe . the sf - 9 transfected cells produced an approximately 85 kda protein and a 110 kda protein which were immunoreactive with anti - flag antibodies . the p2 virus stock were also used to co - infect a 2 liter culture of sf9 cells . the cells were collected 48 hours after infection , centrifuged at low speed to pellet the cells without lysis and stored at - 20 ° c . a cell pellet from 150 mls of this culture was lysed in 7 . 5 ml of lysis buffer ( 50 mm napo 4 ph7 . 2 ; 0 . 5 % np - 40 ; 10 mm imidazole , 25 mm naf , 100 μm na 3 vo 4 ; 0 . 5 mm aebsf ; 1 μg / ml leupeptin ; 1 μg / ml pepstatin a ) and incubated on ice for 15 minutes . the lysate was then centrifuged for 30 minutes at 10 , 000 × g . the supernatant was removed and any dna in the lysate resulting from broken nuclei was sheared by aspirating through an 20 gauge needle . particulate matter was then removed by filtering through a 0 . 8 micron filter followed by a 0 . 2 micron filter . this cleared lysate was adjusted to contain 5 mm β - mercaptoethanol and 0 . 4m nacl . a 1 ml ni - nta - agarose column ( qiagen ) was equilibrated in buffer a ( 0 . 4m nacl ; 5 mm β - mercaptoethanol ; 0 . 1 % triton x - 100 ; 50 mm napo 4 10 mm imidazole ; 25 mm naf , 100 μm na 3 vo 4 ; 0 . 5 mm aebsf ; 1 μg / ml leupeptin ; 1 μg / ml pepstatin a ) prior to loading the cleared lysate . the sample was loaded at a flow rate of 0 . 25 ml / minute , washed 5 ml of buffer a and then eluted in 10 ml of a gradient of 50 to 500 mm imidazole in buffer a . the ability of p110δ to associate with p85 was assessed by western blot analysis . cos cells were transiently transfected with p110δ ( see example 2 ) and association with endogenous p85 was determined by coimmunoprecipitation . as controls , cells were also transfected with flag - tagged p85 dna or empty vector . the cdna encoding the p85 subunit was isolated from human leukocyte cdna by marathon - race pcr . the cdna sequence of p85 was described in otsu , cell , 65 : 91 - 104 ( 1992 ). the p85 cdna was modified for expression as a flag - tagged protein ( pcdna3 : p85 ) in a manner similar to the protocols described herein for p110δ . cos cells were lysed in 3 ml buffer r ( 1 % triton x - 100 , 150 mm nacl , 10 mm tris ph7 . 5 , 1 mm egta , 0 . 5 % np - 40 , 0 . 2 mm na 3 vo 4 , 0 . 2 mm pmsf , 1 × aprotinin , 1 × leupeptin , 1 × pepstatin a ). after 10 minutes at 4 ° c ., the lysates were sheared by passing through a 27 g needle several times . the lysates were clarified by centrifugation at 16 , 000 × g for 10 minutes at 4 ° c ., and immunoprecipitated for 2 hours at 4 ° c . with either 1 μg anti - p110β ( santa cruz laboratories , santa cruz , calif . ), 10 μg anti - flag - m2 ( eastman kodak ), or 1 μg anti - p85 ( santa cruz laboratories ). immune complexes were bound to 60 μl of protein g - sepharose ( pharmacia ) for 30 minutes at 4 ° c . then washed 3 times in 300 μl of buffer r and resuspended in 25 μl pan ( 100 mm nacl , 10 mm pipes ph7 . 0 , 20 μg / ml aprotinin ). 5 μl of each immunoprecipitate was resolved by 8 % sds - page ( novex ), transferred to immobilon - p ( millipore ), blocked one hour at room temperature in 5 % non - fat dried milk in tbs , and detected by western blotting using either anti - p85 rabbit polyclonal antibodies ( santa cruz laboratories ) at 1 μg / ml followed by goat anti - rabbit igg hrp conjugated secondary antibody ( boehringer ) or anti - flag - m2 monoclonal antibody at 10 μg / ml followed by goat anti - mouse igg hrp conjugated secondary antibody ( boehringer ). the westerns showed that anti - flag - m2 antibody recognized immune complexes including flag - tagged p85 and flag - tagged p110δ . while the activation of pi 3 - kinase in a wide range of biological systems has been extensively studied , less is known concerning the cell type specific expression of particular p110 isoforms . the expression of p110δ in human heart , brain , placenta , lung , liver , skeletal muscle , kidney , pancreas , spleen , thymus , prostate , testis , uterus , small intestine , colon , and pbmc was determined by northern blot analysis . 