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Title:
ELONGASE GENE AND USES THEREOF
Document Type and Number:
WIPO Patent Application WO/2008/064096
Kind Code:
A3
Abstract:
The subject invention relates to the identification of a gene involved in the elongation of polyunaturated fatty acids containing unsaturation at the carbon 9 position (i.e., "Δ9-elongase") and to uses thereof. In particular, Δ9-elongase may be utilized, for example, in the conversion of linoleic acid (LA, 18:2n-6) to eicosadienoic acid (EDA, 20:2n-6). The production of dihomo-y-linolenic acid (DGLA, 20:3n-6) from eicosadienoic acid (EDA, 20:2n-6), and arachidonic acid (AA, 20:4n-6) from dihomo-y-linolenic acid (DGLA, 20:3n-6) is then catalyzed by Δ8-desaturase and Δ5-desaturase, respectively. AA or polyunsafurated fatty acids produced therefrom may be added to pharmaceutical compositions, nutritional compositions, animal feeds, as well as other products such as cosmetics.

Inventors:
DAS, Tapas (936 Linkfield Drive, Worthington, OH, 43085, US)
MUKERJI, Pradip (1069 Arcaro Drive, Columbus, OH, 43230, US)
KRISHNAN, Padmavathy (5821 Dena Drive, Hilliard, OH, 43026, US)
LEONARD, Amanda, E. (581 Shadewood, Columbus, OH, 43230, US)
PEREIRA, Suzette, L. (710 Westray Drive, Westerville, OH, 43081, US)
Application Number:
US2007/084902
Publication Date:
October 09, 2008
Filing Date:
November 16, 2007
Export Citation:
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Assignee:
ABBOTT LABORATORIES (Dept. 377/AP6A-1, 100 Abbott Park RoadAbbott Park, IL, 60064, US)
DAS, Tapas (936 Linkfield Drive, Worthington, OH, 43085, US)
MUKERJI, Pradip (1069 Arcaro Drive, Columbus, OH, 43230, US)
KRISHNAN, Padmavathy (5821 Dena Drive, Hilliard, OH, 43026, US)
LEONARD, Amanda, E. (581 Shadewood, Columbus, OH, 43230, US)
PEREIRA, Suzette, L. (710 Westray Drive, Westerville, OH, 43081, US)
International Classes:
C12N15/54; A61K31/20; C12N9/10; C12N15/10; C12N15/82
Domestic Patent References:
WO2002077213A22002-10-03
WO2001059128A22001-08-16
WO2006008099A22006-01-26
WO2007061742A12007-05-31
Foreign References:
US20070118929A12007-05-24
Other References:
QI B ET AL: "Identification of a cDNA encoding a novel C18-DELTA<9> polyunsaturated fatty acid-specific elongating activity from the docosahexaenoic acid (DHA)-producing microalga, Isochrysis galbana<1>" FEBS LETTERS, ELSEVIER, AMSTERDAM, NL, vol. 510, no. 3, 16 January 2002 (2002-01-16), pages 159-165, XP004332633 ISSN: 0014-5793
QI B ET AL: "Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants" NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP, NEW YORK, NY, US, vol. 22, no. 6, 1 June 2004 (2004-06-01), pages 739-745, XP002348313 ISSN: 1087-0156
SAYANOVA O V ET AL: "Eicosapentaenoic acid: biosynthetic routes and the potential for synthesis in transgenic plants" PHYTOCHEMISTRY, PERGAMON PRESS, GB, vol. 65, no. 2, 1 January 2004 (2004-01-01), pages 147-158, XP004483374 ISSN: 0031-9422
NAPIER J A ET AL: "The production of long chain polyunsaturated fatty acids in transgenic plants by reverse-engineering" BIOCHIMIE, MASSON, PARIS, FR, vol. 86, no. 11, 1 November 2004 (2004-11-01), pages 785-792, XP004689087 ISSN: 0300-9084
DREXLER H ET AL: "Metabolic engineering of fatty acids for breeding of new oilseed crops: strategies, problems and first results" JOURNAL OF PLANT PHYSIOLOGY, FISCHER, STUTTGART, DE, vol. 160, no. 7, 1 January 2003 (2003-01-01), pages 779-802, XP004955255 ISSN: 0176-1617
Attorney, Agent or Firm:
WEIDA, Sandra, E. et al. (625 Cleveland Avenue, Dept. 108140 Ds1Columbus, OH, 43215, US)
Download PDF:
Claims:

CLAIMS

1. An isolated nucietc acid molecule or fragment thereof comprising or complementary to a nucleotide sequence encoding a polypeptide having eloRgase activity, wherein the amino acid sequence of said polypeptide has at least 86% sequence identity to the amino acid sequence comprising SEQ ID MO:2.

2. An isolated nucleotide sequence or fragment thereof comprising or complementary to at feast 86% of the nucleotide sequence comprising SEQ SD NO:1.

3. The isolated nucleotide sequence of cϊaim 1 or 2 wherein said sequence encodes a functionally active eloπgase which utilizes a polyunsaturated fatty add as a substrate.

4. The nucleotide sequence of claim 3 wherein the nucleotide sequence is from Tiiraustochytrium aureυm 7087.

5. The nucleotide sequence of claim 3 wherein the nucleotide sequence is from a Thraustochvtrid sp.

6. A purified polypeptide encoded by said nucleotide sequence of ciaim 1, 2 or 3.

7. A purified polypeptide which elongates polyunsaturated fatty acids containing unsaturatioπ at the carbon 9 position and has at least 86% amino acid identity to an amino acid sequence comprising SEQ iD NO:2.

8. A method of producing an efongase comprising the steps of: a) isolating a nucleotide sequence comprising SEQ iD NO:1; b) constructing a vector comprising: i) said isolated nucleotide sequence operabiy linked to ϊi) a regulatory sequence; c) introducing said vector into a host ceil for a time and under conditions sufficient for expression of said elongase.

9. The method of claim 8 wherein said host ceil is selected from the group consisting of a eukaryotic cell and a prokaryotic cell.

10. The method of csatm 9 whereto said prokaryoilc ceii is selected from the grouc consisting of Escherichia co?i. cvaπobacteria and Bacillus subtilis.

11. The method of claim 9 wherein said eυkaryotic eel! ss selected from the group consisting of a mammalian ceii. an insect ceil, a plant cell and a fungaf cell,

12. The method of ciaϊrn 11 wherein said fungal ceIE is selected from the group consisting of Saccharomyces spp., Candida spp., Ui≥omyces spp., YarrowJa spp.. Kiuweromvces spp. = Hansenuia spp. s Aspergillus spp., Penrciilium spp., Neurosppra spp., Trichoderma spp. and PJchia spp.

13. The method of claim 12 wherein said fungal ceti is a yeast cell selected from the group consisting of Saccharomyces spp., Candida spp., Hansenula spp. and Prchia spp.

14. The method of claim 13 wherein said yeast cell is Saccharomyces cerevfeiae.

15. A vector comprising: a} a nucleotide sequence comprising SEQ ID NO:1 operafaly linked to b) a regulatory sequence.

16. A host ceil comprising said vector of claim 15.

17. The host ceiϊ of claim 16, wherein said host ceil is selected from the group consisting of a eukaryotϊc ceii and a prokaryotjc ceiL

18. The host ceil of claim 17, wherein said prokaryotic ceil is selected from the group consisting of Escherichia coii, cyaπobacteria and Bacillus subtiiis.

19. The host ceil of cϊaim 17, wherein said eukaryotic cell is selected from the group consisting of a mamrnaitan cell, an insect cell, a plant ceil and a fungal cell.

20. The host ceil of claim 19, wherein said fungal ceil is selected from the group consisting of Saccharomvces spp., Candida spp., Lipomyces spp., Yarrowia spp., Kiuweromvces spp., Hansenula spp., Aspergillus spp., PenicilSiuro spp., Neurospora spp., Trichoderma spp. and Pichia spp.

21. τ he host ceil of da;m 20 wherein said fungal ceϋ is a yeast ceϋ sefected from xhe group consisting cf Saccharomyces spp , Candida SPP . h'ansenula sop. aid Pichia spp.

22. The host cell of ciairn 21, wherein said host ceil is Saccharomyces csrevisiae.

23. A mammalian celt comprising said vector of ciasm 15, wherein expression of said nucleotide sequence of said vector results in production of aftered levels of EDA and/or ETrA, when said ceϋ is grown in a culture media comprising at Seast one fatty acid seiected from the group consisting of LA and ALA.

24. A plant cefi, plant seed, pfani or p!anl tissue comprising said vector of claim 1S 1 wherein expression of said nucleotide sequence of said vector resutts in production of at Seas! one polyunsaturated fatty acid by said plant ceil, plant or plant tissue.

25. The plant cell, pfant seed, plant or plant tissue of ciasm 24 wherein said polyunsaturated fatty acid is seiected from the group consisting of EDA 1 ALA, and EtrA.

28. One or more pfant oils or fatty acids expressed by said piant ceiS, plant seed, plant or pfant tissue of claim 24.

27. A transgenic piant comprising said vector of claim 15, wherein expression of said nucleotide sequence of said vector results in production of at feast one pofyunsatu rated fatty acid In seeds of said transgenic plant.

28. A method for producing a polyunsaturated fatty acid comprising the steps of: a) isolating a nucleotide sequence comprising SEQ ID NO:1 ; b) constructing a vector comprising said isolated nucleotide sequence; c) introducing said vector into a host celi for a time and under conditions sufficient for expression of δ9-elongase enzyme; and d) exposing said expressed δ9-eiongase enzyme to a substrate polyunsaturated fatty acid in order to convert said substrate to a product polyunsaturated fatty acid.

29 ! he method according to dmm 28, wherein said substrate polyunsaturated fatty acid is LA or ALA and said produci DOsyLnsaiursted fatty aσd is EDA or ETrA, respectively.

30. The method according to claim 28 further comprising the step of exposing said product polyunsaturated fatty acid to at least one desaturase in order to convert said product polyunsaturated fatty acid to another polyunsaturated fatty acid.

31. The method according to claim 30 wherein said product polyunsaturated fatty add ss EDA or ETrA and said anoiher polyunsaturated fatty acid is DGLA or ETA, respectively.

