CA2365096A1 - Polyunsaturated fatty acid (pufa) elongase from caenorhabditis elegans - Google Patents

Polyunsaturated fatty acid (pufa) elongase from caenorhabditis elegans Download PDF

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CA2365096A1
CA2365096A1 CA002365096A CA2365096A CA2365096A1 CA 2365096 A1 CA2365096 A1 CA 2365096A1 CA 002365096 A CA002365096 A CA 002365096A CA 2365096 A CA2365096 A CA 2365096A CA 2365096 A1 CA2365096 A1 CA 2365096A1
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Johnathan A. Napier
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Abstract

A isolated polypeptide comprising a functional long chain polyunsaturated fatty acid (PUFA) elongase.

Description

POLYSATURATED FATTY ACID (PUFA) ELONGASE FROM CAENORHABDITIS ELEGANS
The present invention relates to polyunsaturated fatty acid (PUFA) elongases.
More specifically, the invention relates to a DNA sequence from C. elegans encoding a PUFA
elongase.
Unsaturated fatty acids are essential components required for normal cellular function, being involved in a diverse number of roles ranging from membrane fluidity to acting as signal molecules (Gill, L, Valivety, R. (1997). Trends ~iotechnol. 15, 401-409;
Broun, P., et al (1999) Ann. Rev. Nutr. 19, 197-216). In particular, the class of fatty acids known as the polyunsaturated fatty acids (PUFAs) has attracted considerable interest as pharmaceutical and nutraceutical compounds (Broun supra; Horrobin, D. F. (1990) Reviews in Contemp Pharmacotherpy 1, 1-45).
The synthesis of PUFAs i.e. fatty acids of 18 carbons or more in length and containing two or more double bonds, is thought to be catalyzed in a variety of organisms by a specific fatty acid elongase enzyme. This elongase is responsible for the addition of 2 carbon units to an 18 carbon PUFA, resulting in a 20 carbon fatty acid. An example of this reaction is the elongation of Y-linolenic acid (GLA; 18:306'~~12) to di-homo-y-linolenic acid (DHGLA;
20:348°"°'4) in which the tri-unsaturated 18 carbon fatty acid is elongated by the addition of a two carbon unit to yield the tri-unsaturated 20 carbon fatty acid. Since there is considerable interest in the production of long chain PUFAs of more than 18 carbons in chain length, for example arachidonic acid and eicosapentanoic acid, the identification of this enzyme is of both academic and commercial interest.
At present, there are no examples of identified cloned genes encoding PUFA
elongases, though a number of genes encoding enzymes likely to be involved in other aspects of lipid synthesis have been identified. For example, an Arabidopsis gene (FAE1) has been shown to be required for the synthesis of very long chain monounsaturated fatty acids (such as erucic acid; 20:14") (James, D. W. et al, (1995) Plant Cell7, 309-319).
However, it is clear that this enzyme does not recognize di- and tri-unsaturated 18 carbon fatty acids, for example, linoleic acid, 18:209~'z or a,-linolenic acid, 18:39''2,15 respectively, as substrates, SUBSTITUTE SHEET (RULE 26) and is therefore not involved in the synthesis of long chain PUFAs (Millar &
Kunst (1997), Plant Journal 12, 121-131). This in itself is not surprising; since, of the plant kingdom, only a very few lower plant species, such as the moss Physcomicotrella patens (Girke et al., (1998), Plant J, 15: 39-48); are capable of synthesising long chain PLJFAs, and therefore Arabidopsis would not be expected to contain any such enzymes (Napier et al.
(1997), Biochem J, 328: 717-720; Napier et al., (1999) Trends in Plant Sci 4, 2-5).
A schematic diagram representing a generalized pathway for the product of PLTFAs is shown in Figure 1. Biochemical characterisation of mammalian elongation systems (most notably from liver microsomes) has indicated that a mammalian elongase consists of four subunits, made up of a condensing enzyme, a (3-ketoreductase, a dehydrase and an enoyl reductase (reviewed in Cinti, D. L., et al (1992) Prog. Lipid Res. 31, 1-51).
The Arabidopsis FAEI gene product encodes a polypeptide of 56kDa, which shows very limited homology to condensing enzymes such as chalcone synthase and stillbene synthase (James, D. W. supra). Although FAEI is normally only expressed in seed tissues, ectopic expression in non-seed tissue (or heterologously in yeast) revealed that FAEI
could direct the synthesis of erucic acid (Millar, A. A., Kunst, L. (1997) Plant J. 12, 121-131).
Three fatty acid elongase activities have been characterised from the yeast S.
cerevisiae.
Again, this organism does not synthesis PUFAs, and therefore does not contain genes encoding a PUFA elongase. One gene ELO1, was identified on the basis of a screen to isolate mutants defective in elongation of 14 carbon (i.e. medium) chain saturated fatty acids (Toke & Martin (1996) J Biol Chem 271, 18413-18422). Complementation of elol mutants restored viability, and the ELOl gene product was shown to encode a polypeptide which was responsible for the specific elongation of 14:0 fatty acids to 16:0 fatty acids.
Two related genes were also detected in the genome of S. cerevisiae, and their function determined by disruption. These two genes, subsequently named EL02 and EL03, were shown to be involved in the elongation of the very long chain saturated fatty acids found in sphingolipid molecules (Oh et al (1997), J. Biol Chem 272, 17376-17384). In particular, EL02 was required for elongation of fatty acids up to 24 carbons, and EL03 was required for elongation of the 24 carbon fatty acid to 26 carbons. However, neither gene was essential for viability. Examination of the these three fatty acid elongases revealed the presence of a conserved "histidine box" motif (Shanklin et al., (1994), Biochemistry, 33, 12787-12794) (His-X-X-His-His, where X is any amino acid) towards the centre of the polypeptide sequences. Importantly, there was no detectable homology between the yeast elongases (EL01,2,3) and the plant very long chain mono-unsaturated fatty acid elongase (FAE1) (Oh et al, supra).
In order to identify genes encoding PUFA elongases, it is necessary to study systems in which the synthesis of PUFAs is well documented; a good example of this is the model animal system C. elegans, a small free-living worm (Tanaka et al., (1996), Lipids 31, 1173-1178). C. elegans, like most other animals, and in contrast to higher plants, synthesises PUFAs such as arachidonic acid (AA; ZO:4 05~g~31,14) as precursors to a class of molecules known as the eicosanoids, which in turn serve as precursors for compounds such as prostaglandins and leucotrienes (Horrobin, (1990), Reviews in Contemp Pharmacotherpy, 1:1-45). The presence of AA and other long chain polyunsaturated fatty acids in C. elegans is well documented (Tanaka et al., (1996), Lipids 31, 1173-1178). The complete sequence of the nematode's genome is now publicly available (The C elegans consortium, 1998, Science 282, 2012-2018: Database at http:llwww.sanger.ac.uklProjectslC elganslblast server,shtml).
An object of the invention is to provide an isolated PUFA elongase.
Using the above-mentioned C. elegans genomic sequence, together with suitable search strings, the inventors identified eight related putative open reading frames (ORFs) encoding for PUFA elongases. A number of different search criteria were applied to identify a number of (ORFs) which were likely to encode polypeptides with fatty acid elongase activities. These ORFs were then subject to functional characterisation by heterologous expression in yeast, allowing the identification of a PUFA elongase.
Accordingly, a first aspect of the invention provides an isolated polypeptide comprising a functional long chain polyunsaturated fatty acid (PUFA) elongase i.e. the polypeptide has the function of extending the chain length of an 18 carbon PUFA to 20 carbons in length.
This polypeptide can be used to elevate PUFA levels in animals, thereby providing a ready source of PLTFAs.
The polypeptide may be from a eukaryote.
The polypeptide may comprise at least a portion of the amino acid shown in SEQ
>D. 15, or variants thereof.
For the purposes of the present application, the term "variant" in relation to a certain sequence means a protein or polypeptide which is derived from the sequence through the insertion or deletion of one or more amino acid residues or the substitution of one or more ammo acid residues with amino acid residues having similar properties, e.g.
the replacement of a polar amino acid residue with another polar amino acid residue, or the replacement of a non-polar amino acid residue with another non-polar amino acid residue. In all cases, variants must have an elongase function as defined herein.
A second aspect of the invention provides a polypeptide having at least 60 %
homology to a polypeptide according to a first aspect of the invention. The polypeptide may have at least $0%, or as much as 90% or more homology to a polypeptide according to a first aspect of the invention.
The polypeptide according to either aspect of the invention may include a sequence motif responsible for Endoplasmic Reticulum (ER) - retention. This allows the polypeptide to be specifically located or targeted to the ER of a cell.
The polypeptide may also be able to elongate palmitoleic acid (PA; 16:1~~) to vacceric acid (VA; 18:14"). Thus, the polypeptide is also capable of elongation of a ~9-monounsaturated 16C fatty acid.
Preferably, the polypeptide is from an animal, more preferably, the animal is an invertebrate such as a worm. Where the animal is a worm, it is preferably C. elegans.
Alternatively, the animal is a vertebrate, preferably a mammal such as a human, rat or mouse.

A third aspect of the invention provides an isolated DNA sequence, preferably a cDNA
sequence, encoding a polypeptide according to a first or second aspect of the invention.
This DNA sequence may be used to engineer transgenic organisms.
Preferably, the DNA sequence comprises the sequence shown in SEQ 1D NO: 7 or variants of that sequence due, for example, to base substitutions, deletions, and/or additions.
A fourth aspect of the invention provides an engineered organism, such as a transgenic animal, engineered to express a polypeptide according to a first or second aspect of the invention. The engineered organism may be engineered to express elevated levels of the polypeptide, thereby providing a supply of polypeptide at a reduced cost as a reduced number of organisms need be used.
Preferably, the engineered organism is a mammal such as a rat, mouse or monkey.
A fifth aspect of the invention provides an engineered organism containing a synthetic pathway for the production of a polypeptide according to a first or second aspect of the invention. This has the advantage of allowing greater control over the production of PUFAs by the pathway by an organism.
The pathway may include D'-fatty acid desaturase, and/or 46-fatty acid desaturase.
The engineered organism according to a fourth or fifth aspect of the invention may be a lower eukaryote, such as yeast. Alternatively, the transgenic organism may be a fish.
A sixth aspect of the invention provides a transgenic plant engineered to express a polypeptide according to a first aspect of the invention.
A seventh aspect of the invention provides a transgenic plant containing a DNA
sequence according to a third aspect of the invention.

An eighth aspect of the invention provides a method of producing a PUFA
comprising carrying out an elongase reaction catalysed by a polypeptide according to a first or second aspect of the invention.
The PUFA may be di-homo-gamma-linoleic acid (20:308'"''4), arachidonic acid (20:4058°"''4), eicosapentanoic acid (20:5~5~8~"~'4~"), docosatrienoic acid (22:3~3~'6~'9), docosatetraenoic acid (22:4~'~'°~'3~'6), docosapentaenoic acid (22:50'''°~~3,16,19) or docosahexaenoic acid (22:54''''°,13.16,19)_ The PLJFA may be a 24 carbon fatty acid with at least 4 double bonds.
A ninth aspect of the invention provides a PUFA produced by a method according to an eighth aspect of the invention.
The PLTFA may be used in foodstuffs, dietary supplements or pharmaceutical compositions.
A tenth aspect of the invention provides a foodstuff comprising a PUFA
according to a fifth aspect of the invention. The foodstuff can be fed to an animal.
An eleventh aspect of the invention provides a dietary supplement comprising a PUFA
according to a fifth aspect of the invention. The dietary supplement can be supplied to an animal to augment its PUFA levels.
An twelfth aspect of the invention provides a pharmaceutical composition comprising a polypeptide according to a first or second aspect of the invention or a PLTFA
according to a ninth aspect of the invention.
Preferably, the pharmaceutical composition comprises a pharmaceutically-acceptable diluent, earner, excipient or extender. This allows the composition to be supplied in a form which best suits the pharmaceutical application in question. Fox example, a topical application would preferably be a cream or lotion, whereas if the composition was to be ingested a different form would be more suitable.

