Patent Application: US-53979895-A

Abstract:
a plant δ 6 palmitoyl - acyl carrier protein desaturase , the gene encoding the desaturase , and transgenic plants and plant cells containing the heterologous dna encoding the desaturase are described . the desaturase introduces a double bond at the sixth carbon atom from the carboxyl end of a 16 carbon saturated fatty acid , and is therefore useful in production of plant seeds having a modified fatty acid profile .

Description:
plant material - studies were conducted using developing endosperm dissected from fruits of t . alata bojer ex sims ( black - eyed susan vine ) ( northrup king , minneapolis , minn .). fruits were collected from plants grown either outdoors in pots during summers in east lansing , mich . or under greenhouse conditions with natural illumination . in the latter case , flowers required hand pollination for adequate fruit set . endosperm was frozen in liquid nitrogen following dissection and stored at - 70 ° c . until use in enzyme assays of rna extraction . acyl - acp desaturation assays -- approximately 200 - 300 mg of developing t . alata endosperm was homogenized in 3 ml of buffer consisting of 100 mm tris - hcl , ph 7 . 5 , 2 . 5 mm dithiothreitol , 1 mm isoascorbate , 10 % ( v / v ) glycerol , and 1 . 5 % ( w / v ) polyvinylpolypyrrolidone using an elvehjem tissue grinder . debris and polyvinylpolypyrrolidone were subsequently removed by centrifugation at 14 , 000 × g for 5 min . the supernatant was then passed through two layers of miracloth ( calbiochem ) and spun for an additional 10 min at 30 , 000 × g . the soluble phase was removed while attempting to avoid recovery of the floating fat layer . a portion of contaminating fat was extracted by passing the supernatant through glass wool loosely packed in a pasteur pipette . the supernatant from the 30 , 000 × g spin was further clarified by centrifugation at 100 , 000 × g for 60 min . all centrifugation steps were performed at 5 ° c . the resulting supernatant was used immediately for desaturation assays described below or frozen in aliquots in liquid n 2 and stored at - 70 ° c . until further use . of note , extracts developed a brown color , presumably due to extensive phenolic oxidation , when maintained at - 20 ° c . for longer than 1 - 2 weeks . acyl - acp desaturation assays were based on those previously described ( 8 , 9 ). assays were performed in a total volume of 150 μl in loosely capped 13 × 100 - mm glass tubes and consisted of 1 . 25 mm nadph ( from a freshly prepared stock in 100 mm tricine , ph 8 . 2 ), 3 . 3 mm ascorbate , 0 . 7 mm dithiothreitol , 8000 units of bovine liver catalase ( sigma ), 5 μg of bovine serum albumin ( fraction v ) ( sigma ), 20 μg of spinach ferredoxin ( sigma ), 80 milliunits of spinach ferredoxin : nadph reductase ( sigma ), 33 mm pipes , ph 6 . 0 , and 118 pmol of [ 1 - 14 c ] acyl - acp or - coa . reactions were started with the addition of the 100 , 000 × g supernatant of homogenized t . alata endosperm ( typically 20 - 25 μg of total protein ) and were conducted at room temperature (- 22 ° c .) with shaking ( 100 revolutions / min ). assays were terminated with the addition of 850 μl of 2 . 35 m naoh and carrier fatty acids ( 30 μg of palmitic and petroselinic acid ). the stopped reactions were then heated at 85 ° c . for 1 hour . following acidification with 350 μl of 4m h 2 so 4 , the resulting free fatty acids were recovered by three extractions with 2 . 5 ml of hexane . fatty acids were converted to methyl ester derivatives with 10 % ( w / v ) boron trichloride in methanol ( alltech ) using the method described by morrison and smith ( 24 ). reaction products were then analyzed on 15 % agno 3 tlc plates developed sequentially to heights of 10 and 20 cm in toluene at - 20 ° c . tlc plates were prepared as described previously ( 25 ). radioactivity was detected by autoradiography and quantified by liquid scintillation counting of tlc scrapings in a non - aqueous complete mixture . to confirm the identity of 16 : 1δ 6 produced from palmitoyl - acp , assays were conducted as described above using [ u - 14 c ] palmitoyl - acp as the substrate . the methyl ester derivative of the monounsaturated product was purified by argentation tlc as described above and eluted from tlc scrapings with hexane / ethyl ether ( 2 : 1 , v / v ). the monounsaturated methyl ester was then cleaved at its double bond using permanganate - periodate oxidation ( 26 ). chain lengths of oxidation products were determined relative to [ 14 c ] fatty acid standards by reverse - phase tlc using a mobile phase of acetonitrile / methanol / water ( 75 : 25 : 0 . 