32. The method of claim 30 further composing the step of exposing said another poϊyunsatu rated fatty acid to at least one additional desaturase ϊn order to convert said another polyunsaturated fatty acid to an additional polyunsaturated fatty acid.

33. The method according to ciaϊm 32 wherein said another polyunsaturated fatty acid is DGLA or ETA and said additional polyunsaturated fatty acid is AA or EPA, respectively.

34. The method of claim 32 further comprising the step of exposing said additional polyunsaturated fatty acid to at least one additional desaturase and/or at least one additional efongase in order to convert said additional polyunsaturated fatty acid to a final polyunsaturated fatty acid.

35. The method of claim 34 wherein said additional polyunsaturated fatty acid is AA or EPA and said final polyunsaturated fatty acid is ω3-docosapentaenoιc acid, ω6- docosapentaenoic acid, ADA or DHA, respectively.

36. A composition comprising at least one polyunsaturated fatty acid selected from the group consisting of said product polyunsaturated fatty acid produced according to the method of claim 28, said another polyunsaturated fatty acid produced according to the method of claim 30, said additional polyunsaturated fatty add produced according to the method of ciaim 32, and said final polyunsaturated fatty acid produced according to the method of claim 34.

37 The COF 1 COSiUOn of c.airr 36 wherein sad product oolyunsaruraied fatty ado is at least one polyunsaturated fatly aαα selecteα from :he group consrstmg of EDA and ETrA

38. The composition of claim 36 wheretn said another polyunsaturated fatty add is at ϊeast one polyunsaturated fatty acid selected from the group constsirng of DGLA apd ETA

39. The composition of claim 38 wherein said additional polyunsaturated fatty acid ts at Seast one polyunsaturated fatty add selected from the group consisting of AA and EPA.

40. The composition of claim 36 wherein said final polyunsaturated fatty acid is at least one polyunsaturated fatty acid selected from the group consisting of ω3~ docosapeπiaenoϊc acid, ωβ-docosapentaenoϊc acid, ADA and DHA.

41 A method of preventing or treating a condition in a patient caused by insufπcferst intake of polyunsaturated fatty acids comprising administering to said patient said composition of claim 36 in an amount sufficient to effect said prevention or treatment.

42. A transgenic, non-human mammal whose genome comprises a DNA sequence encoding a δ9-etongase, operably linked to a promoter or regulatory sequence, wherein said DNA sequence comprises SEQ SD NO:1.

43. A fluid produced by said transgenic, non-human mammal of claim 42 wherein said fluid comprises a detectable level of δ9-elongase.

Description:

ELONGASE GENE AND USES THEREOF

BACKGROUMD OF THE JMVEMTiOM

Technical Reid

The subject invention reϊates to the identification of a gene involved in the eSongatfon of iong-ehain polyunsaturated fatty acids (i.e . "elongase") and to uses thereof. In particular, the eϊongase enzyme is utilized in the conversion of one fatty acid to another. For example, ebngase catalyzes the conversion of γ-iϊno!enic acid (GLA. 18:3n-8) to dshomo-y-ϋnofeπic acid (DGLA. 20:3P-6) and the conversion of stearidontc acid (STA, 18:4n-3) to eϊcosatetraenoic acid (ETA, 20:4n-3). Elongase afso catalyzes the conversion of arachidorwc acid (AA 1 20-4π-8) to adrenic acid (ADA, 22:4n-8), the conversion of eicosapentaenoic add (EPA. 2G:5n-3) to ω3- docosapentaenoic acid (22;5n~3), the conversion of finoieic acid (LA 1 18:2n-6) to eicosadieno'ic add (EDA, 2G:2rϊ~δ). and the conversion of α-ttno!enic acid (ALA, 18:3π-3) to eicosatrienoic acid (ETrA, 20:3n-3). ALA, for example, may be utilized in the production of other polyunsaturated fatty acids (PUFAs). such as ETrA. ETrA may then be converted to ETA by a δ8-desaturase. ETA may then be utilized in the production of other polyunsaturated fatty acids, such as EPA, which may be added to pharmaceutical compositions, nutritional compositions, animal feeds, as weil as other products such as cosmetics.

Background Information

The eiongases which have been identified in the past differ in terms of the substrates upon which they act. Furthermore, they are present in both animals and plants. Those found in mammals have the ability to act on saturated, monounsaturated and polyunsaturated fatty acids. In contrast, those found In plants are specific for saturated or monounsaturated fatty acids. Thus, in order to generate polyunsaturated fatty acids in plants, there is a need for a PUFA-specific eSongase.

In both plants and animals, the elongation process is believed to be the result of a four-step mechanism (Lassπer et al., The Plant Ceil 8:281-292 (1996)). CoA is the acyl carrier. Step one involves condensation of malonyi-CoA with a long-chain acyl- CoA to yield carbon dioxide and a β-ketoacyl-CoA in which the acyl moiety has been elongated by two carbon atoms. Subsequent reactions include reduction to β- hydroxyacyl-CoA, dehydration to an enoyl-CoA, and a second reduction to yield the

eioπgaxed acyl-CoA. s he ! n:ua! conoensaiicπ reacroπ is not on'y ihe s absirate- specifse step but also ihe rate-bmSng step

As noted previously, efongases, mere specifically, those which utifee PUFAs as substrates, are critical in the production of tong-chain polyunsaturated fatty acids which have many important functions. For example. PUFAs are important components of ihe piasma membrane of a ceil where they are found in the form of phospholipids. They afso serve as precursors io mammalian prostacyclins, eϊcosanoids, Seukotπeπes and prostaglandins Additionally. PUFAs are necessary for the proper development of the developing infant brain as weif as for tissue formation and repair, in view of the biological significance of PUFAs. attempts are being made to produce them, as welt as intermediates leading to their production, efficiently

A number of enzymes are involved in PUFA biosynthesis including eiongases (ELO) (FIG. 1). For example, [ϊπoieic acid (LA, 18:2n-6) is produced from oleic acid (OA, 18:1n-9) by a δ12-desaturase. Eicosadtenoic acid (EDA, 20:2rs-8) is produced from linoleic add (LA, 18:2n-6) by a δ9-e!oπgase. Dthomo-γ-iinofenic acid (DGLA, 20:3n- 6) is produced from eicosadsβnoic acid (EDA. 2G:2n-6) by a δ8-desatυrase. Arachidonic acid (AA, 20:4n-6) is produced from dihomo-γ-linoleriic add (DGLA, 20:3n-6) by a δ5-desaturase.

It must be noted that animals cannot desaturate beyond the δ9 position and therefore cannot convert oleic add (OA- 18:1n-9) into fϊnoieic acid (LA, 18:2n-6}. Likewise, olinolertic acid (ALA, 18:3π-3) cannot be synthesized by mammais. since they lack δ15-desaturase activity. However, α-iinolenic acid can be converted to stearidonsc acid (STA, 18:4n-3) by a δ6 » desaturase (see PCT publication WO 96/13591 ; see afso U.S. Pat. No. 5,552,306), followed by elongation to eicosatetraenoic acid (ETA, 20:4n-3) in mammals and algae. This polyunsaturated fatty acid (i.e., ETA, 20:4n-3) can then be converted to eicosapentaenoic acid (EPA, 20:5-3) by a δ5-desatυrase. Other eukaryotes, including fungi and piants, have enzymes which desaturate at carbons 12 (see PCT publication WO 94/11516 and U.S. Pat. No. 5,443,974) and 15 (see PCT publication WO 93/11245}. The major polyunsaturated fatty acids of animals therefore are either derived from diet and/or from desaturation and elongation of iinoleic acid or α-linolenic acid, in view of the inability of mammals to produce these essential ioπg-chain fatty acids, it is of significant interest to isolate genes involved in PUFA biosynthesis from species that

naturally produce ihese fatty acids and to express ihese genes in a microbial, piant or animal system which can be aifered to provide production of commercial Quantities of one or more PUFAs. Consequβmfy, there is a definite need for efongase enzymes, the genes encoding the enzymes, as well as recombinant methods of producing the enzymes.

Sn view of the above discussion, a definite need exists for Otis containing ϊevefs of PUFAs beyond those naturally present as we!! as those enriched in noveϊ PUFAs. Such oils can only be made by isolation and expression of elongase genes.

One of the most important long-chain PUFAs is efcosapeπfaenoic add (EPA). EPA rs found in fungi and also in marine oϋs. Docosahexaenoic acid (DHA) is another important Song-chain PUFA. DHA is most often found in fish oil and can also be purified from mammaian brain tissue. Arachidorac acid (AA) is a third important long- chain PUFA. AA is found in filamentous fungi and can also be purified from mammalian tissues inducing the liver and the adrenal glands.

AA, EPA and/or DHA, for example, can be produced via either the alternate delta 8 pathway or the coπventiona! delta 6 pathway (FSG. 1). Elongase, which are active on substrate fatty acids in the conventioπaϊ delta 6 pathway for the production of fong- chain PUFAs 1 particuSariy AA 5 EPA and DHA, have previously been identified. The conventional deita 6 pathway for converting LA to DGLA and ALA to ETA utilizes the δ6-desaturase enzyme to convert LA to GLA, and ALA to STA 1 and the δ6-eiongase enzyme to convert GLA to DGLA, and STA to ETA. However, in certain instances, the alternate delta 8 pathway may be preferred over the conventional delta 6 pathway. For example, if particular residua! omega-δ or omega-3 fatty acid intermediates, such as GLA or STA, are not desired during production of DGLA, ETA, AA, EPA, ω3-docosapentaenoic acid, ωθ-docosapentaenoic acid, ADA and/or DHA, the alternate delta 8 pathway may be used as an alternative to the conventional deita 6 pathway, to bypass GLA and STA formation.

in the present invention, a new source of δ9-elongase has been identified for the production of long-chain PUFAs, in particular DGLA, ETA, AA, EPA, u>3- docosapeπtaeπoic acid, ω6-docosapentaeπσic acid, ADA and/or DHA. Such oils can be made, in part, by isolation and expression of the δ9-eIongase gene. The δ9- elongase enzyme of the present invention converts, for example, LA to EDA. The

production of DGLA from EDA. and AA FOPI DGLA, IS ihe^ catalyzed by a λ8~ desatυrase and a λ5-ctøs8turase respectively.

