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:
[0001]    The present invention relates to an isolated nucleic acid, uses thereof and a method of genetic manipulation of peroxisomal fatty acid transport and/or metabolism, means therefor and products thereof especially for use in regulation of plant growth and in controlling the spectrum of fatty acids which can be utilised by the plant.  
         BACKGROUND TO THE INVENTION  
         [0002]    Fatty acids are major carbon and energy stores in the seeds of many agriculturally important species. For the plant they are essential reserves that support germination, and seedling establishment until the plant can manufacture its own building blocks and energy through photosynthesis. Recent published data shows that blocking the mobilisation of these fatty acids prevents or severely compromises establishment (Hayashi et al., 1998, Germain et al., 2001). Clearly, efficient germination and seedling establishment are of vital importance to farmers. In addition, the fatty acids deposited in seeds renders them important foodstuffs for humans and animals. There is considerable interest in modifying the levels and composition of fatty acids in seeds to improve their nutritional quality and health benefit. Furthermore, there is considerable interest in engineering crop plants to produce novel fatty acids with industrial benefit. These can be used as feedstocks for the chemical and healthcare industries, with the aim of reducing reliance on petrochemical feedstocks, it is therefore desirable to produce these molecules in a more ecologically friendly and sustainable way and to develop non-food crops for European agriculture.  
           [0003]    Plants that are engineered to produce altered fatty acids rarely produce economically viable levels in seeds. The reasons for this are probably complex but one factor may be their turnover, i.e. some proportion is broken down as they are made. Furthermore, if high levels can be achieved this may compromise the ability of these plants to germinal and establish if these altered fatty acids cannot be used efficiently. Clearly this is detrimental to the commercialisation of these plants.  
           [0004]    Fatty acids in seeds are stored in oil bodies in the form of triacylglycerols (3 fatty acid molecules joined to a glycerol backbone) which are laid down during seed development. During germination free fatty acids are released by the action of lipases and the fatty acids enter the β-oxidation pathway which is housed within a specialised organelle the glyoxysome. The fatty acids are then metabolised to produce energy and building blocks for the cell. Control of biochemical pathways frequently resides near the beginning of the pathway and in several cases transport steps have been shown to exert high flux control coefficients. This means that transporting a molecule, for example, from compartment A to compartment B is often an important step in determining the overall rate of the pathway.  
           [0005]    There is some evidence to suggest from human studies that proteins of the ATP Binding Cassette family (referred to as ABCs) are involved in fatty acid transport ABCs are integral membrane proteins that transport a wide variety of molecules across membranes. It is known from the prior art that X-linked adrenoleukodystrophy (X-ALD) is associated with a particular gene mutation (Moser et al., 1993). The clinical symptoms of the disease, results in increasing neurological impairment, progressive mental and physical disability, and eventually death in late childhood or early teens. Biochemically these patients fail to break down very long chain fatty acids. The gene mutated in X-ALD is an ABC transporter closely related to but not identical to another mammalian peroxisomal ABC transporter, PMP70. It is now known that there are 4 of these peroxisomal ABC genes in humans (PMP70, PMP70R, ALD and ALDR. In addition, two homologous genes have been identified in yeast  S. cerevisiae  (PXA1 and PXA2 also known as PAT1 and PAT2) and have been shown to be transporters of fatty acyl CoAs (Hettema et al., 1996). PXA2 is also known as the COMATOSE (CTS) gene (Russell et al., 2000; Footitt et al., 2002).  
           [0006]    Glyoxysomes are cytoplasmic organelles unique to plants. Glyoxysomes are a specialised form of peroxisomes but they also contain enzymes of the glyoxalate cycle. They are abundantly present in the endosperm or cotyledons of oil-rich seeds. It is not known how fatty acids are transported into glyoxysomes.  
           [0007]    It is therefore desirous to identify a transport protein or a regulator protein that is involved with the rate of entry of fatty acids into the degradation pathway; Identification and characterisation of the proteins that transport fatty acids into glyoxysomes would offer an attractive target for biotechnology with a view to repress or promote growth or to alter the spectrum of fatty acids which can be utilised by the plant  
         STATEMENTS OF THE INVENTION  
         [0008]    According to a first aspect of the invention there is provided an isolated nucleic acid comprising a nucleotide sequence which encodes a polypeptide which functions as a fatty acid transporter in plants selected from the group consisting of:  
           [0009]    (i) a nucleic acid sequence depicted in SEQ ID NO: 1,  
           [0010]    (ii) a nucleic acid sequence which is derived from the sequence depicted in SEQ ID NO: 1 according to the degeneracy of the genetic code,  
           [0011]    (iii) derivatives of the sequence depicted in SEQ ID NO: 1, which encodes polypeptides having preferably at least 30% homology to the sequence encoding amino acid sequences depicted in SEQ ID NO: 2 and which sequences function as a fatty acid transporter.  
           [0012]    The nucleic acids of the present invention are conveniently referred to as the CTS gene.  
           [0013]    Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Accordingly, one aspect of the invention pertains to isolated nucleic acid molecules (e.g., cDNAs) comprising a nucleotide sequence encoding a fatty acid transporter protein or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection or amplification of fatty acid transporter—encoding nucleic acid (e.g., DNA or mRNA). In particularly preferred embodiments, the isolated nucleic acid molecule comprises one of the nucleotide sequences set forth in Sequence SEQ ID NO: 1 or the coding region or a complement thereof of one of these nucleotide sequences. In other particularly preferred embodiments, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes to or is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80% or 90%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotide sequence as in Sequence SEQ ID NO: 1, or a portion thereof. In other preferred embodiments, the isolated nucleic acid molecule encodes one of the amino acid sequences set forth in Sequence SEQ ID NO: 2. The preferred fatty acid transporter-gene of the present invention also preferably possess at least one of the fatty acid transporter activities described herein. These may include transport of fatty acids and/or acyl CoAs of varying chain lengths, degree of unsaturation and substitution, and their analogues and derivatives and/or other amphipathic molecules such as 2,4 dichlorophenoxybutyric acid and indole butric acid and their analogues and derivatives.  
           [0014]    In another embodiment, the isolated nucleic acid molecule is at least 15 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 1; Preferably, the isolated nucleic acid molecule corresponds to a naturally-occurring nucleic acid molecule. More preferably, the isolated nucleic acid encodes a naturally-occurring  Arabidopsis thaliana  fatty acid transporter, or a biologically active portion thereof.  
           [0015]    Alternatively, the isolated fatty acid transporter can comprise an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, or is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80%, or 90%, and even more preferably at least about 95%, 96%, 97%, 98,%, or 99% or more homologous, to a nucleotide sequence of SEQ ID NO: 1. It is also preferred that the preferred forms of fatty acid transporters also have one or more of the fatty acid transporter activities described herein Nucleic acid molecules corresponding to natural variants and non- Arabidopsis thaliana  homologues, derivatives or analogues of the  Arabidopsis thaliana  fatty acid transporter cDNA of the invention can be isolated based on their homology to  Arabidopsis thaliana  fatty acid transporter nucleic acid disclosed herein using the  Arabidopsis thaliana  cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising a nucleotide sequence of SEQ BD NO: 1. In other embodiments, the nucleic acid is at least 25, 50, 100, 250 or more nucleotides in length. As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley &amp; Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (═SSC) at about 45° C., 25, followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C. As known by the skilled artisan such hybridization conditions differ depending on the type of the nucleic acid and if for example organic solvents are present in view of the temperature and the concentration of the buffer. The temperature for example differs under “standard hybridization conditions” depending on the type of the nucleic acid between 42° C. and 58C in aqueous buffer with a concentration of 0.1 to 5×SSC (pH 7.2). In the event that organic solvent is present in the before mentioned buffer for example 50% formamide the temperature under standard conditions is about 42° C.  
           [0016]    Preferably hybridisation conditions for DNA:DNA-hybrids are for example 0.1×SSC and 20° C. to 45° C., preferably between 30° C. to 45-C. Preferably hybridisation conditions for DNA:RNA-hybrids are for example 0.1×SSC and 30-C to 55-C, preferably between 45° C. to 55° C. The before mentioned hybridization temperatures are estimated for example for a nucleic acid of about 100 bp (=base pairs) in length with G+C-content of 50% in the absence of formamide. The skilled worker knows how to estimate the necessary hybridization conditions according to textbooks such as the one mentioned above or from the following textbooks Sambrook et al., “Molecular Cloning”, Cold Spring Harbor Laboratory, 1989; Hames and Higgins (eds.), 1985, “nucleic Acids Hybridization: A Practical Approach”, IRL Press at Oxford University Press, Oxford; Brown (ed), 1991, “Essential Molecular Biology: A Practical Approach”, IRL Press at Oxford University Press, Oxford. This is also true for stringent or low stringent hybridization conditions.  
           [0017]    Preferably, an isolated nucleic acid molecule of the invention hybridizes under stringent conditions to a sequence of SEQ ID NO: 1 corresponds to a naturally-occurring nucleic acid molecule. As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein); In one embodiment, the nucleic acid encodes a natural  Arabidopsis thaliana  fatty acid transporter.  
           [0018]    In addition to naturally-occurring variants of the fatty acid transporter sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into a nucleotide sequence of SEQ ID NO: 1, thereby leading to changes in the amino acid sequence of the encoded fatty acid transporter, without altering the functional ability of the fatty acid transporter. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in a sequence of SEQ ID NO: 1. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of one of the fatty acid transporters (SEQ ID NO: 2) without altering the activity of said fatty acid transporter, whereas an “essential” amino acid residue is required for fatty acid transporter activity. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved in the domain having fatty acid transporter activity) may not be essential for activity and thus are likely to be amenable to alteration without altering fatty acid transporter activity.  
