Patent Application: US-48314095-A

Abstract:
the present invention provides purified and isolated polynucleotide sequences encoding human plasma platelet - activating factor acetylhydrolase . also provided are materials and methods for the recombinant production of platelet - activating factor acetylhydrolase products which are expected to be useful in regulating pathological inflammatory events .

Description:
the following examples illustrate the invention . example 1 presents a novel method for the purification of paf - ah from human plasma . example 2 describes amino acid microsequencing of the purified human plasma paf - ah . the cloning of a full length cdna encoding human plasma paf - ah is described in example 3 . identification of a putative splice variant of the human plasma paf - ah gene is described in example 4 . the cloning of genomic sequences encoding human plasma paf - ah is described in example 5 . example 6 desribes the cloning of canine , murine , rodent and macaque cdnas homologous to the human plasma paf - ah cdna . example 7 presents the results of an assay evidencing the enzymatic activity of recombinant paf - ah transiently expressed in cos 7 cells . example 8 describes the expression of human paf - ah in e . coli and s . cerevisiae . example 9 presents a protocol for purification of recombinant paf - ah from e . coli and assays confirming its enzymatic activity . example 10 describes various recombinant paf - ah products including amino acid substitution analogs and amino and carboxy - truncated products . results of a northern blot assay for expression of human plasma paf - ah rna in various tissues and cell lines are presented in example 11 while results of in situ hybridzation are presented in example 12 . example 13 describes the development of monoclonal antibodies specific for human plasma paf - ah . examples 14 , 15 , and 16 respectively describe the in vivo therapeutic effect of administration of recombinant paf - ah products of the invention on acute inflammation , pleurisy and asthma in rats . example 17 presents the results of immunoassays of serum of human patients exhibiting a deficiency in paf - ah activity and describes the identification of a genetic lesion in the patients which is apparently responsible for the deficiency . paf - ah was purified from human plasma in order to provide material for amino acid sequencing . initially , low density lipoprotein ( ldl ) particles were precipitated from plasma with phosphotungstate and solubilized in 0 . 1 % tween 20 and subjected to chromatography on a deae column ( pharmacia , uppsala , sweden ) according to the method of stafforini et al . ( 1987 ), supra , but inconsistent elution of paf - ah activity from the deae column required reevaluation of the solubilization and subsequent purification conditions . tween 20 , chaps ( pierce chemical co ., rockford , ill .) and octyl glucoside were evaluated by centrifugation and gel filtration chromatography for their ability to solubilize ldl particles . chaps provided 25 % greater recovery of solubilized activity than tween 20 and 300 % greater recovery than octyl glucoside . ldl precipitate solubilized with 10 mm chaps was then fractionated on a deae sepharose fast flow column ( an anion exchange column ; pharmacia ) with buffer containing 1 mm chaps to provide a large pool of partially purified paf - ah (&# 34 ; the deae pool &# 34 ;) for evaluation of additional columns . the deae pool was used as starting material to test a variety of chromatography columns for utility in further purifying the paf - ah activity . the columns tested included : blue sepharose fast flow ( pharmacia ), a dye ligand affinity column ; s - sepharose fast flow ( pharmacia ), a cation exchange column ; cu chelating sepharose ( pharmacia ), a metal ligand affinity column ; fractogel s ( em separations , gibbstown , n . j . ), a cation exchange column ; and sephacryl - 200 ( pharmacia ), a gel filtration column . these chromatographic procedures all yielded low , unsatisfactory levels of purification when operated in 1 mm chaps . subsequent gel filtration chromatography on sephacryl s - 200 in 1 mm chaps generated an enzymatically active fraction which eluted over a broad size range rather than the expected 44 kda approximate size . taken together , these results indicated that the ldl proteins were aggregating in solution . different ldl samples were therefore evaluated by analytical gel filtration chromatography for aggregation of the paf - ah activity . samples from the deae pool and of freshly solubilized ldl precipitate were analyzed on superose 12 ( pharmacia ) equilibrated in buffer with 1 mm chaps . both samples eluted over a very broad range of molecular weights with most of the activity eluting above 150 kda . when the samples were then analyzed on superose 12 equilibrated with 10 mm chaps , the bulk of the activity eluted near 44 kda as expected for paf - ah activity . however , the samples contained some paf - ah activity in the high molecular weight region corresponding to aggregates . other samples eluted paf - ah activity exclusively in the approximately 44 kda range when they were subsequently tested by gel filtration . these samples were an ldl precipitate solubilized in 10 mm chaps in the presence of 0 . 5m nacl and a fresh deae pool that was adjusted to 10 mm chaps after elution from the deae column . these data indicate that at least 10 mm chaps is required to maintain non - aggregated paf - ah . increase of the chaps concentration from 1 mm to 10 mm after chromatography on deae but prior to subsequent chromatographic steps resulted in dramatic differences in purification . for example , the degree of paf - ah purification on s - sepharose fast flow was increased from 2 - fold to 10 - fold . paf - ah activity bound the blue sepharose fast flow column irreversibly in 1 mm chaps , but the column provided the highest level of purification in 10 mm chaps . the deae chromatography was not improved with prior addition of 10 mm chaps . chromatography on cu chelating sepharose after the blue sepharose fast flow column concentrated paf - ah activity 15 - fold . it was also determined that paf - ah activity could be recovered from a reduced sds - polyacrylamide gel , as long as samples were not boiled . the activity of material eluted from the cu chelating sepharose column when subjected to sds - polyacrylamide gel electrophoresis coincided with a major protein band when the gel was silver stained . the novel protocol utilized to purify paf - ah for amino acid sequencing therefore comprised the following steps which were performed at 4 ° c . human plasma was divided into 900 ml aliquots in 1 liter nalgene bottles and adjusted to ph 8 . 6 . ldl particles were then precepitated by adding 90 ml of 3 . 85 % sodium phosphotungstate followed by 23 ml of 2m mgcl 2 . the plasma was then centrifuged for 15 minutes at 3600 g . pellets were resuspended in 800 ml of 0 . 2 % sodium citrate . ldl was precipitated again by adding 10 g nacl and 24 ml of 2m mgcl 2 . ldl particles were pelleted by centrifugation for 15 minutes at 3600 g . this wash was repeated twice . pellets were then frozen at - 20 ° c . ldl particles from 5 l of plasma were resuspended in 5 l of buffer a ( 25 mm tris - hcl , 10 mm chaps , ph 7 . 5 ) and stirred overnight . solubilized ldl particles were centrifuged at 3600 g for 1 . 5 hours . supernatants were combined and filtered with whatman 113 filter paper to remove any remaining solids . solubilized ldl supernatant was loaded on a deae sepharose fast flow column ( 11 cm × 10 cm ; 1 l resin volume ; 80 ml / minute ) equilibrated in buffer b ( 25 mm tris - hcl , 1 mm chaps , ph 7 . 5 ). the column was washed with buffer b until absorbance returned to baseline . protein was eluted with an 8 l , 0 - 0 . 5m nacl gradient and 480 ml fractions were collected . this step was necessary to obtain binding to the blue sepharose fast flow column below . fractions were assayed for acetylhydrolase activity essentially by the method described in example 4 . active fractions were pooled and sufficient chaps was added to make the pool about 10 mm chaps . the deae pool was loaded overnight at 4 ml / minute onto a blue sepharose fast flow column ( 5 cm × 10 cm ; 200 ml bed volume ) equilibrated in buffer a containing 0 . 5m nacl . the column was washed with the equilibration buffer at 16 ml / minute until absorbance returned to baseline . paf - ah activity was step eluted with buffer a containing 0 . 5m kscn ( a chaotropic salt ) at 16 ml / minute and collected in 50 ml fractions . this step resulted in greater than 1000 - fold purification . active fractions were pooled , and the pool was adjusted to ph 8 . 0 with 1m tris - hcl ph 8 . 0 . the active pool from blue sepharose fast flow chromatography was loaded onto a cu chelating sepharose column ( 2 . 5 cm × 2 cm ; 10 ml bed volume ; 4 ml / minute ) equilibrated in buffer c 25 mm tris - hcl , 10 mm chaps , 0 . 5m nacl , ph 8 . 0 ( ph 7 . 5 also worked )!, and the column was washed with 50 mi buffer c . paf - ah activity was eluted with 100 ml 50 mm imidazole in buffer c and collected in 10 ml fractions . fractions containing paf - ah activity were pooled and dialyzed against buffer a . in addition to providing a 15 - fold concentration of paf - ah activity , the cu chelating sepharose column gave a small purification . the cu chelating sepharose pool was reduced in 50 mid dtt for 15 minutes at 37 ° c . and loaded onto a 0 . 75 mm , 7 . 5 % polyacrylamide gel . gel slices were cut every 0 . 5 cm and placed in disposable microfuge tubes containing 200 μl 25 mm tris - hcl , 10 mm chaps , 150 mm nacl . slices were ground up and allowed to incubate overnight at 4 ° c . the supernatant of each gel slice was then assayed for paf - ah activity to determine which protein band on sds - page contained paf - ah activity . paf - ah activity was found in an approximately 44 kda band . protein from a duplicate gel was electrotransferred to a pvdf membrane ( immobilon - p , millipore ) and stained with coomassie blue . a photograph of the pvdf membrane is presented in fig1 . as presented in table i below , approximately 200 μg paf - ah was purified 2 × 10 6 - fold from 5 l human plasma . in comparison , a 3 × 10 4 - fold purification of paf - ah activity is described in stafforini et al . ( 1987 ), supra . table 1__________________________________________________________________________ total prot . specific % recoveryvol . activity activity conc . activity of activity fold purificationsample ( ml ) ( cpm × 10 . sup . 6 ) ( cpm × 10 . sup . 9 ) ( mg / ml ) ( cpm × 10 . sup . 6 ) step cum . step cum . __________________________________________________________________________plasma 5000 23 116 62 0 . 37 100 100 1 1ldl 4500 22 97 1 . 76 12 84 84 33 33deae 4200 49 207 1 . 08 46 212 178 3 . 7 124blue 165 881 14 0 . 02 54200 70 126 1190 1 . 5 × 10 . sup . 5cu 12 12700 152 0 . 15 82200 104 131 1 . 5 2 . 2 × 10 . sup . 5sds - page -- -- -- -- -- -- -- ˜ 10 2 . 2 × 10 . sup . 6__________________________________________________________________________ in summary , the following steps were unique and critical for successful purification of plasma paf - ah for microsequencing : ( 1 ) solubilization and chromotography in 10 mm chaps , ( 2 ) chromatography on a blue ligand affinity column such as blue sepharose fast flow , ( 3 ) chromatography on a cu ligand affinity column such as cu chelating sepharose , and ( 4 ) elution of paf - ah from sds - page . for amino acid sequencing , the approximately 44 kda protein band from the paf - ah - containing pvdf membrane described in example 1 was excised and sequenced using an applied biosystems 473a protein sequencer . n - terminal sequence analysis of the ˜ 44 kda protein band corresponding to the paf - ah activity indicated that the band contained two major sequences and two minor sequences . the ratio of the two major sequences was 1 : 1 and it was therefore difficult to interpret the sequence data . to distinguish the sequences of the two major proteins which had been resolved on the sds gel , a duplicate pvdf membrane containing the approximately 44 kda band was cut in half such that the upper part and the lower part of the membrane were separately subjected to sequencing . the n - terminal sequence obtained for the lower half of the membrane was : a search of protein databases revealed this sequence to be a fragment of human serum albumin . the upper half of the same pvdf membrane was also sequenced and the n - terminal amino acid sequence determined was : this sequence did not match any protein in the databases searched and was different from the n - terminal amino acid sequence : which was reported for erythrocyte cytoplasmic paf - ah in stafforini et al . ( 1993 ), supra . the novel sequence ( seq id no : 2 ) was utilized for cdna cloning of human plasma paf - ah as described below in example 3 . a full length clone encoding human plasma paf - ah was isolated from a macrophage cdna library . poly a + rna was harvested from peripheral blood monocyte - derived macrophages . double - stranded , blunt - ended cdna was generated using the invitrogen copy kit ( san diego , calif .) and bstxi adapters were ligated to the cdna prior to insertion into the mammalian expression vector , prc / cmv ( invitrogen ). the resulting plasmids were introduced into e . coli strain xl - 1 blue by electropotation . transformed bacteria were plated at a density of approximately 3000 colonies per agarose plate on a total of 978 plates . plasmid dna prepared separately from each plate was retained in individual pools and was also combined into larger pools representing 300 , 000 clones each . the macrophage library was screened by the polymerase chain reaction utilizing a degenerate antisense oligonucleotide pcr primer based on the novel n - terminal amino acid sequence described in example 2 . the sequence of the primer is set out below in iupac nomenclature and where &# 34 ; i &# 34 ; is an inosine . the codon choice tables of wada et al ., nuc . acids res ., 19s : 1981 - 1986 ( 1991 ) were used to select nucleotides at the third position of each codon of the primer . the primer was used in combination with a primer specific for either the sp6 or t7 promoter sequences , both of which flank the cloning site of prc / cmv , to screen the macrophage library pools of 300 , 000 clones . all pcr reactions contained 100 ng of template cdna , 1 μg of each primer , 0 . 