The search for a iong-chain PUFA-specific δ9~eEongasβ in Thraustochyϊrid sp. began based upon a review of the homologies shared between this gene and by expression screening for PUFA-eloπgase activity.

AH patents and publications referred to herein are hereby incorporated in their entirety by reference.

SOWWARY OF THE EfrJVENTfQN

The present invention encompasses an isolated nucleic acid molecule or fragment thereof comprising or complementary to a nucleotide sequence encoding a pofypeptide OF protein having efongase activity and having at least 86% identity or similarity to an amino add sequence comprising SEQ ID NO:2 (FfG. 3).

Futhermore, the present invention includes an isolated nucleotide sequence or fragment comprising or complementary to at feast 86% of a nucleotide sequence comprising SEQ ID NG: 1 (FfG. 2). in particuiar, the isolated sequence may be represented by SEQ ID NG: 1. The sequence encodes a functionally active elongase which uflzes a polyunsaturated fatty acid as a substrate.

The nucleotide sequence may be from a fungus of. for exampϊe, the family Thraustochvtrids (or Thraustøchytriidae). and may specifically be isolated from, for example, a Thraustochytrid sp., from Thraustochvtrium aureum 7087, or from Thraustochytrid sp. BfCC 7087.

The present invention also includes a purified polypeptide or protein encoded by the above nucleotide sequence as well as a purified polypeptide or protein which elongates polyunsaturated fatty acids containing unsaturation at the carbon 9 position and has at ieast 86% amino acid identity or similarity to the amino acid sequence of the purified protein encoded by the above nucleotide sequence.

Furthermore, the present invention also encompasses a method of producing a δ9- elongase enzyme. This method comprises the steps of: a) isolating the nucleotide sequence comprising SEQ ID NO:1 (FIG. 2); b) constructing a vector comprising: i)

me isσlaied njcseot'de seαueice operebty Isπkeo to iO a D"orρoτer o r some :γpa of regulatory sequence: and c) introducing the vecior into 3 host cef! for a lime and under conditions sufficient for expression of the λ9-eϊongase enzyme, as appropriate. The host eel! may be, for exarrpre, a eukaryotic cell or a prokaryotlc cell. Sn particular, the prokaryotic ceϊf may be, for example, Escherichia cofi, cyanobacteria or Bad [J us subtifis. The eukaryotjc eel! may be, for exampie, a mammalian cell an insect cef!, a plant ceii or a fungal cell. The fungai cell may be. for example. SaccharorQYces spp. = Candida spp., ϋpomyces spp,. Yarrowte spp., Aspergillus spp., Pen id ilium spp., Neurospora spp. r Kluweromyces spp., Haπsenu j a spjx,, Trichoderma spp., or PJchia spp. In particular, the fungal cell may be a yeast celt such as, for example, Saecharomyces spp.. Candida spp,, Hansenuia spp. and PJchia spp. The yeast cell may afso be, for example. SacGharomyces cerevisiae.

Additionally, the present Invention afso encompasses a vector composing: a) a nucleotide sequence as represented by SEQ ID NO:1 (FiG. 2), operably linked to b) a promoter or regulatory sequence. The invention also includes a host cell comprising this vector. The host celϊ may be, for exampie, a eukaryotic ceii or a prokaryotlc ceil. Suitable eukaryotic ceils and prokaryotic cells are as defined above.

Moreover, the present invention afso includes a pϊant ceϊl, plant seed, plant or plant tissue comprising the above vector, wherein expression of the nucleotide sequence of the vector results in production of at feast one polyunsaturated fatty acid by the plant ceii, plant or pfant tissue. The polyunsaturated fatty acid may be, for example, selected from the group consisting of EDA and ETrA. The Invention afso includes one or more plant oils or fatty acids expressed by the above plant ceϊl, plant seed, plant or plant tissue.

Additionally, the present invention a!so encompasses a transgenic plant comprising the above vector, wherein expression of the nucleotide sequence of the vector results in production of at least one polyunsaturated fatty acid in seeds of the transgenic plant.

Aϊso, the invention includes a mammalian ceil comprising the above vector wherein expression of the nucleotide sequence of the vector results in production of altered levels of, for example, EDA and/or ETrA when the cell is grown in a culture media comprising a fatty acid selected from the group consisting of, for exampie, LA and ALA.

It should also be noted that the present invention encompasses a t r ansgenic, non- human mamma! whose genome composes a DNA sequence encoding a λ9- efongase operabfy Sfnked to a promoter or regulatory sequence. The DNA sequence may be represented by SEQ ?D MO:1 (FfG. 2). The present invention also includes a fluid (e.g.. mifk) produced by the transgenic, non-human mammal wherein the fluid comprises a detectable ϊevel of λ9-efoπgase.

Additionally, the present invention lncfudes a method (i.e. "first" method " ) for producing a polyunsaturated fatty acid comprising the steps of: a) isolating the nucleotide sequence represented by SEQ iD NO:1 (FlG. 2); b) constructing a vector comprising the isolated nucleotide sequence; c) introducing the vector into a host ceϊi under time and conditions sufficient for expression of δ9-eiongase enzyme; and d) exposing the expressed λ9-eiongase enzyme Io a substrate polyunsaturated fatty acid in order to convert the substrate to a product polyunsaturated fatty acid. The "substrate" polyunsaturated fatty acid is, for example, LA and ALA, and the "product 1" polyunsaturated fatty acsd is, for example, EDA or ETrA. respectively. This method may further comprise the step of exposing the product polyunsaturated fatty add to at least one desaturase in order to convert the product polyunsaturated fatty acid to another polyunsaturated fatty acid (Le., J second" method). In this method containing the additional step (i.e., "second" method), the product polyunsaturated fatty acid may be, for example, EDA or ETrA, the "another" polyunsaturated fatty acid may be, for example, DGLA or ETA, respectively, and the at least one desaturase may be, for example, δ8-desaturase. The method containing the additional step (i.e., "second" method) may further comprise a step of exposing the another polyunsaturated fatty acid to at feast one additiona! desaturase in order to convert the another polyunsaturated fatty acid to an additional polyunsaturated fatty acid (i.e., "third" method). In this method containing the additionai step (i.e., "third" method}, the another polyunsaturated fatty acid may be, for example, DGLA or ETA, the "additional" polyunsaturated fatty acid may be. for example, AA or EPA, respectively, and the at least one additional desaturase may be, for example, δ5-desaturase. The method containing the additional step (i.e., "third" method) may further comprise a step of exposing the additionai polyunsaturated fatty acid to at least one additional desaturase and/or at least one additiona! elongase in order to in order to convert the additional polyunsaturated fatty acid to a final polyunsaturated fatty acid (i.e., "fourth- method). In this method containing the additional step (i.e., "fourth" method), the

adαkϊoπai polyunsaturated farty acsd nay De for example AA or EPA. ana the "final ' polyunsaturated faity aod may be, for example, uj3-docosapeniaenoic acid, ωS- docosapemaeπoic acid, ADA or DHA.

The present invention aiso encompasses a composϊϊion comprising at least one polyunsaturated fatty acid selected from the group consisting of the product polyunsaturated fatty acid produced according to the methods described above, the another pofyunsattsrated fatty acid produced according to the methods described above, the additional polyunsaturated faity acid produced according to the methods described above and the final polyunsaturated fatty acid produced according io the methods described above. The product polyunsaturated fatty acid may be, for example, EDA or ETrA. The another polyunsaturated fatty acid may be, for example, DGLA or ETA The additional polyunsaturated fatty acid may be, for example. AA or EPA. The Una! polyunsaturated farty acid may be, for example, ω3- docosapentaeπoϊc acid, ω6-docosapentaenoϊc acid, ADA or DHA. The composition may be. for example, a nutritional composition such as an infant formuϊs, a dietary supplement or a dietary substitute and may be administered to a human or an animal and may be administered enteraliy or parenteraiiy. The nutritional composition may further comprise at least one macron utrient selected from the group consisting of coconut oi, soy oil. canoia oil, monogfycerides, digSycerides, triglycerides, glucose, edible lactose, eiectrodiatysed whey τ eiectrodiaiysed skim milk, milk whey, soy protein, protein hydrolysates, sunflower oif, saffiower oil, corn oil, and flax oil It may also comprise at least one vitamin selected from the group consisting of Vitamins A, G 1 D 5 E, and B complex and at least one mineral selected from the group consisting of cateium magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium and iron.

The present invention also includes a pharmaceutical composition comprising 1 ) at least one polyunsaturated fatty acid (PUFA) selected from the group consisting of the "product" PUFA produced according to the methods described above, the "another" PUFA produced according to the methods described above, the "additional PUFA produced according to the methods described above, or the "final" PUFA produced according to the methods described above and 2} a pharmaceutically acceptable carrier. The composition may be administered to a human or an animal, it may also further comprise at least one element selected from the group consisting of a vitamin, a mineral, a salt, a carbohydrate, an amino acid, a free fatty acid, a preservative, an excipient, an anti-histamine, a growth factor, an antibiotic, a diluent, a phospholipid,

an antioxidant and a pbeλo'ύc comDOunc! it nay be adminisierec en τ era!iy parenferaily, topicatiy, rectaiiy, intramuscularly, subcutaneousϊy mtradeπnaliy or by any other appropriate means.

Additionally, the present invention encompasses an awrnai %ed or cosmet i c comprising at least one PUFA sβfected from the group consisting of the product PUFA produced according to the methods described a Dove, ihe another PUFA produced according to the meihods described above, the additional PUFA produced according to the methods described above and the final PUFA produced according to the methods described above. These PLIFAs have been fisted above and are exemplified in Figure 1.

Additionally, the present invention encompasses 3 method of preventing or treating a condition caused by insufficient intake of polyunsaturated fatty acids comprising administering to ihe patient the compositions above In an amount sufficient to effect prevention or treatment

it should aϊso be noted that each nucleotide and amino acid sequence referred to herein has been assigned a particular sequence identification number. The Sequence Listing (which is found herein) lists each such sequence and its corresponding number.

BRIEF DESCRIPHOM OF THE DRAWiMGS

Figure 1 illustrates the fatty acid biosynthetic pathway and the roie of δ9-elongase in this pathway.

Figure 2 illustrates the nucleotide sequence encoding δ9-eiongasβ {pRAT-5A1 } of ThraustQChvtrid sp. BϊCC 7087(SEQ ID NO:1 ).