5 ). inhibition of desaturase activity was examined by supplementing assays with 1 mm kcn ( neutralized ) or 1 mm h 2 o 2 . in the latter case , catalase was omitted from reactions . oxygen dependence of desaturase activity was characterized by purging assay tubes completely with nitrogen prior to and after addition of plant extract , and the reaction tube was tightly capped for the duration of the assay . radiolabeled acyl - acps were synthesized enzymatically using escherichia coli acp according to the method of rock and garwin ( 27 ). the following fatty acids were used in the synthesis of acyl - acps : [ 1 - 14 c ] myristic acid ( american radiolabeled chemicals , st . louis , mo .) ( specific activity 55 mci / mmol ), [ 1 - 14 c ] palmitic acid ( nen dupont ) ( specific activity 58 mci / mmol ), [ u - 14 c ] palmitic acid ( nen dupont ) ( specific activity 800 mci / mmol ), and [ 1 - 14 c ] stearic acid ( american radiolabeled chemicals ) ( specific activity 55 mci / mmol ). [ 1 - 14 c ] palmitoyl - coa ( specific activity 52 mci / mmol ) was purchased from amersham corp . a [ 14 c ] petroselinic acid standard was prepared by incubation of coriander endosperm slices in [ 1 - 14 c ] acetate as described previously ( 25 ). t . alata endosperm cdna library construction -- total rna was isolated from t . alata endosperm using the method of hall et al . ( 28 ). rna was then passed through a cellulose ( sigma cell 50 , sigma ) column in order to reduce amounts of polysaccharides potentially recovered along with the rna . poly ( a ) + rna was enriched by passing total rna once through a column of oligo ( dt ) cellulose ( pharmacia lkb biotechnology inc .) and subsequently used in the construction of a uni - zap xr ( stratagene ) cdna expression library according to the instructions of the manufacturer . a portion of the total amplified library packaged in phage was mass excised ( 29 ) yielding pbluescript ii sk (-) harboring cdna inserts . the recovered plasmid dna was used for cdna isolation by colony hybridization and polymerase chain reaction ( pcr ) amplification as described below . pcr amplification of nucleotide sequences encoding acyl - acp desaturases -- fully degenerate sense and antisense oligonucleotides were prepared that corresponded respectively to the conserved amino acid sequences ( seq id nos 10 and 18 ) gly - asp - met - ile - thr - glu - glu and glu - lys - thr - ile - gln - tyr - leu present in δ 9 18 : 0 -( 13 , 15 - 20 ) and δ 4 16 : 0 - acp desaturases ( 21 ). the sequence of the resulting sense and antisense oligonucleotides ( seq id nos 5 and 6 ) were 5 &# 39 ;- gg ( a / c / g / t ) ga ( c / t ) atgat ( a / c / t ) ac ( a / c / g / t ) ga ( a / g ) ga - 3 &# 39 ; and 5 &# 39 ;- a ( a / g )( a / g ) tattg ( a / g / t ) at ( a / c / g / t ) gt ( c / t ) tt ( c / t ) tc - 3 &# 39 ;, respectively . included on the 5 &# 39 ; terminus of each oligonucleotide was sequence ( 5 &# 39 ;- caucaucaucau - 3 &# 39 ; or 5 &# 39 ;- cuacuacuacua - 3 &# 39 ; seq id nos 7 and 8 ) that allowed for insertion of pcr products into the pamp1 vector ( life technologies , inc .). template for pcr amplification was generated by transformation of the solr strain ( stratagene ) of e . coli with an aliquot of the mass - excised t . alata endosperm cdna library . following growth of transformed e . coli to stationary phase in 3 ml of liquid culture , plasmid dna was purified for use as template in pcr amplification . reactions were performed in a 50 - μl volume and consisted of 10 μm sense and antisense oligonucleotides , 150 - 300 ng of plasmid dna derived from the t . alata cdna library , 2 mm mgcl 2 , 0 . 2 mm dntps , 1 × taq reaction buffer ( life technologies , inc . ), and 5 units of taq polymerase ( life technologies , inc .). temperature conditions for pcr amplification were 5 min at 95 ° c . and 25 cycles of 1 min at 95 ° c ., 1 . 5 min at 55 ° c ., and 1 . 5 min at 72 ° c . this was followed by an additional 10 min extension at 72 ° c . pcr fragments of approximately 215 base pairs were gel - purified , ligated into the pamp1 vector using the cloneamp system ( life technologies , inc .) according to the manufacturer &# 39 ; s protocol , and introduced into e . coli dh5a ( life technologies , inc .). the resulting colonies were screened using colony hybridization as described by sambrook et al . ( 30 ). a &# 34 ; negative &# 34 ; screening protocol was used to reduce the chances of reisolating cdnas ( ptad1 , 2 , and 3 ) encoding δ 9 18 : 0 - acp desaturases that were previously obtained by antibody screening of the t . alata endosperm cdna library ( 19 ). dna probes for library screening were formed by pcr amplification of portions of ptad 1 , 2 , and 3 . primers and pcr reaction conditions were the same as those described above . an equimolar mixture of the pcr products derived from ptad1 , 2 , and 3 was used as template for the synthesis of [∝- 32 p ] dctp random - primed labeled probes . hybridization of plasmids of lysed colonies with radiolabeled probes was carried out in 6 × ssc and 0 . 