Patent Application: US-11810193-A

Abstract:
a modified saccharomyces cerevisiae cell , wherein the cell expresses mink but does not express trk1 and trk2 . also disclosed is a process for detecting modulators of mink , which comprises treating such modified saccharomyces cerevisiae cells with a test compound , assessing growth in the presence of a test compound and determining an increase or decrease in potassium uptake into the saccharomyces cerevisiae cells . mink inhibitors are useful anti - arrhythmic or antifibrillatory agents ; activators , anti - ischemic agents .

Description:
the following definitions apply to the terms as used throughout this specification , unless otherwise limited in specific instances . the terms &# 34 ; deficient &# 34 ; or &# 34 ; deficiency &# 34 ; as used with respect to a gene refers to an allele altered ( e . g ., by homologous recombination , resulting in the insertion of foreign sequences ) such that either no product or only an inoperative fragment of the wild - type product can be expressed . a &# 34 ; deficient &# 34 ; allele within this definition may also comprise a gene deletion , wherein the gene has been deleted in toto ; a gene disruption , wherein the gene is interrupted by another gene or nucleic acid sequence ; a partial deletion , wherein one or more bases are deleted ; a substitution , wherein one or more bases are replaced by other bases ; and other such mutations as will be understood by persons having ordinary skill in the art . such deletions , disruptions , and substitutions may take place in , for example , the coding region , a promoter , or an enhancer . the term &# 34 ; modified &# 34 ; as used with respect to a cell refers to a cell in which the wild - type genome has been altered by addition of one or more heterologous genes , a defidency in one or more wild - type genes , or a combination thereof . such modifications may be carried out by transformation and homologous recombination through techniques well understood by those having ordinary skill in the art . the term &# 34 ; a mink protein &# 34 ; refers to a protein having at least the smallest portion of the full - length wild - type mink protein that can result in measurable potassium ion transport in saccharomyces cerevisiae . human mink protein is preferred . exemplary methods for such measurement of potassium ion transport are described herein . proteins having residues 41 to 72 of fig2 are preferred for the mink protein . the term &# 34 ; a functional derivative or mutant thereof &# 34 ; as used with respect to a protein refers to a protein differing from the subject protein by one or more amino acid residues but still having the biochemical function of the protein and greater than about 90 % sequence homology . in the case of the mink protein , a &# 34 ; functional derivative or mutant thereof &# 34 ; refers to such proteins that have potassium transport activity but do not have the identical amino acid sequence shown in fig2 . such derivatives and mutants may include proteins that differ from the wild - type protein by amino acid substitutions , deletions , disruptions , and the like . such differences may be accomplished by genetic means , using such techniques as site - directed mutagenesis or mutagenic pcr prior to translation , or by chemical means , using proteases and / or ligases after translation . the yeast cell of the present invention begins with a yeast strain having deficient alleles in the trk1 and trk2 genes required for potassium uptake . the modified alleles are genetically stable and recessive , so they can be complemented with activities encoded by heterologous genes introduced into the strain . biosynthetic marker genes ( e . g ., his3 and trp1 ) may be inserted into the loci for the wild - type potassium uptake genes . such modified alleles may then be transformed into yeast to replace the wild - type loci by homologous recombination . the modified alleles are readily detected by scoring for the presence of the biosynthetic marker . the markers inserted into the transporter loci also provide a simple means of transferring these mutations into other genetic backgrounds . a strain deficient in one potassium uptake gene ( e . g ., trk1 ) may be crossed with a strain of opposite mating type