Figure 3 illustrates the translated amino acid sequence of δ9-elongase (pRAT-5A1 ) of Thraυstochytrid sp. BlCC 7087{SEQ \D NO:2).

Figure 4 illustrates the amino acid sequence identity between the ' 660R1 ' EST clone (SEQ ID NO: 17), mouse eiongase MEL04 (SEQ NO: 19), and Thraustochytrid sp. BICC eiongase TELO1 (SEQ ID NO: 18).

Figure b iPustrares t^e physica * nap of the consTituttve yeas: express:cn vector. ρYX242, used for the cloning of λ9-e!ongase gene (SEQ SD NO-I ) for ;he production of encoded eiongase enzyme m yeast.

Figure 6 illustrates the PUFA etoπgase activity of the δ9~efongase gene (pRAT-5A1 ) from Thraustochytπd sp. BICC 7087.

Figure 7 iffustrates ihe amino acid sequence identify between eioπgasβ-encoded proteins from Danio rerio (SEQ !D NO: 20} and pRAT-5A1 (SEQ ID NO: 2)

Figure 8 illustrates the amjno acid sequence identity between δ9~efongase-eπcoded proiesns from lsochrysis galbana (SEQ ED NO: 22} and pRAT-5A1 (SEQ ID NO: 2}

Figure 9 illustrates the amino acid sequence identity between λ9-eSongase~enccded proteins from Pavlova safϊna (SEQ ID NO: 24) and pRAT-5A1 (SEQ ID NO: 2).

Figure 10 illustrates the amino acid sequence identity between oroteiπs encoded by Thraustochytrium sp. FjN-IO (SEQ ID MO: 26) and pRAT-5A1 (SEQ ID NO: 2).

Figure 11 illustrates the amino acid sequence alignment of proteins encoded by pRAT-5A1 (SEQ ID NO:2), pRAT-5A1 mutant 1 (SEQ ID NO: 5} and pRAT-5A1 mutant 2 (SEQ ID NO:6}

Figure 12 illustrates the PUFA eiongase activity of the δ9~e!ongase gene (pRAT-5A1) from Thraustochytrid sp. BfCC 7087, the pRAT-5A1 mutant 1 and the pRAT-5A1 mutant 2.

Figure 13 illustrates the amino acid sequence alignment of proteins encoded by pRAT-5A1 (SEQ ID NO:2), pRAT-5A11 (SEQ ID NO: 15) and pRAT-5B (SEQ ID NO:16).

Figure 14 illustrates the physical map of the plant expression vector, pRS1 , used for the cioning of the pRAT-5A1 encoded protein and for expression studies in pϊant seeds.

F gure 15 illustrates -he f aπy add prcλfe of irar-sgensc seeds expressing oRA ϊ -5.A1 versus non-iransgenlc seeds

DETAILSD DESCRlPTiQW OF THE INVENTION

The subject invention relates to the nucleotide and translated amino acsd sequences of a δ9-elongase gene from Thraustochvtrtd sp., for example. Thraustochytrium aureum 7087, or Thrausiochytrid sp. BlCC 7087. Furthermore, the subject invention also includes uses of the gene and of the enzyme encoded by the gene. For example, the gene and corresponding enzyme may be used \n the production of polyunsaturated fatty adds such as. for instance, EDA. EtrA, DGLA. ETA. AA. EPA 7 {i)3-docosapentaenoϊc acid, ωθ-docosapentaersoic acid, ADA and/or DHA which may be added to pharmaceutical compositions, nutritional compositions and to other valuable products.

The A9-Elonqase Gene and Enzyme Encoded Thereby

As noted above, the enzyme encoded by the δ9-βiongase gene of the presen-t invention is essentiaf in the production, via the alternate delta 8 pathway, of long- chain polyunsaturated fatty adds, having a tength greater than 20 carbons. The nucleotide sequence of the isolated Thraustochvtrid sp. BiCC 7087 A9-eSongase gene (ρRAT-5A1) is shown in Figure 2, and the amino acid sequence of the corresponding purified protein is shown in Figure 3.

The conversion of LA to DGL-A and ALA to ETA using a δ9-elongase enzyme and a δ8-desaturase enzyme is referred to as the alternate delta 8 pathway. The conventional delta 6 pathway for converting LA to DGLA and ALA to ETA utilizes a δ6-desaturase enzyme to convert LA to GLA, and ALA to STA, and a δ6-eSoπgase gene to convert GLA to DGLA, and STA to ETA, respectively. In either pathway, the production of AA or EPA is then catalyzed by, for example, a δ5-desaturase. DHA, for example, may be produced upon the conversion of EPA to ω3-docosapentaenoic acid, and ω3-docosapentaenoϊc acid to DHA, utilizing, for example, a C20-elongase and a δ4-dθsaturase t respectively.

Although, for example, DGLA, ETA, AA, EPA, ω3-docosapentaeπoic acid, ω6- docosapentaenoic acid, ADA and/or DHA can be produced via either the alternate delta 8 pathway or the conventional delta 6 pathway, in certain instances, the

akernaie deka 8 pathway may be preferred over the conventional αelta 8 pathway For example, if particular residual omega-6 or omega-3 fatty acid intermediates such as GLA or STA. are not desired during production of DGLA, ETA. AA, EPA. u>3- docosapenfaeαosc acid, ω6-docosaρentaeπolc add, ADA and/or DHA, the alternate delta 8 pathway may be used as an alternative to the conventional delta 6 pathway, to bypass GLA and STA formation.

As discussed above, δ9-eiongase is a necessary enzyme In ihe alternate delta 8 pathway. EPA, for exampSe, cannot be synthesized via the silemate delta 8 pathway without the δS-eiongase gene and enzyme encoded thereby. The isolated δ9- eioπgase enzyme of the present invention converts, for example, AlA to ETrA. The production of ETA from ETrA, and EPA from ETA. is then catalyzed by, for exampte. a δ8-desaturase and a λS-o'esaturase, respectively. As a result of using ihe alternate delta 8 pathway, the intermediate GLA and STA fatty acids are bypassed.

it should be noted that the present invention afso encompasses nucleotide sequences (and the corresponding encoded proteins) having sequences comprising or complementary Io at least 86%, preferabfy at [east 88%, more preferably at least 90%, more preferably at feast 95% and most preferabfy at least 97% of the πucieotides in sequence (i.e., having sequence identity to) SEQ D NO:1 (i.e., the nucleotide sequence of the δ9-eioπgase gene of Thraustochvtrid sp. BiCC 7087). (All integers between 80% and 100% are also considered to be within the scope of the present invention with respect to percent identity.) Such sequences may be from human sources as well as other non-human sources (e.g., C. eJeqans or mouse).

Furthermore, the present invention afso encompasses fragments and derivatives of the nucleotide sequence of the present invention (i.e., SEQ [D NO:1 (shown in Figure 2)), as we(i as of the sequences from other sources, and having the above-described complementarity or correspondence. Functional equivalents of the above-sequences

(i.e., sequences having δ9-e!ongase) are a!so encompassed by the present invention.

it should also be noted that the present invention also encompasses nucleotide sequences or fragments thereof encoding a polypeptide having elongase activity, wherein the amino acid sequence of said polypeptide has at least 86%, preferably at least 88%, more preferably at least 90%, more preferably at least 95% and most

I l

preferabiy at least 87% seαueπce 'αeniiry TO the am 1 no aαd seαuence comprising SEQ ID NO:2 (Aϊi iniegers between 80% and 1G0% are also considered to be within ihe scope of the present invention with respect to percent identity } Such sequences may be from human sources as weϊϊ as other non-human sources (e.g CX efegans or mouse).

The invention also includes a purified polypeptide which elongates polyunsaturated farty acids contaiπϊng unsaturation at the carbon 9 position and has at least 86% amino acid similarity or 'density preferably at least 88% similarity or identity, more preferabiy at teas! 90% similarity or identify, more preferably at ieast 95% similarity or identity, and most preferably at least 97% similarity or identity to the amino add sequence (Le . SEQ I'D NO:2 (shown in Figure 3)), polypeptide or protein of ihe above-noted proteins which are. in turn, encoded by the above-described nucleotide sequences. AIf iniegers between 80-100% similarity or identity are aiso included within the scope of the invention.

The term "identity" " refers to the reiatedness of two sequences on a nucieotide-by- nudeotide basis over a particular comparison window or segment. Thus, identity is defined as the degree of sameness, correspondence or equivalence between the same strands (either sense or antisense) of two DNA segments. "Percentage of sequence identity is calculated by comparing two optimally aligned sequences over a particular region, determining the number of positions at which the identicai base occurs in both sequence in order to yield the number of matched positions, dividing the number of such positions by the total number of positions in the segment being compared and multiplying the result by 100. Optima! alignment of sequences may be conducted by the algorithm of Smith & Waterman, AppL Math. 2:482 {1981 ), by the algorithm of Needieman & Wunsch, J. MoI. BioL 48:443 (1970), by the method of Pearson & Lipman, Proc. NatS. Acad. Sci. (USA) 85:2444 (1988) and by computer programs which implement the relevant algorithms (e.g., Higgins et ai., CABiOS. 5Lt 51-153 (1989)} » FASTDB (infeiiigenetics), BLAST (National Center for Biomedical information; Altschul et al., Nucleic Acids Research 25:3389-3402 (1997)), PiLEUP (Genetics Computer Group, Madison, Wf) or GAP, BESTFiT, FASTA and TFASTA (Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, Madison, Wl). (See U.S. Patent No. 5,912,120.)

For purposes of the present invention, "complementarity" is defined as the degree of reiatedness between two DNA segments, it is determined by measuring the ability of

:he sense strand of oie DNA segmert to hybrdize wnr me anusenss strand of me other DNA segment, under aopropriate conditions, to form a double heϋx, In the double heisx. adenine appears in one strand, thymine appears in Ehe oϊher strand. Similarly, wherever guanine is found in one strand, cytosine is found in the other. The greater ϊhe re!aiedness between the nucleotide sequences of two DUA segments, the greater the ability to form hybrid duplexes between the strands of the two DNA segments,

"Similarity" " between two amino acid sequences is defined as the presence of a series of identical as weli as conserved amino add residues in both sequences. The higher the degree cf similarity between two amino acid sequences, the higher the correspondence, sameness or equivalence of the two sequences. ("Identity " between two amino acid sequences is defined as ϊhe presence of a seπes of exactly alike or invariant amino acid residues in both sequences.)