           [0019]    In another embodiment, the isolated nucleic acid molecule encodes a protein or portion thereof wherein the protein or portion thereof includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of sequence SEQ ID NO: 2 such that the protein or portion thereof maintains a fatty acid transporter activity. Preferably, the protein or portion thereof encoded by the nucleic acid molecule maintains the ability to participate in the metabolism of compounds necessary for the construction of fatty acids especially PUFAs or cellular membranes of plants or in the transport of molecules across these membranes. In one embodiment, the protein encoded by the nucleic acid molecule is at least about 50%, preferably at least about 60%, and more preferably at least about 70%, 80%, or 90%4 and most preferably at least about 95%, 96%, 97%, 98%, or 99% or more homologous (=identity) to an amino acid sequence of Sequence SEQ ID NO: 2.  
           [0020]    Further, DNAs of which code for proteins of the present invention, or DNAs which hybridize to that of SEQ ID NO:1 but which differ in codon sequence from SEQ ED NO: 1 due to the degeneracy of the genetic code, are also part of this invention. The degeneracy of the genetic code, which allows different nucleic acid sequences to code for the same protein or peptide, is well known in the literature. See, e.g., U.S. Pat. No. 4,757,006 to Toole et al. at Col. 2, Table 1.  
           [0021]    Sequence identity: the similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as a sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologues or orthologues of the protein, and the corresponding cDNA or gene sequence, will possess a relatively high degree of sequence identity when aligned using standard methods. This homology will be more significant when the orthologous proteins or genes or cDNAs are derived from species that are more closely related (e.g., human and chimpanzee sequences), compared to species more distantly related (e.g. human and  C. elegans  sequences).  
           [0022]    Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith &amp; Waterman  Adv. Appl. Math.  2: 482, 1981; Needleman &amp; Wunsch  J. Mol. Biol.  48: 443, 1970; Pearson &amp; Lipman  Proc. Natl. Acad. Sci. USA  85: 2444, 1988; Higgins &amp; Sharp  Gene,  73: 237-244, 1988; Higgins &amp; Sharp  CABIOS  5: 151-153, 1989; Corpet et al.  Nuc. Acids Res.  16, 10881-90, 1988; Huang et al.  Computer Appls. In the Biosciences  8, 155-65, 1992; and Pearson et al.  Meth Mol. Bio. 24, 307-31, 1994. Altschul et al. (J. Mol. Biol. 215:403410, 1990), presents a detailed consideration of sequence alignment methods and homology calculations.  
           [0023]    The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al: J. Mol. Biol. 215:403410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis blastp, blastn, blastx, tblastn and tblastx. By way of example, for comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment may for example be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).  
           [0024]    According to a yet further aspect of the invention there is provided a gene construct comprising an isolated nucleic acid having the sequence SEQ ID NO: 1 as herein before described, wherein the nucleic acid is functionally linked to one or more regulatory signals.  
           [0025]    Accordingly, another embodiment of the invention is a novel gene construct comprising an isolated nucleic acid derived from a plant which encodes a polypeptide which functions as fatty acid transporter or the gene sequence of SEQ ID No. 1, its homologous, derivatives or analogous as defined above which have been functionally linked to one or more regulatory signals, advantageously to increase gene expression. Examples of these regulatory sequences are sequences to which inducers or repressors bind and thus regulate the expression of the nucleic acid. In addition to these novel regulatory sequences, the natural regulation of these sequences in front of the actual structural genes can still be present and, where appropriate, have been genetically modified so that the natural regulation has been switched off and the expression of the genes has been increased. The gene construct can, however, also have a simpler structure, that is to say no additional regulatory signals have been inserted in front of the sequence SEQ ID No. 1 or its homologs, and the natural promoter with its regulation has not been deleted. Instead, the natural regulatory sequence has been mutated so that regulation no longer takes place, and gene expression is enhanced. The gene construct may additionally advantageously comprise one or more so-called enhancer sequences functionally linked to the promoter and making increased expression of the nucleic acid sequence possible. It is also possible to insert at the 3′ end of the DNA sequences additional advantageous sequences, such as further regulatory elements or terminators. The fatty acid transporter genes may be present in one or more copies in the gene construct. It is advantageous for further genes to be present in the gene construct for insertion of further genes into organisms.  
           [0026]    Advantageous regulatory sequences for the novel process are present, for example, in promoters such as cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lacIq-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, I-PR- or l-PL-promoter and are advantageously used in Gram-negative bacteria Further advantageous regulatory sequences are present, for example, in the Gram-positive promoters amy and SPO2, in the yeast or fungal promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plant promoters CaMV/35S [Franck et al., Cell 21 (1980) 285-294], PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, lib4, usp, STLS1, B33, nos or in the ubiquitin or phaseolin promoter. Also advantageous in this connection are inducible promoters such as the promoters described in EP-A-0 388 186 (benzyl sulfonamide inducible), Plant J. 2, 1992: 397-404 (Gatz et al., Tetracyclin inducible), EP-A-0 335 528 (abscisic acid inducible) or WO 93/21334 (ethanol or cyclohexenol inducible). Additional useful plant promoters are the cytosolic FBPase promotor or ST-LSI promoter of the potato (Stockhaus et al., EMBO J. 8, 1989, 2445), the phosphorybosyl phyrophoshate amido transferase promoter of  Glycine max  (gene bank accession No. U87999) or the noden specific promoter described in EP-A-0 249 676. Particularly advantageous promoters are promoters which allow the expression in tissues which are involved in the fatty acid biosynthesis. Most particularly advantageous are seed specific promoters such as usp-, LEB4-, phaseolin or napin promoter. Additional particularly advantageous promoters are seed specific promoters which can be used for monokotyledones or dikotyledones are described in U.S. Pat. No. 5,608,152 (napin promoter from rapeseed), WO 98/45461 (phaseolin promoter from Arobidopsis), U.S. Pat. No. 5,504,200 (phaseolin promoter from  Phaseolus vulgaris ), WO 91/13980 (Bce4 promoter from  Brassica ), Baeumlein et al., Plant J., 2, 2, 1992: 233-239 (LEB4 promoter from leguminosa) said promoters are useful in dikotyledones. The following promoters are useful for example in monokotyledones lpt-2- or lpt-1-promoter from barley (WO 95/15389 and WO 95/23230), hordein promoter from barley and other useful promoters described in WO 99/16890.  
           [0027]    It is possible in principle to use all natural promoters with their regulatory sequences like those mentioned above for the novel process. It is also possible and advantageous in addition to use synthetic promoters.  
           [0028]    The gene construct may, as described above, also comprise further genes which are to be inserted into the organisms. It is possible and advantageous to insert and express in host organisms regulatory genes such as genes for inducers, repressors or enzymes which intervene by their enzymatic activity in the regulation, or one or more or all genes of a biosynthetic pathway. These genes can be heterologous or homologous in origin. The inserted genes may have their own promoter or else be under the control of the promoter of the sequence SEQ ID No. 1 or its homologs.  
           [0029]    The gene construct advantageously comprises, for expression of the other genes present, additionally 3′ and/or 5′ terminal regulatory sequences to enhance expression, which are selected for optimal expression depending on the selected host organism and gene or genes.  
           [0030]    These regulatory sequences are intended to make specific expression of the genes and protein expression possible as mentioned above. This may mean, depending on the host organism, for example that the gene is expressed or overexpressed only after induction, or that it is immediately expressed and/or overexpressed.  
           [0031]    The regulatory sequences or factors may moreover preferably have a beneficial effect on expression of the introduced genes, and thus increase it. It is possible in this way for the regulatory elements to be enhanced advantageously at the transcription level by using strong transcription signals such as promoters and/or enhancers. However, in addition, it is also possible to enhance translation by, for example, improving the stability of the mRNA.  
           [0032]    In addition the inventive gene construct preferably comprises additional gene of different biochemical pathways for example genes for the synthesis of vitamins, carotinoids, sugars such as monosaccharides, oligosaecharides or polysaccharides or fatty acid biosynthesis genes, more preferably the gene construct comprises fatty acid biosynthesis genes such as desaturases, hydroxylases, Acyl-ACP-thioesterases, elongases, acetylenases, synthesases or reductases such as n19-, n17-, n15-, n12-, n9-, n8-, n6-, n5-, n4-desaturases, hydroxylases, elongases, n12-acetylenase. Acyl-ACP-thioesterasen, β-Ketoacyl-ACP-synthases or β-Ketoacyl-ACP-reductases.  
           [0033]    According to a yet further aspect of the invention there is provided a vector comprising the nucleic acid of the present invention or a gene construct of the present invention.  
           [0034]    This aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a fatty acid transporter (or a portion thereof). As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.  
           [0035]    The recombinant expression vectors of the invention comprise at least one inventive nucleic acid or at least one inventive gene construct of the invention in a form suitable, for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence are fused to each other so that both sequences fulfil the proposed function addicted to the sequence used. (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) or see: Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnolgy, CRC Press, Boca Raton, Fla., eds.: Glick and Thompson, Chapter 7, 89-108 including the references therein. Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., fatty acid transporters, mutant forms of fatty acid transporters, fusion proteins, etc.).  