125 mm of each dntp , 10 mm tris - hcl ph 8 . 4 , 50 mm mgcl 2 and 2 . 5 units of taq polymerase . an initial denaturation step of 94 ° c . for four minutes was followed by 30 cycles of amplification of 1 minute at 94 ° c ., 1 minute at 60 ° c . and 2 minutes at 72 ° c . the resulting pcr product was cloned into pbluescript sk ( stratagene , la jolla , calif .) and its nucleotide sequence determined by the dideoxy chain termination method . the pcr product contained the sequence predicted by the novel peptide sequence and corresponds to nucleotides 1 to 331 of seq id no : 7 . the pcr primers set out below , which are specific for the cloned pcr fragment described above , were then designed for identifying a full length clone . pcr reactions utilizing the primers were performed as described above to first screen the cdna pools of 300 , 000 clones and then the appropriate subset of the smaller pools of 3000 clones . three pools of 3000 clones which produced a pcr product of the expected size were then used to transform bacteria . dna from the transformed bacteria was subsequently screened by hybridization using the original cloned pcr fragment as a probe . colonies were blotted onto nitrocellulose and prehybridized and hybridized in 50 % formamide , 0 . 75m sodium chloride , 0 . 075m sodium citrate , 0 . 05m sodium phosphate ph 6 . 5 , 1 % polyvinyl pyrolidine , 1 % ficoll , 1 % bovine serum albumin and 50 ng / ml sonicated salmon sperm dna . the hybridization probe was labeled by random hexamer priming . after overnight hybridization at 42 ° c ., blots were washed extensively in 0 . 03m sodium chloride , 3 mm sodium citrate , 0 . 1 % sds at 42 ° c . the nucleotide sequence of 10 hybridizing clones was determined . one of the clones , clone sah 406 - 3 , contained the sequence predicted by the original peptide sequence of the paf - ah activity purified from human plasma . the dna and deduced amino acid sequences of the human plasma paf - ah are set out in seq id nos : 7 and 8 , respectively . clone sah 406 - 3 contains a 1 . 52 kb insert with an open reading frame that encodes a predicted protein of 441 amino acids . at the amino terminus , a relatively hydrophobic segment of 41 residues precedes the n - terminal amino acid ( the isoleucine at position 42 of seq id no : 8 ) identified by protein microsequencing . the encoded protein may thus have either a long signal sequence or a signal sequence plus an additional peptide that is cleaved to yield the mature functional enzyme . the presence of a signal sequence is one characteristic of secreted proteins . in addition , the protein encoded by clone sah 406 - 3 includes the consensus g × s × g motif ( amino acids 271 - 275 of seq id no : 8 ) that is believed to contain the active site serine of all known mammalian lipases , microbial lipases and serine proteases . see chapus et al ., biochimie , 70 : 1223 - 1224 ( 1988 ) and brenner , nature , 334 : 528 - 530 ( 1988 ). table 2 below is a comparison of the amino acid composition of the human plasma paf - ah of the invention as predicted from seq id no : 8 and the amino acid composition of the purportedly purified material described by stafforini et al . ( 1987 ), supra . table 2______________________________________ clone sah 406 - 3 stafforini et al . ______________________________________ala 26 24asp & amp ; asn 48 37cys 5 14glu & amp ; gln 36 42phe 22 12gly 29 58his 13 24ile 31 17lys 26 50leu 40 26met 10 7pro 15 11arg 18 16ser 27 36thr 20 15val 13 14trp 7 not determinedtyr 14 13______________________________________ the amino acid composition of the mature form of the human plasma paf - ah of the invention and the amino acid composition of the previously purified material that was purportedly the human plasma paf - ah are clearly distinct . when alignment of the hattori et al ., supra nucleotide and deduced amino acid sequences of bovine brain cytoplasmic paf - ah with the nucleotide and amino acid sequences of the human plasma paf - ah of the invention was attempted , no significant structural similarity in the sequences was observed . a putative splice variant of the human paf - ah gene was detected when pcr was performed on macrophage and stimulated pbmc cdna using primers that hybridized to the 5 &# 39 ; untranslated region ( nucleotides 31 to 52 of seq id no : 7 ) and the region spanning the translation termination codon at the y end of the paf - ah cdna ( nucleotides 1465 to 1487 of seq id no : 7 ). the pcr reactions yielded two bands on a gel , one corresponding to the expected size of the paf - ah cdna of example 3 and the other was about 100 bp shorter . sequencing of both bands revealed that the larger band was the paf - ah cdna of example 3 while the shorter band lacked exon 2 ( example 5 below ) of the paf - ah sequence which encodes the putative signal and pro - peptide sequences of plasma paf - ah . the predicted catalytic triad and all cysteines were present in the shorter clone , therefore the biochemical activity of the protein encoded by the clone is likely to match that of the plasma enzyme . genomic human plasma paf - ah sequences were also isolated . the structure of the paf - ah gene was determined by isolating lambda and p1 phage clones containing human genomic dna by dna hybridization under conditions of high stringency . fragments of the phage clones were subcloned and sequenced using primers designed to anneal at regular intervals throughout the cdna clone sah 406 - 3 . in addition , new sequencing primers designed to anneal to the intron regions flanking the exons were used to sequence back across the exon - intron boundaries to confirm the sequences . exon / intron boundaries were defined as the points where the genomic and cdna sequences diverged . these analyses revealed that the human paf - ah gene is comprised of 12 exons . exons 1 , 2 , 3 , 4 , 5 , 6 , and part of 7 were isolated from a male fetal placental library constructed in lamda fix ( stratagene ). phage plaques were blotted onto nitrocellulose and prehybridized and hybridized in 50 % formamide , 0 . 75m sodium chloride , 75 mm sodium citrate , 50 mm sodium phosphate ( ph 6 . 5 ), 1 % polyvinyl pyrolidine , 1 % ficoll , 1 % bovine serum albumin , and 50 ng / ml sonicated salmon sperm dna . the hybridization probe used to identify a phage clone containing exons 2 - 6 and part of 7 consisted of the entire cdna clone sah 406 - 3 . a clone containing exon 1 was identified using a fragment derived from the 5 &# 39 ; end of the cdna clone ( nucleotides 1 to 312 of seq id no : 7 ). both probes were labelled with 32 p by hexamer random priming . after overnight hybridization at 42 ° c ., blots were washed extensively in 30 mm sodium chloride , 3 mm sodium citrate , 0 . 1 % sds at 42 ° c . the dna sequences of exons 1 , 2 , 3 , 4 , 5 , and 6 along with partial surrounding intron sequences are set out in seq id nos : 9 , 10 , 11 , 12 , 13 , and 14 , respectively . the remainder of exon 7 as well as exons 8 , 9 , 10 , 11 , and 12 were subcloned from a p1 clone isolated from a human p1 genomic library . p1 phage plaques were blotted onto nitrocellulose and prehybridized and hybridized in 0 . 75m sodium chloride , 50 mm sodium phosphate ( ph 7 . 4 ), 5 mm edta , 1 % polyvinyl pyrolidine , 1 % ficoll , 1 % bovine serum albumin , 0 . 5 % sds , and 0 . 1 mg / ml total human dna . the hybridization probe , labeled with 32 p by hexamer random priming , consisted of a 2 . 6 kb ecor1 fragment of genomic dna derived from the 3 &# 39 ; end of a lambda clone isolated above . this fragment contained exon 6 and the part of exon 7 present on the phage clone . after overnight hybridization at 65 ° c ., blots were washed as described above . the dna sequences of exons 7 , 8 , 9 , 10 , 11 , and 12 along with partial surrounding intron sequences are set out in seq id nos : 15 , 16 , 17 , 18 , 19 , and 20 , respectively . full length plasma paf - ah cdna clones were isolated from mouse and canine spleen cdna libraries and a partial rodent clone was isolated from a rat thymus cdna library . the clones were identified by low stringency hybridization ( hybridization conditions were the same as described for exons 1 through 6 in example 5 above except that 20 % formamide instead of 50 % formamide was used ). a 1 kb hindiii fragment of the human paf - ah sah 406 - 3 cdna clone ( nucleotides 309 to 1322 of seq id no : 7 ) was used as a probe . in addition , a partial monkey clone was isolated from macaque brain cdna by pcr using primers based on nucleotides 285 to 303 and 851 to 867 of seq id no : 7 . the nucleotide and deduced amino acid sequences of the mouse , canine , rat , and macaque cdna clones are set out in seq id nos : 21 and 27 , 22 and 28 , 23 and 29 , and 24 and 30 , respectively . a comparison of the deduced amino acid sequences of the cdna clones with the human cdna clone results in the amino acid percentage identity values set out in table 3 below . table 3______________________________________ human dog mouse______________________________________dog 80mouse 66 64monkey 92 82 69rat 74 69 82______________________________________ to determine whether human plasma paf - ah cdna clone sah 406 - 3 ( example 3 ) encodes a protein having paf - ah activity , the prc / cmv expression construct was transiently expressed in cos 7 cells . three days following transfection by a deae dextran method , cos cell media was assayed for paf - ah activity . cells were seeded at a density of 300 , 000 cells per 60 mm tissue culture dish . the following day , the cells were incubated in dmem containing 0 . 5 mg / ml deae dextran , 0 . 1 mm chloroquine and 5 - 10 μg of plasmid dna for 2 hours . cells were then treated with 10 % dmso in phosphate - buffered saline for 1 minute , washed with media and incubated in dmem containing 10 % fetal calf serum previously treated with diisopropyl fluorophosphate ( dfp ) to inactivate endogenous bovine serum paf - ah . after 3 days of incubation , media from transfected cells were assayed for paf - ah activity . assays were conducted in the presence and absence of either 10 mm edta or 1 mm dfp to determine whether the recombinant enzyme was calcium - independent and inhibited by the serine esterase inhibitor dfp as previously described for plasma paf - ah by stafforini et al . ( 1987 ), supra . negative controls included cells transfected with prc / cmv either lacking an insert or having the sah 406 - 3 insert in reverse orientation . paf - ah activity in transfectant supernatants was determined by the method of stafforini et al . ( 1990 ), supra , with the following modifications . briefly , paf - ah activity was determined by measuring the hydrolysis of 3 h - acetate from acetyl - 3 h ! paf ( new england nuclear , boston , mass .). the aqueous free 3 h - acetate was separated from labeled substrate by reversed - phase column chromatography over octadecylsilica gel cartridges ( baker research products , phillipsburg , pa .). assays were carried out using 10 μl transfectant supernatant in 0 . 1m hepes buffer , ph 7 . 2 , in a reaction volume of 50 μl . a total of 50 pmoles of substrate were used per reaction with a ratio of 1 : 5 labeled : cold paf . reactions were incubated for 30 minutes at 37 ° c . and stopped by the addition of 40 μl of 10m acetic acid . the solution was then washed through the octadecylsilica gel cartridges which were then rinsed with 0 . 