deficient in another potassium uptake gene ( e . g ., trk2 ). progeny bearing both genetic deficiencies may be identified by scoring for the presence of both markers . a variety of potassium ion channels may be introduced into this strain to assess whether these channels can complement the growth defect on potassium - deficient media . alternatively , the strain may be used to screen gene libraries ( e . g ., human cdna libraries ) for clones that complement the growth defect . this analysis may reveal potassium ion channels that have been described physiologically but have not yet been cloned . each application results in a strain expressing a foreign potassium ion channel , useful in a screen for modulators of the channel . a yeast strain expressing a potassium ion channel can be adapted to natural products screening . a simple screen design involving growth inhibition or potassium ion uptake in agar plates or in liquid culture may detect compounds that modulate channel function . for screening of activators of the potassium ion channel , the screen may include such modifications as metabolic inhibition of calcium , anoxic growth conditions , increased growth rate , or increased potassium ion uptake . a test substance used in the process of this invention may be , for example , a synthetic compound or a natural product . such natural products include extracts from plants , animals and microorganisms . a mutation in the high affinity potassium transporter , trk1 , began with the isolation of the wild type gene from a yeast genomic library . large portions of the 5 &# 39 ; and 3 &# 39 ; ends of the trk1 coding region ( fig3 seq . id . no . 3 ) were amplified in polymerase chain reactions , radiolabeled and hybridized to colonies containing the yeast genomic library . three clones contained the entire trk1 locus ( promoter and coding region ) as determined by hybridization to both 5 &# 39 ; and 3 &# 39 ; fragments . these results were further confirmed by southern blots and restriction mapping . the trk1 gene was subcloned into bacterial vectors , puc19 and psl1180 . a 2350 bp deletion of the coding region was created by digestion with xbai . into this deletion was inserted the yeast biosynthetic gene his3 . the mutant allele , trk1 :: his3 , was used to transform yeast strains to histidine prototrophy . transformants were verified by southern blots . because trk1 encodes the high affinity potassium ion transporter , the mutant strain should have a growth requirement for potassium . as expected , his + transformants grew very poorly on media containing 100 μm potassium chloride . the growth defect was remediated by increasing the potassium concentration to 100 mm . oligonucleotides were used to produce portions of the trk2 coding region ( fig4 seq . id . no . 5 ) corresponding to the 5 &# 39 ; ( 600 bp ) and 3 &# 39 ; ( 800 bp ) ends of the coding region by polymerase chain reaction ( pcr ) amplification of yeast genomic dna . the fragments were assembled in psl1180 . the construct was linearized at the junction of the 5 &# 39 ; and 3 &# 39 ; coding fragments and religated with a fragment containing the yeast trp1 gene to create trk2 :: trp1 . this construct lacks 1170 basepairs of the trk2 coding region and was used to transform a yeast strain to tryptophan prototrophy . transformants were verified by southern blots . since the trk2 locus encodes a low affinity potassium ion transporter , the mutant strain does not have a growth phenotype on potassium ion - deficient media . the high and low affinity transporter mutations were constructed in yeast strains of opposite mating types to facilitate genetic analysis of the mutants and to obtain the double mutant ( trk1 -, trk2 -) strain . such a cross was carried out between the two strains ( mata , trk1 :: his3 × mata , trk2 :: trp1 ). the diploid strain grew vigorously on all media regardless of potassium concentration , indicating that the mutations are recessive . recessive mutations are required for complementation with heterologous genes introduced into this strain . in haploid , the modified alleles marked by the his3 and trp1 biosynthetic marker genes were readily scored and segregated as expected for two unlinked genes . both markers segregated 2 +: 2 -, and their segregation was independent relative to each other . this result was anticipated , given that trk1 maps to chromosome 10 and trk2 maps to chromosome 11 . the trkpheno - types ( inviable on media with low concentrations of potassium ) co - segregated with the appropriate biosynthetic markers , indicating that the introduced mutations mimic the phenotypes described in rodriguez - navarro , a . and ramos , j ., j . bacteriol . 