The definitions of "cørnpiementarity 1" , "identity 1" and "similarity * are weiϊ known Io those of ordinary skill in the art.

"Encoded by" refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 amino acids, more preferably at feast 8 amino actds. and even more preferably at feast 15 amino acids from a polypeptide encoded by the nucleic acscl sequence.

The present invention also encompasses an isolated nucleotide sequence which encodes PUFA efongase activity and that is hybridizable, under moderateiy stringent conditions, to a nucleic acid having a nucleotide sequence comprising or complementary to the nucleotide sequence comprising SEQ ID NO:1 {shown in Figure 2). A nucieic add molecule is "hybridϊzabie" to another nucleic acid molecule when a single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and ionic strength (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York)). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. "Hybridization" requires that two nucleic acids contain complementary sequences. However, depending on the stringency of the hybridization, mismatches between bases may occur. The appropriate stringency for

hybridizing r.udβiC aces depends OP φe iengih of the nucϊeic aασs and the degree of corrtpiemeniafion. Such variables are we if known in the art. More specifically. :he greater the degree of similarity or homology between TWO nucleotide sequences, the greater the value of Tm for hybrids of nucleic adds having those sequences. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et a!., supra). For hybridization with shorter nucleic acids, the position of mismatches becomes more important, and the length of the oϋgoϊiucieo-tde determines its specificity (see Sambrook et si., supra).

As used herein, an "isoϊated nucleic acid fragment or sequence" is a polymer of RNA or DMA thai is singϊe- or double-stranded, optionally containing synthetic, non-natural or aitered nucleotide bases. An isofetecf nucfeic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDMA. genomic DNA or synthetic DMA. (A "fragment " of a specified polynucleotide refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least aboui 8 nucleotides, preferably a! teas! about 8 nucleotides, more preferably at feast about 10 πucfeotides, and even more preferably at teast about 15 nucleotides, and most preferable at least about 25 nucleotides identical or complementary to a region of the specified nucleotide sequence.) Nucleotides (usually found in their 5'-monophosphaie form) are referred to by their singfe letter designation as follows: "A" for adenylate or deoxyadenylate (for RNA or DMA, respectively), "C" for cyttdylate or deoxycytkfytate, "G" for guanylate or deoxyguanyiate, "U" for urϊdylate, " T * for deoxythymidyfete. "FT for purines (A or G), "Y" for pyrimidϊnes (C or T), "1 K" for G or T, "H" for A or C or T, 'T for iπosine, and "N' 1 for any nucleotide.

The terms "fragment or subfragment that is functionally equivalent" and "functionally equivalent fragment or subfragment" are used interchangeably herein. These terms refer to a portion or subsequence of an isolated nucleic acid molecule in which the ability to alter gene expression or produce a certain phenotype is retained whether or not the fragment or subfragment encodes an active enzyme. For example, the fragment or subfragment can be used in the design of chimeric constructs to produce the desired phenotype in a transformed plant. Chimeric constructs can be designed for use in co-sυppression or antisense by unking a nucleic acid fragment or subfragment thereof, whether or not it encodes an active enzyme, in the appropriate orientation relative to a plant promoter sequence.

The terms ' hornoiog/ " 'icmdcgous . 'substantially s'rr.iiar' and " cor-espoπding substantially "1 are used interchangeably herein. They refer to nucleic acid molecufes wherein changes in one or more nucleotide bases does noi afect the ability of the nucieic acid rnofecuSe to mediate gene expression or produce a certain phenoiype. These terms also refer to modifications of the nucfeϊc add molecules o f the instant invention such as deletion or insertion of one or more nucleotides that do not substantia tϊy after the functional properties of the resulting nucleic acid mofecufe relative to the initial, unmodified moϋecuie. If is therefore understood, as those skfFled in the art will appreciate, that the invention encompasses more than the specific exemplary sequences.

"Gene" refers Io a nucleic acid molecule that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non- coding sequences) the coding sequence.

" Native gene" " refers to a gene as found in nature with its own reguiatory sequences. In contrast, "chimeric construct " refers to a combination of nucleic acid molecules that are not normally found together In nature. Accordingly, a chimeric construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that normafiy found in nature. (The term "isolated" means that the sequence is removed from its natural environment.)

A "foreign" gene refers to a gene not normaliy found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric constructs. A "transgene" is a gene that has been introduced into the genome by a transformation procedure.

"Coding sequence" refers to a DNA sequence that codes for a specific amino acid sequence. "Regulatory sequences" refer to nucleotide sequences located upstream (5 * non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Reguiatory sequences may include, but are not limited to, promoters, translation ieader sequences, introπs, and polyadenylation recognition sequences.

'Promoter" refers to a DNA sequence capable of co j τtolPng ihe expression of a coding sequence or functional RNA The promoter seαϋence consists of proxinai and more distal upstream elements, ihe tetter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the ievei or tissue-specificity of a promoter. Promoter sequences can also be located within :he transcribed portions of genes, and/or downstream of the transcribed sequences. Promoters may De derived tn their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. K is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cefi types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonfy referred to as "constitutive promoters." New promoters of various types useful in pEaπt ceifs are constantly being discovered; numerous examples may be found in the compilation by Gkamuro and Goldberg, (1989} Biochemistry of Plants 15:1 -82. ft is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DMA mofecuϊes of some variation may have identical promoter activity.

An "intrσπ * is an intervening sequence in a gene that does not encode a portion of the protein sequence. Thus, such sequences are transcribed into RNA but are then excised and are not translated. The term is also used for the excised RNA sequences. An "exon" is a portion of the sequence of a gene that is transcribed and is found in the mature messenger RNA derived from the gene, but is not necessarily a part of the sequence that encodes the final gene product.

The "translation leader sequence" refers to a DNA sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described {Turner, R. and Foster, G. D. (1995) Molecular Biotechnology 3:225).

he 3 non coαrg seque ni -es " refer c DKA se^'^nces located cowrs r ean of a Godrg seα^eπce aπσ nduαe oosyaderylauoπ recogoπor sequences and Other sequences encoding regulatory signals caoabie o f affecting mRNA processing or gene expression The poSyadeπyiatsoπ ssgnal ss usua^y characterized by affecang the addition of poϊyadenyisc aαd tracts to the 3' end oτ the rrsRNA precursor The use of different 3 1 non-coding sequences is exemplified by lngelbrecht et a! , {1989} Plant Ceil 1 67 - 1 -880

RNA transcript' refers io the proσucϊ resufeng from RNA polymerase-caiafyzed xranscπpoon of a DNA seαueαce When τ he RNA ^ranscnpt >s a perfect complementary copy of tr>e DNA sequence. «t is referred io as the pπmary transcript or tt may be a RNA sequence derived from Dost-iranscπpisortal processing of the pπmary transcript and is referred to as the mature RNA Messenger RNA (mRNA)" refers Io the RNA that is wthoul ϊntrons aπo that can be transiaied into protein by the celi "cDNA "1 refers to a DMA in at ts complementary to and synthesized from a mRNA template using the enzyme reverse transcriptase Tπe cDNA can be ssngle- sϊranded or converted ϊπ»O the double-stranded form using ϊhe KSenow molecufe of DNA polymerase I ' Sense" RNA refers to RNA transcript that snciudes ihe mRNA and can be translated into pro^em wrthtπ a cef! or in vtτro "Antϊsense RNA" refers to an RNA transcript that is complementary to alt or part of a target primary transcript or rnRNA and that blocks the expression of a targei gene (U S Patent No 5,107,065) The compfementaπty of an antiseπse RNA may be with any part of the specific gene transcript, i.e.. at the 5' non-coding sequence, 3 1 non-coding sequence, introrts, or the coding sequence "Functional RNA" refers to aπtiseπse RNA, rϊbozyme RNA 1 or other RNA that may not be translated but yet has an effect on cellular processes The terms "complement" and "reverse complement" are used interchangeably herein with respect to mRNA transcripts, and are meant to define the antisense RNA of the message.

The term "endogenous RNA" refers to any RNA which is encoded by any nucleic acid sequence present m the genome of the host prior to transformation with the recombinant construct of the present invention, whether naturaϋy-occurring or noπ- naturaiiy occurring, i e , introduced by recombinant means, mutagenesis, etc

The term "non-πaturaϋy occurring" means artificial, not consistent with what is normally found in nature.

The term 'operab-y ύr.kecf fetes s :o the associat'on cf ".Ldeic aaά sec^ences on 3 single nude-c ac ! d mo^ecuse so that the funeύon of one is reguiaied by me other For example, a promcier is operabiy ϊinked with a ceding sequence when i! is capable ef regulating the expression of that coding sequence (i e., that the coding sequence is under ihe transcriptional controi of the promoter). Coding sequences can be operabiy fsnked to regulatory sequences in a sense or arrasense orientation, In another example, the complementary RNA regions of the invention can be operabiy linked, either direct'y or indirectly. 5 ' to the target rπRNα, or 3" to rne target rnRMA, or within zhe iarget mRNA, or a first complementary' region ss 5 " and its complement is 3' ϊO the iargei mRNA.

The term ' expression", as used herein, refers to the production of 3 functional end- product. Expression of a gene fnvofves transcription of the gene and translation of the mRNA into a precursor or mature protein. "Antisense inhibition " refers to the production of antisense RNA transcripts capable of suppressing the expression of :he target protein. " Co-suppression" refers to the production of sense RHA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Patent No. 5,231.020).

" Mature" protein refers to a post-translatϊonairy processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed. ' 'Precursor" protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization sϊgnaϊs.