A thirteenth aspect of the invention provides a method of treatment of an animal, such as a mammal, or a plant, comprising supplying to the animal or plant a DNA sequence according to a third aspect of the invention, a foodstuff according to a tenth aspect of the invention, a dietary supplement according to an eleventh aspect of the invention, a pharmaceutical composition according to a twelfth aspect of the invention or a PUFA according to a ninth aspect of the invention.
Preferably, the mammal is a human.
The invention will now be further described, by way of example only, with reference to SEQ
>D1 to 16, and Figures 2 to 11, in which;
SEQ 1D 1 to 8 show the putative ORFs encoding PUFA elongases A to H
respectively; and SEQ ID9 to 16 show the deduced amino acid sequences of the putative ORFs of SEQ ID
NO: 1 to 8 respectively; and Figures 2 to 9 show hydrophobicity plots for each of PUFA elongases A to H
respectively.
Figure 10 shows an amino acid sequence line-up comparing the C'. elegans ORF
F56H11.4 (Z68749) with related sequences.
Figure 11 shows chromatograms of fatty acid methyl esters from transformed yeast.
Introduction to general strategy Initially the C. elegans databases were searched for any sequences which showed low levels of homology to yeast ELO genes (EL02 and EL03) using the TBLASTN programme. A
similar search was carried out using short (20 to 50 amino acid) stretches of ELO genes which were conserved amongst the three ELO polypeptide sequences. C. elegans sequences which were identified by this method were then used themselves as search probes, to identify any related C. elegans genes which the initial search with the yeast sequences failed to identify. This was necessary because the level of homology between the yeast ELO genes and ~ worm genes is always low (see BLAST scores later). To allow for a more sensitive search of worm sequences, a novel approach was adopted to circumvent the major drawback with searches using the BLAST programmes, namely that the search string (i.e.
the input search motif) must be longer than 1 S characters for the algorithm to work.
Thus, if it was desired to search for a short motif (like a histidine box), then the BLAST
programme would not be capable of doing this. A complete list of all the predicted ORFs present in the C.
elegans genome exists as a database called Wormpep, which is freely available from the Sanger WWW site (http://www.sanger.ac.uk/Projects/C_eleganslwebace front end.shtml).
The latest version of Wormpep was down loaded to the hard disc of a Pentium PC, and re-formatted as a Microsoft Word6 document, resulting in a document of about 3,500 pages.
This was then searched using the "Search & Replace" function of Word6, which also allows for the introduction of "wildcard" characters into the search motif. So, for example, it is possible to search both for the short text string HPGG, which would identify any predicted worm ORF present in the Wormpep 3,500 page document containing this motif, or alternatively search with HPGX (where X is a wild card character). Clearly, such (manual) searches of a 3,500 page document are extremely time-consuming and demanding, also requiring visual inspection of each and every identified ORF. For example, searching with a motif such as HXXHH identifies in excess of 300 different ORFs. However, by using a number of different short search strings (as outlined below), and combining these with other methods for identifying putative elongase enzymes, a number of candidate ORFs have been identified.
Database search using the FAE1 polypeptide sequence As a negative control, to demonstrate that the FAEl gene sequence was unlikely to provide a useful search sequence in the identification of C.elegans sequences encoding for PUFA
elongases, the GenBank databases (http://www.ncbi.nlm.nih.gov/Web/Search/index.html) were searched using the Arabidopis FAE1 polypeptide sequence to identify related genes or expressed sequence transcripts (ESTs). GenBank is the NIH genetic sequence database, an annotated collection of all publicly available DNA sequences (Nucleic Acid Research (1998) 26, 1-7). There are approximately 2,162,000,000 bases in 3,044,000 sequence records as of December 1998. The search was carried out using the BLAST2 (Basic Local Alignment Search Tool) algorithm (Altschul et al., (1990) ,I Mol Biol 215,403,410) Although a number of plant ORFs and ESTs were reported as being related, no animal sequences were identified by this search, confirming the observation that FAE1 was unlikely to be a suitable candidate as a search template for PUFA elongases.
Database search using yeast ELO sequences Using the three yeast fatty acid elongase sequences (ELO 1, 2, 3) as probes, a number of putative ORFs in the DNA of C. elegans-derived cosmid sequences which form the C.
elegans genomic sequence database were identified. Moreover, an extensive and time-consuming search of a downloaded copy of the WormPep database (ftp://ftp.sanger.ac.uk./pub/databases/wormpep) using manual search strings in MSWord 6, identified a number of C. elegans ORFs which contained presumptive histidine boxes.
Wormpep contains predicted proteins from the Caenorhabditis elegans genome sequence project, which is carried out jointly by the Sanger Centre in Cambridge, UK
and Genome Sequencing Center in St. Louis, USA. The current Wormpep database, Wormpep 16, contains 16,332 protein sequences (7,120,115 residues). Search strings used included [HXXHH], [HXXXHH], [QXXHH] and [YHH]. Comparison of the data from the two different searches indicated a small (<10) number of putative ORFs as candidate elongases.
The histidine box motifs are shown in bold in SEQ >D 9 to 16.
Hydrophobicity plot analysis Since the fatty acid elongase reaction is predicted to be carried out on the cytosolic face of the endomembrane system (Toke & Martin (1996), supra; Oh et al (1997), supra), the putative C. elegans ORFs were examined for potential membrane spanning domains, via Kyte & Doolittle hydrophobicity plots (J. Mol Biol, (1982), 157, 105-.132).
This revealed a number of ORFs with possible membrane-spanning domains, and also indicated a degree of similarity in the secondary-structure of a number of identified ORFs.
Screening for ER-retention signal sequences The inventors postulated that since fatty acid elongases are expected to be endoplasmic reticulum (ER) membrane proteins, they might be expected to have peptide signals which are responsible for "ER-retention". In the case of ER membrane proteins, this signal often takes the form of a C-terminal motif [K-K-XZ_,-Stop], or similar variants thereof (Jackson et al., (1990), EMBO J., 9, 3153-3162). Further sequence analysis of the C.
elegans putative elongases revealed that 4 ORFs (F41H10.7, F41H10.8, F56H11.4, Y53F4B.c) had C-terminal motifs that exactly matched this search pattern, and that a further 2 ORFs (F11E6.5, C40H1.4) had related sequences. These sequence motifs are underlined in SEQ
m 9 to 13, 15 and 16.
Chromosome mapping Since the inventors had previously observed that C.elegans genes involved in the synthesis of PUFA may exist in tandem (for example the 05 and 06 desaturases required for AA and GLA synthesis, respectively, are < 1 kB apart on chromosome N (Michaelson et al., (1998), FEBS Letts 439, 215-218), the positions of the putative C. elegans elongase ORFs were determined using the Sanger Centre's WebAce C. elegans server (http://www.sanger.ac.uk/Projects/C elegans/webace-front end.shtml).. This indicated that two pairs of putative elongases were in close proximity to each other on the C. elegans chromosome N.
F41H10.7 and F41H10.8 were identified as being approximately 10 Kb apart on chromosome N, and F56H11.3 and F56H11.4 were identified as being approximately 2 Kb apart on chromosome N.
Putative C. elegans fatty acid elongases The positions of the putative OIRF's in the C. elegans genome are shown below i.e.
chromosome number, and map position in centiMorgans, together with the GenBank database accession numbers.
The designations used employ the same method as used on the Sanger Centre's C
elegans database, i.e. ORF C40H1.4 is predicted coding sequence 4 on cosmid C40H1.
Elon ase Cosmid Saner ID GenBank Acc Chromosome Code A C40H 1.4 Z 19154 III

B D2024.3 U41011 N, 7.68 C F11E6.5 281058 IV, 18.8 D F41H10.7* U61954 N, 29.8 E F41H10.8* U61954 N, 29.8 F F56H11.3# 268749 IV, 2.5 G F56H11.4~ 268749 IV, 2.5 H Y53F4B.c 292860 II
* ors indicates genes in tandem Comparison of C. elegans putative elongase ORFs with yeast genes:
Each of the three yeast ELO polypeptides were compared against all of the worm putative elongase translated ORF sequences, and then ranked in order of similarity (as measured by the BLAST score) (Altschul et al (1990), supra) The results are shown below, with the ORF sequences ranked from most similar to least similar, and the BLAST scores are shown in brackets:
Yeast ELO1 (14 to 16 carbon fatty acid elongase) G (262) > E (241) > D (225) > C (219) > A (216) > F (215) > H (197) > B (172) Yeast EL02 (24 carbon sphingolipid elongase) E(231)>C(226)>G(189)>A(181)>F(166)>D(150)>H(141)>B(140) Yeast EL03 (24 to 26 sphingolipid elongase) D(171)>G(163)>F(154)>A(152)>E(150)>C(131)>B(132)>H(128) It is clear from the numeric values of the BLAST scores that the sequences are related, but the levels of homology are low. For comparison, the BLAST score for homology between two related worm proteins, the 05 and the 46 desaturase is in excess of 500.
Analysis of potential sphingolipid ancestry Previously, the inventors had noted the similarities between the fatty acid D6 desaturase and sphingolipid desaturases in plants, and that the two distinct enzymes could have arisen from one ancestral gene. Moreover, it was considered likely that the sphingolipid desaturase predated the fatty acid desaturase, and may in fact have been the ancesteral progenitor.
Therefore it is plausible that the next step in the arachidonic acid biosynthetic pathway has also evolved from the sphingolipid metabolic pathway. It is therefore considered highly significant that some of the C. elegans ORF putative elongases have similarity to sphingolipid enzymes. For this reason, these ORFs are considered to be very clear candidates for PUFA elongases. It has previously been considered that the C.
elegans 05 and D6 fatty acid desaturases have evolved from 1 ancestral gene (Michaelson et al., ( 1998), FEBS Letts 439, 215-218). It is also significant that one pair of C. elegans putative elongase ORFs (F & G) genetically maps close to the 05/06 fatty acid desaturase genes, with both gene pairs being located at the top end of chromosome IV.
Cosmid San eg r ID GenBank Acc Chromosome Encoded Peptide Code W08D2.4 270271 IV, 3.06 06 fatty acid desaturase T13F2.1 281122 IV, 3.06 45 fatty acid desaturase Cloning of Desaturase and Elongase Genes in Yeast Expression Vectors Putative elongases sequences F56H11.4 and F41H10.8 were cloned by PCR into the pYES2 vector (Invitrogen). A C. elegans mixed stage cDNA library was used as a PCR
template. F56H11.4 was amplified using primers:
56h114.for 5'-GCGGGTACCATGGCTCAGCATCCGCTC-3' and;
56h1 l4.rev 5'-GCGGGATCCTTAGTTGTTCTTCTTCTT-3'.
F41H10.8 was amplified using primers:
41h108.for 5'-GCGGGTACCATGCCACAGGGAGAAGTC-3' and;
41h108.rev 5'-GCGGGATCCTTATTCAATTTTTCTTTT-3'.
Amplified sequences were then restricted using Kpnl and BamHI (underlined in the forward and reverse primers, respectively), purified using the Qiagen PCR
purification kit, and ligated into a KpnI/BamHI cut pYes2 vector.
An ORF encoding the Mortierella alpina 45-fatty acid desaturase (Michaelson, L. V., et al (1998) J. Biol. Chem. 273, 19055-19059) was amplified using primers:
MadS.for 5'-GCGAATTCACCATGGGTACGGACCAAGGA-3' and;
MadS.rev 5'-GCGGAGCTCCTACTCTTCCTTGGGACG-3', and restricted using EcoRI and SacI, gel purified as described and ligated into a EcoRIlSacI
cut pESC-TRP vector (Stratagene) to generate pESC/~5.
An ORF encoding the borage O6-fatty acid desaturase (Sayanova, O., et al (1997) Proc.
Natl. Acad. Sci USA 94, 4211-4216) was restricted from pGEM3 using BamHI and XhoI
and ligated into a BamHIlXhoI cut pESC-TRP vector to generate pESC/0''.