1m sodium acetate . the aqueous eluate from each sample was collected and counted in a liquid scintillation counter for one minute . enzyme activity was expressed in counts per minute . as shown in fig2 media from cells transfected with sah 406 - 3 contained paf - ah activity at levels 4 - fold greater than background . this activity was unaffected by the presence of edta but was abolished by 1 mm dfp . these observations demonstrate that clone sail 406 - 3 encodes an activity consistent with the human plasma enzyme paf - ah . pcr was used to generate a protein coding fragment of human plasma paf - ah cdna from clone sah 406 - 3 which was readily amenable to subcloning into an e . coli expression vector . the subcloned segment began at the 5 &# 39 ; end of the human gene with the codon that encodes ile 42 ( seq id no : 8 ), the n - terminal residue of the enzyme purified from human plasma . the remainder of the gene through the native termination codon was included in the construct . the 5 &# 39 ; sense pcr primer utilized was : 3 &# 39 ; and contained an xbai cloning site as well as a translation initiation codon ( underscored ). the 3 &# 39 ; antisense primer utilized was : and encompassed the termination codon of sah 406 - 3 and contained an ecorv cloning site . pcr reactions were performed essentially as described in example 3 . the resulting pcr product was digested with xbai and ecorv and subcloned into a pbr322 vector containing the trp promoter deboer et al ., pnas , 80 : 21 - 25 ( 1983 )! immediately upstream of the cloning site . e . coli strain xl - 1 blue was transformed with the expression construct and cultured in l broth containing 100 μg / ml of carbenicillin . transformants from overnight cultures were pelleted and resuspended in lysis buffer containing 50 mm tris - hcl ph 7 . 5 , 50 mm nacl , 10 mm chaps , 1 mm edta , 100 μg / ml lysozyme , and 0 . 05 trypsin - inhibiting units ( tiu )/ ml aprotinin . following a 1 hour incubation on ice and sonication for 2 minutes , the lysates were assayed for paf - ah activity by the method described in example 4 . e . coli transformed with the expression construct ( designated trp ah ) generated a product with paf - ah activity . see table 6 in example 9 . constructs including three additional promoters , the tacii promoter ( deboer , supra ), the arabinose ( ara ) b promoter from salmonella typhimurium horwitz et al ., gene , 14 : 309 - 319 ( 1981 )!, and the bacteriophage t7 promoter , were also utilized to drive expression of human paf - ah sequences in e . coli . constructs comprising the trp promoter ( puc trp ah ), the tacii promoter ( puc tac ah ), and the arab promoter ( puc ara ah ) were assembled in plasmid puc19 ( new england biolabs , massachusetts ) while the construct comprising the t7 promoter ( pet ah ) was assembled in plasmid pet15b ( novagen , madison , wis .). a construct containing a hybrid promoter , phab / ph , consisting of the arab promoter fused to the ribosome binding sites of the t7 promoter region was also assembled in pet15b . all e . coli constructs produced paf - ah activity within a range of 20 to 50 u / ml / od 600 . this activity corresponded to a total recombinant protein mass of ≧ 1 % of the total cell protein . recombinant human paf - ah was also been expressed in saccharomyces cerevisiae . the yeast adh2 promoter was used to drive rpaf - ah expression and produced 7 u / ml / od 600 ( table 4 below ). table 4______________________________________ enzyme activityconstruct promoter strain ( u / ml / od ) ______________________________________puc tac ah tac e . coli w3110 30puc trp ah trp e . coli w3110 40puc ara ah arab e . coli w3110 20pet ah t7 e . coli bl21 ( de3 ) 50 ( novagen ) phab / ph arab / t7 e . coli xl - 1 34pyep adh2 ah adh2 yeast bj2 . 28 7______________________________________ several e . coli expression constructs were also evaluated which produce paf - ah with extended amino termini . the n - terminus of natural plasma paf - ah was identified as ile 42 by amino acid sequencing ( example 2 ). however , the sequence immediately upstream of ile 42 does not conform to amino acids found at signal sequence cleavage sites i . e ., the &# 34 ;- 3 - 1 - rule &# 34 ; is not followed , as lysine is not found at position - 1 ; see von heijne , nuc . acids res ., 14 : 4683 - 4690 ( 1986 )!. presumably a more classical signal sequence ( m 1 - a 17 ) is recognized by the cellular secretion system , followed by endoproteolytic cleavage . the entire coding sequence for paf - ah beginning at the initiating methionine ( nucleotides 162 to 1487 of seq id no : 7 ) was engineered for expression in e . coli using the trp promoter . as shown in table 5 , this construct made active paf - ah , but expression was at about one fiftieth of the level of the original construct beginning at ile 42 . another expression construct , beginning at val 18 ( nucleotides 213 to 1487 of seq id no : 7 ), produced active paf - ah at about one third the level of the original construct . these results suggest that amino terminal end extensions are not critical or necessary for activity of recombinant paf - ah produced in e . coli . table 5______________________________________ paf - ah activity ( u / ml / od . sub . 600 ) construct lysate media______________________________________puc trp ah 177 . 7 0 . 030puc trp ah met . sub . 1 3 . 1 0 . 003puc trp ah val . sub . 18 54 . 6 0 . 033______________________________________ recombinant human plasma paf - ah ( beginning at ile 42 ) expressed in e . coli was purified to a single coomassie - stained sds - page band by various methods and assayed for activities exhibited by the native paf - ah enzyme . the first purification procedure utilized is similar to that described in example 1 for native paf - ah . the following steps were performed at 4 ° c . pellets from 50 ml paf - ah producing e . coli ( transformed with expression construct trp ah ) were lysed as described in example 8 . solids were removed by centrifugation at 10 , 000 g for 20 minutes . the supernatant was loaded at 0 . 8 ml / minute onto a blue sepharose fast flow column ( 2 . 5 cm × 4 cm ; 20 ml bed volume ) equilibrated in buffer d ( 25 mm tris - hcl , 10 mm chaps , 0 . 5m nacl , ph 7 . 5 ). the column was washed with 100 ml buffer d and eluted with 100 ml buffer a containing 0 . 5m kscn at 3 . 2 ml / minute . a 15 ml active fraction was loaded onto a 1 ml cu chelating sepharose column equilibrated in buffer d . the column was washed with 5 ml buffer d followed by elution with 5 ml of buffer d containing 100 mm imidazole with gravity flow . fractions containing paf - ah activity were analyzed by sds - page . the results of the purification are shown in table 6 wherein a unit equals μmol paf hydrolysis per hour . the purification product obtained at 4 ° c . appeared on sds - page as a single intense band below the 43 kda marker with some diffuse staining directly above and below it . the recombinant material is significantly more pure and exhibits greater specific activity when compared with paf - ah preparations from plasma as described in example 1 . table 6__________________________________________________________________________ total prot specific % recovery foldvolume activity act . conc activity of activity purificationsample ( ml ) ( units / ml ) ( units × 10 . sup . 3 ) ( mg / ml ) ( units / mg ) step cum . step cum . __________________________________________________________________________lysate 4 . 5 989 4451 15 . 6 63 100 100 1 1blue 15 64 960 0 . 07 914 22 22 14 . 4 14 . 4cu 1 2128 2128 0 . 55 3869 220 48 4 . 2 61__________________________________________________________________________ when the same purification protocol was performed at ambient temperature , in addition to the band below the 43 kda marker , a group of bands below the 29 kda marker correlated with paf - ah activity of assayed gel slices . these lower molecular weight bands may be proteolytic fragments of paf - ah that retain enzymatic activity . a different purification procedure was also performed at ambient temperature . pellets ( 100 g ) of paf - ah - producing e . coli ( transformed with the expression construct puc trp ah ) were resuspended in 200 ml of lysis buffer ( 25 mm tris , 20 mm chaps , 50 mm nacl , 1 mm edta , 50 μg / ml benzamidine , ph 7 . 5 ) and lysed by passing three times through a microfluidizer at 15 , 000 psi . solids were removed by centrifugation at 14 , 300 × g for 1 hour . the supernatant was diluted 10 - fold in dilution buffer 25 mm mes ( 2 - n - morpholino ! ethanesulfonic acid ), 10 mm chaps , 1 mm edta , ph 4 . 9 ! and loaded at 25 ml / minute onto an s sepharose fast flow column ( 200 ml ) ( a cation exchange column ) equilibrated in buffer e ( 25 mm mes , 10 mm chaps , 1 mm edta , 50 mm nacl , ph 5 . 5 ). the column was washed with 1 liter of buffer e , eluted with 1m nacl , and the eluate was collected in 50 ml fractions adjusted to ph 7 . 5 with 0 . 5 ml of 2m tris base . fractions containing paf - ah activity were pooled and adjusted to 0 . 5m nacl . the s pool was loaded at 1 ml / minute onto a blue sepharose fast flow column ( 2 . 5 cm × 4 cm ; 20 ml ) equilibrated in buffer f ( 25 mm tris , 10 mm chaps , 0 . 5m nacl , 1 mm edta , ph 7 . 5 ). the column was washed with 100 ml buffer f and eluted with 100 ml buffer f containing 3m nacl at 4 ml / minute . the blue sepharose fast flow chromatography step was then repeated to reduce endotoxin levels in the sample . fractions containing paf - ah activity were pooled and dialyzed against buffer g ( 25 mm tris ph 7 . 5 , 0 . 5m nacl , 0 . 1 % tween 80 , 1 mm edta ). the results of the purification are shown in table 7 wherein a unit equals μmol paf hydrolysis per hour . table 7__________________________________________________________________________ total prot specific % recovery foldvolume activity act . conc activity of activity purificationsample ( ml ) ( units / ml ) ( units × 10 . sup . 3 ) ( mg / ml ) ( units / mg ) step cum . step cum . __________________________________________________________________________lysate 200 5640 1128 57 . 46 98 100 100 1 1s 111 5742 637 3 . 69 1557 57 56 16 16blue 100 3944 394 0 . 84 4676 35 62 3 48__________________________________________________________________________ the purification product obtained appeared on sds - page as a single intense band below the 43 kda marker with some diffuse staining directly above and below it . the recombinant material is significantly more pure and exhibits greater specific activity when compared with paf - ah preparations from plasma as described in example 1 . yet another purification procedure contemplated by the present invention involves the following cell lysis , clarification , and first column steps . cells are diluted 1 : 1 in lysis buffer ( 25 mm tris ph . 7 . 5 , 150 mm nacl , 1 % tween 80 , 2 mm edta ). lysis is performed in a chilled microfluidizer at 15 , 000 - 20 , 000 psi with three passes of the material to yield & gt ; 99 % cell breakage . the lysate is diluted 1 : 20 in dilution buffer ( 25 mm tris ph 8 . 5 , 1 mm edta ) and applied to a column packed with q - sepharose big bead chromatography media ( pharmacia ) and equilibrated in 25 mm tris ph 8 . 5 , 1 mm edta , 0 . 015 % tween 80 . the eluate is diluted 1 : 10 in 25 mm mes ph 5 . 5 , 1 . 2m ammonium sulfate , 1 mm edta and applied to butyl sepharose chromography media ( pharmacia ) equilibrated in the same buffer . paf - ah activity is eluted in 25 mm mes ph . 5 . 5 , 0 . 1 % tween 80 , 1 mm edta . the most remarkable property of the paf acetylhydrolase is its marked specificity for substrates with a short residue at the sn - 2 position of the substrate . this strict specificity distinguishes paf acetylhydrolase from other forms of pla 2 . thus , to determine if recombinant paf - ah degrades phospholipids with long - chain fatty acids at the sn - 2 position , hydrolysis of 1 - palmitoyl - 2 - arachidonoyl - sn - glycero - 3 - phosphocholine ( arachidonoylpc ) was assayed since this is the preferred substrate for a well - characterized form of pla 2 . as predicted from previous studies with native paf - ah , this phospholipid was not hydrolyzed when incubated with recombinant paf - ah . in additional experiments , arachidonoylpc was included in a standard paf hydrolysis assay at concentrations ranging from 0 to 125 μm to determine whether it inhibited the hydrolysis of paf by recombinant paf - ah . there was no inhibition of paf hydrolysis even at the highest concentration of paf - ah , which was 5 - fold greater than the concentration of paf . thus , recombinant paf - ah exhibits the same substrate selectivity as the native enzyme ; long chain substrates are not recognized . moreover , recombinant paf - ah enzyme rapidly degraded an oxidized phospholipid ( glutaroylpc ) which had undergone oxidative cleavage of the sn - 2 fatty acid . native plasma paf - ah has several other properties that distinguish it from other phospholipases including calcium - independence and resistance to compounds that modify sulfhydryl groups or disrupt disulfides . both the native and recombinant plasma paf - ah enzymes are sensitive to dfp , indicating that a serine comprises part of their active sites . an unusual feature of the native plasma paf acetylhydrolase is that it is tightly associated with lipoproteins in circulation , and its catalytic efficiency is influenced by the lipoprotein environment . when recombinant paf - ah of the invention was incubated with human plasma ( previously treated with dfp to abolish the endogenous enzyme activity ), it associated with low and high density lipoproteins in the same manner as the native activity . this result is significant because there is substantial evidence that modification of low density lipoproteins is essential for the cholesterol deposition observed in atheromas , and that oxidation of lipids is an initiating factor in this process . paf - ah protects low density lipoproteins from modification under oxidizing conditions in vitro and may have such a role in vivo . administration of paf - ah is thus indicated for the supression the oxidation of lipoproteins in atherosclerotic plaques as well as to resolve inflammation . these results all confirm that the cdna clone sah 406 - 3 encodes