25 % ( w / v ) non - fat dry milk with shaking for 4 hours at 53 ° c . as described by sambrook et al . ( 30 ). filters were washed three times in 1 × ssc and 0 . 1 % sds at 60 ° c . ( 45 min / wash ) and exposed to autoradiography . plasmid dna was subsequently isolated from 10 colonies which displayed little or no hybridization to the probes . nucleotide sequence of the inserts of these plasmids was obtained by dideoxy chain termination using sequenase 2 . 0 ( united states biochemical inc .) according to the manufacturer &# 39 ; s instructions . two classes of plasmids were identified ( designated pec6 and 7 ), both of which contained inserts encoding portions of apparent acyl - acp desaturases ( based on amino acid identity with known δ 9 18 : 0 - and δ 4 16 : 0 - acp desaturases ). screening of a t . alata endosperm cdna library for a full - length divergent acyl - acp desaturase -- aliquots of the mass excised t . alata endosperm cdna library were used to transform e . coli solr cells . approximately 50 , 000 colonies were screened using colony hybridization as described previously ( 30 ). nucleotide probes for screening were generated by [∝- 32 p ] dctp random - primed hexamer labeling of inserts of pec6 and 7 . hybridization and washing conditions were the same as those described above . colonies containing plasmid dna that strongly hybridized to the probe derived from pec6 were isolated , and nucleotide sequence was obtained from both strands of the longest cdna insert ( the corresponding plasmid was designated ptad4 ) using sequenase 2 . 0 . because of a relative lack of abundance , colonies containing plasmid hybridizing to the pec7 - derived probe were not further characterized . e . coli expression of a putative cdna for δ 6 palmitoyl - acp desaturase -- to determine the activity of the desaturase encoded by ptad4 , the portion of the clone corresponding to the mature peptide ( total protein minus plastid transit peptide ) was expressed in e . coli . this region of the cdna insert of ptad4 was first amplified by pcr using vent dna polymerase ( new england biolabs ). the nucleotide sequence of the sense primer was 5 &# 39 ;- gcttcgactattactcac3 -&# 39 ;( seq id no : 9 ). m - 13 (- 20 ) forward primer was used as the antisense oligonucleotide . the pcr product was blunt - end ligated into the ncol site of the e . coli expression vector pet3d ( novagen ) as described ( 30 ). the ncol - digested vector had been previously treated with the klenow fragment of dna polymerase i to fill - in 5 &# 39 ; protruding ends . the junction between the vector and the 5 &# 39 ; terminus of the insert was sequenced to confirm that the pcr product was ligated into pet3d in the proper reading frame . this construct was subsequently introduced into the e . coli strain bl21 plyss and grown in lb media with carbenicillin ( 125 μg / ml ) and chloroamphenicol ( 30 μg / ml ) selection . at a cell density of od 600 ˜ 0 . 8 , cultures were induced with the addition of isopropyl - 1 - thio - β - d - galactopyranoside to a final concentration of 0 . 5 mm and grown for an additional 4 hours . cells were then washed in 50 mm tris - hcl , ph 7 . 5 , lysed by two freeze - thaw cycles ( using a liquid nitrogen bath for freezing and a 22 ° c . water bath for thawing ). lysates were then incubated with bovine pancreas dnase i ( boehringer mannheim ) ( 20 μg / ml ) for 15 min at 22 ° c . the extract was subsequently centrifuged at 14 , 000 × g for 5 min . the resulting supernatant was used for acyl - acp desaturation assays as described above . radiolabel in the tlc - analyzed reaction products was detected using a bioscan system 200 image scanner . the double bond position of the monounsaturated product was determined by gas chromatography - mass spectrometry analysis of its dimethyl disulfide derivative ( 31 ). in these studies , the desaturation assays described above were scaled up 6 - fold , and reactions were conducted with 2 . 6 nmol of [ 1 - 14 c ] 16 : 0 - acp and 1 . 1 mg of soluble protein of lysed e . coli expressing the t . alata cdna . assays were conducted for 4 hours . high protein concentrations and long incubation periods were used to ensure the synthesis of sufficient amounts of monounsaturated fatty acid for mass spectral analyses . reaction products were converted to fatty acid methyl esters as described above and subsequently reacted with 100 μl of an iodine solution ( 60 mg / ml ethyl ether ) and 350 μl of dimethyl disulfide ( aldrich ). after 3 hours of incubation with shaking ( 250 revolutions / min ) at 37 ° c ., dimethyl disulfide derivatives of unsaturated fatty acid methyl esters were extracted as described previously ( 32 ). these derivatives ( dissolved in hexane ) were then analyzed by gas chromatograph - mass spectrometry using a hewlett packard ph589011 gas chromatograph interfaced with a hp5971 mass selective detector . separation of analytes was achieved using a db23 ( 30 m × 0 . 