32 p - labelled cdna probes were prepared by pcr using 10 ng of plasmid dna template encoding p110δ , as described previously godiska et al , j . neuroimmun ., 58 : 167 - 176 ( 1995 )!. the forward primer was ctgccatgttgctcttgttga ( seq id no : 16 ) and the reverse primer was gagttcgacatcaacatc ( seq id no : 17 ). reactions were heated for 4 minutes at 94 ° c ., followed by 15 cycles of denaturation for 1 minute at 94 ° c ., annealing for 1 minute at 55 ° c . and extension for 2 minutes at 72 ° c . unincorporated nucleotides were removed by passing the reaction over a sephadex g50 column ( boehringer mannheim biochemicals ). a multiple tissue northern blot ( clontech , palo alto , calif .) was probed and washed under stringent conditions according to the manufacturer &# 39 ; s recommendations . the autoradiograph was exposed for 1 - 4 days at - 80 ° c . with intensifying screens . northern blot analysis revealed a single transcript of approximately 5 . 4 kb ( consistent with the size of the composite cdna ). the highest levels of expression were seen in peripheral blood mononuclear cells ( pbmc ) and in spleen and thymus . on prolonged exposure of the autoradiograph , expression of p110δ could also be detected in testes , uterus , colon , and small intestine , but not in other tissues examined including prostate , heart , brain , and liver . in contrast , p110β is expressed at high levels in brain , heart , kidney and liver , but cannot be readily detected in lymphoid tissues such as spleen . p110β is expressed at high levels in the transformed jurkat t cell line ( hu et al . 1993 ). the expression of the p110α isoform has not been well documented . p110 isoforms have been shown to differ with respect to their preferred substrate specificities stephens et al ., current biology , 4 : 203 - 214 ( 1994 )!. in view of their potential for interaction with a common p85 adaptor protein , it is likely that the nature of the phosphorylated lipids generated in response to a particular agonist may be regulated at least in part by the cell / tissue specific expression of the different isoforms of the kinase enzymatic activity . the abundant expression of p110δ in pbl and lymphoid tissues such as spleen and thymus suggests that this isoform may be involved in aspects of leukocyte activation . monoclonal antibodies were generated against the carboxy terminal portion of p110δ ( amino acids 740 - 1044 of seq id no : 2 ) expressed as a fusion protein with glutathione s transferase ( gsi ) pharmacia , alameda , calif .!. five balb / c mice ( charles river biotechnical services , inc ., wilmington , mass ., iacuc # 901103 ) were immunized subcutaneously with 30 ug of antigen in complete freund &# 39 ; s adjuvant cfa ! ( sigma ), a second immunization of 30 ug of antigen in incomplete freunds adjuvant ( ifa ) ( sigma ) was administered on day 22 . a third immunization with 30 ug of antigen in ifa was administered on day 44 . immune serum was collected via retro - orbital bleeding on day 55 and tested by western blotting to determine reactivity to p110δ . all animals showed reactivity towards the immunogen and were immunized a fourth time on day 66 with 30 ug of antigen in ifa . immune serum was collected via retro - orbital bleeding on day 76 and tested by western blotting to determine its reactivity , animal # 2321 showed the highest level of immunoreactivity and was chosen for fusion . on day 367 and 368 mouse # 2321 was injected intraperitoneally with 50 ug of antigen in pbs and a fusion was performed on day 371 . the spleen was removed sterilely and a single - cell suspension was formed by grinding the spleen between the frosted ends of two glass microscope slides submerged in serum free rpmi 1640 , supplemented with 2 mm l - glutamine , 1 mm sodium pyruvate , 100 units / ml penicillin , and 100 μg / ml streptomycin ( rpmi ) ( gibco , canada ). the cell suspension was filtered through sterile 70 - mesh nitex cell strainer ( becton dickinson , parsippany , n . j . ), and washed twice by centrifuging at 200 g for 5 minutes and resuspending the pellet in 20 ml serum free rpmi . thymocytes taken from 3 naive balb / c mice were prepared in the same manner . two × 10 8 spleen cells were combined with 4 × 10 7 ns - 1 cells ( kept in log phase in rpmi with 11 % fetal bovine serum ( fbs ) for three days prior to fusion ), centrifuged and the supernatant was aspirated . the cell pellet was dislodged by tapping the tube and 2 ml of 37 ° c . peg 1500 ( 50 % in 75 mm hepes , ph 8 . 0 ) ( boehringer mannheim ) was added with stirring over the course of 1 minute , followed by adding 14 ml of serum free rpmi over 7 minutes . an additional 16 ml rpmi was added and the cells were centrifuged at 200 g for 10 minutes . after discarding the supernatant , the pellet was resuspended in 200 ml rpmi containing 15 % fbs , 100 mm sodium hypoxanthine , 0 . 