Patent Application: US-41900995-A

Abstract:
an almond n - glycosidase gene and genes associated therewith are described . vectors integrated such genes therein , recombinant microorganisms transformed with said vectors , and a process for preparing the almond n - glycosidase using said recombinant microorganisms are also described .

Description:
to obtain the almond n - glycosidase of the present invention , firstly , the almond n - glycosidase in an acetone powder was subjected to various chromatographies to be highly purified . it was found that the purified almond n - glycosidase protein was composed of a light chain and a heavy chain , the molecular weights of which were 27000 and 60000 on sds - page , respectively , which are not covalently bonded each other . it has not been shown heretofore in the prior art that an almond n - glycosidase is composed of such two polypeptides . in addition , it has been quite unknown whether they are coded on different genes and expressed individually , or coded on the same gene and generated by a cleavage after translation into proteins . therefore , for cloning of a cdna coding the almond n - glycosidase , unlike the case where an usual single chain polypeptide is cloned , it is necessary to consider the relationship between genes for the two polypeptides as above mentioned . for cloning the cdna coding the almond n - glycosidase , for example , the following procedure can be used . firstly , the amino acid sequences of the portions of the highly purified two polypeptides of the almond n - glycosidase , that is , the light chain and the heavy chain are analyzed . the amino acid sequences of these portions can be determined from n - terminal of the proteins directly by edman method . alternatively , the sequences can be determined by hydrolysing the proteins into fragments with a protease such as trypsin and lysyl endopeptidase , isolating and purifying them , then determining individually . on the basis of the amino acid sequences of these portions , synthesized dnas of appropriate length are prepared according to the fluctuation to be used as primers or probes . then , rna is extracted from a tissue abundant in the almond n - glycosidase , for example almond seeds , and poly ( a )- rna is purified therefrom using a carrier such as a latex carrier to which oligo -( dt ) is bound . cdna is synthesized using the poly ( a )- rna as a template by the action of reverse transcriptase . the dna is inserted into a plasmid or a phage vector , and introduced into a host to make a cdna library according to a method , for example okayama - berg method or gubler - hoffmann method . as for procedures to obtain a cdna coding the desired almond n - glycosidase , for example the following two procedures can be applied . one procedure is to screen the cdna library directly using the dna previously synthesized as a probe . alternatively , it is effective to use the dna fragment as a probe when the portion of the desired cdna has been obtained . that is , firstly , the library is cultured on plates , and colonies or plaques grown are transferred to nitrocellulose membranes or nylon membranes , then the dnas are immobilized to the membranes by denaturation . these membranes are incubated in a solution containing the probe dna labelled with for example 32 p to form hybrids ( hereinafter referred this treatment as hybridization ). the incubation temperature is set slightly lower than the tm ( melting temperature ) of the probe used . after hybridization , non - specifically adsorbed matters are washed out , and clones hybridized to the probe are identified by a means such as autoradiography . in addition , in this case , when hybridization to the library dna immobilized to a membrane is carried out using the dnas corresponding to the partial amino acid sequences of the light chain and the heavy chain , respectively as probes , the relationship between the light chain and the heavy chain will be clarified by whether hybridization to the same clone occurs or not . this operation is repeated until the hybridized clones become a single clone . the cdna coding the desired protein is inserted into the clone thus obtained . another procedure is to amplify the cdna coding the desired almond n - glycosidase using the cdna as a template synthesized from the poly ( a )- rna from almond seeds by pcr ( polymerase - chain - reaction ) method . as the primers which are used , a pair of synthesized dnas assumed from the partial amino acid sequence of the almond n - glycosidase ; the synthesized dna and a random primer ( a mixture of the all sequences ); or the synthesized dna and a primer the sequence of which is known , are exemplified . to use the primers the sequence of which are known , it is necessary to add a region to the terminal of the desired dna , to which the primers having known sequences can anneal . to add the region , a known method in which dna ligase is used can be used . the amplification is not always occur by any probe , especially non - specific amplification or no amplification tends to occur because synthesized dna assumed from the partial amino acid sequence contains mixed sequences , therefore it is necessary to investigate what primers that have been prepared according to what partial amino acid sequence should be used , and what combination of the primers should be used . in addition , it is necessary to select the pcr condition suitable to the primers . to amplify the desired dna effectively , it is effective to carry out nested pcr . that is , firstly , pcr is performed among primers which have been prepared , then using the amplified product as a template , the second pcr is performed using primers coding the inner sequences compared with the primers used in the primary pcr . for pcr method , a gene amplifying kit containing taq polymerase , and an automatic gene amplifying apparatus are available from takara shuzo co ., ltd . the desired dna region can be amplified using them . further , dna primers are synthesized on the basis of the sequences of the cdna fragments of the light chain and the heavy chain amplified by pcr . depending on whether amplification can occur using primers from the heavy chain and the light chain or not , it is clarified whether the light chain and the heavy chain are coded on the same gene or not , in other words , the relationship of the location of the light chain and the heavy chain on the gene is clarified . the sequence of the cdna obtained is determined , and identified whether it is the desired gene or not . in case that the clone has been obtained by hybridization , it is cultured , for example in a test tube to extract the plasmid according to a conventional method when the recombinant is e . coli . this plasmid is digested by a restriction enzyme to obtain a fragment for insertion . the fragment is subcloned into a vector such as m13 phage vector to determine the sequence by dideoxy method . when the recombinant is a phage , sequencing can be carried out basically in similar steps . in case that the cdna has been obtained by pcr method , the amplified dna is purified by agarose gel electrophoresis , and the terminal is blunt - ended . the dna is subcloned into a vector such as m13 phage , and the sequence is determined by dideoxy method . as for these experimental methodologies from culture to nucleotide sequencing , see for example t . maniatis et al ., molecular cloning . a laboratory manual ( 1982 ), cold spring harbor laboratory . the molecular weight , and the result obtained by the n - terminal analysis of the almond n - glycosidase , the gene structure and the amino acid sequence can be determined by comparing the determined sequence with the partial amino acid sequence . when the cdna which has been obtained does not contain the coding region of the almond n - glycosidase at all , the whole nucleotide sequence of the coding region can be determined by synthesizing dna primers on the basis of the nucleotide sequence obtained to amplify the lacking portion by pcr , or by further screening the cdna library using the fragment of the cdna obtained as a probe . to obtain a polypeptide or a protein which has an almond n - glycosidase activity , firstly , the almond n - glycosidase gene is connected with a vector that can express them in a suitable host cell , for example bacteria such as e coli , bacillus subtilis , yeasts , animal cells , insect cells , and plant cells , then the transformation is performed thereby to prepare a recombinant organism . by culturing the recombinant organism , the polypeptide having the almond n - glycosidase can be produced . construction of such a vector , and transformation of the host cell using them are preformed by methods known to those skilled in the art . alternatively , a polypeptide without sugar chain can be expressed by using cells as hosts which do not have an ability of synthesizing sugar chains , for example , procaryotes such as e . coli and bacillus subtilis , or mutant cells of yeasts , animal calls , insect cells and plant cells , which have lost an ability of synthesizing sugar chains . the polypeptide or the protein can be accumulated as an insoluble matter ( inclusion body ), depending on the expression system used . in this case , the activity can be recovered by collecting the insoluble matter , and solubilize it under a mild condition , for example with urea , then removing the denaturating reagent . the expression can be confirmed by assaying the almond n - glycosidase activity . for example , the assay can be carried out using dabsyl ovomucoid glycopeptide ( manufactured by genzyme ) as a substrate according to the manual appended . that is , after incubation the enzyme solution with dabsyl ovomucoid at 37 ° c ., assay of the almond glycosidase activity can be performed by separating and detecting the dabsyl peptide produced by liberation of the sugar chain from the substrate , using reverse phase chromatography and thin layer chromatography . alternatively , after labelling the reducing terminal of the sugar chain which has been liberated by pyridylamination , the activity can be assayed by separation and quantification of the labelled sugar chain using high performance liquid chromatography . to purify a polypeptide or a protein which has an almond glycosidase activity from the transformant , common chromatography techniques can be used . for example , when the cultured cells have been broken and the desired polypeptide has been solubilized , the desired polypeptide expressed can be obtained by subjecting the supernatant of the culture to chromatographies such as hydrophobic , ion exchange and gel