A double construct was also generated by ligating the BamHIlXhoI borage 46 insert into the pESC/OS construct described previously, generating pESC/(05,06).
Functional Characterisation in Yeast Elongases and desaturase constructs were introduced in Saccharomyces cerevisiae W303-lA using a lithium acetate based method (Elble, R. (1992) Biotechniques 13, 18-20) and expression of the transgenes was induced by addition of galactose to 2%
(w/v) as described in Napier et al (Napier, J. A., et al (1998) Biochem J330, 611-614;
Michaelson L. V., supra; Michaelson, L. V., (1998) FEBS Letts 439, 215-218). Yeast transformants containing pYES2-derived constructs were grown on synthetic minimal media (SD, the composition of which is defined in Sherman, F ( 1991 ) Methods in Enzymology 194, 3-21 );
synthetic minimal medium minus uracil; pESC-derived constructs were grown on SD
minimal medium minus tryptophan. Co-transformed yeast (containing both pYES2 and pESC derivatives) were grown on SD minimal medium minus uracil and tryptophan.
Prior to induction, cultures were grown in the presence of 2% raffinose and supplemented with 0.5 mM of the appropriate fatty acid substrate in the presence of 1 % tergitol-(NP40) (Sigma). All cultures were then grown for a further 48-h unless indicated.
Fatty Acid Analysis To identify the elongation reaction responsible for the synthesis of di-homo-y-linolenic acid (DHGLA; 20:308"~'4) from GLA, this latter fatty acid was supplied as the (exogenous) substrate.
Lipids were extracted from transformed and control yeast by homogenisation in MeOH-CHC13 using a modification of the method of Bligh and Dyer (Dickenson &
Lester (1999) Biochim Biophys Acta 1426, 347-357). The resulting CHC13 phase was evaporated to dryness under nitrogen gas and the samples were transmethylated with 1M HCl in methanol at 80 °C for 1 hour. Fatty acid methyl esters (FAMES) were extracted in hexane and purified using a small column packed with Florisil. Analysis of FAMES was conducted using a Hewlett Packard 5880A Series Gas Chromatograph equipped with a 25M x 0.32mm RSL-SOOBP bonded capilliary column and a flame ionisation detector.
Fatty acids were identified by comparison of retention times with FAME
standards (Sigma) WO 00/$5330 CA 02365096 2001-09-14 pCT/GB00/01035 separated on the same GC. Quantitation was carried out using peak height area integrals expressed as a total of all integrals (Bligh, E.G. & Dyer, W.J. (1959) Can. J.
Biochem.
Physiol. 37, 911-917).
Total fatty acids extracted from yeast cultures were analysed by gas chromatography (GC) of methyl ester derivatives. Lipids were extracted, transmethylated and the fatty acid methyl esters (FAMEs) analysed as described by Sayanova et al.
Figure 11 shows chromatograms of fatty acid methyl esters from yeast transformed with the control (empty) plasmid pYES2 (Fig. 11A) or with ORF F56H11.4 in pYES2 (Fig.
11B).
Exogenous substrate in the form of GLA was supplied to the cultures. Two novel peaks are observed in (B); these peaks (annotated as 20:3 and 18:1 *) were identified (against known standards) as DHGLA and vaccenic acid, respectively. Detection was by flame ionisation.
One cDNA ORF tested in this manner displayed a high level of elongase activity on the GLA substrate, converting 44% to DHGLA. The identity of this elongation product was confirmed as DHGLA by comparison with a known standard (the standards used were known standards for either DHGLA, AA, EPA or VA from Sigma Chemicals, Ltd.), using GCMS analysis using a Kratos MS80RFA (Napier, J. A., supra; Michaelson, L. V., supra;
Michaelson, L. V., supra). The deduced amino acid sequence of the functional elongase clone identified it as being encoded by the C. elegans gene F56H11.4, and comparison with the yeast ELO genes showed low homology confined to a few short amino acid motifs (see Fig. 10). Some similarity with a mouse gene Cig30 (Tvrdik, P., (1997) J. Biol.
Chem. 272, 31738-31746), which has been implicated in the recruitment of brown adipose tissue in liver tissue, was also observed, as well as a potential human homologue encoded by a gene located on chromosome 4q25, BAC 207d4. The most closely related C. elegans ORFs, F41H10.8 (U61954) and F56H11.3 (Z68749) are also shown, as is part of a related human gene present on chromosome N (present on BAC clone B207d4; AC004050). The GenBank accession numbers are given for all sequences.
The range of fatty acids synthesised by C. elegans can potentially require a number of different elongation reactions (Tanaka, T., (1996) Lipids 31, 1173-1178). The substrate-specificity of the F56H11.4 PUFA elongase was therefore determined using a range of exogenously supplied fatty acids. This revealed that GLA is the major substrate, with a number of other fatty acids being elongated at a lower efficiency (see Table 1 ).
Although most of these substrates are polyunsaturated fatty acids, it was unexpectedly observed that palmitoleoic. acid (PA; 16:1 09) was also elongated by F56H11.4 to yield vaccenic acid (VA; 18:1 0"). The biosynthetic pathway for VA is unclear, but the data indicate that it may be synthesised by elongation of 0~-monounsaturated 16C
fatty acid.
The C. elegans PUFA elongase ORF F56H11.4 maps to the top of chromosome IV (at 4.32 cM) with a related sequence (F56H11.3; 51 % similarity) located 1,824bp downstream.
Another C. elegans gene (F41H10.8) was also observed, which is present on chromosome IV, and which shows a slightly higher level (53%) of similarity to the PUFA
elongase than F56H11.3 (see Fig. 10). However, when a PCR product encoding ORF F41H10.8 was expressed in yeast in a manner identical to that used for F56H11.4, the former failed to direct the elongation of any fatty acids, despite the provision of a range of substrates (see Table II).
In order to reconstitute the PUFA biosynthetic pathway in a heterologous system, the PLTFA elongase F56H11.4 was expressed in yeast in conjunction with either the 06- or OS-fatty acid desaturases previously isolated and characterised by the inventor (Napier, J.
A., supra; Michaelson, L. V., supra). Expression of the OG-fatty acid desaturase and F56H11.4 was carried out in the presence of two different substrates (LA or ALA) while the 4'-fatty acid desaturase and the elongase were expressed in the presence of GLA only.
This demonstrated that was possible to combine a desaturase and an elongase in yeast to generate significant amounts of a final "product" (see Table III). In the case of the elongase and the 4G-fatty acid desaturase, the reactions proved highly efficient with the production of 4.5% of DHGLA from the LA substrate. This resulted from 25%
desaturation of the LA substrate to GLA, which was then elongated to DHGLA at a similar level of efficiency (18%). This is lower than the % conversion observed for GLA when supplied exogenously (see Table I), indicating that the in vivo production of substrates for elongation may be rate-limiting.

If ALA was used as a substrate, 27% of this was initially O6-desaturated to yield octadecatetraenoic acid (OTA; 18:4 06.9,'z,'s) but only 8% of was subsequently elongated to yield eicosatetraenoic acid (20:4 ~8~"~'4~"). Thus, the conversion efficiency of ALA to the final 20-carbon tetraenoic PUFA was only about 2.2%.
Since DHGLA is an n-6 fatty acid, whilst the OTA-derived eicostetraenoic acid is an n-3 type, this demonstrates that the elongase is capable of accepting both forms of essential fatty acid, albeit with different efficiencies. Verification was also provided that the 20C
PUFAs synthesised in the yeast expression system were generated by the O6-desaturation of 18C substrates which were subsequently elongated, as the O6-desaturase showed no activity on 20:2 or 20:3 substrates (see Table IIII).
The combination of the D5-desaturase and the elongase also demonstrated that these two enzymes could work in tandem, although the efficiency of this overall conversion was lower (3.3% AA from GLA) which was due to the previously observed low activity of the OS-desaturase enzyme itself (Michaelson, L. V., supra; Michaelson, L. V., supra). Thus, although nearly 45% of the GLA substrate was elongated to DHGLA, only 7.5% of this was then desaturated to AA (see Table III).
Finally, the production of either AA or eicosapentanoic acid (EPA;
20:5~5~8~"~'4~") in yeast from dienoic or trienoic 18 carbon substrates was achieved via expression of all three enzymes (the two desaturases and the F56H11.4 PUFA elongase) simultaneously.
As shown in Table IV, small but significant amounts of AA were produced when the yeast was supplied with the 18C dienoic fatty acid LA.
GC-Mass Spectroscopy (MS) Analysis Peak identification and confirmation were carried out by GC-MS using a Kratos using known standards (Sigma). The identity of this 20C PLTFA was verified by GCMS, indicating that the conversion efficiency from LA was 0.65%. When ALA was used as a substrate, 12.5% of the (46-desaturated and elongated) eicosatetraenoic n-3 fatty acid was OS-desaturated, resulting in a total conversion of 0.3% of the ALA substrate to EPA (the identity of EPA was confirmed by GCMS).

Expression of C. elegans elongase in plants In order to express C. elegans elongase in plants, the following protocol is an example of a process which can be used to create the transgenic plants. C. elegans ORF
sequence can be subcloned into a plant expression vector pJD330, which comprises a viral 35S
promoter, and a Nos terminator. The resulting cassette or promoter/coding sequence/terminator can then be subcloned into the plant binary transformation vector pain 19, and the resulting plasmid introduced into Agrobacterium tumefaciens. This Agrobacterium strain can then be used to transform Arabidopsis by the vacuum-infiltration of inflorescences, and the seeds harvested and plated onto selective media containing kanamycin. Since pain 19 confers resistance to this antibotic, only transformed plant material will grow.
Resistant lines can therefore be identified and self fertilized to produce homozygous material.
Leaf material can then be analyzed for expression of C. elegans elongase.
Fatty acid methyl ester analysis can be carried out as previously described.