4 mm aminopterin , 16 mm thymidine ( hat ) ( gibco ), 25 units / ml il - 6 ( boehringer mannheim ) and 1 . 5 × 10 6 thymocytes / ml . the suspension was dispensed into ten 96 - well flat bottom tissue culture plates ( corning , united kingdom ) at 200 μl / well . cells were fed on days 2 , 4 , and 6 days post - fusion by aspirating 100 μl from each well with an 18 g needle ( becton dickinson ), and adding 100 μl / well plating medium containing 10 u / ml il - 6 and lacking thymocytes . when cell growth reached 60 - 80 % confluence ( day 8 - 10 ), culture supernatants were taken from each well and screened for reactivity to p110δ by elisa . immulon 4 plates ( dynatech , cambridge , mass .) were coated at 4 ° c . with 50 μl / well with 100 ng / well of p110δ : gst or gst in 50 mm carbonate buffer , ph 9 . 6 . plates were washed 3 × with pbs with 0 . 05 %, tween 20 ( pbst ), blocked 30 minutes at 37 ° c . with 0 . 5 % fish skin gelatin . plates were washed as described above and 50 μl culture supernatant was added . after incubation at 37 ° c . for 30 minutes , 50 μl of horseradish peroxidase conjugated goat anti - mouse igg ( fc ) ( jackson immunoresearch , west grove , pa .) diluted 1 : 10 , 000 in pbst ! was added . plates were incubated at 37 ° c . for 30 minutes , washed 4 × with pbst and 100 μl of substrate , consisting of 1 mg / ml tnb ( sigma ) and 0 . 15 ml / ml 30 % h 2 o 2 in 100 mm citrate , ph 4 . 5 , was added . the color reaction was stopped in 3 minutes with the addition of 50 ml of 15 % h 2 so 4 . a 450 was read on a plate reader ( dynatech ). thirty - six wells showed preferential reactivity to p110δ versus gst . supernatants from these wells were then screened for reactivity to recombinant p110δ by western blotting . ten wells ( 208a , 208b , 208c , 208d , 208e , 208f , 208 g , 208h , 208i , and 208j ) showed reactivity by western blotting and were cloned twice by limiting dilution . selected wells were tested by elisa 7 - 10 days later . activity was retained in all ten lines . monoclonal antibodies produced by the cell lines were isotyped by elisa assay . 208a , 208c , 208d , 208e , 208 g , 208h , 208i were igg 2a , while 208j was igg 1 and 208b was igg2b . an exemplary monoclonal antibody , produced by hybridoma cell line 208f ( atcc hb 12200 ), showed high reactivity with p110δ and recognized a 110 kd protein in pbmc by western analysis . the molecular weight of the 110 kd protein is consistent with the molecular weight of p110δ . elevated levels of 3 &# 39 ; phosphorylated phosphoinositides have been detected in cells transformed with viral oncoproteins . this observation suggests that pi 3 - kinases may play a role in carcinogenesis . chromosomal localization of p110δ provides insights into the role of pi 3 - kinase in carcinogenesis . chromosomal localization studies of p110δ of cancerous cells may identify inappropriate and / or over expression of p110δ . for example , in 90 - 95 % of chronic myelogenous leukaemia there is a reciprocal chromosomal translocation which leads to the transfer of the tyrosine kinase c - abl from chromosome 9 into the ber gene on chromosome 22 . the resultant inappropriate expression of c - abl tyrosine kinase activity is critical for cell transformation and tumorigenesis . chromosomal localization of p110δ is determined by fluorescence in situ hybridization ( fish ) using the complete cdna for p110δ as a probe . in this manner , the role of p110δ in chromosomal translocations observed during tumorigenesis ( e . g . leukemogenesis ) is identified . pi 3 - kinase activity has been reported to be associated with a number of growth factor receptors . in addition , it has been observed that pi 3 - kinase activity increases following cell activation . the antibodies to p110δ disclosed in example 5 are utilized to determine by western blotting and immunoprecipitation the nature of the receptors with which p110δ associates . these antibodies are also useful in elucidating the regulation of pi 3 - kinase enzymatic activity and cellular localization during cell activation . in view of the high levels of expression of p110δ in the immune system , it is likely that growth factor receptors involved in immune activation may associate with or be regulated by p110δ . these receptors include t - cell receptors cd28 and cd2 and cytokine receptors such as il - 1 and il - 4 , and tyrosine kinase coupled receptors such as csf - 1 r . to determine the functional role of p110δ in vivo , the