           [0036]    The recombinant expression vectors of the invention can be designed for expression of fatty acid transporters in prokaryotic or eukaryotic cells, preferably in eukaryotic cells. For example, fatty acid transporter genes can be expressed in bacterial cells such as  C. glutamicum , insect cells (using baculovirus expression vectors), yeast and other fungal cells (see Romanos, M. A. et al. (1992) Foreign gene expression in yeast: a review, Yeast 8: 423488; van den Hondel, C. A. M. J. J. et al. (1991) Heterologous gene expression in filamentous fungi, in: More Gene Manipulations in Fungi, J. W. Bennet &amp; L. L. Lasure, eds., p. 396-428: Academic Press: San Diego; and van den Hondel, C. A. M. J. J. &amp; Punt, P. J. (1991) Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J. F. et al., eds., p. 1-28, Cambridge University Press: Cambridge), algae (Falciatore et al., 1999, Marine Biotechnology.1, 3:239-251), ciliates of the types:  Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena, Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus, Pseudocohnilembus, Euplotes, Engelmaniella , and  Stylonychia , especially of the genus  Stylonychia lemnae  with vectors following a transformation method as described in WO9801572 and multicellular plant cells (see Schmidt, R and Willmitzer, L. (1988), High efficiency  Agrobacterium tumefaciens - mediated  transformation of  Arabidopsis thaliana  leaf and cotyledon explants, Plant Cell Rep.: 583-586); Plant Molecular Biology and Biotechnology, C Press, Boca Raton, Florida, chapter 6/7, S.71-119 (1993); F. P. White, B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds.:Kung und R. Wu, Academic Press (1993), 128-43; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225 (and references cited therein) or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and 17 polymerase.  
           [0037]    Expression of proteins in prokaryotes is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein but also to the C-terminus or fused within suitable regions in the proteins. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase.  
           [0038]    Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. In one embodiment, the coding sequence of the fatty acid transporter is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-X protein. The fusion protein can be purified by affinity chromatography using glutathione-agarose resin. Recombinant fatty acid transporter unfused to GST can be recovered by cleavage of the fusion protein with thrombin.  
           [0039]    Examples of suitable inducible non-fusion  E. coli  expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase tanscription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident 1 prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.  
           [0040]    Other vectors which are useful in prokaryotic organisms are known by a person skilled in the art such vectors are for example in  E. coli  pLG338, pACYC184, pBR-series such as pBR322, pUC-series such as pUC18 or pUC19, M113 mp-series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR90, pIN-III113-B1, lgt11 or pBdCI in  Streptomyces  pIJ101, pIJ364, pIJ702 or pIJ361, in  Bacillus  pUB110, pC194 or pBD214, in  Corynebacterium  pSA77 or pAJ667.  
           [0041]    One strategy to maximize recombinant protein expression is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the bacterium chosen for expression, such as  C. glutamicum  (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.  
           [0042]    In another embodiment, the fatty acid transporter expression vector is a yeast expression vector. Examples of vectors for expression in yeast  S. cerivisae  include pYepSec1 (Baldari, et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.). Vectors and methods for the construction of vectors appropriate for use in other fungi, such as the filamentous fungi, include those detailed in: van den Hondel, C. A. M. J. J. &amp; Punt, P. J. (1991) “Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, J. F. Peberdy, et al., eds., p. 1-28, Cambridge University Press: Cambridge or in: More Gene Manipulations in Fungi [J. W. Bennet &amp; L. L. Lasure, eds., p. 396-428: Academic Press: San Diego]. Additional useful yeast vectors are for example 2 mM, pAG-1, YEp6, YEp13 or pEMBLYe23.  
           [0043]    Alternatively, the fatty acid transporter of the invention can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).  
           [0044]    The above mentioned vectors are only a small overview of possible useful vectors.  
           [0045]    Additional plasmids are well known by the skilled artisan and are described for example in: Cloning Vectors (Eds. Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).  
           [0046]    In another embodiment, the fatty acid transporter of the invention may be expressed in unicellular plant cells (such as algae) see Falciatore et al., 1999, Marine Biotechnology.1 (3):239-251 and references therein and plant cells from higher plants (e.g., the spermatophytes, such as crop plants). Examples of plant expression vectors include those detailed in: Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992) “New plant binary vectors with selectable markers located proximal to the left border”, Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W. (1984) “Binary  Agrobacterium  vectors for plant transformation, Nucl. Acid. Res. 12: 8711-8721; Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds.: Kung und R Wu, Academic Press, 1993, S. 15-38.  
           [0047]    A plant expression cassette preferably contains regulatory sequences capable to drive gene expression in plants cells and which are operably linked so that each sequence can fulfil its function such as termination of transcription such as polyadenylation signals. Preferred polyadenylation signals are those originating from  Agrobacterium tumefaciens  t-DNA such as the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835 ff) or functional equivalents thereof but also all other terminators functionally active in plants are suitable.  
           [0048]    As plant gene expression is very often not limited on transcriptional levels a plant expression cassette preferably contains other operably linked sequences like translational enhancers such as the overdrive-sequence containing the 5′-untranslated leader sequence from tobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al 1987, Nucl. Acids Research 15:8693-8711).  
           [0049]    Plant gene expression has to be operably linked to an appropriate promoter conferring gene expression in a timely, cell or tissue specific manner. Preferrred are promoters driving constitutitive expression (Benfey et al., EMBO J.8 (1989) 2195-2202) like those derived from plant viruses like the 35S CAMV (Franck et al., Cell 21(1980) 285-294), the 19s CaMV (see also U.S. Pat. No. 5,352,605 and WO 84/02913) or plant promoters like those from Rubisco small subunit described in U.S. Pat. No. 4,962,028.  
           [0050]    Additionally vATPase-gene promoters such as an 1153 basepair fragment from Beta-vulgaris (Plant Mol Biol 1999, 39:463-475) can be used to drive ASE gene expression alone or in combination with other PUFA biosynthesis genes.  
           [0051]    Other preferred sequences for use operable linkage in plant gene expression cassettes are targeting-sequences necessary to direct the gene-product in its appropriate cell compartment (for review see Kermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423 and references cited therin) such as the vacuole, the nucleus, all types of plastids like amyloplasts, chloroplasts, chromoplasts, the extracellular space, mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes and other compartments of plant cells.  
           [0052]    Plant gene expression can also be facilitated via a chemically inducible promoter (for rewiew see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89-108).  
           [0053]    Chemically inducible promoters are especially suitable if gene expression is wanted to occur in a time specific manner. Examples for such promoters are a salicylic acid inducible promoter (WO 95/19443), a tetracycline inducible promoter (Gatz et al., (1992) Plant J. 2, 397-404) and an ethanol inducible promoter (WO 93/21334).  
           [0054]    Also promoters responding to biotic or abiotic stress conditions are suitable promoters such as the pathogen inducible PRP1-gene promoter (Ward et al., Plant. Mol. Biol. 22 (1993), 361-366), the heat inducible hsp80-promoter from tomato (U.S. Pat. No. 5,187,267), cold inducible alpha-amylase promoter from potato (WO 96/12814) or the wound-inducible pinII-promoter (EP-A-0 375 091).  
           [0055]    Especially those promoters are preferred which confer gene expression in tissues and organs where lipid and oil biosynthesis occurs in seed cells such as cells of the endosperm and the developing embryo. Suitable promoters are the napin-gene promoter from rapeseed (U.S. Pat. No. 5,608,152), the USP-promoter from  Vicia faba  (Baeumlein et al., Mol Gen Genet, 1991, 225 (3):459-67), the oleosin-promoter from  Arabidopsis  (WO 98/45461), the phaseolin-promoter from  Phaseolus vulgaris  (U.S. Pat. No. 5,504,200), the Bce4-promoter from  Brassica  (WO 91/13980) or the legumin B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2):233-9) as well as promoters conferring seed specific expression in monocot plants like maize, barley, wheat, rye, rice etc. Suitable promoters to note are the lpt2 or lpt1-gene promoter from barley (WO 95/15389 and WO 95/23230) or those described in WO 99/16890 (promoters from the barley hordein-gene, the rice glutelin gene, the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, wheat glutelin gene, the maize zein gene, the oat glutelin gene, the Sorghum kasirin-gene, the rye secalin gene).  
           [0056]    Also especially suited are promoters that confer plastid-specific gene expression as plastids are the compartment where precursors and some end products of lipid biosynthesis are synthesized. Suitable promoters such as the viral RNA-polymerase promoter are described in WO 95/16783 and WO 97/06250 and the cIpP-promoter from  Arabidopsis  described in WO 99/46394.  
           [0057]    Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.  
           [0058]    Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection”, conjugation and transduction are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemical-mediated transfer, or electroporation. Suitable methods for transforming or transfecting host cells including plant cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and other laboratory manuals such as Methods in Molecular Biology, 1995, Vol. 44,  Agrobacterium  protocols, ed: Gartland and Davey, Humana Press, Totowa, N.J.  
           [0059]    The vector is introduced into a microorganism or plant cell (e.g., via  Agrobacterium  mediated gene transfer, biolistic transformation, polyethyleneglycol or other applicable methods) and cells in which the introduced ASE gene has homologously recombined with the endogenous fatty acid transporter gene are selected, using art-known techniques. In case of plant cells the AHAS gene described in Ott et al., J. Mol. Biol. 1996, 263:359-360 is especially suitable for marker gene expression and resistance towards imidazolinone or sulphonylurea type herbicides.  
           [0060]    In another embodiment, recombinant organisms such as microorganisms can be produced which contain selected systems which allow for regulated expression of the introduced gene. For example, inclusion of a fatty acid transporter gene on a vector placing it under control of the lac operon permits expression of the fatty acid transporter gene only in the presence of IPTG. Such regulatory systems are well known in the art. Recombinant organisms means an organism which comprises an inventive nucleic acid sequence, a gene construct or a vector in the cell or inside the genome at an place which is not the “natural” place or an the “natural” place but modified in a manner which is not the natural manner that means the coding sequence is modified and/or the regulatory seqeunce is modified. Modified means that a single nucleotide or one or more codons are changed in comparison to the natural sequence.  