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MPQGEVSFFE VLTTAPFSHE LSKKHIAQTQ YAAFWISMAY VWIFGLKAV
MTNRKPFDLT GPLNLWNAGL AIFSTLGSLA TTFGLLHEFF SRGFFESYIH
IGDFYNGLSG MFTWLFVLSK VAEFGDTLFI ILRKKPLMFL HWYHHVLTMN
YAFMSFEANL GFNTWITWMN FSVHSIMYGY YMLRSFGVKV PAWIAKNITT
MQILQFVITH FILFHVGYLA VTGQSVDSTP GYYWFCLLME ISYWLFGNF
YYQSYIKGGG KKFNAEKKTE KKIE*

F

251 LMYYSLATNQ ARYPSNTPAT LQCLSYTLHL L*

G
MAQHPLVQRL LDVKFDTKRF VAIATHGPKN FPDAEGRKFF ADHFDVTIQA
SILYMVVVFG TKWFMRNRQP FQLTIPLNIW NFILAAFSIA GAVKMTPEFF
GTIANKGIVA SYCKVFDFTK GENGYWVWLF MASKLFELVD TIFLVLRKRP
LMFLHV~IYHHI LTMIYAWYSH PLTPGFNRYG IYLNFVVHAF MYSYYFLRSM
KIRVPGFIAQ AITSLQIVQF IISCAVLAHL GYLMHFTNAN CDFEPSVFKL
AVFMDTTYLA LFVNFFLQSY VLRGGKDKYK AVPKKKNN*

H
MSAEVSERFKVWTGNNETIIYSPFEYDSTLLIESCRCTYQLLILLRQI
YYRDIWSHGNLKACDXLLLAWNGFLAVFSIMGTWRFGIEFYDAVFRXG
FIXSICLAVNPRSPSAFWACMFALSKIAEFGDTMFLVLRKRPVIFLHWYHH
AVVLILSWHAAIELTAPGRWFIFMNYLVHSIMYTYYAITSIGYRXPKIVSMT
VTFLQTLQMLIGVSISCIVLYLKLNGEMCQQSYDNLALSFGIYASFLVLSSFF
NNAYLVKKDKKPDVKKD*

ELONGASE.APP.txt SEQUENCE LISTING
<110> the university of Bristol <120> POLYUNSATURATED FATTY ACID (PUFA) ELONGASE FROM CAENORHABDITIS ELEGANS
<130> >1470003 <140> PCT/GB00/01035 <141> March 20, 2000 <160> 22 <170> Patentln Ver. 2.1 <210> 1 <211> 27 <212> DNA
<213> C. elegans <400> 1 gcgggtacca tggctcagca tccgctc 27 <210> 2 <211> 27 <212> DNA
<213> C. elegans <400> 2 gcgggatcct tagttgttct tcttctt 27 <210> 3 <211> 27 <212> DNA
<213> C. elegans <400> 3 gcgggtacca tgccacaggg agaagtc 27 <210> 4 <211> 27 <212> DNA
<213> c. elegans <400> 4 gcgggatcct tattcaattt ttctttt 27 <210> 5 <211> 29 <212> DNA
<213> C. elegans <400> 5 gcgaattcac catgggtacg gaccaagga 2g <210> 6 <211> 27 <212> DNA

ELONGASE.APP.tXt <213> C. ~legans <400> 6 gcggagctcc tactcttcct tgggacg 27 <210> 7 <211> 876 <212> DNA
<213> c. elegans <400> 7 atggagcttg ccgagttctg gaatgatctc aacaccttca ccatctacgg accgaatcac 60 acagatatga ccacaaaata caaatattca tatcacttcc caggtgaaca ggtggcggat 120 ccgcagtatt ggacgatttt attccagaaa tattggtatc attcgatcac aatatcagtt 180 ctttatttca ttttaattaa ggtgattcaa aagtttatgg agaatcgaaa accattcact 240 ttgaaatacc cattgattct ttggaatgga gctcttgcag cattcagtat aattgccaca 300 ttgcggttct ctattgatcc tctacgatca ctatatgctg aaggattcta caaaactctg 360 tgctattcgt gtaatccaac tgatgtggct gcattttgga gctttgcatt cgctctttcc 420 aagattgttg aacttggaga cactatgttc attattttga gaaaacggcc attgatcttt 480 ttacactact atcatcatgc agcagtgtta atctacactg tccattctgg tgccgagcat 540 actgcagctg gtcgtttcta catcctaatg aactacttcg cacattctct catgtatact 600 tactacacag tttctgccat gggatacaga ttaccgaaat gggtatcaat gactgtcaca 660 actgttcaaa caactcaaat gttagctgga gtcggaataa cttggatggt gtacaaagtg 720 aaaactgaat acaagcttcc ttgtcaacaa tccgtagcca atttgtatct cgcattcgtc 780 atctatgtca catttgccat tcttttcatt caattcttcg tcaaggcata cattatcaag 840 tcgtcgaaga agtcgaaatc ggtgaagaac gaataa <210> 8 <211> 1308 <212> DNA
<213> c. elegans <400> 8 atggcaaaat acgactacaa tccgaagtat gggttagaaa attacagcat attccttccc 60 tttgagacat cttttgatgc atttcgatcg acaacatgga tgcaaaatca ctggtatcaa 120 tcaattacag catctgtcgt gtatgtagcc gtcattttta caggaaagaa ggtggttctc 180 atctacaaaa aatcacgagt tattactttt gagtctagcc ttcagaatgc aattaagaat 240 cgaaaccgaa aatcacttaa tagttctcaa atgtttcaga ttatggaaaa gtacaagccc 300 ttccaactgg acacaccact cttcgtctgg aattcatttt tagccatttt ctcaattctc 360 gggttcctcc gaatgacacc tgaatttgta tggagttggt cagcagaagg aaactcattc 420 aaatattcaa tttgtcattc atcttatgct caaggagtca ctggtttctg gactgaacaa 480 ttcgcaatga gcaaactttt cgagctcatc gacacaatct tcatcgttct tcgtaaacgt 540 ccactcatct tccttcactg gtatcatcat gtaactgtta tgatctacac atggcacgcg 600 tacaaggatc acactgcatc aggacggtgg ttcatttgga tgaattatgg agttcatgct 660 cttatgtatt cctactatgc tcttcgttct ctgaaattcc gtcttccaaa acaaatggca 720 atggttgtta ctactctcca acttgctcaa atggttatgg gagtaatcat cggagtcact 780 gtctaccgta tcaagtcatc gggtgaatac tgccaacaga catgggacaa tttgggatta 840 tgctttggag tttatttcac atatttcctt cttttcgcca acttcttcta ccatgcatat 900 gttaagaaaa acaaccgtac agtaaattat gaaaataatt caaaaaattt ccccgatctc 960 gttttaattt acctgagaaa aaaggtttca agaaaatcga aaaatcggca atgttcagaa 1020 aataattata aaattcaatt ttcatcaaat tttgttaatg ttgatggaaa aaaacataag 1080 aaaacatatg aacttattct tccaagaaga aaaatgacca caattttaac ttttctattt 1140 ggaaaaaatc gaattttttc gaaatatcag aaaaatcgaa aaaacatttc gattcctgtt 1200 gatttcgaaa ttctggagcc aaaagaagat atcaatgcta acatcgctga gccatccatc 1260 acaacgaggt ccgccgccgc acgaagaaaa gttcaaaaag ctgattag 1308 <210> 9 <211> 825 <212> DNA
<213> c. elegans ELONGASE.APP.txt <400> 9 atggcagcag cacaaacaag tccagcagcc acgctcgtcg atgttttgac aaaaccatgg 60 agtctggatc agactgattc ttacatgtct acatttgtac cattatccta taaaatcatg 120 attggttatc tcgtcaccat ctacttcggg caaaaattaa tggctcacag aaaaccattc 180 gatctccaaa atacacttgc tctctggaac ttcgggtttt cactgttctc gggaatcgcc 240 gcctataagc ttattccaga actattcgga gttttcatga aggacgggtt tgtcgcttcc 300 tactgtcaaa acgagaacta ctacaccgat gcatcaactg gattctgggg ctgggccttt 360 gtgatgtcga aagctccaga actaggggat actatgttct tggtccttcg taaaaaacca 420 gttatcttca tgcactggta tcatcatgcc ctcacatttg tctacgcagt agtcacatac 480 tctgagcatc aggcatgggc tcgttggtct ttggctctca accttgccgt ccacactgtt 540 atgtatttct acttcgccgt tcgcgccttg aacatccaaa ctccacgccc agtggcaaag 600 ttcatcacta ctattcaaat tgtccaattt gtcatctcat gctacatttt tgggcatttg 660 gtattcatta agtctgctga ttctgttcct ggttgcgctg ttagctggaa tgtgctatcg 720 atcggaggac tcatgtacat cagttatttg ttcctttttg ccaagttctt ctacaaggcc 780 tacattcaaa aacgctcacc aaccaaaacc agcaagcagg agtag g25 <210> 10 <211> 861 <212> DNA
<213> C. elegans <400> 10 atgtcatcgg acgatcgtgg cactagaacc ttcaagatga tggatcaaat tcttggaaca 60 aacttcactt atgaaggtgc caaagaagtt gctcgaggcc ttgaaggttt ctcagcaaag 120 cttgccgtcg gatatattgc cactattttt ggactgaaat attatatgaa agaccgaaaa 180 gccttcgatc tcagtactcc attaaacatt tggaatggta ttctttcgac attcagctta 240 ttgggattct tattcacttt tcctactttg ttatcagtta tcagaaagga tggatttagt 300 cacacctatt cccatgtctc tgagctttac actgacagta cctctggata ttggatcttc 360 ctttgggtta tctcaaagat tccggaactt ttggatacag tattcattgt tcttcgcaag 420 agaccactta ttttcatgca ctggtaccat cacgcattga ccggttacta tgctcttgtc 480 tgctaccatg aggatgctgt ccatatggtt tgggttgtat ggatgaatta tattattcat 540 gcattcatgt atggatacta tcttctgaaa tctctgaaag ttccaattcc accatcagtt 600 gctcaagcaa tcaccacatc tcaaatggtt caattcgcag ttgccatttt cgcacaagtt 660 catgtttcct ataaacacta tgttgaggga gttgaaggat tagcctactc gttcagagga 720 acagctatcg gatttttcat gcttactacc tacttctatc tatggattca attctacaaa 780 gagcactatc ttaagaatgg aggcaaaaag tacaatttgg caaaggatca ggcaaaaact 840 caaacaaaga aggctaacta a 861 <210> 11 <211> 825 <212> DNA
<213> C. elegans <400> 11 atgccacagg gagaagtctc attctttgag gtgctgacaa ctgctccatt cagtcatgag 60 ctctcaaaaa agcatattgc acagactcag tatgctgctt tctggatctc aatggcatat 120 gttgtcgtta tttttgggct caaggctgtc atgacaaacc gaaaaccatt tgatctcacg 180 ggaccactga atctctggaa tgcgggtctt gctattttct caactctcgg atcacttgcc 240 actacatttg gacttctcca cgagttcttc agccgtggat ttttcgaatc ttacattcac 300 atcggagact tttataatgg actttctgga atgttcacat ggcttttcgt tctctcaaaa 360 gttgctgaat tcggagatac actttttatt attcttcgta aaaagccatt gatgttcctt 420 cattggtatc atcatgtgct tacaatgaat tatgctttta tgtcatttga agctaatttg 480 ggatttaata cttggattac atggatgaat ttctcagttc actcaattat gtatggatat 540 tatatgcttc gttcttttgg tgtcaaggtt ccagcatgga ttgccaagaa tattacaaca 600 atgcaaattc ttcaattcgt tattactcat ttcattcttt tccacgttgg atatttggca 660 gttactggac aatctgttga ctcaactcca ggatattatt ggttctgcct tctcatggaa 720 atctcttatg tcgttctgtt cggaaacttc tactatcaat catacatcaa gggaggtggc 780 aagaagttta atgcagagaa gaagactgaa aagaaaattg aataa 825 ELONGASE.APP.tXt <210> 12 <211> 846 <212> DNA
<213> C. elegans <400> 12 atgtatttga attatttcgc gacggaaatc ttccatcgta gtgcggtttg tgaaacagaa 60 gcttgtcgct cgtcaaaaat aatgattgct gacgtgttca aatggaaatt cgatgcaaac 120 gaattgtgga gtcttttaac gaatcaggat gaagttttcc cgcatattag agcacggcga 180 ttcattcaag aacattttgg tctattcgtc cagatggcaa ttgcatatgt cattttggtg 240 ttctcaatca aaaggttcat gagggatcgt gaaccatttc aactcaccac agctcttcgt 300 ctctggaact tcttcctctc cgtcttctca atttatggtt cctggacaat gtttccattt 360 atggttcaac aaataagact ttatggtctc tacggatgtg gatgcgaagc actttcaaac 420 cttccgagtc aagcagaata ttggcttttc ctgacgatct tgtccaaagc tgtggagttt 480 gttgatacat ttttcttggt tctccggaaa aaaccactca tcttcctaca ctggtatcat 540 catatggcaa catttgtctt cttctgcagt aattacccga ctccatcgtc acaatcacgc 600 gtcggagtta tcgtcaacct gttcgtgcat gccttcatgt acccatacta tttcacccga 660 tcaatgaaca tcaaagttcc tgcgaaaatt tcaatggctg ttacagttct tcaattgact 720 caattcatgt gctttatcta tggatgtact ctcatgtact actcgttggc cactaatcag 780 gcacgatacc cctcaaatac acctgcgaca ctccaatgtt tgtcctacac tctacatttg 840 ctttga 846 <210> 13 <211> 866 <212> DNA
<213> C. elegans <400> 13 atggctcagc atccgctcgt tcaacggctt ctcgatgtca aattcgacac gaaacgattt 60 gtggctattg ctactcatgg gccaaagaat ttccctgacg cagaaggtcg caagttcttt 120 gctgatcact ttgatgttac tattcaggct tcaatcctgt acatggtcgt tgtgttcgga 180 acaaaatggt tcatgcgtaa tcgtcaacca ttccaattga ctattccact caacatctgg 240 aatttcatcc tcgccgcatt ttccatcgca ggagctgtca aaatgacccc agagttcttt 300 ggaaccattg ccaacaaagg aattgtcgat cctactgcaa agtgtttgat ttcacgaaag 360 gagagaatgg atactgggtg tggctcttca tggcttccaa acttttcgaa cttgttgaca 420 ccatcttctt ggttctccgt aaacgtccac tcatgttcct tcactggtat caccatattc 480 tcaccatgat ctacgcctgg tactctcatc cattgacccc aggattcaac agatacggaa 540 tttatcttaa ctttgtcgtc cacgccttca tgtactctta ctacttcctt cgctcgatga 600 agattcgcgt gccaggattc atcgcccaag ctatcacatc tcttcaaatc gttcaattca 660 tcatctcttg cgccgttctt gctcatcttg gttatctcat gcacttcacc aatgccaact 720 gtgatttcga gccatcagta ttcaagctcg cagttttcat ggacacaaca tacttggctc 780 ttttcgtcaa cttcttcctc caatcatatg ttctccgcgg aggaaaagac aagtacaagg 840 cagtgccaaa gaagaagaac aactaa 866 <210> 14 <211> 801 <212> DNA
<213> C. elegans <400> 14 atgtcggccg aagtgtccga acgattcaaa gtttggacag gaaacaatga gaccatcatc 60 tattccccat tcgagtacga ttccacgttg ctcatcgagt catgtcggtg tacttatcag 120 ctgcttatat tattgcgaca aatttattac agagatatat ggagtcacgg aaacctaaaa 180 cttttactag catggaacgg ttttttggca gtgttcagta ttatgggtac atggagattt 240 ggaatcgaat tctacgatgc tgttttcaga agaggcttca tcgattcgat ctgcctggct 300 gtaaatccac gttcaccgtc cgcattctgg gcatgcatgt tcgctctatc gaaaatcgcc 360 gagtttgggg acacgatgtt cttggtgctg aggaaacggc cggttatatt ccttcactgg 420 tatcatcacg ctgttgttct gatcctttct tggcatgctg caatcgaact cacagctcca 480 ggacgctggt ttatttttat gaactatttg gtgcattcaa taatgtatac atactacgca 540 ataacatcaa tcggctatcg tcttcccaaa atcgtttcaa tgactgttac attccttcaa 600 actcttcaaa tgctcattgg tgtcagcatt tcttgcattg tgctttattt gaagcttaat 660 ELONGASE.APP.tXt ggagagatgt gccaacaatc ctacgacaat ctggcgttga gcttcggaat ctacgcctca 720 ttcctggtgc tattctccag tttcttcaac aatgcatatt tggtaaaaaa ggacaagaaa 780 cccgatgtga agaaggatta a <210>

