Patent Application: US-48637604-A

Abstract:
a nucleic acid which encodes a peroxisomal fatty acid transporter , uses thereof and a method of genetic manipulation of peroxisomal fatty acid transport and / or metabolism . the nucleic acid and its products are especially for use in regulation of peroxisomal fatty acid transport in plant and in controlling the spectrum of fatty acids which can be utilised by the plant .

Description:
the yeast and human peroxisomal abc transporter proteins were used to search the publically accessible sequence databases of arabidopsis thaliana using the blast algorithm . one sequence was identified ( t5j17 . 20 ; at4g39850 , from chromosome iv ) which had a significant sequence homology to all the probe sequences and which corresponded to an est ( h1a6t7 ) from a 3 day seedling hypocotyl library . this clone was obtained from the arabidopsis biological resource centre at ohio state university ( usa ), fully sequenced and named cts ( seq id no : 1 ). bioinformatic analysis of the sequence revealed that this encoded a protein of 1337 amino acids with a predicted molecular weight of 149 , 576 ( seq id no : 2 ). functional abc transporters have 4 domains , two sets of 5 or 6 transmembrane spans and two atpase domains which can be on separate polypeptide chains or fused in various combinations . cts is of the type in which all domains are fused where as scpxa2p is of the half transporter type containing one transmembrane domain and one atpase domain . cts contains all the conserved residues typical of an abc transporter . the two half transporter domains of cts are 34 % identical to one another . the highest matches on blastp search ( p & lt ; 7 . 1 e - 67 ) are the mammalian pmp70 and adrenoleukodystrophy proteins , also half transporters that are involved in fatty acid transport into peroxisomes . saccharomyces cerevisiae pxa2p is the 6th most similar sequence ( p = 1 . 5e - 44 ). an alignment of the deduced amino acid sequences of cts with pmp70 , aldp , scpxa1p and scpxa2p is shown in fig1 . the atp binding sites , designated walker a and b motifs are highly conserved in all atpases . the c - sequence , otherwise known as the abc motif is diagnostic of the abc transporter superfamily , as is the eaa sequence . the sequence nseeiafy ( seq id no : 12 ) is diagnostic of the human and yeast peroxisomal abc transporters and the closely related sequence h ( s / a ) siaf ( y / f ) ( seq id no : 13 ) occurs in the two halves of cts . the loop1 region and the sequence pqrpymtlgtlrdq ( seq id no : 14 ) is diagnostic of animal peroxisomal abc transporters . the almost identical sequence pqrpy ( m / t )( a / c ) lgtlrdq ( seq id no : 15 ) occurs in both halves of cts . therefore cts contains sequences which assign it to the peroxisomal sub - class of abc transporters . to determine whether the expression pattern of cts was consistent with its involvement in fatty acid uptake in peroxisomes , a northern blot was performed ( fig2 ). fatty acid oxidation is an on - going process in plant cells due to turn over of membrane lipids by β - oxidation . however higher levels of expression would be expected during and immediately after germination as is seen for the β - oxidation enzymes thiolase ( germain et al ., 2001 ) and acyl coa oxidase ( hooks et al ., 1999 ). consistent with this the cts probe detects a transcript of 4 . 9 kb that is expressed throughout the first 12 days post imbibition but with highest expression at day 1 . to provide experimental evidence for the peroxisomal location of cts , antibodies were raised to the second atpase domain . a fragment corresponding to amino acids 1112 - 1337 was cloned into pet28b ( novagen ) to produce a his tagged recombinant protein . the recombinant protein , which is in inclusion bodies was purified by nta agarose chromatography ( fig3 ). affinity purified antisera detected a protein of ca . 140 kda in membrane fractions from arabidopsis seedlings and tissue culture cells ( fig4 ). further experiments demonstrated the localisation of this protein specifically in peroxisomes / glyoxysomes ( fig7 ). thus far the function of the cts protein is inferred from sequence homology to known peroxisomal fatty acid abc transporters . to obtain functional information for cts knockout mutations in arabidopsis were sought through reverse genetics . primers were designed and sent to the arabidopsis knock out facility at the university of wisconsin ( usa ) to screen their population of t - dna tagged mutants . three alleles of cts were detected ( fig5 ). the location of the t - dna insertions were determined by sequencing the pcr products . t - dna insertions are in exon 2 and introns 1 ( in the 5 ′ utr ) and 4 of cts . the insertions in exon 2 of cts would be predicted to be a null alleles , while those in the introns may or may not give a phenotype depending on whether they are correctly spliced