           [0061]    A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a fatty acid transporter. An alternate method can be applied in addition in plants by the direct transfer of DNA into developing flowers via electroporation or  Agrobacterium  medium gene transfer. Accordingly, the invention further provides methods for producing fatty acid transporters using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a fatty acid transporter has been introduced, or into which genome has been introduced a gene encoding a wild-type or altered fatty acid transporter) in a suitable medium until fatty acid transporter is produced. In another embodiment, the method further comprises isolating fatty acid transporters from the medium or the host cell.  
           [0062]    Host organisms suitable in principle to cover the nucleic acid of the invention, the novel gene construct or the inventive vector are all prokaryotic or eukaryotic organisms. The host organisms advantageously used are organisms such as bacteria, fungi, yeasts, animal or plant cells. Additional advantageously organisms are Fungi, yeasts or plants, preferably fungi or plants, very particularly preferably plants such as oilseed plants containing high amounts of lipid compounds such as rapeseed, primrose, canola, peanut, linseed, soybean, sufflower, sunflower, borage or plants such as maize, wheat, rye, oat, triticale, rice, barley, cotton, manihot, pepper, tagetes, solanaceaous plants such as potato, tobacco, eggplant, and tomato,  Vicia  species, pea, alfalfa, bushy plants (coffee, cacao, tea),  Salix  species, trees (oil palm, coconut) and perennial grasses and forage crops. Particularly preferred plants of the invention are oilseed plants such as soybean, peanut, rapeseed, canola, sunflower, safflower, trees (oil palm, coconut).  
           [0063]    According to a further aspect of the invention there is provided a nucleic acid molecule comprising SEQ ID NO:1 or a part thereof, or homologue thereof, which encodes a peroxisomal ABC (ATP-binding Cassette) transporter.  
           [0064]    Also provided is a nucleic acid encoding an ABC cassette transporter protein especially one involved in fatty acid transport across peroxisomal membranes, the nucleic acid being selected from a group consisting of:  
           [0065]    (a) DNA having the nucleotide sequence given herein in SEQ ID NO: 1 which encodes a protein having the amino acid sequence given herein as SEQ ID NO:2.  
           [0066]    (b) nucleic acid which hybridize to DNA of (a) above (e.g. under stringent conditions); and  
           [0067]    (c) nucleic acids which differ from the DNA of (a) or (b) above due to the degeneracy of the genetic code, and which encodes a protein encoded by a DNA of (a) or (b) above.  
           [0068]    DNAs of the present invention include those coding for proteins homologous to, and having essentially the same biological properties as, the proteins disclosed herein, and particularly the DNA disclosed herein as SEQ ED NO: land encoding the protein given herein SEQ ID NO:2. This definition is intended to encompass natural allelic variations therein. Thus, isolated DNA or cloned genes of the present invention can be of any plant species of origin. Thus, DNAs which hybridise to DNA disclosed herein as SEQ ID NO:1 (or fragments or derivatives thereof which serve as hybridisation probes as discussed below) and which code on expression for a protein associated with fatty acid or acyl CoA transport into peroxisomes (e.g., a protein according to SEQ ID NO:2) are included in the present invention.  
           [0069]    According to a further aspect of the present invention there is provided use of the nucleic acid of the invention and/or protein or polypeptide encoded thereby in any one or more of the following processes: regulating fatty acid transport across peroxisome and/or glyoxisome membranes; regulating growth; regulating seed development and; modulating fatty acid utilisation by the plant.  
           [0070]    According to a yet further aspect of the present invention there is provided a method of regulating any one or more of the following processes: regulating fatty acid transport across peroxisome and/or glyoxisome membranes; regulating growth; regulating seed development and; modulating fatty acid utilisation by the plant comprising genetically engineering a plant cell or tissue or seed so as to enhance or reduce/prevent expression of the nucleic acid of the present invention.  
           [0071]    Such techniques are well-known in the art and include but are not restricted to: reduction of expression of the nucleic acid and encoded protein by antisense expression of part or all of the sequence corresponding to SEQ ID No1, expression of part or all of SEQ ID No1 as double stranded interference RNA (RNAi); or co-supression of the endogenous gene brought about by introduction of additional copies of part or all of SEQ ID No1. Conversely expression may be increased, or altered spatially and temporally, by the introduction of constructs fused to different regulatory sequences as hereinbefore described.  
           [0072]    According to a yet further aspect of the invention there is provided a method of modification of a plant cell to increase or decrease the transport of some or all fatty acids across cell membranes and/or to increase or prevent their breakdown.  
           [0073]    Preferably the modification could take the form of mutating, disabling or deleting the CTS gene. The CTS gene could also be modified to alter its expression levels in specific tissues or at specific times. By example and not by way of limitation, CTS expression could be inhibited in developing seeds but not in germinating seeds. Alternatively, the plant cell may be modified so as to containing an increased number of copies of the nucleic acid according to the invention as compared to the wild-type.  
           [0074]    Mutation of the sequence may be through designed changes or random changes followed by selection to introduce variants of SEQ ID No 1 and SEQ ID No2 with altered substrate specificity or transport rate or regulation, or through the expression of mutants with dominant negative activity.  
           [0075]    The invention therefore includes transgenic plants comprising a nucleic acid molecule of the invention as well as transgenic plants adapted to increase or decrease expression of an active peroxisomal ABC transporter, for example by having increased or decreased numbers of gene copies or modification of transcription control elements  
           [0076]    According to a yet further aspect of the invention there is provided a method of regulating fatty acid levels in plants comprising genetically engineering a plant cell or tissue or seed so as to disrupt, deactivate, disable, mutate, delete, knockout or render transcriptionally ineffective a nucleic acid according to the invention.  
           [0077]    According to a yet further aspect of the invention there is provided a plant cell and/or a plant tissue and/or plant and/or plant seed that does not containing a transcriptionally activated/activatable form of the nucleic acid molecule according to the invention or contains a reduced number of copies of the nucleic acid of the present invention as compared to the wild-type.  
           [0078]    According to a yet further aspect of the invention there is provided a plant generated from a plant cell and/or plant tissue and/or plant and/or plant seed which contains a disrupted, deactivated, disabled, mutated, deleted, knocked-out or rendered transcriptionally ineffective nucleic acid according to the invention as compared to the wild-type.  
           [0079]    According to a yet further aspect of the invention there is provided a plant cell and/or a plant tissue and/or plant and/or plant seed comprising:  
           [0080]    (i) an increased number of copies of the nucleic acid according to the invention as compared to the wild-type;  
           [0081]    (ii) increased transcription of the nucleic acid according to the invention as compared to the wild-type; or  
           [0082]    (iii) a differing number of copies of the nucleic acid according to the invention depending on the time of development of the plant.  
           [0083]    According to a yet further aspect of the invention there is provided a plant generated from a plant cell and/or plant tissue and/or plant and/or plant seed comprising:  
           [0084]    (i) an increased number of copies of the nucleic acid according to the invention as compared to the wild-type;  
           [0085]    (ii) increased transcription of the nucleic acid according to the invention as compared to the wild-type; or  
           [0086]    (iii) a differing number of copies of the nucleic acid according to the invention depending on the time of development of the plant.  
           [0087]    According to a yet further aspect of the invention there is provided a primer comprising any one of SEQ ID NOS: 3 to 10 or parts thereof capable of recognising the nucleic acid of the present invention or homologues thereof, the primer being specific for a nucleic acid according to the invention.  
           [0088]    The following T-DNA knockout lines containing insertions in the CTS gene were screened from Wisconsin knockout population alpha:— 
           [0089]    cts-2; position of insertion in genomic DNA is 16,674 in Accession AL161596 ( Arabidopsis thaliana  DNA chromosome 4, contig fragment No. 92, VERSION AL161596.2). This corresponds to Exon 3 of the gene or position 846 in Sequence ID NO:1 (cDNA) and in codon Thr115 in SEQ ID NO:2 (the derived amino acid sequence). The primers used in the initial PCR screen are preferably H1A6T7 DSR1 (SEQ ID NO:8) AND JL 202 (SEQ ID NO:11).  
           [0090]    F27; position of insertion in genomic DNA is 15,873 in Accession AL161596. This is in Intron-1 which is located in the 5′-UTR. The primers used in the initial PCR screen are preferably H1A6T7 DSF1 (SEQ ID NO:4) and JL 202(SEQ ID NO:11).  
           [0091]    F12; position of insertion in genomic DNA is 17,318 in Accession AL161596. This is in Intron-4. The primers used in the initial PCR screen are preferably H1A6T7 DSF1 (SEQ ID NO:4) and JL 202(SEQ ID NO:11).  
           [0092]    Preferably, the primers of the present invention include:  
           [0093]    Primer H1A6T7 DSF1 Forward primer 5′ at position 15,667 of Accession AL161596, (SEQ ID NO:4); Primer H1A6T7-DSF2 5′ position at 15,640 of Accession ALI 61596, (SEQ ID NO:5); Primer H1A6T7 DSF3 5′ position at 15,320 of Accession AL161596, (SEQ ID NO:6); Primer H1A6T7 DSF 5′ position at 15,542 of Accession AL161596, (SEQ ID NO:3); Primer H1A6T DSR1 Reverse primer 5′ at position 20,611 of Accession AL161596, (SEQ ID NO:8); Primer H1A6T7 DSR2 5′ position at 17,819 of Accession AL161596, (SEQ ID NO:9); Primer H1A6T7 DSR3 5′ position at 21,191 of Accession AL161596, (SEQ ID NO:10); Primer H1A6T7 DSR 5′ position at 19,978 of Accession AL161596 (SEQ ID NO:7) and/or, Primer JL 202 Left Border primer for PCR out of constructs of Wisconsin knockout population alpha (SEQ ID NO:11).  
           [0094]    According to a yet further aspect of the invention there is provided use of a primer comprising any one of SEQ ID-NOS: 3 to 10 or parts thereof capable of recognising the nucleic acid of the present invention or homologues thereof, in identifying a nucleic acid sequence according to the invention.  
           [0095]    According to a yet further aspect of the invention there is provided a method of identifying plant material selected from a plant cell and/or plant tissue and/or plant and/or plant seed comprising a disrupted, deactivated, disabled, mutated, deleted, knocked-out or rendered transcriptionally ineffective nucleic acid according to the invention comprising contacting the plant material with a primer comprising any one of SEQ ID NOS: 3 to 10. 
       