filtration . when the expressed product is accumulated as an insoluble matter , the cells are broken , and the precipitation is collected to be solubilized with a denaturating reagent such as urea . then , the polypeptide or the protein having the desired activity can be obtained after removing the denaturating reagent and refolding . an example of determination of the structure of the almond n - glycosidase gene is shown below . firstly , cdna for the n - terminal of the heavy chain is amplified by pcr method to determine the nucleotide sequence . the n - terminal amino acid sequence of the highly purified heavy chain of the almond n - glycosidase protein is determined . the sequence is shown in seq id no : 4 . a sense mixed primer hnt1 ( seq id no : 5 ) corresponding to the portion of amino acids 3 - 9 , and a sense mixed primer hnt2 ( seq id no : 6 ) corresponding to the portion of amino acids 8 - 14 are synthesized by a dna synthesizer , and purified . inner partial amino acid sequences are shown in seq id no : 7 to 14 . anti sense mixed primers are synthesized on the basis of these sequences , and purified . that is , an anti sense mixed primer hic1 ( seq id no : 15 ) corresponding to the region of amino acids 12 - 19 of the inner sequence of the heavy chain shown in seq id no : 7 , and an anti sense mixed primer hic2 ( seq id no : 16 ) and hic3 ( seq id no : 17 ) corresponding to the region of amino acids 12 - 19 and 2 - 8 , respectively , shown in seq id no : 11 are synthesized . the first chain of the cdna is prepared using d ( t ) 20 m4 ( seq id no : 18 ) as a primer synthesized by a dna synthesizer using the mrna from almond seeds as a template . using the primers and the first chain of the cdna , nested pcr is carried out . the primary pcr is carried out using the first chain of the cdna as a template , and using three combinations of primers , hnt1 and hic1 , hnt1 and hic2 , and hnt1 and hic3 . each reaction mixture is named pcr1 , pcr2 and pcr3 . the secondary pcr is carried out using three reaction mixtures from the primary pcr as templates respectively , and using three combinations of primers to each template , that is , hnt2 and hic1 , hnt2 and hic2 , and hnt2 and hic3 . each reaction mixture is named pcr - 1 , pcr1 - 2 , pcr1 - 3 , pcr2 - 1 , pcr2 - 2 , pcr2 - 3 , pcr3 - 1 , pcr3 - 2 and pcr3 - 3 . a fragment about 500 bp which is specifically amplified n - terminal portion of the heavy chain ( fhn ) is obtained only from pcr2 - 2 of the reaction mixtures from the secondary pcr . this fhn is cloned . for the four clones , inserted sequences of the fhn ( fhn1 - fhn4 ) are determined . these sequences are shown in seq id no : 19 - 22 , respectively . then , cdna containing the c - terminal portion of the heavy chain is amplified by pcr method to determine the nucleotide sequence . for this determination , two primers shown in seq id no : 23 and 24 are designed from the sequences of fhn1 - fhn4 which have been already determined , and synthesized by a dna synthesizer . that is , these two primers are sense primer hi1 and hi2 shown in seq id no : 23 and seq id no : 24 , which correspond to nucleotides 72 - 95 and nucleotides 148 - 164 of fhn1 - fhn4 , respectively , shown in seq id no 19 - 22 . further , a synthesized dna m13 primer m4 ( seq id no : 25 , takara shuzo co ., ltd .) which has the same sequence as nucleotides 1 - 17 of d ( t ) 20m 4 ( seq id no : 18 ) is used as an anti sense primer . using these primers and the first chain of the cdna , nested pcr is carried out . the primary pcr is carried out using the first chain of the cdna as a template , and hi1 ( seq id no : 23 ) and m13 primer m4 ( seq id no : 25 ) as primers . the secondary pcr is carried out using the reaction mixtures from the primary pcr as templates , and hi2 ( seq id no : 24 ) and m13 primer m4 ( seq id no : 25 ) as primers . by the secondary pcr , a dna fragment of approximately 1 . 2 kbp ( fhc ) containing the c - terminal portion of the heavy chain which has been amplified specifically . this fhc is cloned . for the four clones , inserted sequences of the fhc ( fhc1 - fhc4 ) are determined . the sequences are shown in seq id no : 26 - 29 . then , cdna containing the full length of the light chain of the almond n - glycosidase is amplified by pcr to determine the nucleotide sequence . as in the case of the heavy chain , the n - terminal amino acid sequence of the highly purified light chain of the almond n - glycosidase protein is determined . the sequence is shown in seq id no : 30 . on the basis of the amino acid sequence of the light chain ( seq id no : 30 ), sense mixed primers shown in seq id no : 31 - 34 are designed respectively , and synthesized by a dna synthesizer , then purified . also , on the basis of the sequence of the cdna of the heavy chain previously determined , 10 anti sense mixed primers are designed , and synthesized by a dna synthesizer , then purified . that is , sense mixed primers ln1c and ln1t shown in seq id no : 31 and seq id no : 32 which correspond to amino acids 1 - 8 of the n - terminal sequence of the light chain shown in seq id no : 30 ; a sense mixed primer ln2 shown in seq id no : 33 which corresponds amino acids 6 - 12 thereof ; and a primer ln3 shown in seq id no : 34 which corresponds to amino acids 12 - 18 thereof are synthesized . in addition , as an anti sense primer , primer hic4 shown in seq id no : 35 which corresponds to nucleotides 262 - 286 of seq id no : 19 - 22 and to nucleotides 125 - 149 of seq id no : 32 - 35 is synthesized . using these primers and the first chain of the cdna , nested pcr is carried out . when the light chain and the heavy chain exist tandem in this order on the same gene , it is possible that the gene coding the full length of the light chain and the n - terminal portion of the heavy chain is amplified . the primary pcr is carried out using the first chain of the cdna as a template , and using three combinations of primers , ln1c and hic4 , ln1t and hic4 , and ln2 and hic4 . each reaction mixture is named pcr4 , pcr5 and pcr6 . the secondary pcr is carried out using three reaction mixtures from the primary pcr as templates respectively . to templates pcr4 and pcr5 , the secondary pcr is carried out using two combinations 10 of primers , that is , ln2 and hic4 , and ln3 and hic4 . the reaction mixtures are named pcr4 - 1 , pcr4 - 2 , pcr5 - 1 and pcr5 - 2 , respectively . to a template pcr6 , the pcr reaction is carried out using primers ln3 and hic4 , and the reaction mixture named pcr6 - 1 . an amplified dna fragment of approximately 900 bp containing n - termini of the light chain and the heavy chain ( fl ) is obtained only from reaction mixtures pcr4 - 1 and pcr5 - 2 . therefore , it is thought that the light chain and the heavy chain are coded tandem in this order on the same gene . then , fl is cloned . for the six clones , the nucleotide sequences of the fl ( fl1 - fl6 ) inserted are determined . the sequences are shown in seq id no : 36 - 41 , respectively . when no dna fragment containing the light chain is amplified by the method above mentioned , the relation of the location between the light chain and the heavy chain can be investigated after determination using the similar procedure in the case of the heavy chain . the relationship among fhn , fhc and fl above described , and the individual restriction maps are shown in fig1 . the region coding the almond n - glycosidase is determined by applying the result of the analysis of the amino acid sequence of the almond n - glycosidase to the sequences of fhn , fhc and fl . the nucleotide sequence of the cdna which can code the almond n - glycosidase is shown in seq id no : 2 , and the amino acid sequence which can be coded by the nucleotide sequence can code is shown seq id no : 1 . to produce a polypeptide or a protein having an almond n - glycosidase activity , firstly , the dna coding the almond n - glycosidase is connected with a vector . in this case , either a fragment fl or fhc amplified by pcr , or a synthesized dna can be used as a dna coding the almond n - glycosidase . alternatively , the nucleotide sequence can be selected so that it will fit for the sequence determined by the analysis of the partial amino acid sequence of the almond n - glycosidase . the polypeptide having the almond n - glycosidase activity can be produced by introducing the vector for expression of the almond n - glycosidase thus obtained into a host cell , and culturing the transformed cell . as a host cell , the cell above mentioned can be used . for example , a fission yeast such as schizosaccharomyces pombe atcc 38440 can be used . in the examples below , schizosaccharomyces pombe strain t - 1 which is belong to the present applicants and have the same characters as 38400 is used . using these genes obtained as probes , all n - glycosidase genes which have slightly different sequences and are expected to have the similar activity can be obtained by hybridization under a stringent condition . as sources of the gene to be hybridized , dnas and mrnas obtained from bacteria , fungi , plant and animal cells can be used . for example , as for plants , dnas and mrnas obtained from tissues such as seeds and shoots can be used , and as for animals , dnas and mrnas obtained from cultured cells and tissues of internal organs can be used . examples of plants contain plants of family rosaceae such as apricot , plum , peach , cherry and loquat , and plants of different families , for example plants of family leguminosae such as sword bean , horse bean , liquorice , wisteria , pea , clara , and plants of family brassicaceae and family cucurbitaceae , and family solanaceae . such a stringent condition is , for example , as follows : dna which is immobilized to a nylon membrane is hybridized to a probe in a solution containing 6 × ssc ( 1 × ssc consists of sodium chloride 8 . 7 g , and sodium citrate 4 . 41 g in 1 l of water ), 1 % sodium lauryl sulfate , 100 μg / ml salmon sperm dna , and 5 × denhart &# 39 ; s ( containing 0 . 1 % bovine serum albumin , 0 . 1 % polyvinylpyrrolidone and 0 . 