25 mm inner diameter ) column ( j & amp ; w scientific ) with the oven temperature programmed from 185 ° c . ( 3 min hold ) to 230 ° c . at rate of 2 . 5 ° c ./ min . detection of a soluble δ 6 palmitoyl - acp desaturase in t . alata endosperm extracts -- the seed oil of t . alata is composed of nearly 85 % weight of the unusual monounsaturated fatty acid δ 6 hexadecenoic acid ( 16 : 1δ 6 ). to examine the metabolic origin of the double bond of this fatty acid , the 100 , 000 × g supernatant of a homogenate of developing t . alata seed endosperm was incubated with [ 14 c ] 16 : 0 - acp and potential desaturase cofactors . using this assay system , substantial amounts of 16 : 0 - acp desaturase activity were detected in the soluble endosperm extract ( fig1 ). in the absence of a radiolabeled standard for 16 : 1δ 6 , two independent analytical methods indicated that the double bond of the resulting 16 : 1 moiety was positioned at the δ 6 carbon atom : 1 ) the 16 : 1 desaturation product displayed mobility on argentation tlc plates similar to that of the δ 6 monounsaturated fatty acid petroselinic acid ( 18 : 1δ 6 ) when these molecules were analyzed as methyl ester derivatives ( fig1 ) and 2 ) permanganate - periodate oxidation of the methyl ester of the 16 : 1 desaturation product gave rise to a molecule with mobility on reverse - phase tlc equivalent to that of decanoic acid ( 10 : 0 ) ( fig2 product b ) as well as to an acyl moiety containing a lesser number of carbon atoms ( fig2 product a ). substrate properties of the δ 6 acyl - acp desaturase -- to confirm that the δ 6 desaturase identified above is most active with 16 : 0 - acp , assays were conducted using 14 c - saturated acyl - acp substrates containing 14 , 16 , and 18 carbons . as with 16 : 0 - acp ( described above ), δ 6 desaturase activity was also detected when [ 1 - 14 c ] 14 : 0 and 18 : 0 - acp were reacted with a 100 , 000 × g supernatant of a t . alata endosperm homogenate . following derivitization , a portion of the desaturation products - resulting from 18 : 0 - acp comigrated on argentation tlc with the methyl ester of petroselinic acid , which was resolvable in this system from methyl oleic acid ( data now shown ). similarly , the desaturation product arising from 14 : 0 - acp migrated in the expected position for a fatty acid containing a δ 6 double bond ( data not shown ). therefore , it appears that the δ 6 desaturase of t . alata endosperm positions the placement of unsaturation with regard to the carboxyl end of fatty acid substrates . this double bond positioning property has been previously observed with the δ 9 18 : 0 - and δ 4 16 : 0 - acp desaturases ( 22 , 33 ). under the assay conditions used , δ 6 16 : 0 - acp desaturase activity in the 100 , 000 × g supernatant of a t . alata endosperm homogenate was essentially linear over 10 min ( fig3 ). when assays were conducted over this time period , the specific activity of the δ 6 desaturase was approximately 7 - fold higher using [ 1 - 14 c ] 16 : 0 - acp as a substrate rather than either [ 1 - 14 c ] 14 : 0 - or 18 : 0 - acp ( table 1 ). values obtained with the latter substrate , however , were obscured because of the presence of completing δ 9 18 : 0 - acp desaturase activity in the endosperm extract . finally , no desaturase activity was detected when [ 1 - 14 c ] 16 : 0 - coa was presented as a potential substrate . overall , these results indicate that the δ 6 desaturase is most active in vitro with 16 : 0 esterified to acp . table i______________________________________in vitro substrate specificities of acyl - acp or - coa desaturasesof t . alata endospermdesaturase assays were conducted for 10 min using 118 pmol of [ 1 -. sup . 14 c ] acyl - acp or - coa substrate and 23 μg oftotal protein from 100 , 000 × g supernatant of at . alata endosperm homogenate . monounsaturated products . sup . asubstrate δ . sup . 6 δ . sup . 9______________________________________ pmol / min / mg protein14 : 0 - acp 13 nd . sup . b16 : 0 - acp 99 nd16 : 0 - coa nd nd18 : 0 - acp 12 173 . sup . c______________________________________ . sup . a 14 : 1δ . sup . 6 , 18 : 1δ . sup . 6 , and 18 : 1δ . sup . 