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0096]    [0096]FIG. 1 illustrates alignment of peroxisomal ABC transporters. The peptide sequences of Human ALD and PMP70 were aligned with  Arabidopsis  CTS and yeast Pxa1, CTS. Although the  arabidopsis  ABC is a double transporter consisting of 2 sets of trasmembrane domains comprising multiple transmembrane helicesand 2 ATPase domains (format: TM domain-ATPase-TM domain-ATPase) its closest homologs outside the plant kingdom are half transporters (format: TM domain-ATPase). For this alignment the peptide sequence of the  Arabidopsis  abc was arbitrarily divided into two halves: CTSa and CTSb. Conserved domains previously described in the literature are highlighted. Loop 1 is conserved among peroxisomal ABC transporters only. The EAA-like motif, Walker A and B, and the C-sequence are all conserved between prokaryotic and eukaryotic ABC transporters.  
         [0097]    [0097]FIG. 2 illustrates analysis of CTS transcript levels during germination in the light. Seeds were surface sterilized using Milton® and imbibed in the dark at 4C for 4d before plating and incubating at 22C. Data points are days post imbibition (dpi).  
         [0098]    [0098]FIG. 3 shows expression and purification of second ATPase domain of CTS.  
         [0099]    [0099]FIG. 4 illustrates the location of CTS to membrane fractions.  Arabidopsis  cell suspension culture derived from leaf was homogenized and centrifuged at low speed (1000 g) to remove nuclei followed by 20 000 g to sediment membrane fractions. These were then resuspended and loaded onto a sucrose step gradient Fractions of equivalent protein loading were separated by SDS-PAGE followed by immuno-blotting with antisera raised against CTS. Lane 1, SDS-extraction of original homogenate; lane 2, post-nuclear supernatant; lane 3, resuspended membrane pellet; lane 4, membrane recovered at 0.5 M/1.6M sucrose interface and lane 5, 1.6M/2.2M sucrose interface. Concentration of CTS in the membrane fractions is consistent with CTS being a membrane protein.  
         [0100]    [0100]FIG. 5 represents the location of T-DNA insertions in three CTS knockouts cts-2F27 and F12. Exons are shown as dark shaded boxes. Non coding transcribed regions are indicated by light shading.  
         [0101]    [0101]FIG. 6 illustrates the isolation of individual CTS knockout plants. 100 individual plants were screened by PCR for the exon 2 knockout (cts-2) from a pool containing seed from 9 T-DNA tagged lines previously identified by PCR. PCR was carried out using a T-DNA specific primer (JL-202) and gene-specific primer DSR1 (A) or DSR3 (B). A positive plant (lane 5) was identified from this sample of 15 individuals. (C) HindM restriction digest of genomic DNA from wt (lane 1) and the positive plant (lane 2) was probed with CTS probe. Bands of 2.5 kb and 6.5 kb correspond to predicted sizes for wt CTS. The band of 8.7 kb corresponds to a predicted size consistent with a T-DNA insertion in exon 2. Together these data suggest that the positive plant is a heterozygote having one copy of the wt CTS gene and a mutant gene containing a T-DNA insertion. (I) PCR identifying presence of knockout plants in a pool of germinating seedlings from 9 T-DNA tagged lines for knockout F27 (lanes 1-3) and knockout F12 (lanes 66) using a T-DNA specific primer and a range of gene-specific primers.  
         [0102]    [0102]FIG. 7 illustrates CTS localisation in peroxisomes. Sucrose density gradient showing the localisation of peroxisomal markers catalase (closed circles) and 3-ketoacyl thioloase (KAT), ER marker calreticulin (CAL), mitochondrial marker adenine nucleotide translocator (ANT) and chlrophyll (open circles). CTS antigen follows the distribution of catalase and thiolase.  
         [0103]    [0103]FIG. 8 illustrates the profiles of triacyl glycerol-derived fatty acids and acyl CoA levels in wild type and cts-2 mutant: a=Comparison of TAG-derived fatty acids in the wild types and cts-2 mutant in seeds and seedlings 0, 2 and 5 days after sowing; b=profile of TAG-derived fatty acids in imbibed wild type and mutant seeds by chain length; c=as b but profile from 5 day old seedlings; d=comparison of Acyl CoA levels in wild types and cts-2 mutant seedlings 0,2 and 5 days after sowing; e=profile of Acyl CoAs in imbibed wild type and mutant seeds by chain length and: f as e but profile from 5 day old seedlings. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0104]    The yeast and human peroxisomal ABC transporter proteins were used to search the publically accessible sequence databases of  Arabidopsis taliana  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 (H1A677) 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).  
         [0105]    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 whereas 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;7.1e-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 6 th  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 FIG. 1. 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 is diagnostic of the human and yeast peroxisomal ABC transporters and the closely related sequence H(S/A) SIAF(Y/F) occurs in the two halves of CTS. The loop1 region and the sequence PQRPY(M/T)(A/C)LGTLRDQ is diagnostic of animal peroxisomal ABC transporters. The almost identical sequence PQRPY(M/T)(A/C)LGTLRDQ occurs in both halves of CTS. Therefore CTS contains sequences which assign it to the peroxisomal sub-class of ABC transporters.  
         [0106]    To determine whether the expression, pattern of CTS was consistent with its involvement in fatty acid uptake in peroxisomes, a northern blot was performed (FIG. 2). 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 day1.  
         [0107]    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 (FIG. 3). Affinity purified antisera detected a protein of ca.140 kDa in membrane fractions from  Arabidopsis  seedlings and tissue culture cells (FIG. 4). Further experiments demonstrated the localisation of this protein specifically in peroxisomes/glyoxysomes (FIG. 7).  
         [0108]    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 (FIG. 5). 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 (FIG. 6). 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 (FIG. 8). These results provide strong support for the proposed biochemical function of CTS as a fatty acyl CoA transporter.  
         [0109]    Methods  
         [0110]    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.  
         [0111]    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).  
         [0112]    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.  
         [0113]    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 (FIG. 8 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 (FIG. 8 b,c ).  
         [0114]    Total Acyl CoAs increased in both lines over the period 05 days (FIG. 8 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 (FIG. 8 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.  
         [0115]    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 C 2-2  acyl CoAs which are predominantly T-G-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.  
         [0116]    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 FIG. 8, 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.  
         [0117]    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.  
       REFERENCES  
       [0118]    Footitt, S., Slocombe, S P., Laarner, V., Kurup, S., Wu, Y., Larson, T., Graham, I., Baker, A and Holdsworth (2002) EMBO 21, 12, 2912-2922.  
         [0119]    Germain, V., Rylott, E. Larson, T. R, Sherson, S. M., Bechtold, N. Carde, J-P., Bryce J. H.  
         [0120]    Graham, I. A. and Smith, S. M. (2001) Plant J. 28, 1-12.  
         [0121]    Hayashi M, Toriyama, K, Kondo, M. ad Nishimura, M. (1998) Plant Cell 10, 183-195.  
         [0122]    Hettema, E. H., van Roermund, C W T, Distel, B. van den Berg, M. Vilela, C., Rodrigues-Pousada C, Wanders, R J A and Tabak, H F (1996) EMBO J. 15, 3813-3822.  
         [0123]    Hooks, M. A., Kellas, F. and Graham, I. A. (1999) Plant J. 20, 1-3.  
         [0124]    Krysan, P. J., Young, J. C. and Sussman, M. R. (1999)  Plant Cell,  11, 2283-2290.  
         [0125]    Larson, T. R, Graham, I. A. (2001) Plant J. 25:115-125  
         [0126]    Mannaerts, G. P., van Veldhoven, P., van Broekhoven, A., Vandebroek, G., and Debeer, L. J. (1982). Biochem. J. 204, 17-23.  
         [0127]    Mosser, J. Douar, A. M., Sarde, C-O., Kioschis, P. Feil, R Moser, H., Poustka, A-M., Mandel, J-M. and Augbourg, P (1993) Nature 361, 726-730.  
         [0128]    Olsen, J. A. and Lusk, K. R (1994) Phytochemistry 36, 7-9.  
         [0129]    Russell, L., Larner, V., Kurup, S., Bougourd and Holdsworth, M (2000) Development, 127, 3759-3767.  
         [0130]    Tugal, H. B., Pool, M. and Baker, A. (1999)  Plant Physiology,  120, 309-320.  
         [0131]    Zollman B. K., Yoder, A. and Bartel, B. (2000) Genetics 156, 1323-1337  
     