<211>

<212>
PRT

<213> elegans C.

<400>

MetGlu LeuAla GluPheTrpAsn AspLeuAsn ThrPheThr IleTyr GlyPro AsnHis ThrAspMetThr ThrLysTyr LysTyrSer TyrHis PhePro GlyGlu GlnValAlaAsp ProGlnTyr TrpThrIle LeuPhe GlnLys TyrTrp TyrHisSerIle ThrIleSer ValLeuTyr PheIle LeuIle LysVal IleGlnLysPhe MetGluAsn ArgLysPro PheThr LeuLys TyrPro LeuIleLeuTrp AsnGlyAla LeuAlaAla PheSer IleIle AlaThr LeuArgPheSer IleAspPro LeuArgSer LeuTyr AlaGlu GlyPhe TyrLysThrLeu CysTyrSer CysAsnPro ThrAsp ValAla AlaPhe TrpSerPheAla PheAlaLeu SerLysIle ValGlu LeuGly AspThr MetPheIleIle LeuArgLys ArgProLeu IlePhe LeuHis TyrTyr HisHisAlaAla ValLeuIle TyrThrVal HisSer GlyAla GluHis ThrAlaAlaGly ArgPheTyr IleLeuMet AsnTyr PheAla HisSer LeuMetTyrThr TyrTyrThr ValSerAla MetGly TyrArg LeuPro LysTrpValSer MetThrVal ThrThrVal GlnThr ThrGln MetLeu AlaGlyValGly IleThrTrp MetValTyr LysVal LysThr GluTyr LysLeuProCys GlnGlnSer ValAlaAsn LeuTyr LeuAla PheVal IleTyrValThr PheAlaIle LeuPheIle GlnPhe PheVal LysAla TyrIleIleLys SerSerLys LysSerLys SerVal ELONGASE.APP.tXt ' 2?5 280 285 Lys Asn Glu <210>

<211>

<212>
PRT

<213> elegan s c.