"Stabfe transformation" refers to the transfer of a nucleic acid molecule into a genome of a host organism, including both nuciear and organeϋar genomes, resulting in genetically stable inheritance. In contrast, "transient transformation" refers to the transfer of a nucleic acid molecule into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without integration or stable inheritance. Host organisms containing the transformed nucleic acid molecules are referred to as "transgenic" organisms. The preferred method of cell transformation of rice, com and other monocots is the use of particie-accelerated or "gene gun" transformation technology (Klein et al., (1987) Nature (London) 327:70-73; U.S. Patent No. 4,945,050), or an Agrobacterium-mediated method using an appropriate Ti plasmid containing the transgene (Ishida Y. et a!., 1996, Nature

Biotech 14.7^5-750; he "ernt ' transformation ' ?s t-sed here'n refers to both s*able transformation and transient rar-sformatton

Standard recombinant DNA and molecular cfoning techniques used herein are wefi known in the art and are described more fully in Sambrook, J.. Fritsch, E.F and Manϊatϊs, T. Molecular Cloning: A Laboratory Manual: Cofd Spring Harbor Laboratory Press: CoSd Spring Harbor, 1989 (hereinafter "Sambrook " ).

The term ''recombinant" refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of ϊsofafed segments of nucleic acids by genetic engineering techniques,

" 1 PCR" or 'Polymerase Chain Reaction" is a technique for the synthesis of large quantities of specific DNA segments, consists of a series of repetitive cycles (Perkϊn Efmer Cetus instruments, Morwaik, CT). Typicatiy, the double stranded DMA Is heat denatured, the two primers complementary to the 3 1 boundaries of the target segment are annealed at low temperature and then extended at an intermediate temperature. One set of these three consecutive steps is referred to as a cycle.

Polymerase chain reaction ("PCR") is a powerfu! technique used to amplify DMA millions of fold, by repeated replication of a template, in a short period of time. (Muϋis et a!, Cold Spπng Harbor Sγrπp. Quant. Biol. 51:263-273 (1986); Eiiich et af, European Patent Application 50,424; European Patent Application 84,796; European Patent Application 258,017, European Patent Application 237,362; MuSHs, European Patent Application 201 ,184, MuHis et aϊ U.S. Patent No. 4,683,202; Erlich, U.S. Patent No. 4,582,788; and Saiki et ai, U.S. Patent No. 4,683,194). The process utilizes sets of specific in vitro synthesized oligonucleotides to prime DNA synthesis. The design of the primers is dependent upon the sequences of DNA that are desired to be analyzed. The technique is carried out through many cycles (usualiy 20-50) of melting the template at high temperature, allowing the primers to anneal to complementary sequences within the template and then repϊicating the template with DNA polymerase.

The products of PCR reactions are analyzed by separation in agarose gels followed by ethϊdium bromide staining and visualization with UV transillumination. Alternatively, radioactive dNTPs can be added to the PCR in order to incorporate label into the products. In this case the products of PCR are visualized by exposure

of the gβs io x-rsy ' i!m ' he sd^ed aαvamage of radioiabeϋng PCR orcducfs is ;haι the ϊevets of sndivsduai amplification products can be quantitated

The terms "recombinant construct', " expression construcf and " recombinant expression construcf are used interchangeably herein. These terms refer to a functional unit of genetic materia! that can be inserted into the genome of a ceti using standard methodology weft known to one skilfed in the art. Such construct may be itself or may be used in conjunction with a vector. If a vector is used then the choice of vector is dependent upon the method that will be used to transform host plants as is well known to those skilled in the art. For exampie, a piasmϊd vector can be used. The skiϋed artisan is wei! aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cefSs comprising any of the isoϊated nucϊeic acid molecules of the invention. The skiffβd artisan w*!i also recognize that different independeni transformation events will result In different levels and patterns of expression (Jones et af., {1985} EMBO J. 4:2411-2418: De Almeida et a!., (1989) MoL Gen. Genetics 218:78-86), and thus thai muϊtipfe events must be screened in order to obtain ϊines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRMA expression. Western analysis of protein expression, or phenotypic analysis.

Production of the δ9-Eioπqase Enzyme

Once the gene encoding the eϊongase enzyme has been isolated, it may then be introduced into either a prokaryotic or eukaryotic host cell through the use of a vector or construct. The vector, for example, a bacteriophage, cosmid, or ptøsmϊd, may comprise the nucleotide sequence encoding the δ9-e!ongase enzyme, as we!! as any regulatory sequence (e.g., promoter) which is functional in the host ceff and is able to elicit expression of the elongase encoded by the nucleotide sequence. The regulatory sequence is in operable association with or operabiy linked to the nucleotide sequence. (As noted above, regulatory is said to be "operabiy linked" with a coding sequence if the regulatory sequence affects transcription or expression of the coding sequence.) Suitable promoters include, for example, those from genes encoding alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglucoisomerase, phosphoglycerate kinase, acid phosphatase, T7, TP!, lactase, rnetallothionein, cytomegalovirus immediate early, whey acidic protein, giucoamyiase, and promoters activated in the presence of galactose, for example,

GAL/ and GAL/G. Additionally, nucleotide secuences wdic h encode ether oroxeins. oligosaccharides, lipids etc may also be sndudeα within the vecior as α'eii as oiher regulatory sequences such as a polyadenylatfon signal (e.g., the poly-A signal of SV- 4QT-antigen, ova!aiburran or bovine growth hormone). The choice of sequences present in the construct is dependent upon the oesired expression products as we!! as the nature of the host cefϊ.

As noted above, once the vector has been constructed, it may then be introduced into the host ceil of choice by methods known to those of ordinary skiff m the art including, for example, transfection, transformation and eiectroporaiion (see Molecufar Ciontng: A Laboratory Manual, 2 nd ed., Vo!. 1-3, ed. Sambrook et aL Coid Spring Harbor Laboratory Press (1989)). The host cell is then cultured under suitable conditions permitting expression of the genes ieading to the production of the desired PUFA 1 which is then recovered and purified.

Examples of suitable prokaryotϊc host cells include, for example, bacteria such as Escherichia coii, Bacittus subtiiis as well as cysnobacieria such as Spiruifna SPP. (i.e., bSue-greeπ aigae). The eukaryotϊc ceil may be, for example, a mammalian cell, an insect ceil, a plant ce!! or a fungal eel. The fungai ceϊi may be, for example, Saccharomyces spp., Candida spp., ϋpomyces spp., Yarrowia spp., Aspergillus spp., Peniciliium spp., Neurospora spp., KJuyveromycβs spp., Hanseπuia SEE., Trichoderma spp., or Pichia spp. In particular, the fuπgaϊ ceil may be a yeast ceil such as, for example, Saccharomyces spp., Candida spp., Hansenula spp. and Pichia spp. The yeast ceil may also be Saccharomyces cerevisiae.

Expression in a host eel! can be accomplished in a transient or stable fashion. Transient expression can occur from introduced constructs which contain expression signals functional in the host cefl, but which constructs do not replicate and rarely integrate in the host cell, or where the host cell is not proliferating. Transient expression also can be accomplished by inducing the activity of a regulatable promoter operably linked to the gene of interest, although such inducible systems frequently exhibit a Sow basal level of expression. Stable expression can be achieved by introduction of a construct that can integrate into the host genome or that autonomously repϊicates in the host cell. Stable expression of the gene of interest can be selected for through the use of a selectable marker located on or transfected with the expression construct, foliowed by selection for cells expressing the marker. When stable expression results from integration, the site of the constructs integration

can occur ra^dorply within :πe hos: genorre or can be targeted through the jse of constructs containing regions of homology wkh ihe host genome sufficient to target recombination with ihe HOST [OCUS. Where constructs are targeted to an endogenous tocus. all or some of the transcriptional and transiationaf regulatory regions can be provided by ihe endogenous focus

A transgenic marnmsi may aiso be used in order to express the A9-eiongase enzyme and ultimately the PUFA(s) of interest. More specifically, once the above-described construct is created, it may be inserted Into the pronucleus of an embryo. The embryo may then be implanted into a recipient ferπafe. Alternatively, a nuclear transfer method could atso be utilized (Schnieke et a!.. Science 278:2130-2133 (1997))- Gestation and birth are then permitted (see. e.g., U.S. Patent Mo. 5.750.176 and U S. Patent No. 5,700,671), Mϋk, tissue or other fluid sampfes from the offspring should then contain altered levels of PUFAs. as compared to the ieveis norrrsaiy found in ihe non-transgenic animal Subsequent generations may be monitored for production of the altered or enhanced levels of PUFAs and thus incorporation of the gene encoding the desired desaturase enzyme into their genomes. The mammal utilized as the host may be selected from the group consisting of. for example, a mouse, a rat, a rabbi!, a pig, a goat, a sheep, a horse and a cow. However, any mammal may be used provided it has the ability to incorporate DNA encoding the enzyme of interest into its genome.

For expression of a eiongase polypeptide, functional transcriptional and translattonal initiation and termination regions are operabfy finked to the DNA encoding the eiongase polypeptide. Transcriptional and transiatioπal initiation and termination regions are derived from a variety of nonexclusive sources, including the DNA to be expressed, genes known or suspected to be capable of expression in the desired system, expression vectors, chemical synthesis, or from an endogenous locus in a host cell. Expression in a plant tissue and/or plant part presents certain efficiencies, particularly where the tissue or part is one which is harvested early, such as seed, leaves, fruits, flowers, roots, etc. Expression can be targeted to that location with the plant by utilizing specific regulatory sequence such as those of U.S. Patent Nos. 5,463,174, 4,943,674, 5,106,739, 5,175,095, 5,420,034, 5,188,958, and 5,589,379. Alternatively, the expressed protein can be an enzyme which produces a product which may be incorporated, either directly or upon further modifications, into a fluid fraction from the host plant. Expression of a eiongase gene, or aπtisense eiongase transcripts, can alter the ieveis of specific PUFAs, or derivatives thereof, found in

oianf oans and'or part issues " ie eiongase polypeofiαe cocing region may be expressed eiiher by itseif or with other genes. >n order to produce tissues and'or p^aπt parts containing higher proportions of desired PUFAs or in which the PUFA composition more cioseiy resembles that of human breasi milk (Prieto et al, PC " publication WO 95/24494). The ienninalion region may be derived from the 3 ' region of the gene from which the initiation region was obtained or from a different gene. A iarge number of termination regions are known to and have been found to be satisfactory fπ a variety of hosts from the same and different genera and specres. The termination region usually is selected as a matter of convenience rather than because of any particular property