       
       
         1 
         
           
             11  
           
           
             1  
             5073  
             DNA  
             Arabidopsis thaliana  
           
            1 

atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa     60 

aacgacggcc agtgaattgt aatacgactc actatagggc gaattgggta ccgggccccc    120 

cctcgaggtc gacggtatcg ataagcttga tatcgaattc gcggccgcct ctctctctct    180 

ctatctctat ctctcgattt gggggagttc cgtcacggtg gactagtacg tctcgttgcc    240 

gttggtggcg tagtcggaat taatttcctc ggcgttgaga ttcacatggt ctagaattct    300 

agctaagtgg ttgttgttgt tgttacgatt tccgatttct cgagtttttt tttttatatt    360 

tagcttctgt ttcgtttatc cctcccggag acactccttg gtcgaatctc tcatgctgag    420 

gtgttttgga cacttgttgt caagaagaaa ccagttttgg ttctgattaa tcgttggttg    480 

gaaaatatac tcaattccag gccatgcctt cacttcaact attgcagtta actgagcggg    540 

gtcggggtct tgtagcgtca agacggaaat ctatactgct tgcggctggg attgtagctg    600 

ctggtggaac tgctgtttac ctgaaatcaa gggtcgcttc ccggaggcct gattcttcgc    660 

gtctttgcaa tggtcagagt gatgatgatg agactttgga aaagctgact gcaactgatc    720 

aaaatgcaaa gataaccacg aaaaagaaga aaggaggagg attgaagtct cttcaggttc    780 

tgactgctat tcttctctct cagatgggaa aaatgggtgc cagggatctt ttggcactag    840 

tcgccaccgt ggttttcaga acagctttga gcaatagatt ggcaaaagtg caaggtttcc    900 

ttttccgtgc tgctttctta aggcgtgcgc cactgtttct acggctcatc tccgagaata    960 

ttatgttgtg tttcatgcta tcaacattgc actctacttc aaagtacata actggggcat   1020 

tgagtttgcg attcagaaag atattgacca agattatcca ttcacactat tttgagaata   1080 

tggtatatta caaaatatca cacgtggatg gtcggattac gcaccctgaa caaagaattg   1140 

ccagcgatgt accaagattc tcctcagagt tgagcgatct tatactggat gatttgacgg   1200 

cggttactga tggaattttg tatgcatggc gcctgtgttc atatgctagt ccaaaataca   1260 

tcttctggat actggcctat gtactggggg ctgggacggc gataagaaac ttttctcctt   1320 

cttttgggaa attgatgtcc aaggaacagc agttagaagg agagtaccgg caacttcatt   1380 

cacgcttaag gactcattcg gaaagcatag cattctatgg tggggaaacc agggaagaat   1440 

ctcatataca acaaaagttc aagaatcttg ttagccatat gagtcacgtg cttcatgatc   1500 

actggtggtt tggtatgatc caagattttc tgctgaagta tcttggggcc acagttgcag   1560 

ttattctgat tatcgaacca ttcttctctg ggcatctaag acctgacgac tcgaccttag   1620 

gaagagctga gatgcttagc aatataagat atcacactag tgtcattata tctctctttc   1680 

aggcgttggg aacactttct ataagttcca ggcggctcaa ccgactcagt ggttatgctg   1740 

accgaatcca tgagttgatg gctgtctcaa gagaactcag tggtgatgat aaatcgtctt   1800 

tccagagaaa tagaagcaga aattatctaa gtgaagctaa ttatgtagag ttttccgatg   1860 

tcaaggttgt tactccaacc ggaaatgttt tggtggagga tctcaccctt cgagttgagc   1920 

aagggtctaa tcttctgatt acaggtccta atggaagtgg caagagttcc cttttccgag   1980 

tattaggagg tctatggccc ctggtgtctg gacatattgt gaagccagga gttggttctg   2040 

atcttaacaa ggagatcttc tatgtgccgc aacggcctta tatggcagta ggaacacttc   2100 

gtgaccagtt aatatatcct cttacttctg gccaagagag tgaactgctc actgagattg   2160 

gaatggtgga gctattgaaa aatgttgatc tagaatattt attggatcgc taccaacctg   2220 

aaaaagaggt taattggggt gatgaattat ctcttggaga gcaacagaga ttgggtatgg   2280 

ccagactatt ctaccacaaa cccaaatttg caattctaga tgaatgcaca agtgctgtca   2340 

caactgatat ggaagaacgc tttgccgcta aggttcgagc tatgggaact tcttgcataa   2400 

caatctccca tcgtccagcg cttgttgcat tccatgatgt tgttctgtca ttagacggtg   2460 

aaggaggatg gagtgttcat tacaagaggg atgactctgc ccttctgacg gatgctgaaa   2520 

ttgattcagt gaaaagttca gatacagatc ggcaaaatga tgcgatggtt gttcaacgag   2580 

cgtttgctgc agctagaaag gaatctgcta ctaattcaaa ggctcagtcg taccagacac   2640 

agttaattgc aagatcacct gttgtagata aaagtgtagt gttgcctcgt tttcctcaac   2700 

ctcaaacatc ccaaagggct ttaccatcaa gagtagctgc aatgttaaac gtgttgatac   2760 

ccactatatt tgacaagcaa ggagctcaac tgcttgctgt tgcttgcctt gtcgtctcaa   2820 

gaacgctgat ctctgaccga atagcctctt tgaatgggac cactgtgaag tatgtcttgg   2880 

agcaagataa ggcagccttt gttcgtttga ttggtttgag tgttctccaa agtggtgcat   2940 

cttctataat tgctccttca ctaaggcatt taacgcaaag gctagcgtta gggtggagga   3000 

ttcgtttgac tcaacatctg ctaaggaact atttgagaaa taatgcgttt tacaaggttt   3060 

tccacatgtc aggcaatagt attgatgcgg accagagact cactcgtgac ctggaaaagt   3120 

taaccgctga cttgtctgga cttcttactg gaatggtaaa gccatcggtt gacattctct   3180 

ggttcacctg gaggatgaag ttactgactg gtcagagggg agttgccata ctttacacat   3240 

atatgttact tggtcttggt tttctgagac gtgttgctcc cgatttcggt gatctagccg   3300 

gtgaagaaca gcagcttgaa gggaagtttc ggtttatgca cgagaggctg aacactcatg   3360 

ctgaatctat tgcattcttt ggaggtggag ctcgagaaaa ggctatggtt gacaaaaaat   3420 

tcagggccct actggatcat tctctcatgc tcttgaggaa gaaatggttg tatggcatac   3480 

ttgatgattt tgtgacaaag caacttccca ataatgtgac gtggggattg agtttattgt   3540 

atgccctaga acacaaggga gacagagcac ttgtctccac tcaaggtgaa ttggcacatg   3600 

cattgcggta tctagcttct gttgtctccc aaagctttat ggcttttggc gatattcttg   3660 

aactacacaa gaagttcctg gagctctctg gtggtattaa cagaattttt gagctcgatg   3720 

agtttttgga tgcttctcag tcaggtgtta cctcagaaaa tcaaacaagt cgtttggatt   3780 

ctcaagatct actttccttt tcggaggtgg atatcattac ccctgctcag aaattgatgg   3840 

ctagcaagtt gtcgtgtgaa atagtttcag ggaaaagcct gctcgtcaca ggtccaaatg   3900 

gtagtggaaa gacttcagta tttagagtcc ttagagatat ctggcccact gtatgtggaa   3960 

gacttaccaa accatcattg gatatcaaag aacttgggtc agggaatggc atgttttttg   4020 

tcccgcagcg accttataca tgtttaggga cactgagaga tcaaattata taccctctat   4080 

ctaaagaaga agcagagaaa agggcagcaa agttgtacac cagtggagag agctcaacag   4140 

aagctggaag cattctggat tctcatttga aaaccattct ggagaatgtt cggttagttt   4200 