p110δ gene is inactivated in the germline of mammals by homologous recombination . animals in which an endogenous gene has been inactivated by homologous recombination are also known as &# 34 ; knockout &# 34 ; animals . exemplary mammals include rabbits and rodent species such as mice . &# 34 ; knockout &# 34 ; animals can be prepared by homologous recombination methods using the p110δ genomic clone of example 3 . these &# 34 ; knockout &# 34 ; animals allow for the determination of the role of p110δ in immune and proliferative responses . the role of p110δ in immune and proliferative response is determined by analysis of the development of the immune system in these animals ( as determined by pacs analysis of cell populations at different stages of development ), characterization of the effector function of the mature lymphoid populations of these animals both in vivo ( as determined by antibody responses to injected antigens , cytotoxic t cell responses to viruses and or injected tumor cell lines , and the ability to reject allografts ) and in vitro ( as determined by proliferation of lymphocytes in response to allo - antigen , polyclonal activation by mitogens / superantigens , and the ability to elaborate cytokines ). while the present invention has been described in terms of specific embodiments , it is understood that variations and modifications will occur to those skilled in the art . accordingly , only such limitations as appear in the appended claims should be placed on the invention . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 17 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 5220 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( ix ) feature :( a ) name / key : cds ( b ) location : 196 .. 3327 ( xi ) sequence description : seq id no : 1 : 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( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 1044 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 2 : metproproglyvalaspcysprometgluphetrpthrlysgluglu151015asnglnservalvalvalasppheleuleuprothrglyvaltyrleu202530asnpheprovalserargasnalaasnleuserthrilelysglnleu354045leutrphisargalaglntyrgluproleuphehismetleusergly505560proglualatyrvalphethrcysileasnglnthralagluglngln65707580gluleugluaspgluglnargargleucysaspvalglnpropheleu859095provalleuargleuvalalaarggluglyaspargvallyslysleu100105110ileasnserglnileserleuleuileglylysglyleuhisgluphe115120125aspserleucysaspprogluvalasnasppheargalalysmetcys130135140glnphecysgluglualaalaalaargargglnglnleuglytrpglu145150155160alatrpleuglntyrserpheproleuglnleugluproseralagln165170175thrtrpglyproglythrleuargleuproasnargalaleuleuval180185190asnvallysphegluglyserglugluserphethrpheglnvalser195200205thrlysaspvalproleualaleumetalacysalaleuarglyslys210215220alathrvalpheargglnproleuvalgluglnprogluasptyrthr225230235240leuglnvalasnglyarghisglutyrleutyrglyasntyrproleu245250255cysglnpheglntyrilecyssercysleuhisserglyleuthrpro260265270hisleuthrmetvalhisserserserileleualametargaspglu275280285glnserasnproalaproglnvalglnlysproargalalyspropro290295300proileproalalyslysproserservalserleutrpserleuglu305310315320glnpropheargilegluleuileglnglyserlysvalasnalaasp325330335gluargmetlysleuvalvalglnalaglyleuphehisglyasnglu340345350metleucyslysthrvalsersersergluvalservalcysserglu355360365provaltrplysglnargleuglupheaspileasnilecysaspleu370375380proargmetalaargleucysphealaleutyralavalileglulys385390395400alalyslysalaargserthrlyslyslysserlyslysalaaspcys405410415proilealatrpalaasnleumetleupheasptyrlysaspglnleu420425430lysthrglygluargcysleutyrmettrpproservalproaspglu435440445lysglygluleuleuasnprothrglythrvalargserasnproasn450455460thraspseralaalaalaleuleuilecysleuprogluvalalapro465470475480hisprovaltyrtyrproalaleuglulysileleugluleuglyarg485490495hisserglucysvalhisvalthrgluglugluglnleuglnleuarg500505510gluileleugluargargglyserglygluleutyrgluhisglulys515520525aspleuvaltrplysleuarghisgluvalglngluhispheproglu530535540alaleualaargleuleuleuvalthrlystrpasnlyshisgluasp545550555560valalaglnmetleutyrleuleucyssertrpprogluleuproval565570575leuseralaleugluleuleuasppheserpheproaspcyshisval580585590glyserphealailelysserleuarglysleuthraspaspgluleu595600605pheglntyrleuleuglnleuvalglnvalleulystyrglusertyr610615620leuaspcysgluleuthrlyspheleuleuaspargalaleualaasn625630635640arglysileglyhispheleuphetrphisleuargserglumethis645650655valproservalalaleuargpheglyleuileleuglualatyrcys660665670argglyserthrhishismetlysvalleumetlysglnglygluala675680685leuserlysleulysalaleuasnaspphevallysleusersergln690695700lysthrprolysproglnthrlysgluleumethisleucysmetarg705710715720glnglualatyrleuglualaleuserhisleuglnserproleuasp725730735proserthrleuleualagluvalcysvalgluglncysthrphemet740745750aspserlysmetlysproleutrpilemettyrserasnglugluala755760765glyserglyglyservalglyileilephelysasnglyaspaspleu770775780argglnaspmetleuthrleuglnmetileglnleumetaspvalleu785790795800trplysglngluglyleuaspleuargmetthrprotyrglycysleu805810815prothrglyaspargthrglyleuilegluvalvalleuargserasp820825830thrilealaasnileglnleuasnlysserasnmetalaalathrala835840845alapheasnlysaspalaleuleuasntrpleulysserlysasnpro850855860glyglualaleuaspargalaileglugluphethrleusercysala865870875880glytyrcysvalalathrtyrvalleuglyileglyasparghisser885890895aspasnilemetilearggluserglyglnleuphehisileaspphe900905910glyhispheleuglyasnphelysthrlyspheglyileasnargglu915920925argvalpropheileleuthrtyraspphevalhisvalileglngln930935940glylysthrasnasnserglulysphegluargpheargglytyrcys945950955960gluargalatyrthrileleuargarghisglyleuleupheleuhis965970975leuphealaleumetargalaalaglyleuprogluleusercysser980985990lysaspileglntyrleulysaspserleualaleuglylysthrglu99510001005gluglualaleulyshispheargvallyspheasnglualaleuarg101010151020glusertrplysthrlysvalasntrpleualahisasnvalserlys1025103010351040aspasnarggln ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 32 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 15 ( d ) other information : / note = &# 34 ; n = inosine &# 34 ;( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 24 ( d ) other information : / note = &# 34 ; n = inosine &# 34 ;( xi ) sequence description : seq id no : 3 : gcagacggatccggngaygayhknagrcarga32 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 7 amino acids ( b ) type : amino acid ( c ) strandedness : not relevant ( d ) topology : not relevant ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 4 : glyaspaspleuargglnasp15 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 30 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 16 ( d ) other information : / note = &# 34 ; n = inosine &# 34 ;( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 25 ( d ) other information : / note = &# 34 ; n = inosine &# 34 ;( xi ) sequence description : seq id no : 5 : gcagacgaattcrwrnccraartcnryrtg30 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( c ) strandedness : not relevant ( d ) topology : not relevant ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 6 : hisileasppheglyhis15 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 21 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( xi ) sequence description : seq id no : 7 : catgctgaccctgcagatgat21 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 21 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 8 : aacagctgcccactctctcgg21 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 26 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 9 : gggccacatgtagaggcagcgttccc26 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 25 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 10 : ggcccaggcaatggggcagtccgcc25 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 23 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( xi ) sequence description : seq id no : 11 : gatgcggaacggctgctccaggg23 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 23 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( xi ) sequence description : seq id no : 12 : ccagggaccacagggacacagag23 ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 65 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( xi ) sequence description : seq id no : 13 : agttacggatccggcaccatggactacaaggacgacgatgacaagccccctggggtggac60tgccc65 ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 22 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( xi ) sequence description : seq id no : 14 : ccacatgtagaggcagcgttcc22 ( 2 ) information for seq id no : 15 :( i ) sequence characteristics :( a ) length : 8 amino acids ( b ) type : amino acid ( c ) strandedness : not relevant ( d ) topology : not relevant ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 15 : asptyrlysaspaspaspasplys15 ( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 21 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( xi ) sequence description : seq id no : 16 : ctgccatgttgctcttgttga21 ( 2 ) information for seq id no : 17 :( i ) sequence characteristics :( a ) length : 18 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( xi ) sequence description : seq id no : 17 : gagttcgacatcaacatc18__________________________________________________________________________