159 : 940 - 945 ( 1984 ); ramos , j . et al ., arch . microbiol . 143 : 88 - 93 ( 1985 ); gaber , r ., styles , c . and fink , g ., mol . cell biol . 8 : 2848 - 2859 ( 1988 ); and ko , c . and gaber , r ., genetics 125 : 305 - 312 ( 1990 ). three phenotypes for potassium requirements were expected and observed from segregants of this cross , resulting from the following four genotypes : wild type , low affinity mutant , high affinity mutant , and double mutant ( high and low affinity ). physical characterization of the modifications in the four segregants of a tetrad confirmed the segregation results . southern blots and pcr amplification of the mutant loci with primers that flank the trk genes produced the expected products . the double mutant strain was transformed with a plasmid ( pgal ) containing the wild type trk1 gene , the heterologous test gene ( hmink ) or the vector alone . the hmink - transformed strain is a preferred embodiment of this invention and was deposited with the american type culture collection , 12301 parklawn drive , rockville , md . 20852 - 1776 or aug . 27 , 1993 and assigned accession number atcc 74 , 238 . the strain is available upon issuance of a u . s . patent hereon and will be maintained for a period of at least 30 years after the date of deposit , and for a period of at least five years after the most recent request for a sample . transformants were diluted serially and spotted onto plates with either high ( 100 mm ) or low ( 0 . 1 mm ) concentrations of potassium chloride . plates were incubated at 30 ° c . for 2 days . as shown in fig7 strains containing either vector , trk1 , or hmink are able to grow at 100 mm potassium chloride , but only the trk1 or hmink transformed strains grown at 0 . 1 mm potassium chloride . growth measurements of the trk mutants in either potassium - deficient or low ph media are consistent with the phenotypes reported in gaber , r ., styles , c . and fink , g ., mol . cell biol . 8 : 2848 - 2859 ( 1988 ) and ko , c . and gaber , r , genetics 125 : 305 - 312 ( 1990 ). in media supplemented with potassium chloride , the mutant strains grew in a potassium - dependent manner ( fig5 ). in another experiment , the double mutant was transformed with pgal vector including either trk1 or hmink as described above . the transformed strains were then separately challenged with clofilium ( 100 μm ) and dofetilide ( 0 . 1 μm ) at 100 mm and 0 . 1 mm of potassium chloride . as expected , clofilium blocked potassium uptake in the hmink strain but not in the trk1 strain . in addition , the hmink gene used in the transformation was sequenced . this analysis , which included sequencing of both strands , revealed a single nucleotide change resulting in the conversion of amino acid 38 from serine to glycine . this mutation is located in the n - terminus and does not appear to affect hmink function . for further verification , mrna transcripts were expressed in xenopus oocytes . the expression products caused potassium ion currents that are blocked by clofilium ( 50 μm ) and kinetically indistinguishable from hmink currents reported in the literature . this result is consistent with the recent report indicating that residues 41 to 72 define the pore of the protein and are sufficient to form channels in lipid bilayers . ben efraim et al ., biochemistry 32 : 2371 - 2377 ( 1993 ). that report further indicates that the region encompassing residue 38 may not be functionally important . a . trk1 deletion . trk1 dna sequences spanning nucleotides 181 to 4108 were synthesized by pcr amplification using the following oligonucleotides : 5 &# 39 ; atcggcatgcgttgacgatgacgaaagcac 3 &# 39 ; ( seq . id . no . 