a protein with the activities of the the human plasma paf acetylhydrolase . various other recombinant paf - ah products were expressed in e . coli . the products included paf - ah analogs having single amino acid mutations and paf - ah fragments . paf - ah is a lipase because it hydrolyses the phospholipid paf . while no obvious overall similarity exists between paf - ah and other characterized lipases , there are conserved residues found in comparisons of structurally characterized lipases . a serine has been identified as a member of the active site . the serine , along with an aspartate residue and a histidine residue , form a catalytic triad which represents the active site of the lipase . the three residues are not adjacent in the primary protein sequence , but structural studies have demonstrated that the three residues are adjacent in three dimensional space . comparisons of structures of mammalian lipases suggest that the asp residue is generally twenty - four amino acids c - terminal to the active site serine . in addition , the histidine is generally 109 to 111 amino acids c - terminal to the active site serine . by site - directed mutagenesis and pcr , individual codons of the human paf - ah coding sequence were modified to encode alanine residues and were expressed in e . coli . as shown in table 8 below wherein , for example , the abbreviation &# 34 ; s108a &# 34 ; indicates that the serine residue at position 273 was changed to an alanine , point mutations of ser 273 , asp 296 , or his 351 completely destroy paf - ah activity . the distances between active site residues is similar for paf - ah ( ser to asp , 23 amino acids ; ser to his , 78 amino acids ) and other lipases . these experiments demonstrate that ser 273 , asp 296 , and his 351 are critical residues for activity and are therefore likely candidates for catalytic triad residues . cysteines are often critical for the functional integrity of proteins because of their capacity to form disulfide bonds . the plasma paf - ah enzyme contains five cysteines . to determine whether any of the five is critical for enzyme actvity , each cysteine was mutated individually to a serine and the resulting mutants were expressed in e . coli . as shown below in table 8 , a significant but not total loss of paf - ah activity resulted from the conversion of either cys 229 or cys 291 to serine . therefore , these cysteines appear to be necessary for full paf - ah activity . other point mutations had little or no effect on paf - ah catalytic activity . in table 8 , &# 34 ;++++&# 34 ; represent wild type paf - ah activity of about 40 - 60 u / ml / od 600 , &# 34 ;+++&# 34 ; represents about 20 - 40 u / ml / od 600 activity , &# 34 ;++&# 34 ; represents about 10 - 20 u / ml / od 600 activity , &# 34 ;+&# 34 ; represents 1 - 10 u / ml / od 600 activity , and &# 34 ;-&# 34 ; indicates & lt ; 1 u / ml / od 600 activity . table 8______________________________________mutation paf - ah activity______________________________________wild type ++++ s108a ++++ s273a - d296a - d338a ++++ h351a - h395a , h399a ++++ c67s +++ c229s + c291s + c334s ++++ c407s +++ ______________________________________ c - terminal deletions were prepared by digesting the 3 &# 39 ; end of the paf - ah coding sequence with exonuclease iii for various amounts of time and then ligating the shortened coding sequence to plasmid dna encoding stop codons in all three reading frames . ten different deletion constructs were characterized by dna sequence analysis , protein expression , and paf - ah activity . removal of twenty - one to thirty c - terminal amino acids greatly reduced catalytic activity and removal of fifty - two residues completely destroyed activity . see fig3 . similar deletions were made at the amino terminal end of paf - ah . fusions of paf - ah with e . coli thioredoxin at the n - terminus were prepared to facilitate consistent high level expression paf - ah activity lavallie et al ., bio / technology , 11 : 187 - 193 ( 1993 )!. removal of nineteen amino acids from the naturally processed n - terminus ( ile 42 ) completely destroyed enzymatic activity in the fusion protein . see fig3 . a preliminary analysis of expression patterns of human plasma paf - ah mrna in human tissues was conducted by northern blot hybridization . rna was prepared from human cerebral cortex , heart , kidney , placenta , thymus and tonsil using rna stat 60 ( tel - test &# 34 ; b &# 34 ;, friendswood , tex .). additionally , rna was prepared from the human hematopoietic precursor - like cell line , thp - 1 ( atcc tib 202 ), which was induced to differentiate to a macrophage - like phenotype using the phorbol ester phorbolmyristylacetate ( pma ). tissue rna and rna prepared from the premyelocytic thp - 1 cell line prior to and 1 to 3 days after induction were electrophoresed through a 1 . 2 % agarose formaldehyde gel and subsequently transferred to a nitrocellulose membrane . the full length human plasma paf - ah cdna , sah 406 - 3 , was labelled by random priming and hybridized to the membrane under conditions identical to those described in example 3 for library screening . initial results indicate that the paf - ah probe hybridized to a 1 . 8 kb band in the thymus , tonsil , and to a lesser extent , the placental rna . the expression of paf - ah rna in monocytes isolated from human blood and during their spontaneous differentiation into macrophages in culture was also examined . little or no rna was detected in fresh monocytes , but expression was induced and maintained during differentiation into macrophages . there was a concomitant accumulation of paf - ah activity in the culture medium of the differentiating cells . expression of the human plasma paf - ah transcript was also observed in the thp - 1 cell rna at 1 day but not 3 days following induction . thp - 1 cells did not express mrna for paf - ah in the basal state . paf - ah expression in human and mouse tissues was examined by in situ hybridization . human tissues were obtained from national disease research interchange and the cooperative human tissue network . normal mouse brain and spinal cord , and eae stage 3 mouse spinal cords were harvested from s / jlj mice . normal s / jlj mouse embryos were harvested from eleven to eighteen days after fertilization . the tissue sections were placed in tissue tek ii cryomolds ( miles laboratories , inc ., naperville , ill .) with a small mount of oct compound miles , inc ., elkhart , ind .). they were centered in the cryomold , the cryomold filled with oct compound , then placed in a container with 2 - methylbutane c 2 h 5 ch ( ch 3 ) 2 , aldrich chemical company , inc ., milwaukee , wis .! and the container placed in liquid nitrogen . once the tissue and oct compound in the cryomold were frozen , the blocks were stored at - 80 ° c . until sectioning . the tissue blocks were sectioned at 6 μm thickness and adhered to vectabond ( vector laboratories , inc ., burlingame , calif .) coated slides and stored at - 70 ° c . and placed at 50 ° c . for approximately 5 minutes to warm them and remove condensation and were then fixed in 4 % paraformaldehyde for 20 minutes at 4 ° c ., dehydrated ( 70 %, 95 %, 100 % ethanol ) for 1 minute at 4 ° c . in each grade , then allowed to air dry for 30 minutes at room temperature . sections were denatured for 2 minutes at 70 ° c . in 70 % formamide / 2 × ssc , rinsed twice in 2 × ssc , dehydrated and then air dried for 30 minutes . the tissues were hybridized in situ with radiolabeled single - stranded mrna generated from dna derived from an internal 1 kb hindiii fragment of the paf - ah gene ( nucleotides 308 to 1323 of seq id no : 7 ) by in vitro rna transcription incorporation 35 s - utp ( amersham ). the probes were used at varying lengths from 250 - 500 bp . hybridization was carried out overnight ( 12 - 16 hours ) at 50 ° c . ; the 35 s - labeled riboprobes ( 6 × 10 5 cpm / section ), trna ( 0 . 5 μg / section ) and diethylpyrocarbonate ( depc )- treated water were added to hybridization buffer to bring it a final concentration of 5095 formamide , 0 . 3m nacl , 20 mm tris ph 7 . 5 , 1095 dextran sulfate , 1 × denhardt &# 39 ; s solution , 100 mm dithiothretol ( dtt ) and 5 mm edta . after hybridization , sections were washed for 1 hour at room temperature in 4 × ssc / 10 mm dtt , then for 40 minutes at 60 ° c . in 50 % formamide / 1 × ssc / 10 mm dtt , 30 minutes at room temperature in 2 × ssc , and 30 minutes at room temperature in 0 . 1 × ssc . the sections were dehydrated , air dried for 2 hours , coated with kodak ntb2 photographic emulsion , air dried for 2 hours , developed ( after storage at 4 ° c . in complete darkness ) and counterstained with hematoxylin / eosin . in both the mouse and the human brains , strong signal was seen in the purkinje cell layer of the cerebellum , as well as on individual neuronal cell bodies in the dentate nucleus ( one of the four deep nuclei in the cerebellum ). additionally , signal was seen on individual cells in the granular and molecular layers of the grey matter . in the human hippocampus section , individual cells throughout the section , which appear to be neuronal cell bodies , showed strong signal . on both human and mouse brain stem sections , there was strong signal on individual cells in the grey matter . on human cortex sections taken from the cerebral , occipital , and temporal cortexes , and on mouse whole brain sections , individual cells throughout the cortex showed strong signal . there does not appear to be differentiation in the expression pattern in the different layers of the cortex . these in situ hybridization results are different from the results for cerebral cortex obtained by northern blotting . the difference is likely to result from the greater sensitivity of in situ hybridization compared to that of northern blotting . somewhat weak signal was seen on scattered individual cells in the pars distalis of the human tissue section . both normal and crohn &# 39 ; s disease colons displayed signal in the lymphatic aggregations present in the mucosa of the sections , with the level of signal being slightly higher in the section from the crohn &# 39 ; s disease patient . the crohn &# 39 ; s disease colon also had strong signal in the lamina propria . similarly , a high level of signal was observed in a diseased appendix section while the normal appendix exhibited a lower but still detectable signal . the sections from the ulcerative colitis patient showed no evident signal in either the lymphatic aggregations or the lamina propria . strong signal was seen on scattered groups of individual cells within the germinal centers of the tonsil and within the thymus . strong signal was observed on the lymph node section taken from a normal donor , while somewhat weak signal was observed in the lymph nodules of the section from a donor with septic shock . both normal and crohn &# 39 ; s disease small intestine had weak signal in the peyer &# 39 ; s patches and lamina propria in the sections , with the signal on the diseased tissue slightly higher . signal was not observed on any of the spleen ( normal and splenic abcess sections ) or lung ( normal and emphysema sections ) tissues . in both the normal and eae stage 3 spinal cords , there was strong signal in the grey matter of the spinal cord , with the expression being slightly higher in the eae stage 3 spinal cord . in the eae stage 3 spinal cord , cells in the white matter and perivascular cuffs , probably infiltrating macrophages and / or other leukocytes , showed signal which was absent in the normal spinal cord . in the day 11 embryo signal was apparent in the central nervous system in the fourth ventricle , which remained constant throughout the embryo time course as it developed into the cerebellum and brain stem . as the embryos matured , signal became apparent in central nervous system in the spinal cord ( day 12 ), primary cortex and ganglion gasseri ( day 14 ), and hypophysis ( day 16 ). signal was observed in the peripheral nervous system ( beginning on day 14 or 15 ) on nerves leaving the spinal cord , and , on day 17 , strong signal appeared around the whiskers of the embryo . expression was also seen in the liver and lung at day 14 , the gut ( beginning on day 15 ), and in the posterior portion of the mouth / throat ( beginning on day 16 ). by day 18 , the expression pattern had differentiated into signal in the cortex , hindbrain ( cerebellum and brain stem ), nerves leaving the lumber region of the spinal cord , the posterior portion of the mouth / throat , the liver , the kidney , and possible weak signal in the lung and gut . paf - ah mrna expression in the tonsil , thymus , lymph node , peyer &# 39 ; s patches , appendix , and colon lymphatic aggregates is consistent with the conclusions that the probable predominant in vivo source of paf - ah is the macrophage because these tisues all are populated with tissue macrophages that serve as phagocytic and antigen - processing cells . expression of paf - ah in inflamed tissues would be consistent with the hypothesis that a role of monocyte - derived macrophages is to resolve inflammation . paf - ah would be expected to inactivate paf and the pro - inflammatory phospholipids , thus down - regulating the inflammatory cascade of events initiated by these mediators . paf has been detected in whole brain tissue and is secreted by rat cerebellar granule cells in culture . in vitro and in vivo experiments have demonstrated that paf binds a specific receptor in neural tissues and induces functional and phenotypic changes such as calcium mobilization , upregulation of transcription activating genes , and differentiation of the neural precursor cell line , pc12 . these observations suggested a physiologic role for paf in the brain , and consistent with this , recent experiments using hippocampal tissue section cultures and paf analogs and antagonists have implicated paf as an important retrograde messenger in hippocampal long term potentiation . therefore , in addition to its pathological effect in inflammation , paf appears to participate in routine neuronal signalling processes . expression of the extracellular paf - ah in the brain may serve to regulate the duration and magnitude of paf - mediated signalling . monoclonal antibodies specific for recombinant human plasma paf - ah were generated using e . coli produced paf - ah as an immunogen . mouse # 1342 was injected on day 0 , day 19 , and day 40 with recombinant paf - ah . for the prefusion boost , the mouse was injected with the immunogen in pbs , four days later the mouse was sacrificed and its spleen removed sterilely and placed in 10 ml serum free rpmi 1640 . 