As noted above, a plant (e.g.. Glvcjηe max (soybean) or Brassica napus (canofa)) or plant tissue may afso be utilized as a host or host cell, respectively, for expression of the eiongase enzyme which may, in turn, be utilized in the production of polyunsaturated fatty acids, ^lore specifically, desired PUFAS can be expressed in seed. Methods of isolating seed oils are known in the art. Thus, in addition to providing a source for PUFAs. seed oil components may be manspuiafed through the expression of the eiongase gene, as well as perhaps desaturase genes and other eiongase genes, in order Io provide seed oils that can be added to nutritionai compositions, pharmaceutical compositions, animal feeds and cosmetics. Once again, a vector which comprises a DMA sequence encoding the eiongase operably linked to a promoter, will be introduced into the plant tissue or pfant for a time and under conditions sufficient for expression of the eiongase gene. The vector may also comprise one or more genes that encode other enzymes, for example, efongase, δ4- deεaturase, δ5-desaturase, δ6-desaturase, δiO-desaturase, δi2-desaturase, δ15- desaturase, δi7-desaturase s and/or δ19-desaturase. The piant tissue or piant may produce the relevant substrate upon which the enzymes act or a vector encoding enzymes which produce such substrates may be introduced into the plant tissue, piant ceil or plant. In addition, substrate may be sprayed on plant tissues expressing the appropriate enzymes. Using these various techniques, one may produce PUFAs by use of a pfant cell, piant tissue or piant. it should aiso be noted that the invention also encompasses a transgenic plant comprising the above-described vector, wherein expression of the nucleotide sequence of the vector results in production of a polyunsaturated fatty acid in, for example, the seeds of the transgenic plant.

i he regeneration, development snd cb'ivanoπ of plants Torn sfngte plant protop'asi iransformanis or from various transformed expiams is ^eH known in the ad (Weissbach and Weissbach, in: Methods for Plant Mofecϋar Biology, (Eds.), Academic Press. Inc. San Diego, CA, (198S)) This regeneration and growth process typϊcaiϊy includes the steps of selection of transformed cells, eulturing these individualized cefϊs through the usual stages of embryonic development through the rooted piantfei siage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign, exogenous gene that encodes a protein of interest is weil known to the art. Preferably, the regenerated plants are self-poSIϊnaied to provide homozygous transgenic plants. Otherwise, poϋen obtained from the regenerated plants is crossed to seed-grown plants of agronomics * Iy important Sines. Conversely. polSen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide is cuitivated using methods wei! known to one skilled in the art.

There are a variety of methods for the regeneration of plants from plant tissue. The particular method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated.

Methods for transforming dicots. primarily by use of Agrobacterium tumefatiens, and obtaining transgenic plants have been published for cotton (U.S. Patent No. 5,004,863, U.S. Patent No. 5,159,135, U.S. Patent No. 5,518, 908); soybean (U.S. Patent No. 5,569,834, U.S. Patent No. 5,416,011, McCabe et. al., BiolTechπσlogy 6:923 (1988), Christou et al., Plant Physiol. 87:671-674 (1988)); Brassica (U.S. Patent No. 5,463,174); peanut (Cheng et a!., Plant Cell Rep. 15:653-657 (1996), McKentiy et al., Plant Cell Rep. 14:699-703 (1995)); papaya; and pea (Grant et al.. PEant Cell Rep. 15:254-258, (1995)).

Transformation of monocotyledons using electroporatioπ, particle bombardment, and Agrobacterium have also been reported. Transformation and plant regeneration have been achieved in asparagus (Bytebier et a!., Proc. Natl. Acad. Sci. (USA) 84:5354, (1987)); barley (Wan and Lemaux, Plant Physiol 104:37 (1994)}; Zea mays (Rhodes et al., Science 240:204 (1988), Gordon-Kamm et al., Plant Cell 2:603-618

(1990^ t-roirrn ei ai Bioi τ ecfrndogy S'833 π 390} Koziel et a! Biotechnology λ λ 19^, (1993), Arrnsirong ei a! Crop Science 35 550-557 (1S95;>. oat (So g ers et aϊ , BsolTechnology 10. 1 S 89 (1992)}; orchard grass (Horn et al,. Plant Cell Rep, 7 ^69 (1988)); rice (Tonyama et a* . TheorAppl Genet 205 * 34, (1986): Part ef aϊ , Plant MoL BioL 32 1135-1148. (1996): Abediπia et at., Ausi. J Plant Physiol 24:133-141 (1997): Zhang and Wu. Theor AppL Genet 76:835 (1988), Zhang et af. Plant Cell Rep. 7:379. (1988): Batfraw and Half, Plant Sd 36.181-202 (1992): Chtistou ai al . Bio/Technology 9"957 (1991)); rye (De ia Pena et ai.. Nature 325:274 (1987)): sugarcane (Bower and Bϊrcb, PtantJ. 2:409 (1992)); tali fescue (Wang et al . BblTechnoSogy 10:691 (1992)). and wheat (Vasiϊ ef ai.. Bio/Technology 10:667 (1992); U.S. Patent No. 5.631 ,152)

Assays for gene expression based on the transient expression of cloned nudec acid constructs have been developed by introducing ihe nucfeic add moiecutes into plant cells by poiyethyiene glycol treatment, efeciroporaiioπ, or particle bombardment (Marcotte et a!.. Nature 335:454-457 (1988); Marcoite ex at.. Plant Cell 1 "523-532 (1989); McCarty et ai., Cell 66:895-905 (1991); Nation ei a!.. Genes Dev. 6:609-618 (1992); Golf et aϊ., EMBO J, 9:2517-2522 (1990».

Transient expression systems may be used to functionatfy dJssect gene constructs (see generally, Malϊga et al., Methods in Plant Molecular Biofogy, Coid Spring Harbor Press (1995)}. It is understood that any of the nucleic acid moSecules of the present invention can be introduced into a plant cβif in a permanent or transient manner in combination with other genetic eiements such as vectors, promoters, enhancers etc.

fn addition to the above discussed procedures, practitioners are famifiar with the standard resource materials which describe specific conditions and procedures for the construction, manipulation and isolation of macromoiecules (e.g., DNA molecules, piasmids, etc.), generation of recombinant organisms and the screening and isolating of clones, (see for example, Sambrook et ai., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989); Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995); Birren et at., Genome Analysis: Detecting Genes, 1 , Cold Spring Harbor, New York (1998); Birren et al.. Genome Analysis: Analyzing DNA, 2, Cold Spring Harbor, New York (1998); Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer, New York (1997)).

' he stbsFates λ'bc h rray be D-oduced Cy τ he nos * cell β'ther naturally or transgenics!:/, as wed as ihe enzymes which may De encooed by DKA sequences present fn the vector whsch rs subsequently reroαuced mio ihe host cell, am shown in

Figure 1.

In view of ihe above, the present invention encompasses a method of producing ihs ^9-tongase enzyme comprising the steps of: 1) ssoiaiing the nucleotide sequence of ihe gere encoding the eloαgase enzyme; 2) constructing a vector comprising said nucleotide sequence; and 3) introσucing said vector into a host celt under isme and conditions sufficient for ϊhe production of the efongase enzyme.

The present iπvent'on also encompasses a method of producing polyunsaturated fatty acids comprising exposing an add to ihe enzyme such that the eiongase conveys ihe acid to a polyunsaturated fatly acid. For example, when LA is exposed io a A9-etongase enzyme, it is concerted to EDA EDA may then be exposed io. f or example, δ8~desaiurase which converts the EDA to DGLA. The DGLA ihen may be converted io AA by exposing the DGLA to, for exampfe. δ5-desafurase. Thus, λ9- eiongase may be used in ihe production of poϊy unsaturated fatty acids which may be used, in turn, for particular beneficia! purposes, or may be used in the production of other PLfFAs.

Uses of the δ9-Elonqase Gene

As noted above, the isolated eiongase gene and the efongase enzyme encoded thereby have many uses. For example, the gene and corresponding enzyme may be used indirectly or directly in the production of polyunsaturated fatty acids, for exampfe, δ9-elongase may be used in the production of EDA, ETrA 5 DGLA 1 ETA, AA, EPA, ω3-docosapentaenoic add, ωδ-docosapentaeπoϊc acid, ADA and/or DHA. ("Directly" ts meant to encompass the situation where the enzyme directly converts the acid to another acid, the latter of which is utilized in a composition (e.g., the conversion of LA to EDA}. "Indirectly "1 is meant to encompass the situation where an acid is converted to another acid (i.e., a pathway intermediate) by the enzyme (e.g., LA to EDA) and then the latter acid is converted to another acid by use of a non- eioπgase enzyme (e.g., EDA to DGLA by, for example, δ8-desaturase). These polyunsaturated fatty acids (i.e., those produced either directly or indirectly by activity of the eiongase enzyme) may be added to, for example, nutritional compositions,

eharmaceutscai ccnoosiiions, ocsrnei.cs, and arnr-ai feeds a'\ of which are encompassed by the present invention. These uses are described, in detail, below

Nutritional Compositions

The present invention includes nutritions! compositions. Such compositions, for purposes of the present invention, include any food or preparation for human consumption including for enteral or parenteral consumption, which when taken into the body (a) serve to nourish or build up tissues or suppiy energy arid/or (b) maintain. restore or support adequate nuiritional status or metabolic function.

The nutritional composition of the present invention comprises at least one oil or acid produced directly or indirectly by use of the eSoπgase gene, in accordance with the present invention, and may either be in a solid or fsquid form. Additionally, the composition may incϊude edible macronutrients, vitamins and minerals in amounts desired for a particular use. The amount of such ingredients wffl vary depending on whether the composition is intended for use with normal, healthy infants, children or adults having specialized needs such as those which accompany certain rnetabofic conditions (e.g., metaboϊic disorders).

Examples of rnacronutrients which may be added to the composition include but are not limited to edibfe fats, carbohydrates and proteins. Examples of such edible fats include but are not limited to coconut oil, soy oif, and mono- and dsgfycerides. Examples of such carbohydrates include but are not limited to giucose, edible lactose and hydroiyzed search. Additionally, examples of proteins which may be utilized in the nutritional composition of the invention incfude but are not iimrted to soy proteins, efectrodϊaiysed whey, electrodialysed skim milk, milk whey, or the hydroiysates of these proteins.

With respect to vitamins and minerals, the following may be added to the nutritional compositions of the present invention: calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and Vitamins A, E, D, C, and the B complex. Other such vitamins and minerals may also be added.