8 ). the product was digested with bamhi and sphi and cloned into puc19 . the plasmid was digested with xbai to remove nucleotides 610 to 2964 from the coding region . the remaining vector and flanking regions of trk1 was gel purified and ligated with a fragment containing the his3 gene , containing xbai cohesive ends , to create a deletion / insertion mutation . this plasmid was digested with ecori and sphi and then used to transform a haploid yeast strain ( mata ade2 - 1 , can1 - 100 , his3 - 11 , 15 , leu2 - 3 , 112trp1 - 1 , ura3 - 1 ) to hisitidine prototrophy using the lioac procedure . to confirm the disruption of the trk1 locus , his + transformants were assessed by southern blotting and growth on media lacking potassium . b . trk2 deletion . sequences corresponding to the 5 &# 39 ; and 3 &# 39 ; ends of the trk2 coding region were obtained by pcr amplification of yeast genomic dna . the following oligonucleotides were used to produce a portion of the 5 &# 39 ; end spanning nucleotides 1016 to 1639 : the following oligonucleotides were used to produce a portion of the 3 &# 39 ; end spanning nucleotides 2729 to 3584 : the 5 &# 39 ; and 3 &# 39 ; pcr - amplified products were digested with combinations of sali and saci , and saci and bamhi : respectively . fragments were gel purified and then ligated in a three - way ligation with vector psl1180 ( pharmacia ; note that this vector was initially digested with hindiii and ecorv , endfilled and religated to remove a portion of the polylinker . this modified vector was then digested with sali and bamhi and gel - purified for the three - way ligation .) the three - way ligation product was digested with saci and xmai and ligated with the trp1 gene , containing saci and xmai ends , to create an insertion / deletion mutation . this plasmid was digested with sali and bamhi and then used to transform a hapioid yeast strain ( mata ade2 - 1 , can1 - 100 ,. his3 - 11 . 15 , leu2 - 3 . 112 , trp1 - 1 - 1 , ura3 - 1 ) to tryptophan prototrophy . trp + transformants were assessed by southern blotting to confirm the disruption of the trk2 locus . c . to create the double mutant trk1d trk2d , a standard cross between transformants containing the trk1 :: his3 mutation and the trk2 :: trp1 mutation was performed . the resulting diploid was induced to undergo meiosis and tetrads were dissected onto ypd media supplemented with 100 mm kcl . complete tetrads were then assessed for segregation of the markers ( his and trp ) and for growth on media containing either 100 mm , 10 mm , or 0 . 1 mm kcl . in tetratype tetrads , the four genotypes produced three phenotypes with respect to growth on potassium - deficient media . this is the expected result for the segregation of the trk1 and trk2 mutations which are unlinked genes . all colonies arising from spores that received both the his and trp markers grow only on media containing 100 mm kcl , indicating that these colonies were defective in both k + transporters . pgal :: hmink . the following oligonucleotides were used to amplify sequences encoding the human hmink protein from hela cell genomic dna : 5 &# 39 ; gctctagatcatggggaaggcttcgtctc 3 &# 39 ; ( seq . id . no . 14 ). the pcr - amplified product was digested with ecori and xbai gel pudfied and cloned into an identically digested pyes2 vector ( invitrogen ). the plasmid was verified by standard restriction digests and the insert was analyzed by dna sequencing . pgal :: trk1 . the following oligonucleotides were used to amplify sequences encoding the yeast trk1 protein from a plasmid containing the trk1 gene ( this plasmid was isolated from a yeast genomic library by hybridization with 5 &# 39 ; and 3 &# 39 ; dna fragments unique to the trk1 gene ): 5 &# 39 ; cccgctcgagcgatgagtggggattttgtc 3 &# 39 ; ( seq . id . no . 16 ). the pcr - amplified product was digested with bamhi : and xhoi , gel purified and cloned into identically digested pyeura3 vector ( clontech ). the plasmid was verified by restriction digests and complements a trk1 deletion mutation when expressed in vivo . cultures of k + transporter mutant strains containing either pyes2 vector , pgal :: trk1 or pgal :: hmink plasmids were grown in inducing media ( 2 % galactose ) at 30 ° c . with vigorous aeration . a . serial dilutions of each culture were spotted onto agar plates containing either 100 mm or 0 . 