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 a similar manner . ns - 1 myeloma cells , kept in log phase in rpmi with 11 % feud bovine serum ( fbs ) ( hyclone laboratories , inc ., logan , utah ) for three days prior to fusion , were centrifuged at 200 g for 5 minutes , and the pellet was washed twice as described in the foregoing paragraph . one × 10 8 spleen cells were combined with 2 . 0 × 10 7 ns - 1 cells , centrifuged and the supernatant was aspirated . the cell pellet was dislodged by tapping the tube and 1 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 7 ml of serum free rpmi over 7 minutes . an additional 8 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 μm sodium hypoxanthine , 0 . 4 μm aminopterin , 16 μm thymidine ( hat ) ( gibco ), 25 units / ml il - 6 ( boehringer mannheim ) and 1 . 5 × 10 6 thymocytes / ml and plated into 10 corning flat bottom 96 well tissue culture plates ( corning , corning n . y .). on days 2 , 4 , and 6 , after the fusion , 100 μl of medium was removed from the wells of the fusion plates and replaced with fresh medium . on day 8 , the fusion was screened by elisa , testing for the presence of mouse igg binding to recombinant paf - ah . immulon 4 plates ( dynatech , cambridge , mass .) were coated for 2 hours at 37 ° c . with 100 ng / well recombinant paf - ah diluted in 25 mm tris , ph 7 . 5 . the coating solution was aspirated and 200 ul / well of blocking solution 0 . 5 % fish skin gelatin ( sigma ) diluted in cmf - pbs ! was added and incubated for 30 minutes at 37 ° c . plates were washed three times with pbs with 0 . 05 % tween 20 ( pbst ) and 50 μl culture supernatant was added . after incubation at 37 ° c . for 30 minutes , and washing as above , 50 μl of horseradish peroxidase conjugated goat anti - mouse igg ( fc ) ( jackson immunoresearch , west grove , pa .) diluted 1 : 3500 in pbst was added . plates were incubated as above , washed four times with pbst and 100 μl substrate , consisting of 1 mg / ml o - phenylene diamine ( sigma ) and 0 . 1 μl / ml 30 % h 2 o 2 in 100 mm citrate , ph 4 . 5 , was added . the color reaction was stopped in 5 minutes with the addition of 50 μl of 15 % h 2 so 4 . a 490 was read onn a plate reader ( dynatech ). selected fusion wells were cloned twice by dilution into 96 well plates and visually scoring the number of colonies / well after 5 days . hybridomas cloned were 90d1e , 90e3a , 90e6c , 90g11d ( atcc hb 11724 ), and 90f2d ( atcc hb 11725 ). the monoclonal antibodies produced by hybridomas were isotyped using the isostrip system ( boehringer mannheim , indianapolis , ind .). results showed that the monoclonal antibodies produced by hybridomas from fusion 90 were all igg 1 . experimental studies were performed to evaluate the in vivo therapeutic effects of recombinant paf - ah of the invention on acute inflammation using a rat foot edema model henriques et al ., br . j . pharmacol ., 106 : 579 - 582 ( 1992 )!. the results of these studies demonstrated that paf - ah blocks paf - induced edema . parallel studies were done to compare the effectiveness of paf - ah with two commercially available paf antagonists . e . coli transformed with the paf - ah expression vector puc trp ah were lysed in a microfluidizer , solids were centrifuged out and the cell supernatants were loaded onto a s - sepharose column ( pharmacia ). the column was washed extensively with buffer consisting of 50 mm nacl , 10 mm chaps , 25 mm mes and 1 mm edta , ph 5 . 5 . paf - ah was eluted by increasing the nacl concentration of the buffer to 1m . affinity chromatography using a blue sepharose column ( pharmacia ) was then used as an additional purification step . prior to loading the paf - ah preparation on the blue sepharose column , the sample was diluted 1 : 2 to reduce the nacl concentration to 0 . 5m and the ph was adjusted to 7 . 5 . after washing the blue sepharose column extensively with buffer consisting of 0 . 5m nacl , 25 mm tris , 10 mm chaps and 1 mm edta , ph 7 . 5 the paf - ah was eluted by increasing the nacl concentration to 3 . 0m . purity of paf - ah isolated in this manner was generally 95 % as assessed by sds - page with activity in the range of 5000 - 10 , 000 u / ml . additional quality controls done on each paf - ah preparation included determining endotoxin levels and hemolysis activity on freshly obtained rat erythrocytes . a buffer containing 25 mm tris , 10 mm chaps , 0 . 5m nacl , ph 7 . 5 functioned as storage media of the enzyme as well as carrier for administration . dosages used in experiments were based on enzyme activity assays conducted immediately prior to experiments . six to eight - week - old female long evans rats ( charles river , wilmington , mass . ), weighing 180 - 200 grams , were used for all experiments . prior to experimental manipulations , animals were anesthetized with a mixture of the anesthetics ketaset ( fort dodge laboratories , fort dodge , iowa ), rompun ( miles , shawnee mission , kans . ), and ace promazine ( aveco , fort dodge , iowa ) administered subcutaneously at approximately 2 . 5 mg ketaset , 1 . 6 mg rompun , 0 . 2 mg ace promazine per animal per dose . edema was induced in the foot by administration of either paf or zymosan as follows . paf ( sigma # p - 1402 ) was freshly prepared for each experiment from a 19 . 1 mm stock solution stored in chloroform / methanol ( 9 : 1 ) at - 20 ° c . required volumes were dried down under n 2 , diluted 1 : 1000 in a buffer containing 150 mm nacl , 10 mm tris ph 7 . 5 , and 0 . 25 % bsa , and sonicated for five minutes . animals received 50 μl paf ( final dose of 0 . 96 nmoles ) subcutaneously between the hind foot pads , and edema was assessed after 1 hour and again after 2 hours in some experiments . zymosan a ( sigma # a - 8800 ) was freshly prepared for each experiment as a suspension of 10 mg / ml in pbs . animals received 50 μl of zymosan ( final dose of 500 μg ) subcutaneously between the hind foot pads and edema was assessed after 2 hours . edema was quantitated by measuring the foot volume immediately prior to administration of paf or zymosan and at indicated time point post - challenge with paf or zymosan . edema is expressed as the increase in foot volume in milliliters . volume displacement measurements were made on anesthetized animals using a plethysmometer ( ugo basile , model # 7150 ) which measures the displaced water volume of the immersed foot . in order to insure that foot immersion was comparable from one time point to the next , the hind feet were marked in indelible ink where the hairline meets the heel . repeated measurements of the same foot using this technique indicate the precision to be within 5 %. paf - ah was injected locally between the foot pads , or systematically by iv injection in the tail vein . for local administration rats received 100 μl paf - ah ( 4000 - 6000 u / ml ) delivered subcutaneously between the right hind foot pads . left feet served as controls by administration of 100 μl carrier ( buffered salt solution ). for systemic administration of paf - ah , rats received the indicated units of paf - ah in 300 μl of carrier administered iv in the tail vein . controls received the appropriate volume of carrier iv in the tail vein . rats ( n = 4 ) were injected with 100 μl of paf - ah ( 4000 - 6000 u / ml ) subcutaneously between the right foot pads . left feet were injected with 100 μl carrier ( buffered salt solution ). four other rats were injected only with carrier . all rats were immediately challenged with paf via subcutaneous foot injection and foot volumes assessed 1 hour post - challenge . fig4 wherein edema is expressed as average increase in foot volume ( ml )± sem for each treatment group , illustrates that paf - induced foot edema is blocked by local administration of paf - ah . the group which received local paf - ah treatment prior to paf challenge showed reduced inflammation compared to the control injected group . an increase in foot volume of 0 . 08 ml ± 0 . 08 ( sem ) was seen in the paf - ah group as compared to 0 . 63 ± 0 . 14 ( sem ) for the carrier treated controls . the increase in foot volume was a direct result of paf injection as animals injected in the foot only with carrier did not exhibit an increase in foot volume . rats ( n = 4 per group ) were pretreated iv with either paf - ah ( 2000 u in 300 μl carrier ) or carrier alone , 15 minutes prior to paf challenge . edema was assessed 1 and 2 hours after paf challenge . fig5 wherein edema is expressed as average increase in volume ( ml )± sem for each treatment group , illustrates that iv administration of paf - ah blocked paf induced foot edema at one and two hours post challenge . the group which received 2000 u of paf - ah given by the iv route showed a reduction in inflammation over the two hour time course . mean volume increase for the paf - ah treated group at two hours was 0 . 10 ml ± 0 . 08 ( sem ), versus 0 . 56 ml ± 0 . 11 for carrier treated controls . f . comparison of paf - ah protection in edema induced by paf or zymosan rats ( n = 4 per group ) were pretreated iv with either paf - ah ( 2000 u in 300 μl carrier ) or carrier alone . fifteen minutes after pretreatment , groups received either paf or zymosan a , and foot volume was assessed after 1 and 2 hours , respectively . as shown in fig6 wherein edema is expressed as average increase in volume ( ml )± sem for each treatment group , systemic administration of paf - ah ( 2000 u ) was effective in reducing paf - induced foot edema , but failed to block zymosan induced edema . a mean increase in volume of 0 . 08 ± 0 . 02 was seen in the paf - ah treated group versus 0 . 49 ± 0 . 03 for the control group . in two separate experiments , groups of rats ( n = 3 to 4 per group ) were pretreated iv with either serial dilutions of paf - ah or carrier control in a 300 μl volume , 15 minutes prior to paf challenge . both feet were challenged with paf ( as described above ) and edema was assessed after 1 hour . fig7 wherein edema is expressed as average increase in volume ( ml )± sem for each treatment group , illustrates the increase in protection from paf - induced edema in rats injected with increasing dosages of paf - ah . in the experiments , the id 50 of paf - ah given by the iv route was found to be between 40 and 80 u per rat . h . in vivo efficacy of paf - ah as a function of time after administration in two separate experiments , two groups of rats ( n = 3 to 4 per group ) were pretreated iv with either paf - ah ( 2000 u in 300 μl carrier ) or carrier alone . after administration , groups received paf at time points ranging from 15 minutes to 47 hours post paf - ah administration . edema was then assessed 1 hour after paf challenge . as shown in fig8 wherein edema is expressed as average increase in volume ( ml )± sem for each treatment group , administration of 2000 u of paf - ah protects rats from paf induced edema for at least 24 hours . four rats received 2000 u of paf - ah by iv injection in a 300 μl volume . plasma was collected at various time points and stored at 4 ° c . and plasma concentrations of paf - ah were determined by elisa using a double mab capture assay . in brief , monoclonal antibody 90g11d ( example 13 ) was diluted in 50 mm carbonate buffer ph 9 . 6 at 100 ng / ml and immobilized on immulon 4 elisa plates overnight at 4 ° c . after extensive washing with pbs containing 0 . 05 % tween 20 , the plates were blocked for 1 hour at room temperature with 0 . 5 % fish skin gelatin ( sigma ) diluted in pbs . serum samples diluted in pbs with 15 mm chaps were added in duplicate to the washed elisa plate and incubated for 1 hour at room temperature . after washing , a biotin conjugate of monoclonal antibody 90f2d ( example 13 ) was added to the wells at a concentration of 5 μg / ml diluted in pbs and incubated for 1 hour at room temperature . after washing , 50 μl of a 1 : 1000 dilution of extraavidin ( sigma ) was added to the wells and incubated for 1 hour at room temperature . after washing , wells were developed using opd as a substrate and quantitated . enzyme activity was then calculated from a standard curve . fig9 wherein data points represent means ± sem , shows that at one hour plasma enzyme levels approached the predicted concentration based on a 5 - 6 ml plasma volume for 180 - 200 gram rats , mean = 374 u / ml ± 58 . 