1 % ficoll ), at 65 ° c . for 20 hours . to obtain the desired gene coding the n - glycosidase by hybridization , for example , following method can be applied : firstly , chromosomal dna obtained from the desired gene source , or cdna prepared from mrna by reverse transcriptase is connected to a plasmid or a phage vector according to a conventional method to prepare a library . the library is cultured on plates . grown colonies or plaques are transferred 10 onto a nitrocellulose or a nylon membrane , and the dnas are immobilized to the membrane by denaturation . this membrane is incubated in a solution containing a probe previously labelled with for example 32 p to hybridize the dna on the membrane to the probe . a probe used include a gene coding an amino acid sequence described seq id no : 1 or a portion thereof . for example , a gene described in seq id no : 2 or a portion thereof can be used . for example , a gene coding an amino acid sequence described in seq id no : 4 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 19 , 20 , 21 , 22 , 26 , 27 , 28 , 29 , 30 , 36 , 37 , 38 , 39 , 40 or 41 , or a portion thereof can be used . for example , hybridization between dna immobilized to a membrane and a probe is carried out in a solution containing 6 × ssc , 1 % sodium lauryl sulfate , 100 μg / ml salmon sperm dna , and 5 × denhart &# 39 ; s ( containing bovine serum albumin , polyvinylpyrrolidone , ficoll , each 0 . 1 %) at 65 ° c . for 20 hours . after hybridization , non - specifically adsorbed matters are washed out , and hybridized clones are identified by a means such as autoradiography . this operation is repeated until the hybridized clones become a single clone . the gene coding the desired protein is inserted in the clone thus obtained . the gene obtained is sequenced , for example as described below and identified to code the desired n - glycosidase . to determine the nucleotide sequence , in case that the clone has been obtained by hybridization , it is cultured , for example in a test tube to extract the plasmid according to a conventional method when the recombinant is e . coli . this plasmid is digested by a restriction enzyme to obtain a fragment for insertion . the fragment is subcloned into a vector such as m13 phage vector to determine the sequence by dideoxy method . when the recombinant is a phage , sequencing can be carried out basically in similar steps . these experimental methods from culture to nucleotide sequencing are described in , for example , t . maniatis et al ., molecular cloning a laboratory manual ( 1982 ) cold spring harbor laboratory . to identify whether the gene obtained codes the desired almond n - glycosidase , the structure and the amino acid sequence can be known , by comparing the nucleotide sequence determined with the almond n - glycosidase gene of the present invention and with the amino acid sequence described in seq id no : 1 . when the gene which has been obtained does not contain the coding region of the n - glycosidase at all , the whole nucleotide sequence of the coding region which hybridizes to the almond n - glycosidase gene of the present invention can be determined by synthesizing dna primers on the basis of the nucleotide sequence obtained to amplify the lacking portion by pcr , or by further screening the cdna library using the fragment of the cdna obtained as a probe . to obtain a polypeptide having a n - glycosidase activity using genetic engineering , firstly , according to a standard method , the almond n - glycosidase gene is connected with a vector that can express them in a suitable host cell , for example bacteria such as e coli , bacillus subtilis , yeasts , mammalian cells , insect cells , and plant cells . the recombinant vector is introduced into a host cell to prepare a recombinant cell . by culturing the recombinant cell , a polypeptide having a n - glycosidase activity can be produced . alternatively , a polypeptide without sugar chain can be expressed by using cells as hosts which do not have an ability of synthesizing sugar chains , for example , procaryotes such as e . coli and bacillus subtilis , or mutant cells of yeasts , mammalian cells , insect cells and plant cells , which have lost an ability of synthesizing sugar chains . the polypeptide or the protein can be accumulated as an insoluble matter ( inclusion body ), depending on the expression system used . in this case , the activity can be recovered by collecting the insoluble matter , and solubilize it under a mild condition , for example with urea , then removing the denaturating reagent . the expression can be confirmed by assaying the almond n - glycosidase activity . for example , the assay can be carried out using dabsyl ovomucoid glycopeptide ( manufactured by genzyme ) as a substrate according to the manual appended . that is , after incubation the enzyme solution with dabsyl ovomucoid at 37 ° c ., assay of the almond glycosidase activity can be performed by separating and detecting the dabsyl peptide produced by liberation of the sugar chain from the substrate , using reverse phase chromatography and thin layer chromatography . to purify a polypeptide or a protein which has an almond n - glycosidase activity from the transformant , common chromatography techniques can be used . for example , when the cultured cells have been broken and the desired polypeptide has been solubilized , the desired polypeptide expressed can be obtained by subjecting the supernatant of the culture to chromatographies such as hydrophobic , ion exchange and gel filtration . when the expressed product is accumulated as an insoluble matter , the cells are broken , and the precipitation is collected to be solubilized with a denaturating reagent such as urea . then , the polypeptide or the protein having the desired activity can be obtained after removing the denaturating reagent and refolding . thus , according to the present invention , a primary structure of an almond n - glycosidase gene is elucidated , and it is possible to provide a procedure of genetic engineering for preparing it . also , similar n - glycosidase genes are provided using the genes of the present invention . moreover , by said procedure of genetic engineering , it is possible to produce an almond n - glycosidase having no sugar chain . the following examples further illustrate the present invention in detail . however , they are not to construed to limit the scope of the present invention . two kg of a commercial almond meal ( manufactured by sigma ) was emerged in 10 mm bis - tris buffer solution ( ph 6 . 8 ) containing 2 mm phenyl metanesulfonyl fluoride ( pmsf ) and 1 mm 1 , 2 - epoxy - 3 -( p - nitrophenoxy ) propane ( epnp ) with stirring for 20 hour at 5 ° c ., then the mixture was filtered through a filter cloth , and centrifuged to obtain a supernatant . after precipitating by adding ammonium sulfate to the supernatant to 80 % saturation , dialysis of the precipitation was performed against 10 mm mes buffer solution ( ph 6 . 8 ). the dialyzed solution was applied to a column of an ion exchange resin de52 ( manufactured by whatman ) equilibrated with 10 mm mes buffer solution ( ph 6 . 8 ). the wash - through fraction with the same buffer solution was subjected to ion exchange chromatography on cm - sepharose cl - 6b ( manufactured by pharmacia ), and eluted with a gradient of potassium chloride ( 0 to 0 . 5m ). the active fraction eluted was dialyzed against 10 mm mops buffer solution ( ph 7 . 0 ). a precipitation which generated during the dialysis was removed by centrifugation . the dialyzed solution was subjected to ion exchange column chromatography on mono s ( manufactured by pharmacia ), and eluted with a gradient of potassium chloride ( 0 to 0 . 4m ). the active fraction eluted was dialyzed against 10 mm tris buffer solution ( ph 8 . 8 ). the dialyzed solution was subjected to ion exchange column chromatography on mono q ( manufactured by pharmacia ), and eluted with a gradient of potassium chloride ( 0 to 0 . 5m ). the active fraction eluted was diluted four times with 10 mm tris buffer solution ( ph 8 . 8 ). the diluted active fraction was again subjected to ion exchange column chromatography on mono q , and eluted with a gradient of potassium chloride ( 0 to 0 . 5m ). after concentration of the active fraction eluted by ultrafiltration using centricon 10 ( manufactured by amicon ) of exclusive molecular weight 10000 , the concentrate was subjected to gel permeation column chromatography on superrose 12hr ( manufactured by pharmacia ), and eluted with 10 mm tris buffer solution ( ph 8 . 8 ) containing 0 . 1m sodium chloride to obtain the active fraction . by above operations , the almond n - glycosidase was purified to a homogeneous protein . the purified almond n - glycosidase was composed of two polypeptide chains , a light chain and a heavy chain , which showed molecular weights 27000 and 60000 on sds - page , respectively . ( 1 - 2 ) analysis of the n - terminal amino acid sequence and the inner partial amino acid sequence after dissociating the purified almond n - glycosidase to a light chain and a heavy chain by sds - page , these polypeptides were electrophoretically blotted ( electroblotted ) to a polyvinylidene difluoride ( pvdf ) membrane using sartoblot ii ( manufactured by sartorius ), and sections containing the light chain and the heavy chain on the membrane were cut out separately to obtain samples for determination of the n - terminal amino acid sequences . the amino acid sequences of the samples prepared were determined from the n - terminal one after another by edman method using a gas phase peptide sequencer ( model 477 , manufactured by applied biosystem ). the n - terminal amino acid sequence of the heavy chain obtained is shown in seq id no : 4 , and that of the light chain is shown in seq id no : 30 . determination of the inner amino acid sequence of the heavy chains was carried out as described below . firstly , the purified almond n - glycosidase as a sample was subjected to hplc using an asahipack c4 - p50 column ( manufactured by asahi kasei ). by elution with a gradient of distilled water to acetonitrile containing 0 . 1 % tfa , 500 pmol of the heavy chain was taken out , and solidified in a small sample tube under a reduced pressure . then , the small sample tube containing the heavy chain was inserted into a test tube containing pyridine 5 μl , 4 - vinylpyridine 1 μl , tributyl phosphine 1 μl , and distilled water 5 μl , and the test tube was sealed under vacuum . then , by heating at 100 ° for 5 minutes , cysteine residues in the heavy chain was pyridylethylated under gas phase . the heavy chain thus s - pyridylethylated was dissolved in 50 μl of 10 mm tris buffer solution ( ph 9 . 0 ). one pmol of lysyl endopeptidase ( manufactured by pierce ) was added to the mixture , and digestion was carried out at 37 ° c . for 7 hours . then , the digested sample . was subjected to hplc using a cosmosil 5c18ar column ( manufactured by nakarai ). purification of the peptide fragment was carried out by eluting with a gradient of distilled water containing 0 . 1 % tfa to acetonitrile containing 0 . 