9 were identified by the mobilities of these fatty acids on argentation tlc plates . . sup . b not detected . . sup . c assay conditions were adjusted only for the linear measurement of δ . sup . 6 desaturase activity . therefore this value may underestimate the specific activity of δ . sup . 9 18 : 0acp desaturase . cofactors and inhibitors of δ 6 palmitoyl - acp desaturase activity -- additional in vitro assays were conducted to compare the functional properties of the δ 6 16 : 0 - acp desaturase with those previously determined for the δ 9 18 : 0 - acp desaturase ( 7 , 8 , 10 ). in this regard , virtually no δ 6 16 : 0 - acp desaturase activity was detected in the 100 , 000 × g supernatant of t . alata endosperm homogenates when assays were conducted in the absence of ferredoxin or molecular oxygen ( fig4 ). δ 6 16 : 0 - acp desaturase activity was also reduced when catalase was omitted from assays . furthermore , the inclusion of 1 mm kcn or h 2 o 2 in reactions resulted in the loss of most of the desaturase activity . such catalytic properties of the t . alata δ 6 16 : 0 - acp desaturase were similar to those previously described for the δ 9 18 : 0 - acp desaturase ( 7 , 8 , 10 ). isolation of a cdna encoding a diverged acyl - acp desaturase from t . alata endosperm -- based on functional similarities of the δ 6 16 : 0 - and δ 9 18 : 0 - acp desaturases described above , we examined whether these enzymes are also structurally related . to address this question , attempts were made to isolate a cdna for the δ 6 16 : 0 - acp desaturase using δ 9 18 : 0 - acp desaturase - derived probes . as a first approach , a cdna expression library prepared from poly ( a )* rna of t . alata endosperm was screened with antibodies against the δ 9 18 : 0 - acp desaturase of avocado ( 13 ). this method was previously used to obtain a cdna for the δ 4 16 : 0 - acp desaturase of coriander endosperm ( 21 ). in the present study , however , antibody screening of the t . alata endosperm expression library yielded only cdnas for three apparent isoforms of the δ 9 18 : 0 - acp desaturase , which were designated ptad1 , 2 , and 3 ( 19 ). as an alternative approach , pcr amplification of a δ 6 16 : 0 - acp desaturase - specific nucleotide probe was attempted using degenerate sense and antisense oligonucleotides prepared against two conserved amino acid sequences in δ 9 18 : 0 - and δ 4 16 : 0 - acp desaturases . one of the sequences , seq id no : 10 ( gly - asp - met - ile - thr - glu - glu ) is encoded by the cdna for the δ 4 16 : 0 - acp desaturase and all known cdnas for the δ 9 18 : 0 - acp desaturase . the second sequence ( seq id no : 11 ) ( glu - lys - thr - ile - gln - tyr - leu ) is also encoded by the δ 4 16 : 0 - acp desaturase of cdna and all known δ 9 18 : 0 - acp desaturase cdnas except that of safflower ( 14 ). products of approximately 215 base pairs obtained following one round of pcr amplification of the total t . alata cdna library ( in plasmid form ) were screened after subcloning into the pamp1 vector . to delineate products of the previously isolated cdnas ptad1 , 2 , and 3 , colonies containing pcr - derived clones were screened in a negative manner with random - labeled probes for ptad1 , 2 , and 3 and conditions of moderate to high stringency . one of the resulting clones ( pec6 ) that displayed weak or no hybridization to these probes encoded an amino acid sequence that was somewhat diverged from those of known δ 9 18 : 0 - acp desaturases . when the t . alata endosperm library was screened with a random - labeled probe prepared from the insert of pec6 , & gt ; 0 . 1 % of the total cdnas examined strongly hybridized to this probe . the longest of a selected portion of these cdnas ( the corresponding plasmid was designated ptad4 ) contained 1279 base pairs and had an open - reading frame corresponding to a 387 - amino - acid polypeptide with considerable identity to known δ 4 16 : 0 - and δ 9 18 : 0 - acp desaturases ( fig5 ). based on similarity of flanking bases to the consensus sequence proposed by lutcke et al . ( 34 ), the translational start site of the cdna insert of ptad4 likely occurs at nucleotide 17 . in addition , from homology with δ 4 16 : 0 - and δ 9 18 : 0 - acp desaturases , the mature peptide encoded by ptad4 likely begins at amino acid 33 . as such , the 32 amino acids preceding this residue correspond to a putative plastid transit peptide as is present in all acyl - acp desaturases characterized to date . interestingly , the cdna insert of ptad4 lacks nucleotide sequence for 6 - 7 amino acids found near the amino terminus of all previously characterized δ 9 18 : 0 - acp desaturases . this region is also altered in the cdna for the coriander δ 4 16 : 0 - acp desaturase ( 21 ) as compared to cdnas for δ 9 18 : 0 - acp desaturases . in