2 . beyond one hour plasma levels steadily declined , reaching a mean plasma concentration of 19 . 3 u / ml ± 3 . 4 at 24 hours , which is still considerably higher than endogenous rat paf - ah levels which have been found to be approximately 4 u / ml by enzymatic assays . groups of rats ( n = 4 per group ) were pretreated with one of three potential antiinflammatories : the paf antagonist cv3988 ( biomol # l - 103 ) administered ip ( 2 mg in 200 μl etoh ), the paf antagonist alprazolam ( sigma # a - 8800 ) administered ip ( 2 mg in 200 μl etoh ), or paf - ah ( 2000 u ) administered iv . control rats were injected iv with a 300 μl volume of carrier . the paf antagonists were administered ip because they are solubilized in ethanol . rats injected with either cv3988 or alprazolam were challenged with paf 30 minutes after administration of the paf antagonist to allow the paf antagonist to enter circulation , while paf - ah and carrier - treated rats were challenged 15 minutes after enzyme administration . rats injected with paf - ah exhibited a reduction in paf - induced edema beyond that afforded by the established paf antagonists cv3988 and alprazolam . see fig1 wherein edema is expressed as average increase in volume ( ml )± sem for each treatment group . in summary , paf - ah is effective in blocking edema mediated by paf in vivo . administration of paf - ah can be either local or systemic by iv injection . in dosing studies , iv injections in the range of 160 - 2000 u / rat were found to dramatically reduce paf mediated inflammation , while the id 50 dosage appears to be in the range of 40 - 80 u / rat . calculations based on the plasma volume for 180 - 200 gram rats predicts that a plasma concentration in the range of 25 - 40 u / ml should block paf - elicited edema . these predictions are supported by preliminary pharmacokinetic studies . a dosage of 2000 u of paf - ah was found to be effective in blocking paf mediated edema for at least 24 hours . at 24 hours following administration of paf - ah plasma concentrations of the enzyme were found to be approximately 25 u / ml . paf - ah was found to block paf - induced edema more effectively than the two known paf antagonists tested . collectively , these results demonstrate that paf - ah effectively blocks paf induced inflammation and may be of therapeutic value in diseases where paf : is the primary mediator . recombinant paf - ah of the invention was tested in a second in vivo model , paf - induced pleurisy . paf has previously been shown to induce vascular leakage when introduced into the pleural space henriques et al ., supra !. female rats ( charles river , 180 - 200 g ) were injected in the tail vein with 200 μl of 1 % evans blue dye in 0 . 9 % with 300 μl recombinant paf - ah ( 1500 μmol / ml / hour , prepared as described in example 14 ) or with an equivalent volume of control buffer . fifteen minutes later the rats received an 100 μl injection of paf ( 2 . 0 nmol ) into the pleural space . one hour following paf challenge , rats were sacrificed and the pleural fluid was collected by rinsing the cavity with 3 ml heparinized phosphate buffered saline . the degree of vascular leak was determined by the quantity of evans blue dye in the pleural space which was quantitated by absorbance at 620 nm . rats pretreated with paf - ah were found to have much less vascular leakage than control animals ( representing more than an 80 % reduction in inflammation ). the foregoing results support the treatment of subjects suffering from pleurisy with recombinant paf - ah enzyme of the invention . recombinant paf - ah enzyme of the invention was also tested for efficacy in a model of antigen - induced eosinophil recruitment . the accumulation of eosinophils in the airway is a characteristic feature of late phase immune responses which occur in asthma , rhinitis and eczema . balb / c mice ( charles river ) were sensitized by two intraperitoneal injections consisting of 1 μg of ovalbumin ( ova ) in 4 mg of aluminum hydroxide ( imject alum , pierce laboratories , rockford , ill .) given at a 2 week interval . fourteen days following the second immunization , the sensitized mice were challenged with either aerosolized ova or saline as a control . prior to challenge mice were randomly placed into four groups , with four mice / group . mice in groups1 and 3 were pretreated with 140 μl of control buffer consisting of 25 mm tris , 0 . 5m nacl , 1 mm edta and 0 . 1 % tween 80 given by intravenous injection . mice in groups 2 and 4 were pretreated with 750 units of paf - ah ( activity of 5 , 500 units / ml given in 140 μl of paf - ah buffer ). thirty minutes following administration of paf - ah or buffer , mice in groups 1 and 2 were exposed to aerosolized pbs as described below , while mice in groups 3 and 4 were exposed to aerosolized ova . twenty - four hours later mice were treated a second time with either 140 μl of buffer ( groups 1 and 3 ) or 750 units of paf - ah in 140 μl of buffer ( groups 2 and 4 ) given by intravenous injection . eosinophil infiltration of the trachea was induced in the sensitized mice by exposing the animals to aerosolized ova . sensitized mice were placed in 50 ml conical centrifuge tubes ( corning ) and forced to breath aerosolized ova ( 50 mg / ml ) dissolved in 0 . 9 % saline for 20 minutes using a nebulizer ( model 646 , devilbiss corp ., somerset , pa .). control mice were treated in a similar manner with the exception that 0 . 9 % saline was used in the nebulizer . forty - eight hours following the exposure to aerosolized ova or saline , mice were sacrificed and the tracheas were excised . tracheas from each group were inbeded in oct and stored at - 70 ° until sections were cut . to evaluate eosinophil infiltration of the trachea , tissue sections from the four groups of mice were stained with either luna solution and hematoxylin - eosin solution or with peroxidase . twelve 6 μm thick sections were cut from each group of mice and numbered accordingly . odd numbered sections were stained with luna stain as follows . sections were fixed in formal - alcohol for 5 minutes at room temperature , rinsed across three changes of tap water for 2 minutes at room temperature then rinsed in two changed of dh 2 o for 1 minute at room temperature . tissue sections were stained with luna stain 5 minutes at room temperature ( luna stain consisting of 90 ml weigert &# 39 ; s iron hematoxylin and 10 ml of 1 % biebrich scarlet ). stained slides were dipped in 1 % acid alcohol six times , rinsed in tap water for 1 minute at room temperature , dipped in 0 . 5 % lithium carbonate solution five times and rinsed in running tap water for 2 minutes at room temperature . slides were dehydrated across 70 %- 95 %- 100 % ethanol 1 minute each , at room temperature , then cleared in two changes of xylene for 1 minute at room temperature and mounted in cytoseal 60 . for the peroxidase stain , even numbered sections were fixed in 4 ° c . acetone for 10 minutes and allowed to air dry . two hundred μl of dab solution was added to each section and allowed to sit 5 minutes at room temperature . slides were rinsed in tap water for 5 minutes at room temperature and 2 drops of 1 % osmic acid was applied to each section for 3 - 5 seconds . slides were rinsed in tap water for 5 minutes at room temperature and counterstained with mayers hematoxylin at 25 ° c . at room temperature . slides were then rinsed in running tap water for 5 minutes and dehydrated across 70 %- 95 %- 100 % ethanol 1 minute each at room temperature . slides were cleared through two changes of xylene for 1 minute each at room temperature and mounted in cytoseal 60 . the number of eosinophils in the submucosal tissue of the trachea was evaluated . trachea from mice from groups 1 and 2 were found to have very few eosinophils scattered throughout the submucosal tissue . as expected tracheas from mice in group 3 , which were pretreated with buffer and exposed to nebulized ova , were found to have large numbers of eosinophils throughout the submucosal tissue . in contrast , the tracheas from mice in group 4 , which were pretreated with paf - ah and exposed to nebulized ova were found to have very few eosinophils in the submucosal tissue comparable to what was seen in the two control groups , groups 1 and 2 . thus , therapeutic treatment with paf - ah of subjects exhibiting a late phase immune response involving the accumulation of eosinophils in the airway , such as that which occurs in asthma , rhinitis , and eczema , is indicated . nearly four percent of the japanese population has low or undetectable levels of paf - ah activity in their plasma . this deficiency has been correlated with severe respiratory symptoms in asthmatic children miwa et al ., j . clin . invest ., 82 : 1983 - 1991 ( 1988 )! who appear to have inherited the deficiency in an autosomal recessive manner . to determine if the deficiency arises from an inactive but present enzyme or from an inability to synthesize paf - ah , plasma from multiple patients deficient in paf - ah activity was assayed both for paf - ah activity ( by the method described in example 10 for transfectants ) and for the presence of paf - ah using the monoclonal antibodies 90g11d and 90f2d ( example 13 ) in a sandwich elisa as follows . immulon 4 flat bottom plates ( dynatech , chantilly , va .) were coated with 100 ng / well of monoclonal antibody 90g11d and stored overnight . the plates were blocked for 1 hour at room temperature with 0 . 5 % fish skin gelatin ( sigma ) diluted in cmf - pbs and then washed three times . patient plasma was diluted in pbs containing 15 mm chaps and added to each well of the plates ( 50 μl / well ). the plates were incubated for 1 hour at room temperature and washed four times . fifty μl of 5 μμg / ml monoclonal antibody 90f2d , which was biotinylated by standard methods and diluted in pbst , was added to each well , and the plates were incubated for 1 hour at room temperature and then washed three times . fifty μl of extraavidin ( sigma ) diluted 1 / 1000 in cmf - pbst was subsequently added to each well and plates were incubated for 1 hour at room temperature before development . a direct correlation between paf - ah activity and enzyme levels was observed . an absence of activity in a patient &# 39 ; s serum was reflected by an absence of detectable enzyme . similarly , plasma samples with half the normal activity contained half the normal levels of paf - ah . these observations suggested that the deficiency of paf - ah activity was due to an inability to synthesize the enzyme or due to an inactive enzyme which the monoclonal antibodies did not recognize . further experiments revealed that the deficiency was due to a genetic lesion in the human plasma paf - ah gene . genomic dna from paf - ah deficient individuals was isolated and used as template for pcr reactions with paf - ah gene specific primers . each of the coding sequence exons were initially amplified and sequenced from one individual . a single nucleotide change within exon 9 was observed ( a g to t at position 996 of seq id no : 7 ). the nucleotide change results in an amino acid substitution of a phenylalanine for a valine at position 279 of the paf - ah sequence ( v279f ). exon 9 was amplified from genomic dna from an additional eleven paf - ah deficient individuals who were found to have the same point mutation . to test whether this mutation crippled the enzyme , an e . coli expression construct containing the mutation was generated by methods similar to that described in example 10 . when introduced into e . coli , the expression construct generated no paf - ah activity while a control construct lacking the mutation was fully active . this amino acid substitution presumably results in a structural modification which causes the observed deficiency of activity and lack of immunoreactivity with the paf - ah antibodies of the invention . paf - ah specific antibodies of the invention may thus be used in diagnostic methods to detect abnormal levels of paf - ah in serum ( normal levels are about 1 to 5 u / ml ) and to follow the progression of treatment of pathological conditions with paf - ah . moreover , identification of a genetic lesion in the paf - ah gene allows for genetic screening for the paf - ah deficiency exhibited by the japanese patients . the mutation causes the gain of a restriction endonuclease site ( mac ii ) and thus allows for the simple method of restriction fragment length polymorphism ( rflp ) analysis to differentiate between active and mutant alleles . see lewin , pp . 136 - 141 in genes v , oxford university press , new york , n . y . ( 1994 ). screening of genomic dna from twelve paf - ah deficient patients was carried out by digestion of the dna with maeii , southern blotting , and hybridization with an exon 9 probe ( nucleotides 1 - 396 of seq id no : 17 ). all patients were found to have rflps consistent with the mutant allele . 