<400>

MetAla LysTyrAsp TyrAsnPro LysTyrGly LeuGluAsn TyrSer IlePhe LeuProPhe GluThrSer PheAspAla PheArgSer ThrThr TrpMet GlnAsnHis TrpTyrGln SerIleThr AlaSerVal ValTyr ValAla ValIlePhe ThrGlyLys LysValVal LeuIleTyr LysLys SerArg ValIleThr PheGluSer SerLeuGln AsnAlaIle LysAsn ArgAsn ArgLysSer LeuAsnSer SerGlnMet PheGlnIle MetGlu LysTyr LysProPhe GlnLeuAsp ThrProLeu PheValTrp AsnSer PheLeu AlaIlePhe SerIleLeu GlyPheLeu ArgMetThr ProGlu PheVal TrpSerTrp SerAlaGlu GlyAsnSer PheLysTyr SerIle cysHis SerSerTyr AlaGlnGly ValThrGly PheTrpThr GluGln PheAla MetSerLys LeuPheGlu LeuIleAsp ThrIlePhe IleVal LeuArg LysArgPro LeuIlePhe LeuHisTrp TyrHisHis ValThr ValMet IleTyrThr TrpHisAla TyrLysAsp HisThrAla SerGly ArgTrp PheIleTrp MetAsnTyr GlyValHis AlaLeuMet TyrSer TyrTyr AlaLeuArg SerLeuLys PheArgLeu ProLysGln MetAla MetVal ValThrThr LeuGlnLeu AlaGlnMet ValMetGly ValIle IleGly ValThrVal TyrArgIle LysSerSer GlyGluTyr cysGln S
ELONGASE.APP.tXt Gln Thr Trp Asp Asn Leu Gly Leu Cys Phe Gly Val Tyr Phe Thr Tyr Phe Leu Leu Phe Ala Asn Phe Phe Tyr His Ala Tyr Val Lys Lys Asn Asn Arg Thr Val Asn Tyr Glu Asn Asn Ser Lys Asn Phe Pro Asp Leu Val Leu Ile Tyr Leu Arg Lys Lys Val Ser Arg Lys Ser Lys Asn Arg Gln Cys Ser Glu Asn Asn Tyr Lys Ile Gln Phe Ser Ser Asn Phe Val Asn Val Asp Gly Lys Lys His Lys Lys Thr Tyr Glu Leu Ile Leu Pro Arg Arg Lys Met Thr Thr Ile Leu Thr Phe Leu Phe Gly Lys Asn Arg Ile Phe Ser Lys Tyr Gln Ly5 Asn Arg Lys Asn Ile Ser Ile Pro Val Asp Phe Glu Ile Leu Glu Pro Lys Glu Asp Ile Asn Ala Asn Ile Ala Glu Pro Ser Ile Thr Thr Arg Ser Ala Ala Ala Arg Arg Lys Val Gln Lys Ala Asp <210> 17 <211> 274 <212> PRT
<213> C. elegans <400> 17 Met Ala Ala Ala Gln Thr Ser Pro Ala Ala Thr Leu Val Asp Val Leu Thr Lys Pro Trp Ser Leu Asp Gln Thr Asp Ser Tyr Met Ser Thr Phe Val Pro Leu Ser Tyr Lys Ile Met Ile Gly Tyr Leu Val Thr Ile Tyr Phe Gly Gln Lys Leu Met Ala His Arg Lys Pro Phe Asp Leu Gln Asn Thr Leu Ala Leu Trp Asn Phe Gly Phe Ser Leu Phe Ser Gly Ile Ala Ala Tyr Lys Leu Ile Pro Glu Leu Phe Gly Val Phe Met Lys Asp Gly Phe Val Ala Ser Tyr Cys Gln Asn Glu Asn Tyr Tyr Thr Asp Ala Ser ELONGASE.APP.txt Thr Gly Phe Trp Gly Trp Ala Phe Val Met Ser Lys Ala Pro Glu Leu Gly Asp Thr Met Phe Leu Val Leu Arg Lys Lys Pro Val Ile Phe Met His Trp Tyr His His Ala Leu Thr Phe Val Tyr Ala Val Val Thr Tyr Ser Glu His Gln Ala Trp Ala Arg Trp Ser Leu Ala Leu Asn Leu Ala Val His Thr Val Met Tyr Phe Tyr Phe Ala Val Arg Ala Leu Asn Ile Gln Thr Pro Arg Pro Val Ala Lys Phe Ile Thr Thr Ile Gln Ile Val Gln Phe Val Ile Ser Cys Tyr Ile Phe Gly His Leu Val Phe Ile Lys Ser Ala Asp Ser Val Pro Gly Cys Ala Val Ser Trp Asn Val Leu Ser Ile Gly Gly Leu Met Tyr Ile Ser Tyr Leu Phe Leu Phe Ala Lys Phe Phe Tyr Lys Ala Tyr Ile Gln Lys Arg Ser Pro Thr Lys Thr Ser Lys Gln Glu <210> 18 <211> 286 <212> PRT
<213> C. elegans <400> 18 Met Ser Ser Asp Asp Arg Gly Thr Arg Thr Phe Lys Met Met Asp Gln Ile Leu Gly Thr Asn Phe Thr Tyr Glu Gly Ala Lys Glu Val Ala Arg Gly Leu Glu Gly Phe Ser Ala Lys Leu Ala Val Gly Tyr Ile Ala Thr Ile Phe Gly Leu Lys Tyr Tyr Met Lys Asp Arg Lys Ala Phe Asp Leu Ser Thr Pro Leu Asn Ile Trp Asn Gly Ile Leu Ser Thr Phe Ser Leu Leu Gly Phe Leu Phe Thr Phe Pro Thr Leu Leu Ser Val Ile Arg Lys Asp Gly Phe Ser His Thr Tyr Ser His Val Ser Glu Leu Tyr Thr Asp Ser Thr Ser Gly Tyr Trp Ile Phe Leu Trp Val Ile Ser Lys Ile Pro ELONGASE.APP.tXt ' lI5 120 125 Glu Leu Leu Asp Thr Val Phe Ile Val Leu Arg Lys Arg Pro Leu Ile Phe Met His Trp Tyr His His Ala Leu Thr Gly Tyr Tyr Ala Leu Val Cys Tyr His Glu Asp Ala Val His Met Val Trp Val Val Trp Met Asn Tyr Ile Ile His Ala Phe Met Tyr Gly Tyr Tyr Leu Leu Lys Ser Leu Lys Val Pro Ile Pro Pro Ser Val Ala Gln Ala Ile Thr Thr Ser Gln Met Val Gln Phe Ala Val Ala Ile Phe Ala Gln Val His Val Ser Tyr Lys His Tyr Val Glu Gly Val Glu Gly Leu Ala Tyr Ser Phe Arg Gly Thr Ala Ile Gly Phe Phe Met Leu Thr Thr Tyr Phe Tyr Leu Trp Ile Gln Phe Tyr Lys Glu His Tyr Leu Lys Asn Gly Gly Lys Lys Tyr Asn Leu Ala Lys Asp Gln Ala Lys Thr Gln Thr Lys Lys Ala Asn <210> 19 <211> 274 <212> PRT
<213> C. elegans <400> 19 Met Pro Gln Gly Glu Val Ser Phe Phe Glu Val Leu Thr Thr Ala Pro Phe Ser His Glu Leu Ser Lys Lys His Ile Ala Gln Thr Gln Tyr Ala Ala Phe Trp Ile Ser Met Ala Tyr Val Val Val Ile Phe Gly Leu Lys Ala Val Met Thr Asn Arg Lys Pro Phe Asp Leu Thr Gly Pro Leu Asn Leu Trp Asn Ala Gly Leu Ala Ile Phe Ser Thr Leu Gly Ser Leu Ala Thr Thr Phe Gly Leu Leu His Glu Phe Phe Ser Arg Gly Phe Phe Glu Ser Tyr Ile His Ile Gly Asp Phe Tyr Asn Gly Leu Ser Gly Met Phe Thr Trp Leu Phe Val Leu Ser Lys Val Ala Glu Phe Gly Asp Thr Leu ELONGASE.APP.tXt Phe Ile Ile Leu Arg Lys Lys Pro Leu Met Phe Leu His Trp Tyr His His Val Leu Thr Met Asn Tyr Ala Phe Met Ser Phe Glu Ala Asn Leu Gly Phe Asn Thr Trp Ile Thr Trp Met Asn Phe Ser Val His Ser Ile Met Tyr Gly Tyr Tyr Met Leu Arg Ser Phe Gly Val Lys Val Pro Ala Trp Ile Ala Lys Asn Ile Thr Thr Met Gln Ile Leu Gln Phe Val Ile Thr His Phe Ile Leu Phe His Val Gly Tyr Leu Ala Val Thr Gly Gln Ser Val Asp Ser Thr Pro Gly Tyr Tyr Trp Phe Cys Leu Leu Met Glu Ile Ser Tyr Val Val Leu Phe Gly Asn Phe Tyr Tyr Gln Ser Tyr Ile Lys Gly Gly Gly Lys Lys Phe Asn Ala Glu Lys Lys Thr Glu Lys Lys Ile Glu <210> 20 <211> 281 <212> PRT
<213> C. elegans <400> 20 Met Tyr Leu Asn Tyr Phe Ala Thr Glu Ile Phe His Arg Ser Ala Val Cys Glu Thr Glu Ala Cys Arg Ser Ser Lys Ile Met Ile Ala Asp Val Phe Lys Trp Lys Phe Asp Ala Asn Glu Leu Trp Ser Leu Leu Thr Asn Gln Asp Glu Val Phe Pro His Ile Arg Ala Arg Arg Phe Ile Gln Glu His Phe Gly Leu Phe Val Gln Met Ala Ile Ala Tyr Val Ile Leu Val Phe Ser Ile Lys Arg Phe Met Arg Asp Arg Glu Pro Phe Gln Leu Thr Thr Ala Leu Arg Leu Trp Asn Phe Phe Leu Ser Val Phe Ser Ile Tyr Gly Ser Trp Thr Met Phe Pro Phe Met Val Gln Gln Ile Arg Leu Tyr ELONGASE.APP.tXt Gly Leu Tyr Gly Cys Gly Cys Glu Ala Leu Ser Asn Leu Pro Ser Gln Ala Glu Tyr Trp Leu Phe Leu Thr Ile Leu Ser Lys Ala Val Glu Phe Val Asp Thr Phe Phe Leu Val Leu Arg Lys Lys Pro Leu Ile Phe Leu His Trp Tyr His His Met Ala Thr Phe Val Phe Phe Cys Ser Asn Tyr Pro Thr Pro Ser Ser Gln Ser Arg Val Gly Val Ile Val Asn Leu Phe Val His Ala Phe Met Tyr Pro Tyr Tyr Phe Thr Arg Ser Met Asn Ile Lys Val Pro Ala Lys Ile Ser Met Ala Val Thr Val Leu Gln Leu Thr Gln Phe Met Cys Phe Ile Tyr Gly Cys Thr Leu Met Tyr Tyr Ser Leu Ala Thr Asn Gln Ala Arg Tyr Pro Ser Asn Thr Pro Ala Thr Leu Gln Cys Leu Ser Tyr Thr Leu His Leu Leu <210> 21 <211> 288 <212> PRT
<213> C. elegans <400> 21 Met Ala Gln His Pro Leu Val Gln Arg Leu Leu Asp Val Lys Phe Asp Thr Lys Arg Phe Val Ala Ile Ala Thr His Gly Pro Lys Asn Phe Pro Asp Ala Glu Gly Arg Lys Phe Phe Ala Asp His Phe Asp Val Thr Ile Gln Ala Ser Ile Leu Tyr Met Val Val Val Phe Gly Thr Lys Trp Phe Met Arg Asn Arg Gln Pro Phe Gln Leu Thr Ile Pro Leu Asn Ile Trp Asn Phe Ile Leu Ala Ala Phe Ser Ile Ala Gly Ala Val Lys Met Thr Pro Glu Phe Phe Gly Thr Ile Ala Asn Lys Gly Ile Val Ala Ser Tyr Cys Lys Val Phe Asp Phe Thr Lys Gly Glu Asn Gly Tyr Trp Val Trp Leu Phe Met Ala Ser Lys Leu Phe Glu Leu Val Asp Thr Ile Phe Leu ELONGASE.APP.tXt 130 ' 135 140 Val Leu Arg Lys Arg Pro Leu Met Phe Leu His Trp Tyr His His Ile Leu Thr Met Ile Tyr Ala Trp Tyr Ser His Pro Leu Thr Pro Gly Phe Asn Arg Tyr Gly Ile Tyr Leu Asn Phe Val Val His Ala Phe Met Tyr Ser Tyr Tyr Phe Leu Arg Ser Met Lys Ile Arg Val Pro Gly Phe Ile Ala Gln Ala Ile Thr Ser Leu Gln Ile Val Gln Phe Ile Ile Ser Cys Ala Val Leu Ala His Leu Gly Tyr Leu Met His Phe Thr Asn Ala Asn Cys Asp Phe Glu Pro Ser Val Phe Lys Leu Ala Val Phe Met Asp Thr Thr Tyr Leu Ala Leu Phe Val Asn Phe Phe Leu Gln Ser Tyr Val Leu Arg Gly Gly Lys Asp Lys Tyr Lys Ala Val Pro Lys Lys Lys Asn Asn <210> 22 <211> 269 <212> PRT
<213> C. elegans <400> 22 Met Ser Ala Glu Val Ser Glu Arg Phe Lys Val Trp Thr Gly Asn Asn Glu Thr Ile Ile Tyr Ser Pro Phe Glu Tyr Asp Ser Thr Leu Leu Ile Glu Ser Cys Arg Cys Thr Tyr Gln Leu Leu Ile Leu Leu Arg Gln Ile Tyr Tyr Arg Asp Ile Trp Ser His Gly Asn Leu Lys Ala Cys Asp Xaa Leu Leu Leu Ala Trp Asn Gly Phe Leu Ala Val Phe Ser Ile Met Gly Thr Trp Arg Phe Gly Ile Glu Phe Tyr Asp Ala Val Phe Arg Xaa Gly Phe Ile Xaa Ser Ile Cys Leu Ala Val Asn Pro Arg Ser Pro Ser Ala Phe Trp Ala Cys Met Phe Ala Leu Ser Lys Ile Ala Glu Phe Gly Asp ELONGASE.APP.tXt Thr Met PheLeu ValLeuArg LysArgProVal IlePheLeu HisTrp Tyr His HisAla ValValLeu IleLeuSerTrp HisAlaAla IleGlu Leu Thr AlaPro GlyArgTrp PheIlePheMet AsnTyrLeu ValHis Ser Ile MetTyr ThrTyrTyr AlaIleThrSer IleGlyTyr ArgXaa Pro Lys IleVal SerMetThr ValThrPheLeu GlnThrLeu GlnMet Leu Ile GlyVal SerIleSer CysIleValLeu TyrLeuLys LeuAsn Gly Glu MetCys GlnGlnSer TyrAspAsnLeu AlaLeuSer PheGly Ile Tyr AlaSer PheLeuVal LeuSerSerPhe PheAsnAsn AlaTyr Leu Val LysLys AspLysLys ProAspValLys LysAsp gcgggtacca~ggctcagcatccgctco PRIMER1.GBS.tXt PRIMER2.GBS.tXt gcgggatcct~agttgttcttcttctto PRIMER3.GBS.tXt gcgggtaccatgccacagggagaagtco PRIMER4.GBS.tXt gcgggatccttattcaatttttctttto PRIMERS.GBS.tXt gcgaattcaccatgggtacggaccaagga~