atctcttgga aagagacgta ggtggttggg atgctactac caattgggaa gacatattat   4260 

ctcttggaga gcaacagaga ttaggcatgg cacgtttatt ctttcacagg ccgaagtttg   4320 

gagtccttga tgaatgcaca aatgcgacga gtgttgatgt tgaggaacag ctctatagag   4380 

ttgcacgaga catgggagtc actttcataa cctcatcaca acggccggct ctgatcccat   4440 

tccattcctt ggagctaagg ctgattgatg gagaaggaaa ctgggagctc cgttcgatcg   4500 

aacagacaac agagtgaact cagcaaaaca tttttagaaa ggtctatata gttgttaaag   4560 

aaaaaagtaa taaagttaaa gccattagac gatgcaagct atatggtatg tagtatatgg   4620 

attcttcctc gatcgcaagg agtggaagag aatgcgtcga tgctagtgct tttgttagaa   4680 

ttggaggatt tgatttgatt ctagatatat ataaatgtag gcgattgaat tggtggagca   4740 

ttttgagttc tcctatggag tatggtctta gctttgaaca aacaaagaat atagtgatca   4800 

ctcaaataat gtacagttcg tttcaatttc ctttgttggg attagttttt ctatcttata   4860 

attaaaagaa tgaaattgaa gtgggcggcc gcgaattcct gcagcccggg ggatccacta   4920 

gttctagagc ggccgccacc gcggtggagc tccagctttt gttcccttta gtgagggtta   4980 

atttcgagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg tatccgctca   5040 

caattccaca caacatacga gccggaagca taa                                5073 

 
           
             2  
             1336  
             PRT  
             Arabidopsis thaliana  
           
            2 

Met Pro Ser Leu Gln Leu Leu Gln Leu Thr Glu Arg Gly Arg Gly Leu 
1               5                   10                  15 

Val Ala Ser Arg Arg Lys Ser Ile Leu Leu Ala Ala Gly Ile Val Ala 
            20                  25                  30 

Ala Gly Gly Thr Ala Val Tyr Leu Lys Ser Arg Val Ala Ser Arg Arg 
        35                  40                  45 

Pro Asp Ser Ser Arg Leu Cys Asn Gly Gln Ser Asp Asp Asp Glu Thr 
    50                  55                  60 

Leu Glu Lys Leu Thr Ala Thr Asp Gln Asn Ala Lys Ile Thr Thr Lys 
65                  70                  75                  80 

Lys Lys Lys Gly Gly Gly Leu Lys Ser Leu Gln Val Leu Thr Ala Ile 
                85                  90                  95 

Leu Leu Ser Gln Met Gly Lys Met Gly Ala Arg Asp Leu Leu Ala Leu 
            100                 105                 110 

Val Ala Thr Val Val Phe Arg Thr Ala Leu Ser Asn Arg Leu Ala Lys 
        115                 120                 125 

Val Gln Gly Phe Leu Phe Arg Ala Ala Phe Leu Arg Arg Ala Pro Leu 
    130                 135                 140 

Phe Leu Arg Leu Ile Ser Glu Asn Ile Met Leu Cys Phe Met Leu Ser 
145                 150                 155                 160 

Thr Leu His Ser Thr Ser Lys Tyr Ile Thr Gly Ala Leu Ser Leu Arg 
                165                 170                 175 

Phe Arg Lys Ile Leu Thr Lys Ile Ile His Ser His Tyr Phe Glu Asn 
            180                 185                 190 

Met Val Tyr Tyr Lys Ile Ser His Val Asp Gly Arg Ile Thr His Pro 
        195                 200                 205 

Glu Gln Arg Ile Ala Ser Asp Val Pro Arg Phe Ser Ser Glu Leu Ser 
    210                 215                 220 

Asp Leu Ile Leu Asp Asp Leu Thr Ala Val Thr Asp Gly Ile Leu Tyr 
225                 230                 235                 240 

Ala Trp Arg Leu Cys Ser Tyr Ala Ser Pro Lys Tyr Ile Phe Trp Ile 
                245                 250                 255 

Leu Ala Tyr Val Leu Gly Ala Gly Thr Ala Ile Arg Asn Phe Ser Pro 
            260                 265                 270 

Ser Phe Gly Lys Leu Met Ser Lys Glu Gln Gln Leu Glu Gly Glu Tyr 
        275                 280                 285 

Arg Gln Leu His Ser Arg Leu Arg Thr His Ser Glu Ser Ile Ala Phe 
    290                 295                 300 

Tyr Gly Gly Glu Thr Arg Glu Glu Ser His Ile Gln Gln Lys Phe Lys 
305                 310                 315                 320 

Asn Leu Val Ser His Met Ser His Val Leu His Asp His Trp Trp Phe 
                325                 330                 335 

Gly Met Ile Gln Asp Phe Leu Leu Lys Tyr Leu Gly Ala Thr Val Ala 
            340                 345                 350 

Val Ile Leu Ile Ile Glu Pro Phe Phe Ser Gly His Leu Arg Pro Asp 
        355                 360                 365 

Asp Ser Thr Leu Gly Arg Ala Glu Met Leu Ser Asn Ile Arg Tyr His 
    370                 375                 380 

Thr Ser Val Ile Ile Ser Leu Phe Gln Ala Leu Gly Thr Leu Ser Ile 
385                 390                 395                 400 

Ser Ser Arg Arg Leu Asn Arg Leu Ser Gly Tyr Ala Asp Arg Ile His 
                405                 410                 415 

Glu Leu Met Ala Val Ser Arg Glu Leu Ser Gly Asp Asp Lys Ser Ser 
            420                 425                 430 

Phe Gln Arg Asn Arg Ser Arg Asn Tyr Leu Ser Glu Ala Asn Tyr Val 
        435                 440                 445 

Glu Phe Ser Asp Val Lys Val Val Thr Pro Thr Gly Asn Val Leu Val 
    450                 455                 460 

Glu Asp Leu Thr Leu Arg Val Glu Gln Gly Ser Asn Leu Leu Ile Thr 
465                 470                 475                 480 

Gly Pro Asn Gly Ser Gly Lys Ser Ser Leu Phe Arg Val Leu Gly Gly 
                485                 490                 495 

Leu Trp Pro Leu Val Ser Gly His Ile Val Lys Pro Gly Val Gly Ser 
            500                 505                 510 

Asp Leu Asn Lys Glu Ile Phe Tyr Val Pro Gln Arg Pro Tyr Met Ala 
        515                 520                 525 

Val Gly Thr Leu Arg Asp Gln Leu Ile Tyr Pro Leu Thr Ser Gly Gln 
    530                 535                 540 