this case , the coding sequence for 15 amino acids is absent in the δ 4 16 : 0 - acp desaturase cdna relative to the castor δ 9 18 : 0 - acp desaturase cdna ( 13 ) ( fig5 ). the ptad4 - encoded peptide also contains 2 less amino acids at its carboxyl terminus than both the δ 4 16 : 0 - and δ 9 18 : 0 - acp desaturases . despite these differences , the interior regions of the putative desaturase encoded by ptad4 share significant identity with portions of the primary structures of δ 4 16 : 0 - and δ 9 18 : 0 - acp desaturases , and the spacing between conserved regions of amino acids is the same in all three desaturase types . overall , the mature peptide encoded by the cdna insert of ptad4 shares 66 % identity with the castor δ 9 18 : 0 - acp desaturase and 57 % identity with the coriander δ 4 16 : 0 - acp desaturase , disregarding any missing amino acids . activity of an e . coli - expressed cdna for a diverged acyl - acp desaturase of t . alata endosperm -- to determine the activity of the desaturase corresponding to the cdna insert of ptad4 , the mature peptide - encoding region of this clone was expressed in e . coli with expression driven by the t7 rna polymerase promoter of the vector pet3d ( novagen ). when assayed with [ 1 - 14 c ] 16 : 0 - acp , crude extracts of isopropyl - 1 - thio - β - d - galacto - pyranoside - induced recombinant e . coli catalyzed the synthesis of [ 14 c ] 16 : 1 ( fig6 ). in addition , the methyl ester of the 16 : 1 product displayed mobility on argentation tlc plates similar to that of a methyl petroselinic acid ( 18 : 1δ 6 ) standard , suggesting that this monounsaturated product is a δ 6 isomer . detectable acyl - acp desaturase activity was absent in extracts of e . coli harboring the pet3d vector without cdna insert or in uninducted recombinant e . coli . furthermore , like the activity found in t . alata endosperm extracts , the desaturase expressed in e . coli displayed an in vitro substrate preference for 16 : 0 - acp and exhibited no detectable activity in the absence of reduced ferredoxin ( fig7 ). the [ 14 c ] 16 : 0 moiety produced in vitro from the e . coli - expressed desaturase was conclusively identified as a δ 6 isomer through gas chromatography - mass spectrometry analysis of its dimethyl disulfide derivative ( fig8 ). in the mass spectrum shown , the ions 145 , 177 , 187 , and 364 m / z are diagnostic for a [ 1 - 14 c ] 16 : 1δ 6 moiety . significant amounts of non - radiolabeled or [ 12 c ] 16 : 1δ 6 were also detected among the desaturase assay products . this was indicated by the presence of the additional ions 143 , 175 , and 362 m / z in the mass spectrum of [ 1 - 14 c ] 16 : 1δ 6 as well as by an enrichment in the abundance of ions 187 m / z ( fig8 ). it is unlikely that the non - radiolabeled 16 : 1δ 6 resulted from in vivo synthesis in e . coli . in this regard , e . coli does not normally produce 16 : 1δ 6 ( 35 ). furthermore , gas chromatographic analysis of fatty acids of e . coli expressing the ptad4 - encoded desaturase failed to detect any 16 : 1δ 6 in the bacterial lipids ( data now shown ). given the relatively high concentrations of e . coli protein used in these assays , unlabeled 16 : 1δ 6 likely arose from the in vitro desaturation of endogenous e . coli 16 : 0 - acp present in crude bacterial extracts . of note , expression levels of the t . alata cdna in e . coli appeared to be low relative to that often obtained with dna inserts placed behind the t7 rna polymerase promoter ( 36 ). the expressed protein , for example , could not be distinguished on coomassie - stained sds - polyacrylamide gels of either the total soluble or insoluble protein fractions of lysed e . coli ( data not shown ). also suggestive of low expression levels in e . coli , the specific activity of δ 6 16 : 0 - acp desaturase in recombinant e . coli extracts ( fig7 ) was typically half of that detected in t . alata endosperm homogenates ( table i ). the results presented here demonstrate the involvement of a novel soluble δ 6 16 : 0 - acp desaturase in the synthesis of δ 6 hexadecenoic acid in the endosperm of t . alata . the activity of this enzyme has several properties similar to those previously described for the δ 9 18 : 0 - acp desaturase . these include the requirement of reduced ferredoxin for detectable in vitro activity , the stimulation of activity by catalase , and the inhibition of activity by potassium cyanide and hydrogen peroxide . the existence of a δ 6 16 : 0 - acp desaturase in t . alata endosperm was confirmed by the isolation of a cdna for this enzyme . while the amino acid sequence deduced from this cdna shares some identity with δ 9 18 : 0 - and δ 4 16 : 0 - acp desaturases , these findings , together with