PRIMER6.GBS.tXt gcggagctcctactcttccttgggacgo SEQID1.GBS.tXt atggagcttgccgagttctggaatgatctcaacaccttcaccatctacggaccgaatcac acagatatgaccacaaaatacaaatattcatatcacttcccaggtgaacaggtggcggat ccgcagtattggacgattttattccagaaatattggtatcattcgatcacaatatcagtt ctttatttcattttaattaaggtgattcaaaagtttatggagaatcgaaaaccattcact ttgaaatacccattgattctttggaatggagctcttgcagcattcagtataattgccaca ttgcggttctctattgatcctctacgatcactatatgctgaaggattctacaaaactctg tgctattcgtgtaatccaactgatgtggctgcattttggagctttgcattcgctctttcc aagattgttgaacttggagacactatgttcattattttgagaaaacggccattgatcttt ttacactactatcatcatgcagcagtgttaatctacactgtccattctggtgccgagcat actgcagctggtcgtttctacatcctaatgaactacttcgcacattctctcatgtatact tactacacagtttctgccatgggatacagattaccgaaatgggtatcaatgactgtcaca actgttcaaacaactcaaatgttagctggagtcggaataacttggatggtgtacaaagtg aaaactgaatacaagcttccttgtcaacaatccgtagccaatttgtatctcgcattcgtc atctatgtcacatttgccattcttttcattcaattcttcgtcaaggcatacattatcaag tcgtcgaagaagtcgaaatcggtgaagaacgaataao SEQID2.GBS.tXt atggcaaaatacgactacaatccgaagtatgggttagaaaattacagcatattccttccc tttgagacatcttttgatgcatttcgatcgacaacatggatgcaaaatcactggtatcaa tcaattacagcatctgtcgtgtatgtagccgtcatttttacaggaaagaaggtggttctc atctacaaaaaatcacgagttattacttttgagtctagccttcagaatgcaattaagaat cgaaaccgaaaatcacttaatagttctcaaatgtttcagattatggaaaagtacaagccc ttccaactggacacaccactcttcgtctggaattcatttttagccattttctcaattctc gggttcctccgaatgacacctgaatttgtatggagttggtcagcagaaggaaactcattc aaatattcaatttgtcattcatcttatgctcaaggagtcactggtttctggactgaacaa ttcgcaatgagcaaacttttcgagctcatcgacacaatcttcatcgttcttcgtaaacgt ccactcatcttccttcactggtatcatcatgtaactgttatgatctacacatggcacgcg tacaaggatcacactgcatcaggacggtggttcatttggatgaattatggagttcatgct cttatgtattcctactatgctcttcgttctctgaaattccgtcttccaaaacaaatggca atggttgttactactctccaacttgctcaaatggttatgggagtaatcatcggagtcact gtctaccgtatcaagtcatcgggtgaatactgccaacagacatgggacaatttgggatta tgctttggagtttatttcacatatttccttcttttcgccaacttcttctaccatgcatat gttaagaaaaacaaccgtacagtaaattatgaaaataattcaaaaaatttccccgatctc gttttaatttacctgagaaaaaaggtttcaagaaaatcgaaaaatcggcaatgttcagaa aataattataaaattcaattttcatcaaattttgttaatgttgatggaaaaaaacataag aaaacatatgaacttattcttccaagaagaaaaatgaccacaattttaacttttctattt ggaaaaaatcgaattttttcgaaatatcagaaaaatcgaaaaaacatttcgattcctgtt gatttcgaaattctggagccaaaagaagatatcaatgctaacatcgctgagccatccatc acaacgaggtccgccgccgcacgaagaaaagttcaaaaagctgattago SEQID3.GBS.txt atggcagcagcacaaacaagtccagcagccacgctcgtcgatgttttgacaaaaccatgg agtctggatcagactgattcttacatgtctacatttgtaccattatcctataaaatcatg attggttatctcgtcaccatctacttcgggcaaaaattaatggctcacagaaaaccattc gatctccaaaatacacttgctctctggaacttcgggttttcactgttctcgggaatcgcc gcctataagcttattccagaactattcggagttttcatgaaggacgggtttgtcgcttcc tactgtcaaaacgagaactactacaccgatgcatcaactggattctggggctgggccttt gtgatgtcgaaagctccagaactaggggatactatgttcttggtccttcgtaaaaaacca gttatcttcatgcactggtatcatcatgccctcacatttgtctacgcagtagtcacatac tctgagcatcaggcatgggctcgttggtctttggctctcaaccttgccgtccacactgtt atgtatttctacttcgccgttcgcgccttgaacatccaaactccacgcccagtggcaaag ttcatcactactattcaaattgtccaatttgtcatctcatgctacatttttgggcatttg gtattcattaagtctgctgattctgttcctggttgcgctgttagctggaatgtgctatcg atcggaggactcatgtacatcagttatttgttcctttttgccaagttcttctacaaggcc tacattcaaaaacgctcaccaaccaaaaccagcaagcaggagtago atgtcatcgg'acgatc t SEQzo4.GBS.txt g ggcactagaaccttcaagatgatggatcaaattcttggaaca aacttcacttatgaaggtgccaaagaagttgctcgaggccttgaaggtttctcagcaaag cttgccgtcggatatattgccactatttttggactgaaatattatatgaaagaccgaaaa gccttcgatctcagtactccattaaacatttggaatggtattctttcgacattcagctta ttgggattcttattcacttttcctactttgttatcagttatcagaaaggatggatttagt cacacctattcccatgtctctgagctttacactgacagtacctctggatattggatcttc ctttgggttatctcaaagattccggaacttttggatacagtattcattgttcttcgcaag agaccacttattttcatgcactggtaccatcacgcattgaccggttactatgctcttgtc tgctaccatgaggatgctgtccatatggtttgggttgtatggatgaattatattattcat gcattcatgtatggatactatcttctgaaatctctgaaagttccaattccaccatcagtt gctcaagcaatcaccacatctcaaatggttcaattcgcagttgccattttcgcacaagtt catgtttcctataaacactatgttgagggagttgaaggattagcctactcgttcagagga acagctatcggatttttcatgcttactacctacttctatctatggattcaattctacaaa gagcactatcttaagaatggaggcaaaaagtacaatttggcaaaggatcaggcaaaaact caaacaaagaaggctaactaao SEQIDS.GBS.txt atgccacagggagaagtctcattctttgaggtgctgacaactgctccattcagtcatgag ctctcaaaaaagcatattgcacagactcagtatgctgctttctggatctcaatggcatat gttgtcgttatttttgggctcaaggctgtcatgacaaaccgaaaaccatttgatctcacg ggaccactgaatctctggaatgcgggtcttgctattttctcaactctcggatcacttgcc actacatttggacttctccacgagttcttcagccgtggatttttcgaatcttacattcac atcggagacttttataatggactttctggaatgttcacatggcttttcgttctctcaaaa gttgctgaattcggagatacactttttattattcttcgtaaaaagccattgatgttcctt cattggtatcatcatgtgcttacaatgaattatgcttttatgtcatttgaagctaatttg ggatttaatacttggattacatggatgaatttctcagttcactcaattatgtatggatat tatatgcttcgttcttttggtgtcaaggttccagcatggattgccaagaatattacaaca atgcaaattcttcaattcgttattactcatttcattcttttccacgttggatatttggca gttactggacaatctgttgactcaactccaggatattattggttctgccttctcatggaa atctcttatgtcgttctgttcggaaacttctactatcaatcatacatcaagggaggtggc aagaagtttaatgcagagaagaagactgaaaagaaaattgaataaa SEQID6.GBS.txt atgtatttgaattatttcgcgacggaaatcttccatcgtagtgcggtttgtgaaacagaa gcttgtcgctcgtcaaaaataatgattgctgacgtgttcaaatggaaattcgatgcaaac gaattgtggagtcttttaacgaatcaggatgaagttttcccgcatattagagcacggcga ttcattcaagaacattttggtctattcgtccagatggcaattgcatatgtcattttggtg ttctcaatcaaaaggttcatgagggatcgtgaaccatttcaactcaccacagctcttcgt ctctggaacttcttcctctccgtcttctcaatttatggttcctggacaatgtttccattt atggttcaacaaataagactttatggtctctacggatgtggatgcgaagcactttcaaac cttccgagtcaagcagaatattggcttttcctgacgatcttgtccaaagctgtggagttt gttgatacatttttcttggttctccggaaaaaaccactcatcttcctacactggtatcat catatggcaacatttgtcttcttctgcagtaattacccgactccatcgtcacaatcacgc gtcggagttatcgtcaacctgttcgtgcatgccttcatgtacccatactatttcacccga tcaatgaacatcaaagttcctgcgaaaatttcaatggctgttacagttcttcaattgact caattcatgtgctttatctatggatgtactctcatgtactactcgttggccactaatcag gcacgatacccctcaaatacacctgcgacactccaatgtttgtcctacactctacatttg ctttgao SEQID7.GBS.txt atggctcagcatccgctcgttcaacggcttctcgatgtcaaattcgacacgaaacgattt gtggctattgctactcatgggccaaagaatttccctgacgcagaaggtcgcaagttcttt gctgatcactttgatgttactattcaggcttcaatcctgtacatggtcgttgtgttcgga acaaaatggttcatgcgtaatcgtcaaccattccaattgactattccactcaacatctgg aatttcatcctcgccgcattttccatcgcaggagctgtcaaaatgaccccagagttcttt ggaaccattgccaacaaaggaattgtcgatcctactgcaaagtgtttgatttcacgaaag gagagaatggatactgggtgtggctcttcatggcttccaaacttttcgaacttgttgaca ccatcttcttggttctccgtaaacgtccactcatgttccttcactggtatcaccatattc tcaccatgatctacgcctggtactctcatccattgaccccaggattcaacagatacggaa tttatcttaactttgtcgtccacgccttcatgtactcttactacttccttcgctcgatga agattcgcgtgccaggattcatcgcccaagctatcacatctcttcaaatcgttcaattca tcatctcttgcgccgttcttgctcatcttggttatctcatgcacttcaccaatgccaact gtgatttcgagccatcagtattcaagctcgcagttttcatggacacaacatacttggctc ttttcgtcaacttcttcctccaatcatatgttctccgcggaggaaaagacaagtacaagg cagtgccaaagaagaagaacaactaa~

SEQID8.GBS.tXt atgtcggccgaagtgtccgaacgattcaaagtttggacaggaaacaatgagaccatcatc tattccccattcgagtacgattccacgttgctcatcgagtcatgtcggtgtacttatcag ctgcttatattattgcgacaaatttattacagagatatatggagtcacggaaacctaaaa cttttactagcatggaacggttttttggcagtgttcagtattatgggtacatggagattt ggaatcgaattctacgatgctgttttcagaagaggcttcatcgattcgatctgcctggct gtaaatccacgttcaccgtccgcattctgggcatgcatgttcgctctatcgaaaatcgcc gagtttggggacacgatgttcttggtgctgaggaaacggccggttatattccttcactgg tatcatcacgctgttgttctgatcctttcttggcatgctgcaatcgaactcacagctcca ggacgctggtttatttttatgaactatttggtgcattcaataatgtatacatactacgca ataacatcaatcggctatcgtcttcccaaaatcgtttcaatgactgttacattccttcaa actcttcaaatgctcattggtgtcagcatttcttgcattgtgctttatttgaagcttaat ggagagatgtgccaacaatcctacgacaatctggcgttgagcttcggaatctacgcctca ttcctggtgctattctccagtttcttcaacaatgcatatttggtaaaaaaggacaagaaa cccgatgtgaagaaggattaao SEQID9.GBS.txt MELAEFWNDL~ITFTIYGPNHTDMTTKYKYSYHFPGEQVADPQYWTILFQKYWYHSITISV
LYFILIKVIQKFMENRKPFTLKYPLILWNGALAAFSIIATLRFSIDPLRSLYAEGFYKTL
CYSCNPTDVAAFWSFAFALSKIVELGDTMFIILRKRPLIFLHYYHHAAVLIYTVHSGAEH
TAAGRFYILMNYFAHSLMYTYYTVSAMGYRLPKWVSMTVTTVQTTQMLAGVGITWMVYKV
KTEYKLPCQQSVANLYLAFVIYVTFAILFIQFFVKAYIIKSSKKSKSVKNEO

SEQID10.GBS.tXt MAKYDYNPKItGLENYSIFLPFETSFDAFRSTTWMQNHWYQSITASVVYVAVIFTGKKWL
IYKKSRVITFESSLQNAIKNRNRKSLNSSQMFQIMEKYKPFQLDTPLFVWNSFLAIFSIL
GFLRMTPEFVWSWSAEGNSFKYSICHSSYAQGVTGFWTEQFAMSKLFELIDTIFIVLRKR
PLIFLHWYHHVTVMIYTWHAYKDHTASGRWFIWMNYGVHALMYSYYALRSLKFRLPKQMA
MVVTTLQLAQMVMGVIIGVTVYRIKSSGEYCQQTWDNLGLCFGVYFTYFLLFANFFYHAY
VKKNNRTVNYENNSKNFPDLVLIYLRKKVSRKSKNRQCSENNYKIQFSSNFVNVDGKKHK
KTYELILPRRKMTTILTFLFGKNRIFSKYQKNRKNISIPVDFEILEPKEDINANIAEPSI
TTRSAAARRKVQKADD