05 % tfa . the peptide fragments obtained were analyzed by a gas phase peptide sequencer ( model 477 , manufactured by applied biosystems ) to determine the inner amino acid sequence of the heavy chain shown in seq id no : 7 - 14 . almond fruits were harvested , and soon freezed at - 80 ° c . to be stored . seven stored fruits were broken in frozen state , and the seeds were separated from the pulp . the seeds were milled with liquid nitrogen in a mortar , and the whole rna was extracted using a rna extraction kit ( manufactured by amersham ) according to the manual to obtain 176 μg of whole rna . the whole rna was treated with oligotex dt 30 ( manufactured by takara shuzo co ., ltd .) to obtain 2 . 9 μg of poly ( a ) rna . using 0 . 7 μg of this poly ( a ) rna as a template and using a cdna synthesis kit ( manufactured by amersham ), for first strand cdna synthesis , priming was accomplished by d ( t ) 20 m4 shown in seq id no : 18 . primers shown in seq id no : 5 , 6 and 15 - 17 were designed from the n - terminal amino acid sequence and the inner amino acid sequence of the heavy chain determined in example 1 ( 1 - 2 ), and synthesized by a dna synthesizer ( model 394 , manufactured by applied biosystems ). that is , sense mixed primers hnt1 and hnt2 shown seq id no : 5 and 6 respectively which correspond to amino acids 3 - 9 and 8 - 14 respectively of the n - terminal sequence of the heavy chain shown in seq id no : 4 ; an anti sense mixed primer hic1 shown in seq id no : 15 which corresponds to amino acids 12 - 19 of the inner sequence of the heavy chain shown in seq id no : 7 ; and anti sense mixed primers hic2 and hic3 shown in seq id no : 16 and 17 respectively which correspond to amino acids 12 - 19 and 2 - 8 respectively of the inner sequence of the heavy chain shown in seq id no : 11 were synthesized . using these primers , nested pcr reaction was carried out . the primary pcr was carried out in a mixture , which was prepared by taking 1 / 20 of the cdna ( 1 μl ) as a template prepared in example 1 ( 1 - 3 ) into a 0 . 5 ml tube for pcr , and adding 10 μl of 10 × buffer solution contained in geneamp ™ pcr reagent kit ( manufactured by takara shuzo co ., ltd . ), 16 μl of 1 . 25 mm dntp mixture , 1 μl of the sense mixed primer ( 500 pmol / μl ), 1 μl of the anti sense mixed primer ( 500 pmol / μl ), 0 . 5 μl of 5 units / μl amplitaq ™, and sterilized water to 100 μl . after overlaying 100 μl of mineral oil ( manufactured by sigma ) on this solution , pcr reaction was carried out by an automatic gene amplifier ( thermal cycler , manufactured by takara shuzo co ., ltd .). the amplification was performed in using the program set to denaturate at 94 ° c . or 2 minutes for a cycle , denature at 94 ° c . for 1 minute , anneal at 55 ° c . for 2 minutes and extend at 55 ° c . for 2 minutes for a total of 30 cycles , then elongate at 60 ° c . for 7 minutes . in this case , the pcr was carried out using three combinations of a sense mixed primer and an anti sense mixed primer , that is , hnt1 and hic1 , hnt1 and hic2 , and hnt1 and hic3 . these reaction mixtures were named pcr1 , pcr2 and pcr3 , respectively . then , using these reaction mixtures , the secondary pcr was carried out . using these three reaction mixtures as templates ( each 1 μl ), the secondary pcr was carried out under the same conditions as the primary pcr using three combinations of a sense mixed primer and an anti sense mixed primer , that is , hnt2 and hic1 , hnt2 and hic2 , and hnt2 and hic3 . the reaction mixtures obtained were named pcr1 - 1 , pcr1 - 2 , pcr1 - 3 , pcr2 - 1 , pcr2 - 2 , pcr2 - 3 , pcr3 - 1 , pcr3 - 2 and pcr3 - 3 . after reactions , upper mineral oil was removed , then 5 μl of each reaction mixture was resolved on an agarose gel electrophoresis , and visualized by ethydium bromide fluorescence to confirm the products amplified . from the results , a specific amplification of dna was found in pcr2 - 2 . this amplified dna fragment of approximately 500 bp was named fhn . the remainder of the reaction mixture from example 1 ( 1 - 4 ) was concentrated by precipitation with ethanol , and the whole precipitation was subjected to agarose gel electrophoresis . after staining with ethydium bromide , a section containing the desired fhn dna fragment was cut out from the gel under ultraviolet , and the dna fragment was extracted and purified from the portion using easytrap ™ ( manufactured by takara shuzo co ., ltd .). then , after phosphorylation of the 5 &# 39 ;- terminal thereof using megalabel ™ ( manufactured by takara shuzo co ., ltd . ), it was blunt - ended using dna blunting kit ( manufactured by takara shuzo co ., ltd . ), and ligated to the hinc ii - digested puc19 plasmid using dna ligation kit ( manufactured by takara shuzo co ., ltd .). using an aliquot of the ligation reaction mixture , e . coli strain jm109 was transformed to obtain a recombinant e . coli containing the fhn dna fragment amplified by pcr in the hinc ii site . four clones of the recombinant e . coli obtained were cultured in liquid medium , and the plasmid dnas were prepared by alkaline lysis method . these four inserted fragments in the plasmids were named fhn1 , fhn2 , fhn3 and fhn4 , and the whole sequences thereof were determined by dideoxy method . each sequence is shown in seq id no : 19 - 22 . in each sequence , nucleotides 1 - 20 was from the sense mixed primer hnt2 , nucleotides 466 - 485 was from the anti sense mixed primer hic2 . when the whole nucleotide sequences of the inserted fragments were translated into amino acids , they coincided well with the n - terminal sequence ( seq id no : 4 ) and the inner sequence ( seq id no : 11 ) of the heavy chain . however , among the nucleotide sequences of fhn1 to fhn4 , differences were found at nucleotides 22 , 36 , 39 , 170 and 245 . ( 1 - 6 ) amplification of the cdna of the c - terminal portion of the heavy chain by pcr primers shown in seq id no : 23 and 24 were designed from the n - terminal portion of the heavy chain determined , and synthesized by a dna synthesizer . that is , sense primers hi1 and hi2 , shown in seq id no 23 and 24 respectively which correspond to nucleotides 72 - 95 and 148 - 164 of fhn1 to fhn4 shown in seq id no : 19 - 22 , were synthesized . also , pcr was carried out using a synthesized dna m13 primer m4 ( seq id no : 25 ) as an anti sense primer which has the same nucleotide sequence as nucleotides 1 - 17 of d ( t ) 20 m4 ( seq id no : 18 ). using these primers , nested pcr was carried out . firstly , the primary pcr was carried out as follows : 16 μl of 1 . 25 mmdntp mixture , 1 μl of 10 pmol / μl hi1 , and 1 μl of 100 pmol / μl m13 primer m40 were taken into a 0 . 5 ml tube for pcr , and a pellet of ampliwax ™ pcr gem100 ( manufactured by takara shuzo co ., ltd .) was added thereto , then the mixture was incubated at 80 ° c . for 6 minutes . then , by incubating the mixture at 25 ° c . for 2 minutes to form a wax layer . further , after adding 1 / 20 ( 1 μl ) of the cdna prepared in example 1 ( 1 - 3 ) as a template , 0 . 5 μl of 5 units / μl amplitaq ™, 10 μl of 10 x buffer solution for amplification , and 70 . 5 μl of sterilized water , pcr was carried out by an automatic gene amplifier ( thermal cycler ) ( hereinafter , referred this pcr method as hot start pcr method ). the reaction was carried out as follows : after melting the wax and mixing the upper and lower layers at 94 ° c . for 2 minutes , a cycle , which consists of denaturation at 94 ° c . for 1 . 5 minutes , annealing of the primer at 55 ° c . for 1 minute , extension at 72 ° c . for 2 . 5 minutes , was repeated 30 times , then further elongation at 60 ° c . for 7 minutes and solidification of the wax at 4 ° c . were carried out . the secondary pcr was carried out under the same conditions as the primary pcr using 1 μl of the reaction mixture from the primary pcr and 1 μl of 100 pmol / μl hi2 . after the reaction , 5 μl of the reaction mixture was subjected to agarose gel electrophoresis , and the dnas were stained with ethydium bromide . a band of the approximately 1 . 2 kbp cdna of the almond n - glycosidase which contains the c - terminal portion of the heavy chain amplified specifically was confirmed . this dna fragment amplified by pcr was named fhc . the remainder of the reaction mixture from example 1 ( 1 - 6 ) was purified as example 1 ( 1 - 4 ), and ligated to the hinc ii - digested puc19 plasmid . by this plasmid , e . coli strain jm109 was transformed to obtain a recombinant e . coli containing fhc dna fragment amplified by pcr in the hinc ii site . four clones of the recombinant e . coli obtained were cultured in liquid medium , the plasmid dnas were prepared by alkali lysis method . these four fragments inserted in the plasmids were named fhc1 , fhc2 , fhc3 and fhc4 , respectively . to determine the whole nucleotide sequences from these inserted fragments , each plasmid was digested by restriction endonucleases pst i and hind iii . after subjecting the dna fragments obtained to agarose gel electrophoresis , the dna fragments were ligated to the pst i or hind iii - digested m13p18 or m13p19 , and e . coli jm109 were transformed with the ligation mixtures . further , the recombinant e . coli were cultured to prepare ssdna . these dnas were sequenced by dideoxy method , and by joining these sequences the whole nucleotide sequences of fhc1 , fhc2 , fhc3 and fhc4 were determined . each sequence is shown in seq id no : 26 - 29 . when the whole nucleotide sequences of the inserted fragments were translated into amino acid sequences , these fragments contained coding region of the c - terminal and a non - translated region of the heavy chain , and coincided well with the inner amino acid sequences of the heavy chain ( seq id no 7 - 14 ). comparing the nucleotide sequences of fhc1 to fhc4 one another , fhc1 has the same nucleotide sequence as fhc3 , and the others have differences in nucleotides 98 , 464 , 500 , 794 , 823 , 877 , 884 and 891 . the 139 bp of the 5 &# 39 ;- termini of fhc1 to fhc 4 overlapped the 139 bp of the 3 &# 39 ;- termini of fhn1 to fhn4 . thus , the whole sequence of the heavy chain cdna was determined by fhn1 to fhn4 and fhc1 to fhc4 . sense mixed primers shown in seq id no : 31 - 34 were designed from the n - terminal partial dna sequence of the light chain determined in example 1 ( 1 - 2 ), and anti sense primers were designed from the cdna sequence of the heavy chain determined in example 1 ( 1 - 2 ) and ( 1 - 3 ). these primers were synthesized by a dna synthesizer and purified . that is , as sense mixed primers , primers ln1c and ln1t shown in seq id no : 31 - 32 respectively which correspond to the amino acids 1 - 8 of the n - terminal sequence of the light chain ; primer ln2 shown in seq id no : 33 which corresponds to amino acids 6 - 12 ; and primer ln3 shown in seq id no : 34 which corresponds to amino acids 12 - 18 were synthesized . also , as an anti sense primer , primer hic4 shown in seq id no : 35 which corresponds to nucleotides 262 - 286 of seq id no : 19 - 22 and nucleotides 125 - 149 of seq id no : 26 - 29 was synthesized . using these primers , nested pcr was carried put . according to hot start pcr above described , amplification by the primary pcr was carried out using 1 / 20 ( 1 μl ) of the cdna as a template prepared in example 1 ( 1 - 3 ). the reaction was conducted under following conditions : after melting the wax and mixing the upper and lower layers at 94 ° c . for 2 minutes , a cycle , which consists of denaturation at 94 ° c . for 1 minute , annealing of the primer at 55 ° c . for 1 minute , extension at 72 ° c . for 2 . 