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 : 30 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 17 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 1 : phelysaspleuglyglugluasnphelysalaleuvalleuileala151015phe ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 16 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 2 : ileglnvalleumetalaalaalaserpheglyglnthrlysilepro151015 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 11 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 3 : metlysproleuvalvalphevalleuglygly1510 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 32 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( ix ) feature :( a ) name / key : modified - site ( b ) location : group ( 13 , 21 , 27 )( c ) other information : / note =&# 34 ; the nucleotide at each ofthese positions is an inosine .&# 34 ;( 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( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 441 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 8 : metvalproprolysleuhisvalleuphecysleucysglycysleu151015alavalvaltyrpropheasptrpglntyrileasnprovalalahis202530metlysserseralatrpvalasnlysileglnvalleumetalaala354045alaserpheglyglnthrlysileproargglyasnglyprotyrser505560valglycysthraspleumetpheasphisthrasnlysglythrphe65707580leuargleutyrtyrproserglnaspasnaspargleuaspthrleu859095trpileproasnlysglutyrphetrpglyleuserlyspheleugly100105110thrhistrpleumetglyasnileleuargleuleupheglysermet115120125thrthrproalaasntrpasnserproleuargproglyglulystyr130135140proleuvalvalpheserhisglyleuglyalapheargthrleutyr145150155160seralaileglyileaspleualaserhisglypheilevalalaala165170175valgluhisargaspargseralaseralathrtyrtyrphelysasp180185190glnseralaalagluileglyasplyssertrpleutyrleuargthr195200205leulysglngluglugluthrhisileargasngluglnvalarggln210215220argalalysglucysserglnalaleuserleuileleuaspileasp225230235240hisglylysprovallysasnalaleuaspleulyspheaspmetglu245250255glnleulysaspserileaspargglulysilealavalileglyhis260265270serpheglyglyalathrvalileglnthrleusergluaspglnarg275280285pheargcysglyilealaleuaspalatrpmetpheproleuglyasp290295300gluvaltyrserargileproglnproleuphepheileasnserglu305310315320tyrpheglntyrproalaasnileilelysmetlyslyscystyrser325330335proasplysgluarglysmetilethrileargglyservalhisgln340345350asnphealaaspphethrphealathrglylysileileglyhismet355360365leulysleulysglyaspileaspserasnvalalaileaspleuser370375380asnlysalaserleualapheleuglnlyshisleuglyleuhislys385390395400asppheaspglntrpaspcysleuilegluglyaspaspgluasnleu405410415ileproglythrasnileasnthrthrasnglnhisilemetleugln420425430asnserserglyileglulystyrasn435440 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 504 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : exon ( b ) location : 185 .. 311 ( xi ) sequence description : seq id no : 9 : gcgcgctctagtagagccgggccacacacgctcctccccgtacctcctccagcatcaccg60aggggaaggagagggtcgggccacaaggcgcgctaggcggacccaggacacagcccgcgc120gcagcccacccgcccggccgcctgccaggagctgctgcggccgcgcagccagggggacag180gcgggctggtcggaggctcgcagtgctgtcggcgagaagcagtcgggtttggagcgcttg240ggtcgcgttggtgcgcggtggaacgcgcccagggaccccagttcccgcgagcagctccgc300gccgcgcctgagtgaggaggggccccgggggcgaggcgggagtgggaggaagggcacggt360cgccgcgctggaggtcgggaccccggagcgcgaccggccggggtgggctcgctgagtcgc420acccgctctgctggccggacctgggctcacagtcctgcagccctcggaaacagcgctagg480atccttcgggagaggagagatgac504 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 417 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : exon ( b ) location : 145 .. 287 ( xi ) sequence description : seq id no : 10 : gtaccaatctaaaacccagcacagaaaaatacatgttttattttttccaagtgttactag60tacctcagcctttcttgatttgtcagcttatttaaggcctcttcattgcatacttctttt120ttcttttaatcatctgcttcgaaggagactaagctgaaactgctgctcagctcccaagat180ggtgccacccaaattgcatgtgcttttctgcctctgcggctgcctggctgtggtttatcc240ttttgactggcaatacataaatcctgttgcccatatgaaatcatcaggtaagaggtgtat300ttgttcaaggtcttgagcaactgatctgtcgccatacttcaagtgggccccaagaagttg360cacatctgcacatctaaacaagtcctatttaaaggcttatggagatcctgtattctc417 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 498 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : exon ( b ) location : 251 .. 372 ( xi ) sequence description : seq id no : 11 : cattaggaggtaacagtccaaggcagctgagagaaaggctatgtctactttcatctcttt60accctccaaaacccctacacagtgtttcaaacagagagaccctcaataattgcatatctt120acttgttaggttgagaaagaaagaaggccagaaactatgggaagtaacttgattccgttg180gaattcttttgcataataaaatctgatatgtaatggatgacaaatgagataatatttacc240tgtttttcagcatgggtcaacaaaatacaagtactgatggctgctgcaacgtttggccaa300actaaaatcccccggggaaatgggccttattccgttggttgtacagacttaatgtttgat360cacactaataaggtaatgctttgatttatacaacttatcctgatactctaatattgtctg420tcgctatggaccactagaaggtgttcaaatgtgaccttgccctcacctgagaatgactca480ttttcgaatttgtattgt498 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 433 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : exon ( b ) location : 130 .. 274 ( xi ) sequence description : seq id no : 12 : cagcagcctaaagtcttagactttgtgaacacagaggtattgagtcccactaattaatat60cgaaaatagctgctggaatatgtttgagacacaacttctctaaaagtgcattaatttctt120tcttaacagggcaccttcttgcgtttatattatccatcccaagataatgatcaccttgac180accctttggatcccaaataaagaatatttttggggtcttagcaaatttcttggaacacac240tggcttatgggcaacattttgaggttactctttggtaagatttctgttgatccttctttg300taggctcttgcatgtatgaaaaccttgaaaacaacaagaacttcaagtagttaagaccaa360agtagatttttcttcagtccaaatagctcctaaaatgataaggaaagtatttctttaaag420cccaggcaactac433 ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 486 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : exon ( b ) location : 164 .. 257 ( xi ) sequence description : seq id no : 13 : ttggtgggtatctagtagcagtctttttaatgaatctactattcatccataaaaaagtag60atataaatcagatgggtctgcattttatgctaatgagatatgaattaaattcactagcaa120cactcagagaaaaccttaactataaccttccattgttgtctaggttcaatgacaactcct180gcaaactggaattcccctctgaggcctggtgaaaaatatccacttgttgttttttctcat240ggtcttggggcattcaggtaatgtttgagaggttgaacaattttggcttccaggaataaa300tgacaatttttttattcaagaaagaaatagcagagtttggaatgtcatgcaggcccttgt360ctggaggagttggggttcctcaataattggctgtgggtctattgatcagtcctagacctg420tctggtcaagtagttttttccctactatcagctcattgggattagcctcacagcagagaa480gaaagg486 ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 363 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : exon ( b ) location : 113 .. 181 ( xi ) sequence description : seq id no : 14 : ccccaggctctactacagggtgtaatggcctccatgttcccagttttattagtgactcag60ccttgtaattcatgactggtagttgtaattcttccctctttttgttttgaaggacacttt120attctgctattggcattgacctggcatctcatgggtttatagttgctgctgtagaacaca180ggtatgttacctgatataattgggctctttggccaactacagggaatgtcaatgctcata240actatgtttctaattttcataaaagtttatttaaaatgttgatggaactttcaagtatgg300taacatcatgagcaaaaaaggagattgagttttatcgacttaaaagacttaaaagcacct360aac363 ( 2 ) information for seq id no : 15 :( i ) sequence characteristics :( a ) length : 441 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : exon ( b ) location : 68 .. 191 ( xi ) sequence description : seq id no : 15 : gaactgagaaacatggtcagatgaggaagggaaggagcatgcataaataattttgcttgt60attatagagatagatctgcatctgcaacttactatttcaaggaccaatctgctgcagaaa120taggggacaagtcttggctctaccttagaaccctgaaacaagaggaggagacacatatac180gaaatgagcaggtacattgcagtgaaaggagaggtggttggtgacctaaaagcatgtaca240aaaggatgacatttgttaatttaattttacacctggcaagttatgctcctagctctccta300tttcccattcccaaaagatctgtcaatagattcctggagcagtaaaattcccttaatgga360atatctagttcatagtaaaaacaaaggcaaatacaaaaatttgggagatgacagtgaata420ttcagaattcctcgagccggg441 ( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 577 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : exon ( b ) location : 245 .. 358 ( xi ) sequence description : seq id no : 16 : ggttaagtaaatcgtctgaagtcacatagtaggtaaggcaaaacagagccaggatttgga60ctaaggctatacctatgtgcaaagctggggcctgtgtcattatggtagcaagtaatagtc120actaatcagatttccagtttataactgaccaacgatttttcccaaatacagcttctacct180aaactttaaaataagtgttataactttttactttgtcatttccttcttctaataattata240ttaggtacggcaaagagcaaaagaatgttcccaagctctcagtctgattcttgacattga300tcatggaaagccagtgaagaatgcattagatttaaagtttgatatggaacaactgaaggt360aagctataaaaagtaatttttctcttgtcctacagttctttattgttttttgtcatttaa420ttttctgcctatattgcaaggtacaatatgataaagggctgcaaccagcccctccccaat480gcgcacacacagacacacaaagcagtacaggtaaagtattgcagcaatgaagaatgcatt540atcttggactagatatgaatgcaaagttagtcagttt577 ( 2 ) information for seq id no : 17 :( i ) sequence characteristics :( a ) length : 396 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : exon ( b ) location : 108 .. 199 ( xi ) sequence description : seq id no : 17 : atcaatgtatttaccatccccatgaaatgaacaattatatgattgacaaatcatttcttc60taacaccacgaaatagctataaatttatatcatgctttttcaaataggactctattgata120gggaaaaaatagcagtaattggacattcttttggtggagcaacggttattcagactctta180gtgaagatcagagattcaggtaagaaaataagatagtaaagcaagagaatagtaaattat240tggaagaaattatattgtgagatataatttttattcaaattcttagtgaaggaaggggat300ctcttggagtttataaggctattcttttgcccccataaaatactctatatacattttcct360aggctaaaacatctcctctcctgctattaaaatctc396 ( 2 ) information for seq id no : 18 :( i ) sequence characteristics :( a ) length : 519 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : exon ( b ) location : 181 .. 351 ( xi ) sequence description : seq id no : 18 : cttacaaagttaatcatatccctttcccacattgaagtatgatacctctttattccaatc60agataacccataataaactggtatggtgcgtgtccaccaatcctagcattattaggatgt120cctcaatgttggctagtatgtaaccagtttaatttcatcattgtcaacaaatatctacag180atgtggtattgccctggatgcatggatgtttccactgggtgatgaagtatattccagaat240tcctcagcccctcttttttatcaactctgaatatttccaatatcctgctaatatcataaa300aatgaaaaaatgctactcacctgataaagaaagaaagatgattacaatcaggtaagtatt360agtgacttatttcattatgtgaaacaaacttgaagcttgggtaaatatcaatcgatatca420tttggtaactattaaagaattgctgaattggttgtttagactttcaataaggagagaatt480agataatctcagtttctaagtacatttagtctactcttt519 ( 2 ) information for seq id no : 19 :( i ) sequence characteristics :( a ) length : 569 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : exon ( b ) location : 156 .. 304 ( xi ) sequence description : seq id no : 19 : tgaaacacatctaagtagatcaaattacaagttttatttcttctttggttttcagtaaac60agaccaacaagaccagtacctttccttacactctaactaaaaaaataataattttatcaa120acaatgtgacttttaaatgtcttgttctcttttaggggttcagtccaccagaattttgct180gacttcacttttgcaactggcaaaataattggacacatgctcaaattaaagggagacata240gattcaaatgtagctattgatcttagcaacaaagcttcattagcattcttacaaaagcat300ttaggtaagaaactatttttttcatgacctaaaccgagatgaatctcgaggacaaagctg360tctatcttaatacagctttagtactatttaaactatttccagttggtttacaatggaaca420aagcagtatatcaatttgaaaacagaaatttgagaaagtcaattttgctgctttacatct480ctatatcatagaaagcaaatcaactgttaaaggtaatattctttgtatgagctagagtga540ctcatgtgaggatatcgaacgacggtgct569 ( 2 ) information for seq id no : 20 :( i ) sequence characteristics :( a ) length : 469 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : exon ( b ) location : 137 .. 253 ( xi ) sequence description : seq id no : 20 : gatacagaggcacatcgtctctaccatcctaacggaacttgtgtaatttgtaaatcttta60ttgccacctaggggcatccaaactgtttaatgctctcaaaagtttaatatgttgattaac120actttatattttataggacttcataaagattttgatcagtgggactgcttgattgaagga180gatgatgagaatcttattccagggaccaacattaacacaaccaatcaacacatcatgtta240cagaactcttcaggaatagagaaatacaattaggattaaaataggttttttaaaagtctt300gtttcaaaactgtctaaaattatgtgtgtgtgtgtgtgtgtgtgtgtgtgagagagagag360agagagagagagagagaattttaatgtattttcccaaaggactcatattttaaaatgtag420gctatactgtaatcgtgattgaagcttggactaagaattttttcccttt469 ( 2 ) information for seq id no : 21 :( i ) sequence characteristics :( a ) length : 1494 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( ix ) feature :( a ) name / key : cds ( b ) location : 117 .. 1436 ( xi ) sequence description : seq id no : 21 : ggcacgagctaggatctgactcgctctggtggcattgctgcgctcagggttctgggtatc60cgggagtcagtgcagtgaccagaacatcaaactgaagccactgctcagctcctaag116atggtaccactcaaactgcaggcgcttttctgcctcctctgctgcctc164metvalproleulysleuglnalaleuphecysleuleucyscysleu151015ccatgggtccatccttttcactggcaagacacatcttcttttgacttc212protrpvalhisprophehistrpglnaspthrserserpheaspphe202530aggccgtcagtaatgtttcacaagctccaatcggtgatgtctgctgcc260argproservalmetphehislysleuglnservalmetseralaala354045ggctctggccatagtaaaatccccaaaggaaatggatcgtaccccgtc308glyserglyhisserlysileprolysglyasnglysertyrproval505560ggttgtacagatctgatgttcggttatgggaatgagagcgtcttcgtg356glycysthraspleumetpheglytyrglyasngluservalpheval65707580cgtttgtactacccagctcaagatcaaggtcgcctcgacactgtttgg404argleutyrtyrproalaglnaspglnglyargleuaspthrvaltrp859095atcccaaacaaagaatattttttgggtcttagtatatttcttggaaca452ileproasnlysglutyrpheleuglyleuserilepheleuglythr100105110cccagtattgtaggcaatattttacacctcttatatggttctctgaca500proserilevalglyasnileleuhisleuleutyrglyserleuthr115120125actcctgcaagctggaattctcctttaaggactggagaaaaatacccg548thrproalasertrpasnserproleuargthrglyglulystyrpro130135140ctcattgtcttttctcatggtctcggagccttcaggacgatttattct596leuilevalpheserhisglyleuglyalapheargthriletyrser145150155160gctattggcattggcttggcatctaatgggtttatagtggccactgtc644alaileglyileglyleualaserasnglypheilevalalathrval165170175gaacacagagacagatctgcatcggcaacttacttttttgaagaccag692gluhisargaspargseralaseralathrtyrphephegluaspgln180185190gtggctgcaaaagtggaaaacaggtcttggctttacctgagaaaagta740valalaalalysvalgluasnargsertrpleutyrleuarglysval195200205aaacaagaggagtcggaaagtgtccggaaagaacaggttcagcaaaga788lysglngluglusergluservalarglysgluglnvalglnglnarg210215220gcaatagaatgttcccgggctctcagtgcgattcttgacattgaacat836alaileglucysserargalaleuseralaileleuaspilegluhis225230235240ggagacccaaaagagaatgtactaggttcagcttttgacatgaaacag884glyaspprolysgluasnvalleuglyseralapheaspmetlysgln245250255ctgaaggatgctattgatgagactaaaatagctttgatgggacattct932leulysaspalaileaspgluthrlysilealaleumetglyhisser260265270tttggaggagcaacagttcttcaagcccttagtgaggaccagagattc980pheglyglyalathrvalleuglnalaleusergluaspglnargphe275280285agatgtggagttgctcttgatccatggatgtatccggtgaacgaagag1028argcysglyvalalaleuaspprotrpmettyrprovalasngluglu290295300ctgtactccagaaccctccagcctctcctctttatcaactctgccaaa1076leutyrserargthrleuglnproleuleupheileasnseralalys305310315320ttccagactccaaaggacatcgcaaaaatgaaaaagttctaccagcct1124pheglnthrprolysaspilealalysmetlyslysphetyrglnpro325330335gacaaggaaaggaaaaatgattacaatcaagggctcaggcaccagaac1172asplysgluarglysasnasptyrasnglnglyleuarghisglnasn340345350tttgacgactttacttttgtaactggcaaaataattggaaacaagctg1220pheaspaspphethrphevalthrglylysileileglyasnlysleu355360365acactgaaaggagaaatcgattccagagtagccatcgacctcaccaac1268thrleulysglygluileaspserargvalalaileaspleuthrasn370375380aaagcttcgatggctttcttacaaaagcatttagggcttcagaaagac1316lysalasermetalapheleuglnlyshisleuglyleuglnlysasp385390395400tttgatcagtgggaccctctggtggaaggagatgatgagaacctgatt1364pheaspglntrpaspproleuvalgluglyaspaspgluasnleuile405410415cctgggtcaccctttgacgcagtcacccaggccccggctcagcaacac1412proglyserpropheaspalavalthrglnalaproalaglnglnhis420425430tctccaggatcacagacccagaattagaagaacttgcttgttacacagttgcct1466serproglyserglnthrglnasn435440tttaaaagtagagtgacatgagagagag1494 ( 2 ) information for seq id no : 22 :( i ) sequence characteristics :( a ) length : 2191 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( ix ) feature :( a ) name / key : cds ( b ) location : 92 .. 