those previously obtained for petroselinic acid biosynthesis ( 19 , 21 ), indicate that natural variations in the primary structures of acyl - acp desaturases can give rise to novel enzymes with altered substrate recognition and double bond positioning properties . the major difference between the primary structures of the mature δ 6 16 : 0 -, δ 4 16 : 0 , and δ 9 18 : 0 - acp desaturases occurs in a region near their amino termini . in this region , the t . alata δ 6 16 : 0 - acp desaturase contains 6 less amino acids than the castor δ 9 18 : 0 - acp desaturase . similarly , this portion of the coriander δ 4 16 : 0 - acp desaturase lacks 15 amino acids relative to the castor δ 9 18 : 0 - acp desaturase . without intending to be limited to any particular theory , one possibility is that differences in recognition of substrate chain length ( 16 : 0 - acp versus 18 : 0 - acp ) and / or double bond positioning of these desaturases are associated with this divergence in the primary structures of these enzymes . alternatively , this region of the amino terminus of δ 9 18 : 0 - acp desaturase may not contribute significantly to the catalytic properties of this enzyme . as such , while again not intending to be limited to any particular theory , if the δ 4 and δ 6 16 : 0 - acp desaturases evolved from the δ 9 18 : 0 - acp desaturase , then there may have been little selective pressure to maintain this region intact in the variant 16 : 0 - acp desaturases . ultimately , an understanding of how differences in the amino acid sequences of δ 4 16 : 0 -, δ 6 16 : 0 -, and δ 9 18 : 0 - acp desaturases contribute to variations in their functional properties will require comparisons of the three - dimensional structures of these enzymes . in this regard , elucidation of the crystal structure of the castor δ 9 18 : 0 - acp desaturase is currently in progress ( 37 ). with such information , it will be possible to overlap amino acid sequences of the δ 4 and δ 6 16 : 0 - acp desaturases onto the three - dimensional structure of δ 9 18 : 0 - acp desaturase to more precisely identify residues associated with the different substrate recognition and double bond positioning properties of these enzymes . this could eventually lead to the design of &# 34 ; tailor - made &# 34 ; desaturases that are capable of inserting double bonds into a variety of positions of acyl moieties of a range of carbon chain lengths . an interesting observation from the studies described above was the lack of detectable amounts of 16 : 1δ 6 in lipids of e . coli expressing the t . alata cdna . similarly , thompson et al . ( 14 ) reported that expression of the safflower δ 9 18 : 0 - acp desaturase cdna did not lead to the in vivo production of oleic acid in recombinant e . coli . the latter result can be explained by the fact that e . coli contains little 18 : 0 - acp ( 38 ). however , 16 : 0 - acp is a major component of the acyl - acp pool of e . coli . therefore it is unlikely that the lack of 16 : 1δ 6 synthesis in e . coli expressing the t . alata cdna is due to the presence of insufficient substrate for the desaturase . in addition , e . coli has been reported to contain ferredoxin ( 39 ), the apparent electron donor for the δ 6 16 : 0 - acp desaturase . however , as proposed by thompson et al . ( 14 ), e . coli ferredoxin may not functionally interact with plant acyl - acp desaturases . alternatively , e . coli may not have adequate amounts of ferredoxin in a reduced form as required for δ 6 16 : 0 - acp desaturase activity . in addition to 16 : 1δ 6 , t . alata seed contains the unusual fatty acid 18 : 1δ 8 , which composes about 2 % weight of the oil of this tissue ( 23 ). we have previously shown that petroselinic acid ( 18 : 1δ 6 ) is formed by elongation of 16 : 1δ 4 - acp in umbelliferae endosperm ( 22 ). in an analogous manner , we predict that 18 : 1δ 8 arises from the elongation of 16 : 1δ 6 - acp rather than from the δ 8 desaturation of 18 : 0 - acp . unlike the synthesis of petroselinic acid , though , elongation of 16 : 1 - acp in t . alata endosperm is likely not a major pathway as the ratio of amounts of 16 : 1δ 6 : 18 : 1δ 8 in this tissue is approximately 40 : 1 . in contrast , the ratio of amounts of 16 : 1δ 4 : 18 : 1δ 6 in endosperm of the umbellifarae coriander is more than 1 : 500 ( 22 , 25 ). finally , significant efforts have been directed toward the development of transgenic crops that produce high value specialty oils ( 4 , 40 , 41 ). using methodologies currently well known in the art , transgenic plants could be produced ( 42 , 43 ) which would contain and express the δ 6 16 : 0 - acp desaturase gene and which would produce high levels of 16 : 1δ 6 . in this regard , oils rich in 16 : 1δ 