5 minutes , was repeated 30 times , then further elongation at 60 ° c . for 7 minutes and solidification of the wax at 4 ° c . were carried out . in this case , using three combinations of sense mixed primers and an anti sense primer , ln1c and hic4 , ln1t and hic4 , and ln2 and hic4 , the pcr was carried out . the reaction mixtures were named pcr4 , pcr5 and pcr6 , respectively . then , the secondary pcr was carried out using these three reaction mixtures ( each 1 μl ) as templates . to templates pcr4 and pcr5 , using two combinations of a sense mixed primer and an anti sense mixed primers , that is , ln2 and hic4 , and ln3 and hic4 , and to pcr6 , using a combination of ln3 and hic4 the pcr was carried out under the same conditions as the primary pcr . the reaction mixtures were named pcr4 - 1 , pcr4 - 2 , pcr5 - 1 , pcr5 - 2 and pcr6 - 1 . 5 μl of the each reaction mixture was subjected to agarose gel electrophoresis , and the dna were stained with ethydium bromide to identify the products amplified . from the results , amplification of the specific dna of the same size was found in pcr4 - 1 and pcr5 - 1 . this amplified dna fragment of about 900 bp was named fl . after concentration of the whole reminder of the pcr by ethanol precipitation , the concentrate was subjected to agarose gel electrophoresis to isolate fragment fl . fragment fl was phosphorylated at the 5 &# 39 ;- terminal and blunt - ended , then ligated with the hinc ii - digested puc19 plasmid . using an aliquot of the ligation reaction mixture , e . coli strain m109 was transformed to obtain a recombinant e . coli containing fl dna fragment amplified by pcr in the hincii site . six clones of the recombinant e . coli were cultured in liquid medium , and plasmid dnas were prepared by alkali lysis method . these six inserts in the plasmid dna were named fl1 , fl2 , fl3 , fl4 , fl5 and fl6 , respectively . in order to determine the whole nucleotide sequences of these inserts , a plasmid which lacked an ecori fragment of about 380 bp was prepared after digestion of each plasmid by a restriction enzyme eco ri . also , a plasmid lacking a fragment of about 220 bp was prepared by a restriction enzyme pst i . these plasmids were sequenced by dideoxy method , and these sequences were connected together to determine the whole nucleotide sequences of fl1 , fl2 , fl3 , fl4 , fl5 and fl6 . each sequence is shown seq id no : 36 - 41 . nucleotides 1 - 20 in sequences of fl1 - fl6 ( seq id no : 36 - 41 ) was the sequence from the sense mixed primer ln2 ( seq id no : 33 ). nucleotides 499 - 774 , and nucleotides 626 - 774 coincided well with nucleotides 1 - 276 of the pcr fragment fhn1 - fhn4 ( seq id no : 19 - 22 ) of the n - terminal portion of the heavy chain , and with nucleotides 1 - 149 of the pcr fragment fhc1 - fhc4 ( seq id no : 26 - 29 ) of the n - terminal portion of the heavy chain , respectively . these relationships are shown in fig1 . further , when the whole nucleotide sequences of fl1 - fl6 ( seq id no : 36 - 41 ) were translated into amino acid sequences , it was found that they contained a sequence coinciding well with the n - terminal sequence of the light chain ( seq id no : 30 ) and the n - terminal sequence of the heavy chain ( seq id no : 4 ) determined in example 1 ( 1 - 2 ). thus , it was found that the amino acid sequences of the light chain and the heavy chain were coded in the same frame on a cdna . by comparing with fl1 - fl6 ( seq id no : 36 - 41 ) one another , it was found that fl1 and fl3 , and fl4 and fl6 contained the same nucleotide sequences , respectively , and that the others were different at nucleotides 205 , 439 , 520 and 723 one another . ( 2 - 1 ) preparation of a dna fragment coding a mature n - terminal polypeptide of the almond n - glycosidase fragment fl which codes the n - terminal portion of the heavy chain lacks nucleotide sequences corresponding to ln1c ( seq id no : 31 ) and ln1t ( seq id no : 32 ) used as primers in the primary amplification by pcr . fragments which compensate these portions were prepared by pcr . an amino acid sequence of the light chain determined in example 1 ( 1 - 2 ) and ( 1 - 9 ), a sense primer angns ( seq id no : 42 ) from the nucleotide sequence of fragment fl , and an anti sense primer angna ( seq id no : 43 ) were designed , and synthesized by a dna synthesizer to be purified . the sense primer angns ( seq id no : 42 ) contains nucleotides 1 - 20 of fl1 shown in seq id no : 36 in its nucleotides 30 - 49 , which corresponds to the n - terminal of the heavy chain ( amino acids 6 - 12 in the amino acid sequence ) shown in seq id no : 30 . in addition , the sense primer angns ( seq id no : 42 ) is a synthesized dna which contains a nucleotide sequence corresponding to amino acids 1 - 5 of the n - terminal amino acid sequence of the light chain ( seq id no : 30 ) in its nucleotides 15 - 29 , and further contains recognition sequences for nco i and bam hi in its nucleotides 1 - 14 . the anti sense primer ( seq id no : 43 ) is a synthesized dna which contains a sequence complementary to nucleotides 148 - 172 of fl1 shown in seq id no : 36 . the amplification by pcr was carried out according to hot start pcr method above described , using 100 pmol of the sense primer angns , 100 pmol of the anti sense primer angna , and 10 ng of the plasmid containing fl1 obtained in example 1 ( 1 - 9 ). the reaction was conducted under following conditions : after melting the wax and mixing the upper and lower layers at 94 ° c . for 2 minutes , a cycle , which consists of denaturation at 94 ° c . for 1 minute , annealing of the primer at 55 ° c . for 1 minute , extension at 72 ° c . for 1 minute , was repeated 25 times , then further elongation at 60 ° c . for 7 minutes and solidification of the wax at 4 ° c . were carried out . after concentration of the whole reaction mixture by ethanol precipitation , agarose gel electrophoresis was carried out . then , a dna fragment of about 200 bp was extracted from the gel and purified . this fragment was digested by restriction enzymes nco i and xba i , then subjected to agarose gel electrophoresis again to cut out a portion of the gel containing a dna fragment angn of about 140 bp , and the fragment was extracted from the gel and purified . ( 2 - 2 ) construction of an expression vector for fission yeast schizosaccharomyces pombe the plasmid dna containing fhc1 ( seq id no : 26 ) obtained in example 1 ( 1 - 7 ) was digested by restriction enzymes xba i and kpn i , and agarose gel electrophoresis was conducted to cut out a portion of the gel containing a fragment fhc1 - xba i / kpn i of about 1 kbp . this fragment was extracted from the gel and purified . this fragment was inserted to a xba i - kpn i site of a plasmid ptvl119n ( manufactured by takara shuzo co ., ltd .) to obtain a plasmid ptang - hc . the plasmid containing fl1 ( seq id no : 36 ) obtained in example 1 ( 1 - 9 ) was digested by restriction enzymes nde i and xba i . agarose gel electrophoresis was conducted , and a portion of the gel containing a fragment fl1 - nde i / xba i of about 660 bp was cut out , and the fragment was extracted from the gel . a plasmid obtained by digesting ptang - hc by restriction enzymes nco i and xba i and dephosphorylating it by alkali phosphatase bap c75 ( manufactured by takara shuzo co ., ltd . ), fragment fl1 - nde i / xba i , and fragment angn obtained in example 2 ( 2 - 1 ) were ligated together to obtain a plasmid ptang ( see fig2 ). further , ptang was digested by restriction enzymes saci and bamhi , and agarose gel electrophoresis was conducted . a portion of the gel , which contained a fragment tang - saci / bamhi of about 1 . 6 kbp containing from the mature n - terminal of the almond n - glycosidase gene to the whole length thereof , was cut out , and the fragment was extracted from the gel . this fragment tang - sac i / bam hi and a plasmid pet23a ( manufactured by novagen ) were digested by sac i and bam hi , and then dephosphorylated . by ligation of these fragments , a plasmid petang was obtained . petang was digested by restriction enzymes bam hi and xho i , then blunted by a dna blunting kit . agarose gel electrophoresis was conducted to obtain a fragment etang bam hi - xho i of about 1 . 8 kbp . this fragment was inserted into an expression vector prep1 for schizosaccharomyces pombe k . maundrell et al ., the journal of biological chemistry , 265 : 10857 - 10864 ( 1990 )!. plasmid prep1 is a shuttle vector which contains an ampicillin resistant marker , a yeast resistant marker leu2 , a schizosaccharomyces pombe autonomous replication sequence ars , and a promotor nmt 1 which is induced by lack of thiamine . after digestion of plasmid prep1 by a restriction enzyme bam hi , the digested plasmid was blunt - ended by a dna blunting kit . fragment etang bam hi - xho i was introduced into the blunted fragment to transform e . coli strain hb101 . plasmid was prepared from the recombinant , and a plasmid , which was proved to have an insert in correct direction by digestion analysis using restriction enzymes , was selected ( see fig3 ). the plasmid obtained was named prang . e . coli transformed with plasmid prang was named escherichia coli hb101 / prang , and it has been deposited to the national institute of bioscience and human - technology agency of industrial science and technology , under the accession number ferm bp - 4949 since 28st feb . 