Glu Ser Glu Leu Leu Thr Glu Ile Gly Met Val Glu Leu Leu Lys Asn 
545                 550                 555                 560 

Val Asp Leu Glu Tyr Leu Leu Asp Arg Tyr Gln Pro Glu Lys Glu Val 
                565                 570                 575 

Asn Trp Gly Asp Glu Leu Ser Leu Gly Glu Gln Gln Arg Leu Gly Met 
            580                 585                 590 

Ala Arg Leu Phe Tyr His Lys Pro Lys Phe Ala Ile Leu Asp Glu Cys 
        595                 600                 605 

Thr Ser Ala Val Thr Thr Asp Met Glu Glu Arg Phe Ala Ala Lys Val 
    610                 615                 620 

Arg Ala Met Gly Thr Ser Cys Ile Thr Ile Ser His Arg Pro Ala Leu 
625                 630                 635                 640 

Val Ala Phe His Asp Val Val Leu Ser Leu Asp Gly Glu Gly Gly Trp 
                645                 650                 655 

Ser Val His Tyr Lys Arg Asp Asp Ser Ala Leu Leu Thr Asp Ala Glu 
            660                 665                 670 

Ile Asp Ser Val Lys Ser Ser Asp Thr Asp Arg Gln Asn Asp Ala Met 
        675                 680                 685 

Val Val Gln Arg Ala Phe Ala Ala Ala Arg Lys Glu Ser Ala Thr Asn 
    690                 695                 700 

Ser Lys Ala Gln Ser Tyr Gln Thr Gln Leu Ile Ala Arg Ser Pro Val 
705                 710                 715                 720 

Val Asp Lys Ser Val Val Leu Pro Arg Phe Pro Gln Pro Gln Thr Ser 
                725                 730                 735 

Gln Arg Ala Leu Pro Ser Arg Val Ala Ala Met Leu Asn Val Leu Ile 
            740                 745                 750 

Pro Thr Ile Phe Asp Lys Gln Gly Ala Gln Leu Leu Ala Val Ala Cys 
        755                 760                 765 

Leu Val Val Ser Arg Thr Leu Ile Ser Asp Arg Ile Ala Ser Leu Asn 
    770                 775                 780 

Gly Thr Thr Val Lys Tyr Val Leu Glu Gln Asp Lys Ala Ala Phe Val 
785                 790                 795                 800 

Arg Leu Ile Gly Leu Ser Val Leu Gln Ser Gly Ala Ser Ser Ile Ile 
                805                 810                 815 

Ala Pro Ser Leu Arg His Leu Thr Gln Arg Leu Ala Leu Gly Trp Arg 
            820                 825                 830 

Ile Arg Leu Thr Gln His Leu Leu Arg Asn Tyr Leu Arg Asn Asn Ala 
        835                 840                 845 

Phe Tyr Lys Val Phe His Met Ser Gly Asn Ser Ile Asp Ala Asp Gln 
    850                 855                 860 

Arg Leu Thr Arg Asp Leu Glu Lys Leu Thr Ala Asp Leu Ser Gly Leu 
865                 870                 875                 880 

Leu Thr Gly Met Val Lys Pro Ser Val Asp Ile Leu Trp Phe Thr Trp 
                885                 890                 895 

Arg Met Lys Leu Leu Thr Gly Gln Arg Gly Val Ala Ile Leu Tyr Thr 
            900                 905                 910 

Tyr Met Leu Leu Gly Leu Gly Phe Leu Arg Arg Val Ala Pro Asp Phe 
        915                 920                 925 

Gly Asp Leu Ala Gly Glu Glu Gln Gln Leu Glu Gly Lys Phe Arg Phe 
    930                 935                 940 

Met His Glu Arg Leu Asn Thr His Ala Glu Ser Ile Ala Phe Phe Gly 
945                 950                 955                 960 

Gly Gly Ala Arg Glu Lys Ala Met Val Asp Lys Lys Phe Arg Ala Leu 
                965                 970                 975 

Leu Asp His Ser Leu Met Leu Leu Arg Lys Lys Trp Leu Tyr Gly Ile 
            980                 985                 990 

Leu Asp Asp Phe Val Thr Lys Gln  Leu Pro Asn Asn Val  Thr Trp Gly 
        995                 1000                 1005 

Leu Ser  Leu Leu Tyr Ala Leu  Glu His Lys Gly Asp  Arg Ala Leu 
    1010                 1015                 1020 

Val Ser  Thr Gln Gly Glu Leu  Ala His Ala Leu Arg  Tyr Leu Ala 
    1025                 1030                 1035 

Ser Val  Val Ser Gln Ser Phe  Met Ala Phe Gly Asp  Ile Leu Glu 
    1040                 1045                 1050 

Leu His  Lys Lys Phe Leu Glu  Leu Ser Gly Gly Ile  Asn Arg Ile 
    1055                 1060                 1065 

Phe Glu  Leu Asp Glu Phe Leu  Asp Ala Ser Gln Ser  Gly Val Thr 
    1070                 1075                 1080 

Ser Glu  Asn Gln Thr Ser Arg  Leu Asp Ser Gln Asp  Leu Leu Ser 
    1085                 1090                 1095 

Phe Ser  Glu Val Asp Ile Ile  Thr Pro Ala Gln Lys  Met Ala Ser 
    1100                 1105                 1110 

Lys Leu  Ser Cys Glu Ile Val  Ser Gly Lys Ser Leu  Leu Val Thr 
    1115                 1120                 1125 

Gly Pro  Asn Gly Ser Gly Lys  Thr Ser Val Phe Arg  Val Leu Arg 
    1130                 1135                 1140 

Asp Ile  Trp Pro Thr Val Cys  Gly Arg Leu Thr Lys  Pro Ser Leu 
    1145                 1150                 1155 

Asp Ile  Lys Glu Leu Gly Ser  Gly Asn Gly Met Phe  Phe Val Pro 
    1160                 1165                 1170 

Gln Arg  Pro Tyr Thr Cys Leu  Gly Thr Leu Arg Asp  Gln Ile Ile 
    1175                 1180                 1185 

Tyr Pro  Leu Ser Lys Glu Glu  Ala Glu Lys Arg Ala  Ala Lys Leu 
    1190                 1195                 1200 

Tyr Thr  Ser Gly Glu Ser Ser  Thr Glu Ala Gly Ser  Ile Leu Asp 
    1205                 1210                 1215 

Ser His  Leu Lys Thr Ile Leu  Glu Asn Val Arg Leu  Val Tyr Leu 
    1220                 1225                 1230 

Leu Glu  Arg Asp Val Gly Gly  Trp Asp Ala Thr Thr  Asn Trp Glu 
    1235                 1240                 1245 

Asp Ile  Leu Ser Leu Gly Glu  Gln Gln Arg Leu Gly  Met Ala Arg 
    1250                 1255                 1260 

Leu Phe  Phe His Arg Pro Lys  Phe Gly Val Leu Asp  Glu Cys Thr 
    1265                 1270                 1275 

Asn Ala  Thr Ser Val Asp Val  Glu Glu Gln Leu Tyr  Arg Val Ala 
    1280                 1285                 1290 

Arg Asp  Met Gly Val Thr Phe  Ile Thr Ser Ser Gln  Arg Pro Ala 
    1295                 1300                 1305 

Leu Ile  Pro Phe His Ser Leu  Glu Leu Arg Leu Ile  Asp Gly Glu 
    1310                 1315                 1320 

Gly Asn  Trp Glu Leu Arg Ser  Ile Glu Gln Thr Thr  Glu 
    1325                 1330                 1335 

 
           
             3  
             29  
             DNA  
             Arabidopsis thaliana  
           
            3 

ctctctctct atctctatct ctcgatttg                                       29 

 
           
             4  
             29  
             DNA  
             Arabidopsis thaliana  
           
            4 

tctagctaag tggttgttgt tgttgttac                                       29 

 
           
             5  
             29  
             DNA  
             Arabidopsis thaliana  
           
            5 

gccgttgaga ttcacatggt ctagaattc                                       29 

 
           
             6  
             29  
             DNA  
             Arabidopsis thaliana  
           
            6 

atgaaacggt ggctagagtt acctgagta                                       29 

 
           
             7  
             29  
             DNA  
             Arabidopsis thaliana  
           
            7 

tacactttta tctacaacag gtgatcttg                                       29 

 
           
             8  
             29  
             DNA  
             Arabidopsis thaliana  
           
            8 

gcaattatag aagatgcacc actttggag                                       29 

 
           
             9  
             29  
             DNA  
             Arabidopsis thaliana  
           
            9 

catagaatgc tatgctttcc gaatgagtc                                       29 

 
           
             10  
             29  
             DNA  
             Arabidopsis thaliana  
           
            10 

cccaatgaga tctttagtgt ctctagcca                                       29 

 
           
             11  
             29  
             DNA  
             Arabidopsis thaliana  
           
            11 

cccaatgaga tctttagtgt ctctagcca                                       29