1 mm . kcl . b . serial dilutions of strains containing pgal :: trk1 or pgal :: hmink plasmids were spotted onto agar plates containing 100 μm clofilium or 1 μm dofetilide at both kcl concentrations . all plates were incubated at 30 ° c . and scored after 2 days . cultures of strains containing the pgal :: hmink plasmid or the pgal :: trk1 plasmid were grown in inducing media ( 2 % galactose ) at 30 ° c . with vigorous aeration . inhibitors were added to cultures over a range of concentrations . samples were removed at various intervals and the optical density at 600 nm was determined . growth measurements were normalized to values obtained for growth in the absence of drug and are plotted as relative growth versus concentration ( fig6 ). similar cultures grown in inducing medium were embedded in agar and assessed for growth in presence of the compounds listed in table 1 . plates were scored after a 48 - hour incubation at 30 ° c . ; &# 34 ;+&# 34 ; denotes growth inhibition . table 1______________________________________ inhibition in agar hmink trk1compound activity strain strain______________________________________clofilium class iii aa ( i . sub . ks and i . sub . kr ) + - dofetilide class iii aa ( i . sub . kr ) - - e - 4031 class iii aa ( i . sub . kr ) - - quinidine class i aa ( na . sup .+ and k . sup .+) + + tetrodotoxin na . sup .+ channel blocker - - tea cl nonselective k . sup .+ inhibitor + - diltiazem calcium entry blocker - - ______________________________________ ( aa denotes antiarrhythmic ; + indicates compound produced a zone on inhibition ) __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 16 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 3707 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 .. 3707 ( xi ) sequence description : seq id no : 1 : 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( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 1235 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 2 : 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( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 2669 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 .. 2669 ( xi ) sequence description : seq id no : 3 : 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( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 889 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 4 : metprothralalysargthrserserargalaserleualaleupro151015pheglnleuargleuvalhislyslyssertrpglyhisargleuarg202530asppheileserglypheleulyssercysargproilealalystyr354045valpheproasnpheilevalvalhistyriletyrleuilethrleu505560serileileglyserileleuleutyrprocyslysasnthralaphe65707580ileaspvalleupheleualaalaglyalaserthrglnglyglyleu859095alathrlysserthrasnasppheasnleutyrglnglnilevalval100105110tyrvalilethrleuleuserthrproileleuilehisglypheleu115120125alaphevalargleutyrtrpphegluargtyrpheaspasnilearg130135140aspileserlysglnasnphelysleuargargthrmetthrleugln145150155160glnarggluleuserglyserserglyasnalaalaargserargser165170175phelysaspasnleupheargglylysphevalserarggluasppro180185190argglnseralaseraspvalprometaspserproaspthrserala195200205leuserserileserproleuasnvalserserserlysglugluser210215220seraspthrglnserserproproasnpheserserlysargglnpro225230235240seraspvalaspproargaspiletyrlysserilemetmetleugln245250255lysglnglnglulysserasnalaasnserthraspserpheserser260265270gluthrasnglyproalapheilevalglngluarghisgluargarg275280285alaprohiscysserleulysarghisservalleuprosersergln290295300gluleuasnlysleualaglnthrlysserpheglnlysleuleugly305310315320leuargargaspgluglyasphisasptyrpheaspglyalaprohis325330335lystyrmetvalthrlyslyslyslysileserargthrglnsercys340345350asnileprothrtyrthralaserproserprolysthrserglygln355360365valvalgluasnhisargasnleualalysseralaproserserphe370375380valaspgluglumetserpheserproglngluserleuasnleugln385390395400pheglnalahisproprolysprolysargarggluglyaspilegly405410415hisprophethrargthrmetserthrasntyrleusertrpglnpro420425430thrpheglyargasnservalpheileglyleuthrlysglnglnlys435440445glugluleuglyglyvalglutyrargalaleuargleuleucyscys450455460ileleumetvaltyrtyrileglypheasnileleualaphevalthr465470475480ilevalprotrpalacysthrarghishistyrsergluileilearg485490495argasnglyvalserprothrtrptrpglyphephethralametser500505510alapheserasnleuglyleuserleuthralaaspsermetvalser515520525pheaspthralaprotyrproleuilephemetmetphepheileile530535540ileglyasnthrglypheproilemetleuargpheileiletrpile545550555560metphelysthrserargaspleuserglnphelysgluserleugly565570575pheleuleuasphisproargargcysphethrleuleupheproser580585590glyprothrtrptrpleuphethrthrleuvalvalleuasnalathr595600605asptrpileleupheileileleuasppheasnseralavalvalarg610615620glnvalalalysglytyrargalaleumetglyleupheglnserval625630635640cysthrargthralaglypheasnvalvalaspleuserlysleuhis645650655proserileglnvalsertyrmetleumetmettyrvalservalleu660665670proleualaileserileargargthrasnvaltyrglugluglnser675680685leuglyleutyraspserglyglnaspaspgluasnilethrhisglu690695700aspaspilelysgluthrasphisaspglygluserglugluargasp705710715720thrvalserthrlysserlysprolyslysglnserprolysserphe725730735valglyalahisleuargargglnleuserpheaspleutrptyrleu740745750pheleuglyleupheileilecysilecysgluglyarglysileglu755760765aspvalasnlysproasppheasnvalphealaileleuphegluval770775780valseralatyrglythrvalglyleuserleuglytyrproasnthr785790795800asnthrserleuseralaglnphethrvalleuserlysleuvalile805810815ilealametleuileargglyargasnargglyleuprotyrthrleu820825830aspargalailemetleuproserasplysleugluglnileasparg835840845leuglnaspmetlysalalysglylysleuleualalysvalglyglu850855860aspprometthrthrtyrvallyslysargserhislysleulyslys865870875880ilealathrlysphetrpglylyshis885 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 398 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 .. 398 ( xi ) sequence description : seq id no : 5 : atgcccaggatgatcctgtctaacaccacagcggtgacgccctttctg48metproargmetileleuserasnthrthralavalthrpropheleu151015accaagctgtggcaggagacagttcagcagggtggcaacatgtcgggc96thrlysleutrpglngluthrvalglnglnglyglyasnmetsergly202530ctggcccgcaggtccccccgcagcagtgacggcaagctggaggccctc144leualaargargserproargserseraspglylysleuglualaleu354045tacgtcctcatggtactgggattcttcggcttcttcaccctgggcatc192tyrvalleumetvalleuglyphepheglyphephethrleuglyile505560atgctgagctacatccgctccaagaagctggagcactcgaacgaccca240metleusertyrileargserlyslysleugluhisserasnasppro65707580ttcaacgtctacatcgagtccgatgcctggcaagagaaggacaaggcc288pheasnvaltyrilegluseraspalatrpglnglulysasplysala859095tatgtccaggcccgggtcctggagagctacaggtcgtgctatgtcgtt336tyrvalglnalaargvalleuglusertyrargsercystyrvalval100105110gaaaaccatctggccatagaacaacccaacacacaccttcctgagacg384gluasnhisleualailegluglnproasnthrhisleuprogluthr115120125aagccttccccatg398lysproserpro130 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 132 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 6 : metproargmetileleuserasnthrthralavalthrpropheleu151015thrlysleutrpglngluthrvalglnglnglyglyasnmetsergly202530leualaargargserproargserseraspglylysleuglualaleu354045tyrvalleumetvalleuglyphepheglyphephethrleuglyile505560metleusertyrileargserlyslysleugluhisserasnasppro65707580pheasnvaltyrilegluseraspalatrpglnglulysasplysala859095tyrvalglnalaargvalleuglusertyrargsercystyrvalval100105110gluasnhisleualailegluglnproasnthrhisleuprogluthr115120125lysproserpro130 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 29 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 7 : ccggatccatgcattttagaagaacgatg29 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 30 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 8 : atcggcatgcgttgacgatgacgaaagcac30 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 30 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 9 : gcggatccgtcgacttcatttccgggttct30 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 34 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 10 : gctaggagctcccgggttggcgcttacttgagaa34 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 25 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 11 : gtcttaaacgctacggattggattc25 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 22 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 12 : gcttcccccaaaactttgttgc22 ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 35 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 13 : gcgaattcaaaaaaaatgatcctgtctaacaccac35 ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 29 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 14 : gctctagatcatggggaaggcttcgtctc29 ( 2 ) information for seq id no : 15 :( i ) sequence characteristics :( a ) length : 36 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 15 : cgggatccaaaaaatgcattttagaagaacgatgag36 ( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 30 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 16 : cccgctcgagcgatgagtggggattttgtc30__________________________________________________________________________