out from the transcript . single heterozygous plants have been obtained for each t - dna insertion ( fig6 ). heterozygous cts - 2 plants were allowed to self fertilise and homozygous progeny selected . homozygous seeds and seedlings were subjected to triacyl glycerol and acyl coa profiling ( fig8 ). these results provide strong support for the proposed biochemical function of cts as a fatty acyl coa transporter . the cts - 2 mutant allele was obtained by pcr - based screening of the wisconsin - alpha gene knockout lines for insertions in the cts gene ( krysan et al ., 1999 ). the sequence of the cts rna was obtained using the cdna clone h1a6t7 ( abrc , ohio state univ .). total rna was extracted , separated by denaturing agarose gel electrophoresis and transferred to a hybridisation membrane . the membrane was hybridised with 32p - labelled clone h1a6t7 as described by hooks et al ., ( 1999 ) and detected by phophoimaging . a polyclonal antibody was raised against a c - terminal fragment of cts ( amino acids 1112 - 1337 ) expressed in e . coli strain bl21 ( de3 ) plyss ( novagen ) using a pet28b ( novagen ) construct containing an nhei - bamhi fragment of cdna clone h1a6t7 ( embl accession aj311341 ). the antibody was affinity - purified using the recombinant fragment ( tugal et al ., 1999 ). acyl coas and total lipids were extracted from five replicate 3 - 10 mg tissue samples and analyzed according to larson and graham ( 2001 ). an aliquot of the total lipid extract was used for triacylglycerol ( tag ) determination . a 1 ml 100 mg bed volume bond elut ® ( varian , surrey , uk ) spe column was prepared by elution with 2 × 1 ml methanol , 3 × 1 ml hexane , and then 100 μl sample loaded in hexane . tags were eluted with 1 . 5 ml 2 : 3 ( v / v ) chloroform : hexane , dried under vacuum , transmethylated to fatty acid methyl esters , and analyzed as described previously ( larson and graham , 2001 ). specificity for tag separation was optimized so that the diacylglycerol , dipalmitin ( sigma ) was excluded from the spe eluate . to provide further information on the specific nature of the block in lipid breakdown and hence the function of the cts protein , the levels of triacyl glycerol ( tag ) and acyl coas were measured in the cts - 2 mutant and corresponding ws wild type . all lines were germinated in the presence of 1 % sucrose ( and the testa of the cts - 2 mutants was ruptured to allow germination ) to ensure that seedlings were at similar morphological stages of development . the summed changes in fatty acid content of extracted tag indicate similar levels of tag - derived fatty acids in imbibed seeds of the wild type and mutant on day 0 ( fig8 a ). higher apparent tag levels per seed ( ling ) in the mutant reflects bigger seed size and is not seen when the data is expressed on a fresh weight basis ). tag fatty acid levels only declined slightly after 2 days germination even in wild type seedlings , presumably due to the presence of sucrose as an alternative energy source . however by day 5 , tag derived fatty acids had decreased by 95 . 8 % in the wild types but by only 32 . 3 % in the mutant . all tag - derived fatty acid chain lengths were mobilised after 5 days germination for wild type , but the cts - 2 mutant retained high levels of the same tags ( fig8 b , c ). total acyl coas increased in both lines over the period 0 - 5 days ( fig8 d ), but this is much more dramatic in the mutant . by day 5 some of the increase in both wild type and mutant seedlings may reflect new lipid synthesis ( e . g . for membrane biogenesis ). however the most striking observation is the retention of 20 : 1 and to a lesser extent 20 : 0 and 22 : 1 coa &# 39 ; s in seedlings of the cts - 2 mutant ( fig8 e , f ). as c20 fatty acids are only very minor components of non - storage lipids in arabidopsis , these data demonstrate that there is a severe block in carbon flux from stored triacyl glycerols during germination in the mutant . analysis of tag - derived fatty acids and acyl coas demonstrates that while some lipid is mobilised in the cts - 2 mutant , catabolism is inhibited before β - oxidation , resulting in an increased acyl coa pool . this is particularly pronounced for c20 and c22 acyl coas which are predominantly tag - derived . these data argue strongly that the primary defect in the cts - 2 mutant is in transport of fatty acyl coas into peroxisomes . 