1423 ( xi ) sequence description : seq id no : 22 : ccgcgcgctccggccgggggaccctggttccggcgagcggctcagcgcggcgcccggaag60tttaagctgaaaccactgctcagcttccaagatgttgccacccaaactgcat112metleuproprolysleuhis15gcgcttttctgcctctgcagctgcctcacactggttcatcctattgac160alaleuphecysleucyssercysleuthrleuvalhisproileasp101520tggcaagacctaaatcctgttgcccatattagatcatcagcatgggcc208trpglnaspleuasnprovalalahisileargserseralatrpala253035aataaaatacaagctctgatggctgctgcaagtattaggcaaagtaga256asnlysileglnalaleumetalaalaalaserileargglnserarg40455055attcccaaaggaaatggatcttattctgtcggttgtacagatttgatg304ileprolysglyasnglysertyrservalglycysthraspleumet606570tttgattatactaataagggcacctttttgcgtttgtattatccatcg352pheasptyrthrasnlysglythrpheleuargleutyrtyrproser758085caagaggatgaccactctgacacgctttggatcccaaacaaagaatat400glngluaspasphisseraspthrleutrpileproasnlysglutyr9095100ttttttggtcttagtaaatatcttggaacaccctggcttatgggcaaa448phepheglyleuserlystyrleuglythrprotrpleumetglylys105110115atattgagcttcttttttggttcagtgacaactcctgcgaactggaat496ileleuserphephepheglyservalthrthrproalaasntrpasn120125130135tcccctctgaggactggtgaaaaatatccactgattgttttttctcat544serproleuargthrglyglulystyrproleuilevalpheserhis140145150ggtcttggagcattccggacaatttattctgctattggcattgatcta592glyleuglyalapheargthriletyrseralaileglyileaspleu155160165gcatcacatgggttcatcgttgctgctatagaacacagagatggatcc640alaserhisglypheilevalalaalailegluhisargaspglyser170175180gcctctgcgacttactatttcaaggaccagtctgctgcagaaataggg688alaseralathrtyrtyrphelysaspglnseralaalagluilegly185190195aacaaatcttggtcttatcttcaagaactaaaaccaggggatgaggag736asnlyssertrpsertyrleuglngluleulysproglyaspgluglu200205210215atacatgttcgaaatgagcaggtacagaaaagggcaaaggagtgctcc784ilehisvalargasngluglnvalglnlysargalalysglucysser220225230caagctctcaacttgattctggacattgatcatggaaggccaattaag832glnalaleuasnleuileleuaspileasphisglyargproilelys235240245aatgtactagacttagagtttgatgtggaacaactgaaggactctatt880asnvalleuaspleuglupheaspvalgluglnleulysaspserile250255260gacagggataaaatagcagtaattggacattcttttggtggagccaca928aspargasplysilealavalileglyhisserpheglyglyalathr265270275gttcttcaggctcttagtgaagaccagagatttaggtgcgggattgcc976valleuglnalaleusergluaspglnargpheargcysglyileala280285290295ttggatgcatggatgcttccactggatgatgcaatatattccagaatc1024leuaspalatrpmetleuproleuaspaspalailetyrserargile300305310cctcagcccctcttttttattaactcggaacggttccaatttcctgag1072proglnproleuphepheileasnsergluargpheglnpheproglu315320325aatatcaaaaaaatgaaaaaatgctactcacctgacaaagaaagaaaa1120asnilelyslysmetlyslyscystyrserproasplysgluarglys330335340atgattacaatcaggggttcagtccatcagaactttgctgatttcact1168metilethrileargglyservalhisglnasnphealaaspphethr345350355tttacaactggcaaaatagttggatacatattcacattaaaaggagat1216phethrthrglylysilevalglytyrilephethrleulysglyasp360365370375atagattcaaatgtagcaattgatctttgcaacaaagcttcattggca1264ileaspserasnvalalaileaspleucysasnlysalaserleuala380385390tttttacaaaagcatttaggactgcggaaagattttgatcagtgggat1312pheleuglnlyshisleuglyleuarglysasppheaspglntrpasp395400405tctttgattgaaggaaaagacgaaaatcttatgccagggaccaacatt1360serleuilegluglylysaspgluasnleumetproglythrasnile410415420aacatcaccaacgaacatgacactctacagaactctccagaagcagag1408asnilethrasngluhisaspthrleuglnasnserproglualaglu425430435aaatcgaatttagattaaaagcacttttttaaagatcttgtttaaaaactgtcaa1463lysserasnleuasp440aaaatgtgtgtatgacttttaatatattttctcaaataactcatattggaaaatgtaggc1523tatcccataaaagtgattgaagcttggactaggaggtttttttctttaaagaaagattgg1583tgtctatcgaaatcatgccagcctaaattttaattttactaaaatgatgctgtgtcaaaa1643ttaataactacttttacattctttaatggacaagtataacaggcacaaggctaatgaaaa1703cgtgttgcaatgacataacaatccctaaaaatacagatgttcttgcctcttttttctatt1763ataattgagttttagcaacatgttatgctaggtagaatttggaagcacttccctttgact1823tttggtcatgataagaaaaattagatcaagcaaatgataaaagcagtgttttaccaagga1883ttagggatactgaacaatttcactatggtaactgaatggggagtgaccaagggtaaaaat1943attaaagccaaggcaaaggcagcagattagaatggattaaagagagtttataatttgttt2003gcatttacttgatggtttatctcatggattcatgagtcaagaaaggtgcgtaggacaggc2063cagggattccagttataacacattattcacccaaagggttctttaattctgtatgagtat2123tgggagtggattagcacaatagaggcatatgttgctttaaaaaaaaaaaaaaaaaaaaaa2183aaaaaaaa2191 ( 2 ) information for seq id no : 23 :( i ) sequence characteristics :( a ) length : 517 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( ix ) feature :( a ) name / key : cds ( b ) location : 2 .. 514 ( xi ) sequence description : seq id no : 23 : ggggcattcttttggaggagcaacagtttttcaagccctaagtgaa46glyhisserpheglyglyalathrvalpheglnalaleuserglu151015gaccagagattcagatgtgggattgcccttgatccgtggatgtttccc94aspglnargpheargcysglyilealaleuaspprotrpmetphepro202530gtgagtgaggagctgtactccagagttcctcagcctctcttctttatc142valserglugluleutyrserargvalproglnproleuphepheile354045aactctgccgaattccagactccaaaggacattgcaaaaatgaaaaac190asnseralaglupheglnthrprolysaspilealalysmetlysasn505560ttctaccagcctgacaaggaaaggaaaatgattacgatcaagggctca238phetyrglnproasplysgluarglysmetilethrilelysglyser657075gtgcaccagaattttgctgacgggacttttgtaactggcaaaataatt286valhisglnasnphealaaspglythrphevalthrglylysileile80859095ggaaacaagctgtcactgaaaggagacatagactccagagttgccata334glyasnlysleuserleulysglyaspileaspserargvalalaile100105110gacctcaccaacaaggcttccttggctttcttacaaaaacatttagga382aspleuthrasnlysalaserleualapheleuglnlyshisleugly115120125cttcataaagactttgatcagtgggactgtctggtggagggagagaac430leuhislysasppheaspglntrpaspcysleuvalgluglygluasn130135140gagaacctcatcccggggtcaccctttgatgtagtcacccagtccccg478gluasnleuileproglyserpropheaspvalvalthrglnserpro145150155gctctgcagagttctcccggatcacacaaccagaattag517alaleuglnserserproglyserhisasnglnasn160165170 ( 2 ) information for seq id no : 24 :( i ) sequence characteristics :( a ) length : 580 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( ix ) feature :( a ) name / key : cds ( b ) location : 1 .. 580 ( xi ) sequence description : seq id no : 24 : caagtactgatggctgctgcaagctttggcgaacgtaaaatccctaag48glnvalleumetalaalaalaserpheglygluarglysileprolys151015ggaaatgggccttattccgttggttgtacagacttaatgtttgattac96glyasnglyprotyrservalglycysthraspleumetpheasptyr202530actaaaaagggcaccttcttgcgtttatattatccatcccaagatgat144thrlyslysglythrpheleuargleutyrtyrproserglnaspasp354045gatcgccttgacaccctttggatcccaaataaggagtatttttggggt192aspargleuaspthrleutrpileproasnlysglutyrphetrpgly505560cttagcaagtatcttggaaaacactggcttatgggcaacattttgagt240leuserlystyrleuglylyshistrpleumetglyasnileleuser65707580ttactctttggttcagtgacaactcctgcaaactggaattcccctctg288leuleupheglyservalthrthrproalaasntrpasnserproleu859095aggcctggtgaaaaatacccacttgttgttttttctcatggtcttgga336argproglyglulystyrproleuvalvalpheserhisglyleugly100105110gcattcaggacaatttattctgctattggcattgacctggcatctcat384alapheargthriletyrseralaileglyileaspleualaserhis115120125gggtttatagttgctgctgtagaacacagagatagatctgcatctgca432glypheilevalalaalavalgluhisargaspargseralaserala130135140acttactatttcaagaaccaatctgctgcagaaatagggaaaaagtct480thrtyrtyrphelysasnglnseralaalagluileglylyslysser145150155160tggctctaccttagaaccctgaaagaagaggaggagatacatatacga528trpleutyrleuargthrleulysglugluglugluilehisilearg165170175aataagcaggtacgacaaagagcaaaagaatgttcccaagctctcagt576asnlysglnvalargglnargalalysglucysserglnalaleuser180185190ctga580leu ( 2 ) information for seq id no : 25 :( i ) sequence characteristics :( a ) length : 41 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( xi ) sequence description : seq id no : 25 : tattctagaattatgatacaagtattaatggctgctgcaag41 ( 2 ) information for seq id no : 26 :( i ) sequence characteristics :( a ) length : 32 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( xi ) sequence description : seq id no : 26 : attgatatcctaattgtatttctctattcctg32 ( 2 ) information for seq id no : 27 :( i ) sequence characteristics :( a ) length : 440 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 27 : metvalproleulysleuglnalaleuphecysleuleucyscysleu151015protrpvalhisprophehistrpglnaspthrserserpheaspphe202530argproservalmetphehislysleuglnservalmetseralaala354045glyserglyhisserlysileprolysglyasnglysertyrproval505560glycysthraspleumetpheglytyrglyasngluservalpheval65707580argleutyrtyrproalaglnaspglnglyargleuaspthrvaltrp859095ileproasnlysglutyrpheleuglyleuserilepheleuglythr100105110proserilevalglyasnileleuhisleuleutyrglyserleuthr115120125thrproalasertrpasnserproleuargthrglyglulystyrpro130135140leuilevalpheserhisglyleuglyalapheargthriletyrser145150155160alaileglyileglyleualaserasnglypheilevalalathrval165170175gluhisargaspargseralaseralathrtyrphephegluaspgln180185190valalaalalysvalgluasnargsertrpleutyrleuarglysval195200205lysglngluglusergluservalarglysgluglnvalglnglnarg210215220alaileglucysserargalaleuseralaileleuaspilegluhis225230235240glyaspprolysgluasnvalleuglyseralapheaspmetlysgln245250255leulysaspalaileaspgluthrlysilealaleumetglyhisser260265270pheglyglyalathrvalleuglnalaleusergluaspglnargphe275280285argcysglyvalalaleuaspprotrpmettyrprovalasngluglu290295300leutyrserargthrleuglnproleuleupheileasnseralalys305310315320pheglnthrprolysaspilealalysmetlyslysphetyrglnpro325330335asplysgluarglysasnasptyrasnglnglyleuarghisglnasn340345350pheaspaspphethrphevalthrglylysileileglyasnlysleu355360365thrleulysglygluileaspserargvalalaileaspleuthrasn370375380lysalasermetalapheleuglnlyshisleuglyleuglnlysasp385390395400pheaspglntrpaspproleuvalgluglyaspaspgluasnleuile405410415proglyserpropheaspalavalthrglnalaproalaglnglnhis420425430serproglyserglnthrglnasn435440 ( 2 ) information for seq id no : 28 :( i ) sequence characteristics :( a ) length : 444 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 28 : metleuproprolysleuhisalaleuphecysleucyssercysleu151015thrleuvalhisproileasptrpglnaspleuasnprovalalahis202530ileargserseralatrpalaasnlysileglnalaleumetalaala354045alaserileargglnserargileprolysglyasnglysertyrser505560valglycysthraspleumetpheasptyrthrasnlysglythrphe65707580leuargleutyrtyrproserglngluaspasphisseraspthrleu859095trpileproasnlysglutyrphepheglyleuserlystyrleugly100105110thrprotrpleumetglylysileleuserphephepheglyserval115120125thrthrproalaasntrpasnserproleuargthrglyglulystyr130135140proleuilevalpheserhisglyleuglyalapheargthriletyr145150155160seralaileglyileaspleualaserhisglypheilevalalaala165170175ilegluhisargaspglyseralaseralathrtyrtyrphelysasp180185190glnseralaalagluileglyasnlyssertrpsertyrleuglnglu195200205leulysproglyaspglugluilehisvalargasngluglnvalgln210215220lysargalalysglucysserglnalaleuasnleuileleuaspile225230235240asphisglyargproilelysasnvalleuaspleuglupheaspval245250255gluglnleulysaspserileaspargasplysilealavalilegly260265270hisserpheglyglyalathrvalleuglnalaleusergluaspgln275280285argpheargcysglyilealaleuaspalatrpmetleuproleuasp290295300aspalailetyrserargileproglnproleuphepheileasnser305310315320gluargpheglnpheprogluasnilelyslysmetlyslyscystyr325330335serproasplysgluarglysmetilethrileargglyservalhis340345350glnasnphealaaspphethrphethrthrglylysilevalglytyr355360365ilephethrleulysglyaspileaspserasnvalalaileaspleu370375380cysasnlysalaserleualapheleuglnlyshisleuglyleuarg385390395400lysasppheaspglntrpaspserleuilegluglylysaspgluasn405410415leumetproglythrasnileasnilethrasngluhisaspthrleu420425430glnasnserproglualaglulysserasnleuasp435440 ( 2 ) information for seq id no : 29 :( i ) sequence characteristics :( a ) length : 171 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 29 : glyhisserpheglyglyalathrvalpheglnalaleusergluasp151015glnargpheargcysglyilealaleuaspprotrpmetpheproval202530serglugluleutyrserargvalproglnproleuphepheileasn354045seralaglupheglnthrprolysaspilealalysmetlysasnphe505560tyrglnproasplysgluarglysmetilethrilelysglyserval65707580hisglnasnphealaaspglythrphevalthrglylysileilegly859095asnlysleuserleulysglyaspileaspserargvalalaileasp100105110leuthrasnlysalaserleualapheleuglnlyshisleuglyleu115120125hislysasppheaspglntrpaspcysleuvalgluglygluasnglu130135140asnleuileproglyserpropheaspvalvalthrglnserproala145150155160leuglnserserproglyserhisasnglnasn165170 ( 2 ) information for seq id no : 30 :( i ) sequence characteristics :( a ) length : 193 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 30 : glnvalleumetalaalaalaserpheglygluarglysileprolys151015glyasnglyprotyrservalglycysthraspleumetpheasptyr202530thrlyslysglythrpheleuargleutyrtyrproserglnaspasp354045aspargleuaspthrleutrpileproasnlysglutyrphetrpgly505560leuserlystyrleuglylyshistrpleumetglyasnileleuser65707580leuleupheglyservalthrthrproalaasntrpasnserproleu859095argproglyglulystyrproleuvalvalpheserhisglyleugly100105110alapheargthriletyrseralaileglyileaspleualaserhis115120125glypheilevalalaalavalgluhisargaspargseralaserala130135140thrtyrtyrphelysasnglnseralaalagluileglylyslysser145150155160trpleutyrleuargthrleulysglugluglugluilehisilearg165170175asnlysglnvalargglnargalalysglucysserglnalaleuser180185190leu__________________________________________________________________________