SEQID11.GBS.tXt MAAAQTSPAA'fLVDVLTKPWSLDQTDSYMSTFVPLSYKIMIGYLVTIYFGQKLMAHRKPF
DLQNTLALWNFGFSLFSGIAAYKLIPELFGVFMKDGFVASYCQNENYYTDASTGFWGWAF
VMSKAPELGDTMFLVLRKKPVIFMHWYHHALTFVYAVVTYSEHQAWARWSLALNLAVHTV
MYFYFAVRALNIQTPRPVAKFITTIQIVQFVISCYIFGHLVFIKSADSVPGCAVSWNVLS
IGGLMYISYLFLFAKFFYKAYIQKRSPTKTSKQE~

SEQID12.GBS.txt MSSDDRGTRl'~FKMMDQILGTNFTYEGAKEVARGLEGFSAKLAVGYIATIFGLKYYMKDRK
AFDLSTPLNIWNGILSTFSLLGFLFTFPTLLSVIRKDGFSHTYSHVSELYTDSTSGYWIF
LWVISKIPELLDTVFIVLRKRPLIFMHWYHHALTGYYALVCYHEDAVHMVWVVWMNYIIH
AFMYGYYLLKSLKVPIPPSVAQAITTSQMVQFAVAIFAQVHVSYKHYVEGVEGLAYSFRG
TAIGFFMLTTYFYLWIQFYKEHYLKNGGKKYNLAKDQAKTQTKKAN~

SEQID13.GBS.tXt MPQGEVSFFE~VLTTAPFSHELSKKHIAQTQYAAFWISMAYVVVIFGLKAVMTNRKPFDLT
GPLNLWNAGLAIFSTLGSLATTFGLLHEFFSRGFFESYIHIGDFYNGLSGMFTWLFVLSK
VAEFGDTLFIILRKKPLMFLHWYHHVLTMNYAFMSFEANLGFNTWITWMNFSVHSIMYGY
YMLRSFGVKVPAWIAKNITTMQILQFVITHFILFHVGYLAVTGQSVDSTPGYYWFCLLME
ISYWLFGNFYYQSYIKGGGKKFNAEKKTEKKIEO

SEQID14.GBS.txt MYLNYFATEI~FHRSAVCETEACRSSKIMIADVFKWKFDANELWSLLTNQDEVFPHIRARR
FIQEHFGLFVQMAIAYVILVFSIKRFMRDREPFQLTTALRLWNFFLSVFSIYGSWTMFPF
MVQQIRLYGLYGCGCEALSNLPSQAEYWLFLTILSKAVEFVDTFFLVLRKKPLIFLHWYH
HMATFVFFCSNYPTPSSQSRVGVIVNLFVHAFMYPYYFTRSMNIKVPAKISMAVTVLQLT
QFMCFIYGCTLMYYSLATNQARYPSNTPATLQCLSYTLHLLD

SEQID15.GBS.tXt MAQHPLVQRL'~LDVKFDTKRFVAIATHGPKNFPDAEGRKFFADHFDVTIQASILYMVWFG
TKWFMRNRQPFQLTIPLNIWNFILAAFSIAGAVKMTPEFFGTIANKGIVASYCKVFDFTK
GENGYWVWLFMASKLFELVDTIFLVLRKRPLMFLHWYHHILTMIYAWYSHPLTPGFNRYG
IYLNFVVHAFMYSYYFLRSMKIRVPGFIAQAITSLQIVQFIISCAVLAHLGYLMHFTNAN
CDFEPSVFKLAVFMDTTYLALFVNFFLQSYVLRGGKDKYKAVPKKKNNO

SEQID16.GBS.txt MSAEVSERFICVWTGNNETIIYSPFEYDSTLLIESCRCTYQLLILLRQIYYRDIWSHGNLK
ACDXLLLAWNGFLAVFSIMGTWRFGIEFYDAVFRXGFIXSICLAVNPRSPSAFWACMFAL
SKIAEFGDTMFLVLRKRPVIFLHWYHHAWLILSWHAAIELTAPGRWFIFMNYLVHSIMY
TYYAITSIGYRXPKIVSMTVTFLQTLQMLIGVSISCIVLYLKLNGEMCQQSYDNLALSFG
IYASFLVLSSFFNNAYLVKKDKKPDVKKD~

Claims (40)

Claims
1. An isolated polypeptide comprising a functional long chain polyunsaturated fatty acid (PUFA) elongase as herein defined.
2. A polypeptide according to claim 1 wherein the polypeptide is from a eukaryote.
3. A polypeptide according to claim 1 or claim 2 wherein the polypeptide has at least a portion of the amino acid sequence shown in SEQ ID 15, or variants thereof.
4. A polypeptide having at least 60% homology to a polypeptide according to claim 3 and having a PUFA elongase function.
5. A polypeptide according to claim 4 having at least 80% homology.
6. A polypeptide according to claim 5 having at least 90% homology.
7. A polypeptide according to any preceding claim wherein the polypeptide sequence includes a sequence motif responsible for Endoplasmic Reticulum (ER) -retention.
8. A polypeptide according to any preceding claim wherein the polypeptide is capable of elongating palmitoleic acid (PA; 16:1.DELTA.9) to vacceric acid (VA;
18:1.DELTA.11).
9. A polypeptide according to any preceding claim wherein the polypeptide is from an animal.
10. A polypeptide according to claim 9 wherein the animal is an invertebrate.
11. A polypeptide according to claim 10 wherein the invertebrate is a worm.
12. A polypeptide according to claim 11 wherein the worm is C. elegans.
13. A polypeptide according to claim 9 wherein the animal is a vertebrate.
14. A polypeptide according to claim 13 wherein the vertebrate is a mammal.
15. A polypeptide according to claim 14 wherein the mammal is a human, rat or mouse.
16. A DNA sequence encoding a polypeptide according to any preceding claim.
17. A DNA sequence according to claim 16 wherein the DNA comprises the sequence shown in SEQ ID 7 or variants of that sequence due to base substitutions, deletions and/or additions.
18. An engineered organism engineered to express a polypeptide according to any one of claims 1 to 15.
19. An engineered organism according to claim 18 wherein the animal is a mammal.
20. An engineered organism according to claim 19 wherein the mammal is a rat, mouse or monkey.
21. An engineered organism containing a synthetic pathway for the production of a polypeptide according to any one of claims 1 to 15.
22. An engineered organism according to claim 21 wherein the pathway includes .DELTA.5-fatty acid desaturase.
23. An engineered organism according to claim 21 or 22 wherein the pathway includes .DELTA.6-fatty acid desaturase.
24. An engineered organism according to any one of claims 21 to 23 wherein the animal is a lower eukaryote.
25. An engineered organism according to claim 24 wherein the lower eukaryote is a yeast.
26. An engineered organism according to claim 18 wherein the animal is a fish.
27. A transgenic plant engineered to express a polypeptide according to any one of claims 1 to 15.
28. A transgenic plant containing a DNA sequence according to claim 16 or 17.
29. A method of producing a PUFA comprising carrying out an elongase reaction catalysed by a polypeptide according to any one of claims 1 to 15.
30. A method according to claim 29 wherein the PUFA is di-homo-gamma-linoleic acid (20:3.DELTA.8,11,14), arachidonic acid (20:4.DELTA.5,8,11,14), eicosapentanoic acid (20:5.DELTA.5,8,11,14,17), docosatrienoic acid (22:3.DELTA.3,16,19), docosatetraenoic acid (22:4.DELTA.7,10,13,16), docosapentaenoic acid (22:5.DELTA.7,10,13,16,19) or docosahexaenoic acid (22:4.DELTA.4,7,10,13,16,19).
31. A method according to claim 29 wherein the PITFA is a 24 carbon fatty acid with at least 4 double bonds.
32. A PUFA produced by a method according to any one of claims 29 to 31.
33. A foodstuff comprising a PUFA according to claim 32.
34. A dietary supplement comprising a PUFA according to claim 32.
35. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 15.
36. A pharmaceutical composition comprising a PUFA according to claim 32.
37. A pharmaceutical composition according to claim 35 or claim 36 wherein the composition comprises a pharmaceutically-acceptable diluent, carrier, excipient or extender.
38. A method of elevating the PUFA levels of an animal or a plant by supplying to the animal or plant a polypeptide according to any of claims 1 to 15, a DNA
sequence according to claim 16 or claim 17, a foodstuff according to claim 33, a dietary supplement according to claim 34, a pharmaceutical composition according to any of claims 35 to 37 or a PUFA according to claim 32.
39. A method of treatment according to claim 38 wherein the animal is a mammal.
40. A method of treatment according to claim 39 wherein the mammal is a human.
CA002365096A 1999-03-18 2000-03-20 Polyunsaturated fatty acid (pufa) elongase from caenorhabditis elegans Abandoned CA2365096A1 (en)

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GB9906307.5 1999-03-18
GBGB9906307.5A GB9906307D0 (en) 1999-03-18 1999-03-18 Novel polypeptides
GB0003869A GB0003869D0 (en) 2000-02-18 2000-02-18 Elongase II
GB0003869.5 2000-02-18
PCT/GB2000/001035 WO2000055330A1 (en) 1999-03-18 2000-03-20 Polysaturated fatty acid (pufa) elongase from caenorhabditis elegans

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US6403349B1 (en) * 1998-09-02 2002-06-11 Abbott Laboratories Elongase gene and uses thereof
GB0107510D0 (en) * 2001-03-26 2001-05-16 Univ Bristol New elongase gene and a process for the production of -9-polyunsaturated fatty acids
CA2449479A1 (en) * 2001-06-05 2002-12-12 Exelixis, Inc. Map3ks as modifiers of the p53 pathway and methods of use
US7705202B2 (en) 2002-03-16 2010-04-27 The University Of York Transgenic plants expressing enzymes involved in fatty acid biosynthesis
MX353906B (en) * 2003-08-01 2018-02-02 Basf Plant Science Gmbh Method for the production of multiply-unsaturated fatty acids in transgenic organisms.
DK1756280T3 (en) 2004-04-22 2015-02-02 Commw Scient Ind Res Org SYNTHESIS OF CHAIN, polyunsaturated fatty acids BY RECOMBINANT CELLS
EP2357243B1 (en) 2004-04-22 2018-12-12 Commonwealth Scientific and Industrial Research Organisation Synthesis of long-chain polyunsaturated fatty acids by recombinant cells
DK1766023T3 (en) 2004-06-04 2010-12-13 Fluxome Sciences As Metabolically engineered cells for the production of polyunsaturated fatty acids
EP2059588A4 (en) 2006-08-29 2010-07-28 Commw Scient Ind Res Org Synthesis of fatty acids
US7892792B2 (en) 2008-06-27 2011-02-22 Indian Institute Of Science Cells expressing Pichia cytochrome C
CN113957105B (en) 2008-11-18 2024-11-01 联邦科学技术研究组织 Enzymes and methods for producing omega-3 fatty acids
UA127917C2 (en) 2012-06-15 2024-02-14 Коммонвелт Сайнтіфік Енд Індастріел Рісерч Організейшн A recombinant brassica napus cell containing long-chain polyunsaturated fatty acids, a transgenic brassica napus plant and seeds, a method for producing an extracted plant lipid, a food product and an ethyl ester of polyunsaturated fatty acids
US9725399B2 (en) 2013-12-18 2017-08-08 Commonwealth Scientific And Industrial Research Organisation Lipid comprising long chain polyunsaturated fatty acids
KR102527795B1 (en) 2014-06-27 2023-05-02 커먼웰쓰 사이언티픽 앤 인더스트리알 리서치 오거니제이션 Lipid comprising docosapentaenoic acid
CN104920307B (en) * 2015-06-19 2018-01-26 安徽省农业科学院农业工程研究所 A kind of Caenorthaditis elegans ws123 fixing means suitable for single-particle microbeam device

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