18 : 2 and 18 : 3 coas do not accumulate to a similar extent , which may reflect their use in the synthesis of structural lipids by er mediated pathways . in contrast c20 : 1 is not a component of structural lipids and may accumulate because it lacks a synthetic sink route . it is possible that the accumulation of 20 : 1 coa , or depletion of free coenzyme a results in the inhibition of lipolysis and therefore the release of further fatty acids from storage tag . the accumulation of acyl coas argues that these are the substrates of the cts protein , and suggests that unlike x - ald patients cts mutants retain vlcfa synthetase activity . the substantial accumulation of long and very long chain acyl coa &# 39 ; s in the cts - 2 mutant is consistent with the activation of these fatty acids by acyl coa synthetases on the cytoplasmic side of the peroxisomal membrane , as reported for s . cerevisiae ( hettema et al ., 1996 ), mammals ( mannaerts et al ., 1982 ) and plants ( olsen and lusk 1994 ). the finding that all fatty acid chain lengths are mobilized in wild type and retained in the mutant argues that the transporter has broad substrate specificity with respect to acyl chain length . acyl coa &# 39 ; s are amphipathic molecules , as are the substrates for many abc transporters . oilseeds can be engineered to produce economically valuable unusual fatty acids . however , the exact fate of the unusual fatty acid , once it is made , is not known . what is known is that not enough of the valuable fatty acid ends up in seed oil . one factor that appears to limit novel oil yield is that the fatty acids comprising them are broken down before being incorporated into oil ( eccleston and ohlrogge 1998 ). cts , the nucleic acid of the present invention , is an excellent candidate to be involved at the beginning of this process . as indicated by the data in fig8 , mutating the function of cts reduces or abolish the entry of some or all fatty acids into the glyoxisome / peroxisome and therefore prevent their breakdown . we predict this would allow their accumulation in seeds or other plant tissues . however , the plant still needs to be able to utilise endogenous fatty acids for germination and growth . blocking β - oxidation through the mutation of thiolase results in germination but subsequent growth is dependent on exogenously supplied sucrose ( germain et al ., 2001 ). it should be possible to alter the expression levels of cts in specific tissues or at specific times , for example inhibiting its activity in developing seeds but not in germinating seeds . alternatively if we can determine and then alter its substrate specificity we may be able to allow accumulation of desired novel oils but not prevent germination and seedling establishment using endogenous fatty acids . an additional problem is that when high levels of novel oils can be achieved , oilseeds cannot use these foreign oils as an efficient source of fuel for young seedlings to grow , resulting in non - viable seed . for example , expressing a form of cts that cannot transport the novel oils in the developing seed but switching on a form which can use the novel oil as a substrate during germination might overcome this problem . these proteins with altered substrate specificity might arise as a resulted of targeted or random mutation of the gene followed by an appropriate selection , or the use of the arabidopsis protein to isolate homologues which may have different substrate specificities from species which naturally accumulate high levels of the desired novel oils . therefore the protein of the present invention has the potential to increase the accumulation of novel oils in seeds to economically viable levels . mutants in β - oxidation show resistance to 2 , 4 - dichlorophenoxybutyric acid ( 2 , 4 db , hayashi et al ., 1998 and indole - 3 - butyric acid ( iba , zollman et al 2000 ) and β - oxidation is clearly impaired in the cts - 2 mutant , most likely as a consequence of a defect in transport of fatty acyl coas into the peroxisome for metabolism . cts maps to the same location on chromosome iv as the ped3 mutant ( hayashi et al ., 1998 ). iba and 2 , 4 db are amphipathic molecules and cts is a good candidate to mediate their uptake into peroxisomes . if a dominant negative or rnai version of cts could be expressed under the control of a regulated promoter , it could be used as a selectable marker for plant transformation . the expression of the dominant negative form of the protein would most likely confer resistance to iba or 2 , 4 db . resistance results in a ‘ long root ’ phenotype and would allow plants expressing this marker to be selected on medium containing iba or 2 , 4 db , sucrose and the inducing molecule for the regulated promoter . the advantage is that the selectable marker is of plant origin and unlike , for example , herbicide resistance , confers selection only in the presence of three separate molecules which is not going to occur in nature . it does not confer antibiotic resistance , so there can be no danger of disseminating resistance in the environment . the use of a regulated promoter results in expression of the selection only under defined conditions . footitt , s ., slocombe , s p ., laarner , v ., kurup , s ., wu , y ., larson , t ., graham , i ., baker , a and holdsworth ( 2002 ) embo 21 , 12 , 2912 - 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