1994 . expression of the almond n - glycosidase polypeptide by fission yeast schizosaccharomyces pombe the uracil , leucine and adenine auxotroph schizosaccharomvces pombe t - 1 was transformed with the prang plasmid . the transformant was named schizpsaccharomyces pombe / prang . transformation was performed using 1 μg of plasmid prang by lithium acetate method of okazaki et al . nucleic acids research , 18 : 6485 ( 1990 )!. a leu + transformant was isolated , and the plasmid was identified . the transformant containing prang was named schizosaccharomvcespombe / prang . the schizosaccharomvces pombe / prang cells were cultured in a minimal medium supplemented with thiamine , uracil and adenine c . king et al ., handbook of genetics pp395 - 446 ( 1974 ) prenum press !. cells grown to logarithmic growth phase were collected by centrifugation , and transferred to a minimal medium which was supplemented with uracil and adenine and did not contain thiamine . the cells were cultured to stationary phase . cells were collected by centrifugation , and suspended in a acetate buffer solution for cell homogenization ( containing 2 mm 4 -( 2 - aminoethyl ) benzenesulfonyl fluoride , 73 μm pepstatin a , 5 mm o - phenanthroline , and 5 mm edta ). glass beads ( 0 . 45 mm ) were added , and cells were homogenated by vigorously stirring for 5 min with intermittent cooling . the homogenate was centrifuged , and the supernatant ( the extract ) was separated from the precipitate . then , assay of n - glycosidase activity in the extract and the precipitate was performed using dabsyl ovomucoid glycopeptide as a substrate . the activity was found in the extract . as described hereinabove , according to the present invention , the nucleotide sequence and the amino acid sequence of the almond n - glycosidase have been elucidated , whereby it is possible to provide an almond n - glycosidase gene . thus , a genetic process advantageous industrially for preparing polypeptides having the almond n - glycosidase activity is provided . similar n - glycodidase genes are also provided . in addition , on the basis of the nucleotide sequence of the almond n - glycosidase , probe dnas and primers for pcr can be synthesized to obtain similar n - glycosidases using them . further , on the basis of the amino acid sequence , antibodies can be prepared . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 43 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 571 ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( ix ) feature :( d ) other information : xaa at position 27 is pro or ser . xaa at position 74 is arg or gly . ( d ) other information : xaa at position 152 is lys or glu . xaa at position 179 is ile pr val . ( d ) other information : xaa at position 488 is arg or gln . xaa at position 506 is glu or gly . ( d ) other information : xaa at position 511 is ile or val . ( xi ) sequence description : seq id no : 1 : gluprothrproleuhisaspthrproprothrvalphepheglu151015valthrlysproilegluvalprolysthrlysxaacyssergln202530leuileleuglnhisaspphealatyrthrtyrglyglnalapro354045valphealaasntyrthrproproseraspcysproserglnthr505560pheserthrilevalleuglutrplysalathrcysargxaaarg657075glnpheaspargilepheglyvaltrpleuglyglyvalgluile808590leuargsercysthralagluproargproasnglyilevaltrp95100105thrvalglulysaspilethrargtyrtyrserleuleulysser110115120asnglnthrleualavaltyrleuglyasnleuileasplysthr125130135tyrthrglyiletyrhisvalasnileserleuhisphetyrpro140145150alaxaaglulysleuasnserpheglnglnlysleuaspasnleu155160165alaserglytyrhissertrpalaaspleuileleuproxaaser170175180argasnleuproleuasnaspglyleutrpphegluvalglnasn185190195serasnaspthrgluleulysgluphelysileproglnasnala200205210tyrargalavalleugluvaltyrvalserphehisgluasnasp215220225gluphetrptyrserasnleuproasnglutyrilealaalaasn230235240asnleuserglythrproglyasnglyprophearggluvalval245250255valserleuaspglygluvalvalglyalavaltrpprophethr260265270valilephethrglyglyileasnproleuleutrpargproile275280285thralaileglyserpheaspleuprothrtyraspilegluile290295300thrpropheleuglylysileleuaspglylysserhislysphe305310315glypheasnvalthrasnalaleuasnvaltrptyrvalaspala320325330asnleuhisleutrpleuasplysglnserthrlysthrglugly335340345lysleuserlyshisserserleuproleuvalvalserleuval350355360seraspphelysglyleuasnglythrpheleuthrargthrser365370375argservalserserthrglytrpvallyssersertyrglyasn380385390ilethrthrargserileglnaspphetyrtyrserasnsermet395400405valleuglylysaspglyasnmetglnilevalasnglnlysile410415420ilepheasnaspservaltyrileasnleuprosersertyrval425430435hisserleuthrserhislysthrpheproleutyrleutyrthr440445450asppheleuglyglnglyasnglythrtyrleuleuilethrasn455460465valaspleuglypheileglulyslysserglyleuglypheser470475480asnserserleuargasnleuxaaseralagluglyasnmetval485490495vallysasnasnleuvalvalserglyleuxaaserthrglngln500505510xaatyrargtyraspglyglylysphecystyrpheargasnile515520525serserserasntyrthrileleutyrasplysvalglyserlys530535540cysasnlyslysserleuserasnleuaspphevalleuserarg545550555leutrppropheglyalaargmetasnphealaglyleuargphe560565570thr ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 1713 ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna to mrna ( xi ) sequence description : seq id no : 2 : 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( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 10 ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( v ) fragment type : n - terminal fragment ( xi ) sequence description : seq id no : 3 : ileaspproargvalvalxaaalaxaaleu1510 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 20 ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 4 : leualaserglytyrhissertrpalaaspleuileleuproile151015serargasnleupro20 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 20 ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( synthesized dna )( xi ) sequence description : seq id no : 5 : wsnggntaycaywsntgggc20 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 20 ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( synthesized dna )( xi ) sequence description : seq id no : 6 : tgggcngayytnathytncc20 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 21 ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 7 : asnasnleuvalvalserglyleuglyserthrglnglnvaltyr151015argtyraspglyglylys20 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 18 ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 8 : phexaatyrpheargxaaileserserserxaatyrthrileleu151015tyrasplys ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 24 ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 9 : sersertyrglyxaailethrthrargserileglnaspphetyr151015tyrserasnsermetvalleuglylys20 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 22 ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 10 : ileilephexaaaspserxaatyrxaaasnleuprosersertyr151015valhisxaaleuxaaxaahis20 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 24 ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 11 : pheglyphexaavalthrasnalaleuasnvaltrptyrvalasp151015alaasnleuhisleutrpleuasplys20 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 22 ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 12 : serglyleuglypheserxaaserserleuargasnleuglnser151015alagluglyasnmetvalval20 ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 20 ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 13 : glyleuxaaglythrpheleuthrargthrserargservalser151015xaathrglyxaaval20 ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 21 ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 14 : thrpheproleutyrleutyrthrxaapheleuglyglnglyxaa151015xaathrxaaleuleuile20 ( 2 ) information for seq id no : 15 :( i ) sequence characteristics :( a ) length : 23 ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( dna )( xi ) sequence description : seq id no : 15 : ccrtcrtanckrtanacytgytg23 ( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 22 ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( dna )( xi ) sequence description : seq id no : 16 : tgnarrttngcrtcnacrtacc22 ( 2 ) information for seq id no : 17 :( i ) sequence characteristics :( a ) length : 20 ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( dna )( xi ) sequence description : seq id no : 17 : gcrttngtnacrttraancc20 ( 2 ) information for seq id no : 18 :( i ) sequence characteristics :( a ) length : 37 ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( dna )( xi ) sequence description : seq id no : 18 : gttttcccagtcacgactttttttttttttttttttt37 ( 2 ) information for seq id no : 19 :( i ) sequence characteristics :( a ) length : 485 ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna to mrna ( xi ) sequence description : seq id no : 19 : 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( 2 ) information for seq id no : 20 :( i ) sequence characteristics :( a ) length : 485 ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna to mrna ( xi ) sequence description : seq id no : 20 : 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( 2 ) information for seq id no : 21 :( i ) sequence characteristics :( a ) length : 485 ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna to mrna ( xi ) sequence description : seq id no : 21 : 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( 2 ) information for seq id no : 22 :( i ) sequence characteristics :( a ) length : 485 ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna to mrna ( xi ) sequence description : seq id no : 22 : 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( 2 ) information for seq id no : 23 :( i ) sequence characteristics :( a ) length : 24 ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( dna )( xi ) sequence description : seq id no : 23 : aagttcagaattcaaatgatacgg24 ( 2 ) information for seq id no : 24 :( i ) sequence characteristics :( a ) length : 27 ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( dna )( xi ) sequence description : seq id no : 24 : tgttggaggtgtatgtttcatttcacg27 ( 2 ) information for seq id no : 25 :( i ) sequence characteristics :( a ) length : 17 ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( dna )( xi ) sequence description : seq id no : 25 : gttttcccagtcacgac17 ( 2 ) information for seq id no : 26 :( i ) sequence characteristics :( a ) length : 1435 ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna to mrna ( xi ) sequence description : seq id no : 26 : 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( 2 ) information for seq id no : 27 :( i ) sequence characteristics :( a ) length : 1318 ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna to mrna ( xi ) sequence description : seq id no : 27 : tgttggaggtgtatgtttcatttcacgagaatgatgaattttggtat47leugluvaltyrvalserphehisgluasnaspgluphetrptyr151015tcaaatcttcctaatgagtacatagctgcaaacaaccttagcggt92serasnleuproasnglutyrilealaalaasnasnleusergly202530acacccggaaatgggccttttagggaggttgtggtcagtctagat137thrproglyasnglyprophearggluvalvalvalserleuasp354045ggtgaggttgttggtgcagtctggccttttactgtgattttcact182glygluvalvalglyalavaltrpprophethrvalilephethr505560ggagggatcaatcctcttttatggagaccaattactgcaattggc227glyglyileasnproleuleutrpargproilethralailegly657075tcattcgatcttccgacttatgatatcgaaattacaccattttta272serpheaspleuprothrtyraspilegluilethrpropheleu808590gggaagatattagatgggaagagccacaagttcgggtttaatgtt317glylysileleuaspglylysserhislyspheglypheasnval95100105acaaatgccttaaatgtttggtacgttgatgcaaatttgcatctt362thrasnalaleuasnvaltrptyrvalaspalaasnleuhisleu110115120tggttggacaaacagagcacaaaaactgaaggaaagctttcgaaa407trpleuasplysglnserthrlysthrgluglylysleuserlys125130135catagtagcttgccccttgttgtttccctggtttcagatttcaag452hisserserleuproleuvalvalserleuvalseraspphelys140145150ggtttaaatggcacatttttgacaaggacaagcaggtccgtgtca497glyleuasnglythrpheleuthrargthrserargservalser155160165tcaactggatgggtgaagtcttcctatgggaatatcacaacccgt542serthrglytrpvallyssersertyrglyasnilethrthrarg170175180tcaattcaagacttctattacagtaattcaatggtcctggggaaa587serileglnaspphetyrtyrserasnsermetvalleuglylys185190195gatggtaatatgcagatagtcaaccagaagatcattttcaatgac632aspglyasnmetglnilevalasnglnlysileilepheasnasp200205210tcagtttatattaacctgccatcctcctatgttcactcactgaca677servaltyrileasnleuprosersertyrvalhisserleuthr215220225tcacacaaaacatttccactttatttgtacactgacttcttagga722serhislysthrpheproleutyrleutyrthrasppheleugly230235240caaggaaatggaacttatttattgattacaaatgtggacttggga767glnglyasnglythrtyrleuleuilethrasnvalaspleugly245250255tttattgagaagaagtctggtttgggattctcgaacagctctctc812pheileglulyslysserglyleuglypheserasnserserleu260265270agaaatctgcggagtgctgagggcaatatggttgtgaaaaacaat857argasnleuargseralagluglyasnmetvalvallysasnasn275280285ttggttgtgagtggattggggagcactcagcaaatctatagatat902leuvalvalserglyleuglyserthrglnglniletyrargtyr290295300gatggtggtaaattctgttacttcagaaatataagcagctcaaac947aspglyglylysphecystyrpheargasnileserserserasn305310315tacacaatactctatgacaaggtggggagcaaatgcaacaaaaaa992tyrthrileleutyrasplysvalglyserlyscysasnlyslys320325330tcgttgtctaatttggattttgtcttaagcagactgtggcctttt1037serleuserasnleuaspphevalleuserargleutrpprophe335340345ggtgctcgaatgaattttgctggtctccgatttacatgagaacaat1083glyalaargmetasnphealaglyleuargphethr350355gaggaaagtctagctcatccacattcatgtatcttcctgtttgttggtacctcaaataaa1143tgtgtattctgtactcaatttcattttgtggggattcttctatattgtagttgaggtatc1203tgcactgcacccaagtgattctggttttacttggatttggaatgattactaaaaagtgga1263tcaggtcagcttgtgctaaaaaaaaaaaaaaaaaaaaagtcgtgactgggaaaac1318 ( 2 ) information for seq id no : 28 :( i ) sequence characteristics :( a ) length : 1426 ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna to mrna ( xi ) sequence description : seq id no : 28 : ttgttggaggtgtatgtttcatttcacgagaatgatgaattttggtat47leugluvaltyrvalserphehisgluasnaspgluphetrptyr151015tcaaatcttcctaatgagtacatagctgcaaacaaccttagcggt92serasnleuproasnglutyrilealaalaasnasnleusergly202530acacctggaaatgggccttttagggaggttgtggtcagtctagat137thrproglyasnglyprophearggluvalvalvalserleuasp354045ggtgaggttgttggtgcagtctggccttttactgtgattttcact182glygluvalvalglyalavaltrpprophethrvalilephethr505560ggagggatcaatcctcttttatggagaccaattactgcaattggc227glyglyileasnproleuleutrpargproilethralailegly657075tcattcgatcttccgacttatgatatcgaaattacaccattttta272serpheaspleuprothrtyraspilegluilethrpropheleu808590gggaagatattagatgggaagagccacaagttcgggtttaatgtt317glylysileleuaspglylysserhislyspheglypheasnval95100105acaaatgccttaaatgtttggtacgttgatgcaaatttgcatctt362thrasnalaleuasnvaltrptyrvalaspalaasnleuhisleu110115120tggttggacaaacagagcacaaaaactgaaggaaagctttcgaaa407trpleuasplysglnserthrlysthrgluglylysleuserlys125130135catagtagcttgccccttgttgtttccctggtttcagatttcaag452hisserserleuproleuvalvalserleuvalseraspphelys140145150ggtttaaatgggacatttttgacaaggacaagcaggtccgtgtca497glyleuasnglythrpheleuthrargthrserargservalser155160165tccactggatgggtgaagtcttcctatgggaatatcacaacccgt542serthrglytrpvallyssersertyrglyasnilethrthrarg170175180tcaattcaagacttctattacagtaattcaatggtcctggggaaa587serileglnaspphetyrtyrserasnsermetvalleuglylys185190195gatggtaatatgcagatagtcaaccagaagatcattttcaatgac632aspglyasnmetglnilevalasnglnlysileilepheasnasp200205210tcagtttatattaacctgccatcctcctatgttcactcactgaca677servaltyrileasnleuprosersertyrvalhisserleuthr215220225tcacacaaaacatttccactttatttgtacactgacttcttagga722serhislysthrpheproleutyrleutyrthrasppheleugly230235240caaggaaatggaacttatttattgattacaaatgtggacttggga767glnglyasnglythrtyrleuleuilethrasnvalaspleugly245250255tttattgagaagaagtctggtttgggtttctcgaacagctctctc812pheileglulyslysserglyleuglypheserasnserserleu260265270agaaatctgcagagtgctgagggcaatatggttgtgaaaaacaat857argasnleuglnseralagluglyasnmetvalvallysasnasn275280285ttggttgtgagtggattggggagcactcagcaagtctatagatat902leuvalvalserglyleuglyserthrglnglnvaltyrargtyr290295300gatggtggtaaattctgttacttcagaaatataagcagctcaaac947aspglyglylysphecystyrpheargasnileserserserasn305310315tacacaatactctatgacaaggtggggagcaaatgcaacaaaaaa992tyrthrileleutyrasplysvalglyserlyscysasnlyslys320325330tcgttgtctaatttggattttgtcttaagcagactgtggcctttt1037serleuserasnleuaspphevalleuserargleutrpprophe335340345ggtgctcgaatgaattttgctggtctccgatttacatgagaacaat1083glyalaargmetasnphealaglyleuargphethr350355gaggaaagtctagctcatccacattcatgtatcttcctgtttgttggtacctcaaataaa1143tgtgtattctgtactcaatttcattttgtggggattcttctagattgtagttgaggtatc1203tgcactgcacccaagtgattctggttttacttggatttggaatgattactaagaagtgga1263tcaggtcagcttgtcagtataatcaaagtttatgcaggaaattttaaaccattatttttt1323ggctttcgcattttttggcttttaaatttttaatatgatcaatgcattaaaaatccaaag1383ggttacctaaaaaaaaaaaaaaaaaagtcgtgactgggaaaac1426 ( 2 ) information for seq id no : 29 :( i ) sequence characteristics :( a ) length : 1358 ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna to mrna ( xi ) sequence description : seq id no : 29 : tgttggaggtgtatgtttcatttcacgagaatgatgaattttggtat47leugluvaltyrvalserphehisgluasnaspgluphetrptyr151015tcaaatcttcctaatgagtacatagctgcaaacaaccttagcggt92serasnleuproasnglutyrilealaalaasnasnleusergly202530acacccggaaatgggccttttagggaggttgtggtcagtctagat137thrproglyasnglyprophearggluvalvalvalserleuasp354045ggtgaggttgttggtgcagtctggccttttactgtgattttcact182glygluvalvalglyalavaltrpprophethrvalilephethr505560ggagggatcaatcctcttttatggagaccaattactgcaattggc227glyglyileasnproleuleutrpargproilethralailegly657075tcattcgatcttccgacttatgatatcgaaattacaccattttta272serpheaspleuprothrtyraspilegluilethrpropheleu808590gggaagatattagatgggaagagccacaagttcgggtttaatgtt317glylysileleuaspglylysserhislyspheglypheasnval95100105acaaatgccttaaatgtttggtacgttgatgcaaatttgcatctt362thrasnalaleuasnvaltrptyrvalaspalaasnleuhisleu110115120tggttggacaaacagagcacaaaaactgaaggaaagctttcgaaa407trpleuasplysglnserthrlysthrgluglylysleuserlys125130135catagtagcttgccccttgttgtttccctggtttcagatttcaag452hisserserleuproleuvalvalserleuvalseraspphelys140145150ggtttaaatggcacatttttgacaaggacaagcaggtccgtgtca497glyleuasnglythrpheleuthrargthrserargservalser155160165tcaactggatgggtgaagtcttcctatgggaatatcacaacccgt542serthrglytrpvallyssersertyrglyasnilethrthrarg170175180tcaattcaagacttctattacagtaattcaatggtcctggggaaa587serileglnaspphetyrtyrserasnsermetvalleuglylys185190195gatggtaatatgcagatagtcaaccagaagatcattttcaatgac632aspglyasnmetglnilevalasnglnlysileilepheasnasp200205210tcagtttatattaacctgccatcctcctatgttcactcactgaca677servaltyrileasnleuprosersertyrvalhisserleuthr215220225tcacacaaaacatttccactttatttgtacactgacttcttagga722serhislysthrpheproleutyrleutyrthrasppheleugly230235240caaggaaatggaacttatttattgattacaaatgtggacttggga767glnglyasnglythrtyrleuleuilethrasnvalaspleugly245250255tttattgagaagaagtctggtttgggattctcgaacagctctctc812pheileglulyslysserglyleuglypheserasnserserleu260265270agaaatctgcagagtgctgagggcaatatggttgtgaaaaacaat857argasnleuglnseralagluglyasnmetvalvallysasnasn275280285ttggttgtgagtggattggagagcacccagcaagtctatagatat902leuvalvalserglyleugluserthrglnglnvaltyrargtyr290295300gatggtggtaaattctgttacttcagaaatataagcagctcaaac947aspglyglylysphecystyrpheargasnileserserserasn305310315tacacaatactctatgacaaggtggggagcaaatgcaacaaaaaa992tyrthrileleutyrasplysvalglyserlyscysasnlyslys320325330tcgttgtctaatttggattttgtcttaagcagactgtggcctttt1037serleuserasnleuaspphevalleuserargleutrpprophe335340345ggtgctcgaatgaattttgctggtctccgatttacatgagaacaat1083glyalaargmetasnphealaglyleuargphethr350355gaggaaagtctagctcatccacattcatgtatcttcctgtttgttggtacctcaaataaa1143tgtgtattccgtactcaatttcattttgtggggattcttctatattgtagttgaggtatc1203tgcactgcacccaagtgattctggttttacttggatttggaatgattactaaaaagtgga1263tcaggtcagcttgtgctatgttagtatcatcaaagtttatgcaggaaattttaaaccatt1323aaaaaaaaaaaaaaaaaagtcgtgactgggaaaac1358 ( 2 ) information for seq id no : 30 :( i ) sequence characteristics :( a ) length : 20 ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 30 : gluprothrproleuhisaspthrproprothrvalphepheglu151015valthrlysproile20 ( 2 ) information for seq id no : 31 :( i ) sequence characteristics :( a ) length : 23 ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( dna )( xi ) sequence description : seq id no : 31 : garccnacnccnctncaygayac23 ( 2 ) information for seq id no : 32 :( i ) sequence characteristics :( a ) length : 23 ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( dna )( xi ) sequence description : seq id no : 32 : garccnacnccnttncaygayac23 ( 2 ) information for seq id no : 33 :( i ) sequence characteristics :( a ) length : 20 ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( dna )( xi ) sequence description : seq id no : 33 : caygayacnccnccnacngt20 ( 2 ) information for seq id no : 34 :( i ) sequence characteristics :( a ) length : 20 ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( dna )( xi ) sequence description : seq id no : 34 : gtnttyttygargtnacnaa20 ( 2 ) information for seq id no : 35 :( i ) sequence characteristics :( a ) length : 25 ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( dna )( xi ) sequence description : seq id no : 35 : aacaacctcaccatctagactgacc25 ( 2 ) information for seq id no : 36 :( i ) sequence characteristics :( a ) length : 774 ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna to mrna ( xi ) sequence description : seq id no : 36 : catgataccccgccaactgtattttttgaagtcaccaaacccatt45hisaspthrproprothrvalphephegluvalthrlysproile151015gaagtaccaaaaaccaagccgtgttcccagctcattctccagcat90gluvalprolysthrlysprocysserglnleuileleuglnhis202530gactttgcctacacatatggccaagctccagtctttgcaaactac135aspphealatyrthrtyrglyglnalaprovalphealaasntyr354045acccctccttccgattgcccatctcaaactttctccacaattgtc180thrproproseraspcysproserglnthrpheserthrileval505560cttgaatggaaagctacctgcagaggaaggcaatttgaccgcatt225leuglutrplysalathrcysargglyargglnpheaspargile657075ttcggggtttggcttggtggggttgagattctcaggagctgcaca270pheglyvaltrpleuglyglyvalgluileleuargsercysthr808590gcagaaccaaggcctaatgggattgtttggactgtcgagaaggac315alagluproargproasnglyilevaltrpthrvalglulysasp95100105atcacaaggtactattcactgcttaagagtaatcaaacacttgct360ilethrargtyrtyrserleuleulysserasnglnthrleuala110115120gtttatcttggcaatttgatagataaaacctacactgggatttat405valtyrleuglyasnleuileasplysthrtyrthrglyiletyr125130135catgtgaatataagccttcatttttaccctgctaaagagaaattg450hisvalasnileserleuhisphetyrproalalysglulysleu140145150aattcttttcagcaaaagttggataatttggcatctgggtaccat495asnserpheglnglnlysleuaspasnleualaserglytyrhis155160165tcttgggctgatttgattttacccgtttcgagaaatctgcctttg540sertrpalaaspleuileleuprovalserargasnleuproleu170175180aatgatgggttgtggtttgaagttcagaattcaaatgatacggaa585asnaspglyleutrpphegluvalglnasnserasnaspthrglu185190195ttgaaggagttcaagattccacaaaatgcttatagggctgtgttg630leulysgluphelysileproglnasnalatyrargalavalleu200205210gaggtgtatgtttcatttcacgagaatgatgaattttggtattca675gluvaltyrvalserphehisgluasnaspgluphetrptyrser215220225aatcttcctaatgagtacatagctgcaaacaaccttagcggtaca720asnleuproasnglutyrilealaalaasnasnleuserglythr230235240cccggaaatgggccttttagggaggttgtggtcagtctagatggt765proglyasnglyprophearggluvalvalvalserleuaspgly245250255gaggttgtt774gluvalval ( 2 ) information for seq id no : 37 :( i ) sequence characteristics :( a ) length : 774 ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna to mrna ( xi ) sequence description : seq id no : 37 : catgacacgccgccgaccgtattttttgaagtcaccaaacccatt45hisaspthrproprothrvalphephegluvalthrlysproile151015gaagtaccaaaaaccaagccgtgttcccagctcattctccagcat90gluvalprolysthrlysprocysserglnleuileleuglnhis202530gactttgcctacacatatggccaagctccagtctttgcaaactac135aspphealatyrthrtyrglyglnalaprovalphealaasntyr354045acccctccttccgattgcccatctcaaactttctccacaattgtc180thrproproseraspcysproserglnthrpheserthrileval505560cttgaatggaaagctacctgcagaggaaggcaatttgaccgcatt225leuglutrplysalathrcysargglyargglnpheaspargile657075ttcggggtttggcttggtggggttgagattctcaggagctgcaca270pheglyvaltrpleuglyglyvalgluileleuargsercysthr808590gcagaaccaaggcctaatgggattgtttggactgtcgagaaggac315alagluproargproasnglyilevaltrpthrvalglulysasp95100105atcacaaggtactattcactgcttaagagtaatcaaacacttgct360ilethrargtyrtyrserleuleulysserasnglnthrleuala110115120gtttatcttggcaatttgatagataaaacctacactgggatttat405valtyrleuglyasnleuileasplysthrtyrthrglyiletyr125130135catgtgaatataagccttcatttttaccctgctaaagagaaattg450hisvalasnileserleuhisphetyrproalalysglulysleu140145150aattcttttcagcaaaagttggataatttggcatctgggtaccat495asnserpheglnglnlysleuaspasnleualaserglytyrhis155160165tcttgggctgatttgattttacccgtttcgagaaatctgcctttg540sertrpalaaspleuileleuprovalserargasnleuproleu170175180aatgacgggttgtggtttgaagttcagaattcaaatgatacggaa585asnaspglyleutrpphegluvalglnasnserasnaspthrglu185190195ttgaaggagttcaagattccacaaaatgcttatagggctgtgttg630leulysgluphelysileproglnasnalatyrargalavalleu200205210gaggtgtatgtttcatttcacgagaatgatgaattttggtattca675gluvaltyrvalserphehisgluasnaspgluphetrptyrser215220225aatcttcctaatgagtacatagctgcaaacaaccttagcggtaca720asnleuproasnglutyrilealaalaasnasnleuserglythr230235240cccggaaatgggccttttagggaggttgtggtcagtctagatggt765proglyasnglyprophearggluvalvalvalserleuaspgly245250255gaggttgtt774gluvalval ( 2 ) information for seq id no : 38 :( i ) sequence characteristics :( a ) length : 774 ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna to mrna ( xi ) sequence description : seq id no : 38 : cacgatacaccgccgacagtattttttgaagtcaccaaacccatt45hisaspthrproprothrvalphephegluvalthrlysproile151015gaagtaccaaaaaccaagccgtgttcccagctcattctccagcat90gluvalprolysthrlysprocysserglnleuileleuglnhis202530gactttgcctacacatatggccaagctccagtctttgcaaactac135aspphealatyrthrtyrglyglnalaprovalphealaasntyr354045acccctccttccgattgcccatctcaaactttctccacaattgtc180thrproproseraspcysproserglnthrpheserthrileval505560cttgaatggaaagctacctgcagaggaaggcaatttgaccgcatt225leuglutrplysalathrcysargglyargglnpheaspargile657075ttcggggtttggcttggtggggttgagattctcaggagctgcaca270pheglyvaltrpleuglyglyvalgluileleuargsercysthr808590gcagaaccaaggcctaatgggattgtttggactgtcgagaaggac315alagluproargproasnglyilevaltrpthrvalglulysasp95100105atcacaaggtactattcactgcttaagagtaatcaaacacttgct360ilethrargtyrtyrserleuleulysserasnglnthrleuala110115120gtttatcttggcaatttgatagataaaacctacactgggatttat405valtyrleuglyasnleuileasplysthrtyrthrglyiletyr125130135catgtgaatataagccttcatttttaccctgctaaagagaaattg450hisvalasnileserleuhisphetyrproalalysglulysleu140145150aattcttttcagcaaaagttggataatttggcatctgggtaccat495asnserpheglnglnlysleuaspasnleualaserglytyrhis155160165tcttgggctgatttgattttacccgtttcgagaaatctgcctttg540sertrpalaaspleuileleuprovalserargasnleuproleu170175180aatgatgggttgtggtttgaagttcagaattcaaatgatacggaa585asnaspglyleutrpphegluvalglnasnserasnaspthrglu185190195ttgaaggagttcaagattccacaaaatgcttatagggctgtgttg630leulysgluphelysileproglnasnalatyrargalavalleu200205210gaggtgtatgtttcatttcacgagaatgatgaattttggtattca675gluvaltyrvalserphehisgluasnaspgluphetrptyrser215220225aatcttcctaatgagtacatagctgcaaacaaccttagcggtaca720asnleuproasnglutyrilealaalaasnasnleuserglythr230235240cccggaaatgggccttttagggaggttgtggtcagtctagatggt765proglyasnglyprophearggluvalvalvalserleuaspgly245250255gaggttgtt774gluvalval ( 2 ) information for seq id no : 39 :( i ) sequence characteristics :( a ) length : 774 ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna to mrna ( xi ) sequence description : seq id no : 39 : cacgacacgccgccgaccgtattttttgaagtcaccaaacccatt45hisaspthrproprothrvalphephegluvalthrlysproile151015gaagtaccaaaaaccaagtcgtgttcccagctcattctccagcat90gluvalprolysthrlyssercysserglnleuileleuglnhis202530gactttgcctacacatatggccaagctccagtctttgcaaactac135aspphealatyrthrtyrglyglnalaprovalphealaasntyr354045acccctccttccgattgcccatctcaaactttctccacaattgtc180thrproproseraspcysproserglnthrpheserthrileval505560cttgaatggaaagctacctgcagaggaaggcaatttgaccgcatt225leuglutrplysalathrcysargglyargglnpheaspargile657075ttcggggtttggcttggtggggttgagattctcaggagctgcaca270pheglyvaltrpleuglyglyvalgluileleuargsercysthr808590gcagaaccaaggcctaatgggattgtttggactgtcgagaaggac315alagluproargproasnglyilevaltrpthrvalglulysasp95100105atcacaaggtactattcactgcttaagagtaatcaaacacttgct360ilethrargtyrtyrserleuleulysserasnglnthrleuala110115120gtttatcttggcaatttgatagataaaacctacactgggatttat405valtyrleuglyasnleuileasplysthrtyrthrglyiletyr125130135catgtgaatataagccttcatttttaccctgctgaagagaaattg450hisvalasnileserleuhisphetyrproalagluglulysleu140145150aattcttttcagcaaaagttggataatttggcatctgggtaccat495asnserpheglnglnlysleuaspasnleualaserglytyrhis155160165tcttgggctgatttgattttacccatttcgagaaatctgcctttg540sertrpalaaspleuileleuproileserargasnleuproleu170175180aatgatgggttgtggtttgaagttcagaattcaaatgatacggaa585asnaspglyleutrpphegluvalglnasnserasnaspthrglu185190195ttgaaggagttcaagattccacaaaatgcttatagggctgtgttg630leulysgluphelysileproglnasnalatyrargalavalleu200205210gaggtgtatgtttcatttcacgagaatgatgaattttggtattca675gluvaltyrvalserphehisgluasnaspgluphetrptyrser215220225aatcttcctaatgagtacatagctgcaaacaaccttagcggtaca720asnleuproasnglutyrilealaalaasnasnleuserglythr230235240cctggaaatgggccttttagggaggttgtggtcagtctagatggt765proglyasnglyprophearggluvalvalvalserleuaspgly245250255gaggttgtt774gluvalval ( 2 ) information for seq id no : 40 :( i ) sequence characteristics :( a ) length : 774 ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna to mrna ( xi ) sequence description : seq id no : 40 : cacgacacaccgccgactgtattttttgaagtcaccaaacccatt45hisaspthrproprothrvalphephegluvalthrlysproile151015gaagtaccaaaaaccaagccgtgttcccagctcattctccagcat90gluvalprolysthrlysprocysserglnleuileleuglnhis202530gactttgcctacacatatggccaagctccagtctttgcaaactac135aspphealatyrthrtyrglyglnalaprovalphealaasntyr354045acccctccttccgattgcccatctcaaactttctccacaattgtc180thrproproseraspcysproserglnthrpheserthrileval505560cttgaatggaaagctacctgcagaagaaggcaatttgaccgcatt225leuglutrplysalathrcysargargargglnpheaspargile657075ttcggggtttggcttggtggggttgagattctcaggagctgcaca270pheglyvaltrpleuglyglyvalgluileleuargsercysthr808590gcagaaccaaggcctaatgggattgtttggactgtcgagaaggac315alagluproargproasnglyilevaltrpthrvalglulysasp95100105atcacaaggtactattcactgcttaagagtaatcaaacacttgct360ilethrargtyrtyrserleuleulysserasnglnthrleuala110115120gtttatcttggcaatttgatagataaaacctacactgggatttat405valtyrleuglyasnleuileasplysthrtyrthrglyiletyr125130135catgtgaatataagccttcatttttaccctgctgaagagaaattg450hisvalasnileserleuhisphetyrproalagluglulysleu140145150aattcttttcagcaaaagttggataatttggcatctgggtaccat495asnserpheglnglnlysleuaspasnleualaserglytyrhis155160165tcttgggctgatttgattttacccatttcgagaaatctgcctttg540sertrpalaaspleuileleuproileserargasnleuproleu170175180aatgatgggttgtggtttgaagttcagaattcaaatgatacggaa585asnaspglyleutrpphegluvalglnasnserasnaspthrglu185190195ttgaaggagttcaagattccacaaaatgcttatagggctgtgttg630leulysgluphelysileproglnasnalatyrargalavalleu200205210gaggtgtatgtttcatttcacgagaatgatgaattttggtattca675gluvaltyrvalserphehisgluasnaspgluphetrptyrser215220225aatcttcctaatgagtacatagctgcaaacaaccttagcggtaca720asnleuproasnglutyrilealaalaasnasnleuserglythr230235240cctggaaatgggccttttagggaggttgtggtcagtctagatggt765proglyasnglyprophearggluvalvalvalserleuaspgly245250255gaggttgtt774gluvalval ( 2 ) information for seq id no : 41 :( i ) sequence characteristics :( a ) length : 774 ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna to mrna ( xi ) sequence description : seq id no : 41 : cacgatacaccgccgactgtattttttgaagtcaccaaacccatt45hisaspthrproprothrvalphephegluvalthrlysproile151015gaagtaccaaaaaccaagccgtgttcccagctcattctccagcat90gluvalprolysthrlysprocysserglnleuileleuglnhis202530gactttgcctacacatatggccaagctccagtctttgcaaactac135aspphealatyrthrtyrglyglnalaprovalphealaasntyr354045acccctccttccgattgcccatctcaaactttctccacaattgtc180thrproproseraspcysproserglnthrpheserthrileval505560cttgaatggaaagctacctgcagaggaaggcaatttgaccgcatt225leuglutrplysalathrcysargglyargglnpheaspargile657075ttcggggtttggcttggtggggttgagattctcaggagctgcaca270pheglyvaltrpleuglyglyvalgluileleuargsercysthr808590gcagaaccaaggcctaatgggattgtttggactgtcgagaaggac315alagluproargproasnglyilevaltrpthrvalglulysasp95100105atcacaaggtactattcactgcttaagagtaatcaaacacttgct360ilethrargtyrtyrserleuleulysserasnglnthrleuala110115120gtttatcttggcaatttgatagataaaacctacactgggatttat405valtyrleuglyasnleuileasplysthrtyrthrglyiletyr125130135catgtgaatataagccttcatttttaccctgctaaagagaaattg450hisvalasnileserleuhisphetyrproalalysglulysleu140145150aattcttttcagcaaaagttggataatttggcatctgggtaccat495asnserpheglnglnlysleuaspasnleualaserglytyrhis155160165tcttgggctgatttgattttacccgtttcgagaaatctgcctttg540sertrpalaaspleuileleuprovalserargasnleuproleu170175180aatgatgggttgtggtttgaagttcagaattcaaatgatacggaa585asnaspglyleutrpphegluvalglnasnserasnaspthrglu185190195ttgaaggagttcaagattccacaaaatgcttatagggctgtgttg630leulysgluphelysileproglnasnalatyrargalavalleu200205210gaggtgtatgtttcatttcacgagaatgatgaattttggtattca675gluvaltyrvalserphehisgluasnaspgluphetrptyrser215220225aatcttcctaatgagtacatagctgcaaacaaccttagcggtaca720asnleuproasnglutyrilealaalaasnasnleuserglythr230235240cccggaaatgggccttttagggaggttgtggtcagtctagatggt765proglyasnglyprophearggluvalvalvalserleuaspgly245250255gaggttgtt774gluvalval ( 2 ) information for seq id no : 42 :( i ) sequence characteristics :( a ) length : 49 ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( dna )( xi ) sequence description : seq id no : 42 : aaaccatgggatccgaaccgactccgctgcatgataccccgccaactgt49 ( 2 ) information for seq id no : 43 :( i ) sequence characteristics :( a ) length : 25 ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( dna )( xi ) sequence description : seq id no : 43 : tggagaaagtttgagatgggcaatc25__________________________________________________________________________