6 may have properties suitable for industrial use . like petroselinic acid , 16 : 1δ 6 can be oxidatively cleaved at its double bond to yield adipic acid , a precursor of nylon 6 , 6 . in addition , high palmitic acid ( 16 : 0 ) mutants of crop plants including soybean ( 44 ) and brassica campestris ( 45 ) are available that could serve as appropriate backgrounds for transgenic expression of the cdna for the t . alata δ 6 16 : 0 - acp desaturase . still the success of such research would likely require additional studies to determine whether enzymes other than δ 6 16 : 0 - acp desaturase are specialized for the synthesis and metabolism of 16 : 1δ 6 in t . alata endosperm . for example , a petroselinoyi - acp - specific thioesterase has been identified in umbelliferae endosperm extracts that efficiently releases petroselinic acid from acp and , as a result , makes this fatty acid available for subsequent storage in triacylglycerol ( 46 ). a related enzyme may also be required for high levels of 16 : 1δ 6 accumulation in transgenic plants . 2 . wada , h ., gombos , z ., and murata , n . 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( 1992 ) plant mol . biol . 20 , 151 - 155 . 18 . taylor , m . a ., smith , s . b ., davies , h . v ., and burch , l . r . ( 1992 ) plant physiol . 100 , 533 - 534 . 19 . cahoon , e . b ., becker , c . k . shanklin , j ., and ohlrogge , j . b . ( 1994 ) plant physiol ., in press . 20 . singh , s ., mckinney , s ., green , a . ( 1994 ) plant physiol . 104 , 1075 . 21 . cahoon , e . b ., shanklin , j ., and ohlrogge , j . b . ( 1992 ) proc . natl . acad . sci . u . s . a . 89 , 11184 - 11188 . 22 . cahoon , e . b ., and ohlrogge , j . b . ( 1994 ) plant physiol . 104 , 827 - 838 . 23 . spencer , g . f ., kleiman , r ., miller , r . w ., and earle , f . r . ( 1971 ) lipids 6 , 712 - 714 . 24 . morrison , w . r . and smith , l . m . ( 1964 ) j . lipid res . 5 , 600 - 608 . 25 . cahoon , e . b ., and ohlrogge , j . b . ( 1994 ) plant physiol . 104 , 845 - 855 . 26 . christie , w . w . ( 1982 ) lipid analysis , 2nd ed ., pergammon press , oxford . 27 . rock , c . o ., and garwin , j . l . 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( 1987 ) in escherichia coli and salmonella typhimurium cellular and molecular biology ( neihardt , f . c ., ed ) pp . 474 - 497 , american society of microbiology , washington , d . c . 36 . studier , f . w ., rosenberg , a . h ., dunn , j . j ., and dubendorff , j . w . ( 1990 ) methods enzymol . 185 , 60 - 89 . 37 . schneider , g ., lindqvist , y ., shanklin , j ., and somerville , c . ( 1992 ) j . mol . biol . 225 , 561 - 564 . 38 . rock , c . o ., and jackowski , s . ( 1982 ) j . biol . chem . 257 , 10579 - 10765 . 39 . knoell , h . e ., and knappe , j . ( 1974 ) eur . j . biochem . 50 , 245 - 252 . 42 . schilperoort , r . a ., hoekema , a ., and hooykaas , p . j . j . ( 1990 ) u . s . pat . no . 4 , 940 , 838 . 43 . tomes , d ., bidney , d ., buising c . m . ( 1994 ) u . s . pat . no . 5 , 322 , 783 . 44 . bubeck , d . m ., fehr , w . r . and hammond , e . g . ( 1980 ) crop . sci . 29 , 652 - 656 . 45 . perrson , c . 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( 1994 ) plant physiol . 104 , 839 - 844 . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 12 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 1258 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( xi ) sequence description : seq id no : 1 : 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( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 5 : ggngayatgathacngarga20 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 6 : arrtattgdatngtyttytc20 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 12 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 7 : caucaucaucau12 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 12 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 8 : cuacuacuacua12 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 18 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 9 : gcttcgactattactcac18 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 7 amino acids ( b ) type : amino acid ( c ) strandedness :( d ) topology : linear ( xi ) sequence description : seq id no : 10 : glyaspmetilethrgluglu15 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 7 amino acids ( b ) type : amino acid ( c ) strandedness :( d ) topology : linear ( xi ) sequence description : seq id no : 11 : ilegluglnthriletyrleu15 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 7 amino acids ( b ) type : amino acid ( c ) strandedness :( d ) topology : linear ( xi ) sequence description : seq id no : 12 : glulysthrileglntyrleu15__________________________________________________________________________