The components utilized in the nutritional compositions of the present invention will be of semi-purified or purified origin. By semi-purified or purified is meant a materia! which has been prepared by purification of a natural material or by synthesis.

Exanoles cf πutπϋonaϊ compositio-s of :he present invention indude but are noi ifmiied to Man! formulas, dietary supplements, dietary substitutes, ana rehydration compositions. Nutritional compositions of particular interest include but are net limited to those utilized for enteral and parenteral supplementation for infants, specialty infant formulas, supplements " 'or the eiderfy. and supplements for those with gastrointestinai difficulties and/or mafabsoφtioπ

The nutritional composition of the present invention may also be added to food even when supplementation of the diet is not required. For example, the composition may be added to food of any type including but not limited Io margarines, modified butters, cheeses, milk, yogurt, chocolate, candy, snacks, safad oils, cooking oils, cooking fats, meats, fish and beverages.

In a preferred embodiment of the present invention, the nutritional composition is an enteral nuirittonaϊ product, more preferably, an adult or pediatric enteraϊ nutritional product. This composition may be administered to adults or children experiencing stress or having specialized needs due to chronic or acute disease states. The cornposϊϋon may comprise, in addition to polyunsaturated fatty adds produced in accordance with the present invention, macronutrients, vitamins and minerals as described above. The rnacronutrients may be present in amounts equivalent to those present in human milk or on an energy basis, i.e., on a per calorie basis.

Methods for formulating liquid or solid enteral and parenteral nutritional formulas are wei known in the art. (See also the Examples below.)

The enteral formula, for example, may be sterilized and subsequently utilized on a ready-to-feed (RTF) basis or stored in a concentrated liquid or powder. The powder can be prepared by spray drying the formula prepared as indicated above, and reconstituting it by rehydrattng the concentrate. Adult and pediatric nutritional formulas are weil known in the art and are commercially avaiϊable {e.g., Sϊmilac®, Ensure®, Jevϊty® and Aϋmeπtum® from Ross Products Division, Abbott Laboratories, Columbus, Ohio). An oil or acid produced in accordance with the present invention may be added to any of these formulas.

The energy density of the πutritiona! compositions of the present invention, when in liquid form, may range from about 0.6 Kcal to about 3 Kcal per mi. When in solid or powdered form, the nutritional supplements may contain from about 1.2 to more than

9 Kcaϊs per gram, creferabiy aboui 3 to 7 Keats oer gm in gene r af. the osmolality of a liquid product should he iess than 703 mOsm and. more preferably. «ss than 660 mOsm.

The nutritional formula may incfi.de macronuments. vitamins, and minerals, as noied above, in addition to the PUFAs produced in accordance with the present invention. The presence of these additional components heips the individual ingest the minimum daily requirements of these elements. In addition to the provision of PLfFAs, it may also be desirable to add zinc, copper, folic acid and antioxidants io the composition. It is believed that these substance boos! a stressed immune system and wifS therefore provide further benefits to the individual receiving the composition, A pharmaceutical composition may also be supplemented with these elements .

in a more preferred embodiment the nutritional composiiron comprises, in addition to antioxidants and at Seas! one PUFA. a source of carbohydrate wherein at least 5 weight percent of the carbohydrate is indigestible oligosaccharide. In a more preferred embodiment, the nutritional composition additionally comprises protein, taurine, and carnitine.

As noted above, the PUFAs produced in accordance with the present invention, or derivatives thereof, may be added to a dietary substitute or supplement particularly an infant formula, for patients undergoing intravenous feeding or for preventing or treating malnutrition or other conditions or disease states. As background, it shoufd be noted that human breast milk has a fatty acid profile comprising from about 0.15% to about 0.36% as DHA, from about 0.03% to about 0.13% as EPA, from about 0.30% to about 0.88% as AA 5 from about 0.22% to about 0.67% as DGLA, and from about 0.27% to about 1.04% as GLA. Thus, fatty acids such as AA, EPA and/or DHA, produced in accordance with the present invention, can be used to after, for example, the composition of infant formulas in order to better replicate the PUFA content of human breast milk or to alter the presence of PUFAs normally found in a non-human mammal's mitk. In particular, a composition for use in a pharmacologic or food supplement, particularly a breast milk substitute or supplement, will preferably comprise one or more of AA, EPA, DGLA 1 and DHA. More preferably, the oil will comprise from about 0.3 to 30% AA, and from about 0.2 to 30% DGLA.

Parenteral nutritional compositions comprising from about 2 to about 30 weight percent fatty acids calculated as triglycerides are encompassed by the present

iπvenι.0" C'he- \τ.ar\\rs irarticuiariy fat-solubie /namms such as vriarππ A, D c and L-carnrjne ca-1 optsonaHy be mcsuded When deseed a preservative sucii as aipha-tocopbeso! may be added in an amount αf abour 0 1 % by wesghi

In addition, the ratios of AA and DGLA can be adapted for a particular given end use When formulated as a breast milk supptemeni or substitute, a composition which comprises one or more of AA. DGLA and GLA will be provided in a ratio of about 1 : 19:30 to aboui 6:1 ,02, r espeetϊveiy. For example, the breast mϋk of animals can vary in ratios of AA 1 DGLA. GLA ranging from 1-19 30 to 6'1 1 Q 2, which includes intermediate ratios which are preferably about 1:^:1. 1:2:1, 1:1:4. When produced together in a host ceil, adjusting the rate and percent of conversion of a precursor substrate such as EDA and DGLA to AA can be used to precisely conϋro! the PUFA ratios. For example, a 5% to 10% conversion rate of DGLA to AA can be used to produce an AA to DGLA ratio of about 1:19, whereas a conversion rate of about 75% TO 80% can be used to produce an AA Io DGLA ratio of about 6:1. Therefore, whether ^n a celt culture system or in a host animal, regulating the timing, extent and specificity of efongasβ expression, as well as the expression of desaturases and other eϊongases. can be used to modulate PUFA ϊevefe and ratios. The PUFAs/acsds produced in accordance with the present invention (e.g., AA and EPA) may then be combined with other PUFAs/acids (e.g., DGLA) in :hβ desired concentrations and ratios.

Additionally, PUFA produced in accordance with the present invention or host ceils containing them may also be used as animal food supplements to aϊter an animal's tissue or milk fatty acid composition to one more desirable for human or animal consumption.

Pharmaceutical Compositions

The present invention also encompasses a pharmaceutical composition comprising one or more of the acids and/or resulting oils produced using the elongase gene described herein, in accordance with the methods described herein. More specifically, such a pharmaceutical composition may comprise one or more of the acids and/or oils as well as a standard, weli-knσwn, non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle such as, for example, phosphate buffered saline, water, ethanoi, polyols, vegetable oils, a wetting agent or an emulsion such as a water/oii emulsion. The composition may be in either a liquid or solid form. For example, the composition may be in the form of a tablet, capsule, ingestible liquid or

Do-vvαer njectifaie. or :opicai ointmen: cr cream Proper fluidity can oe maintained, for example, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants, it may aiso be desirable io inciude isotonic agents, for example, sugars, sodium chloride and the like. Besides such inert diluents, the composition can aiso include adjuvants, such as wetting agents. emuisffy'ng and suspending agents, sweetening agents, flavoring agents and perfuming agents

Suspensions, in addition to the active COmPOUnGs 1 may comprise suspending agents such as, for exampie, ethoxyiated isosiearyi alcohols, pαlyoxyethyϊeπe sorbitol and sorbifaπ esters, rπicrocr/staϋine ceϊfutose, afuminum metahydroxide, bentonite, agar- agar and tragacarrth or mixtures of these substances.

Solid dosage forms such as tablets and capsules can be prepared using techniques well known in the art. For example, PUFAs produced in accordance with the present invention can be tabfeted with conventional tabfet bases such as factose, sucrose, and cornstarch in combination with binders such as acacia, cornstarch or gelatin, disintegrating agents such as potato starch or alginsc acid, and a lubricant such as stearic acid or magnesium stearate. Capsules can be prepared by incorporating these excipients into a geEaiin capsule afong with antioxidants and the relevant PUFA(s). The antioxidant and PUFA components should fit within the guidelines presented above.

For intravenous administration, the PUFAs produced in accordance with the present invention or derivatives thereof may be incorporated into commercial formulations such as ϊntraiϊpids™. The typical normal adult plasma fatty acid profile comprises 6.64 to 9.46% AA, 1.45 to 3.11 % of DGLA, and 0.02 to 0.08% of GLA. These PUFAs or their metabolic precursors can be administered aSone or in combination with other PUFAs in order to achieve a norma! fatty acid profile in a patient. Where desired, the individual components of the formulations may be provided individually, in kit form, for single or multiple use. A typical dosage of a particular fatty acid is from 0.1 mg to 20 g (up to 100 g) daily and is preferably from 10 mg to 1 , 2, 5 or 10 g daily.

Possible routes of administration of the pharmaceutical compositions of the present invention include, for example, enteral (e.g., oral and rectal) and parenteral. For example, a liquid preparation may be administered, for example, oraily or rectally. Additionally, a homogenous mixture can be completely dispersed in water, admixed

under sierye conditions with phys i ologically accepiabie e'X'ems. preservaiives, buffers or propeilants in order to form 3 spray or Inha'a^t

The route of adminisiratton wϊii. of course, depend upon ihe desired effect For exampie, ff the composition is being utilized to treat rough, dry. or aging skin, to treat injured or burned skin, or to treat skin or hair affected by a disease or condfϊion. ft may perhaps be applied topically.

The dosage of the composition to be administered to the patient may be determined by one of ordinary skill In ihe art and depends upon various factors such as weight of the patient, age of the patient, immune status of the patsent, efc.

WiIh respect to form, the composition may be, for example, a solution, a dispersion, a suspension, an emulsion or a sterile powder which is then reconstituted.

The present invention also includes the treatment of various disorders by use of the pharmaceutical and/or πutπtiona! compositions described herein. In particular, the compositions of the present invention may be used to treat restenosis after angioplasty. Furthermore, symptoms of inflammation, rheumatoid arthritis, asthma and psoriasis may also be treated with the compositions of the invention. Evidence also indicates that PUFAs may be involved in calcium metabolism; thus, the compositions of the present invention may, perhaps, be utilized in the treatment or prevention of osteoporosis and of kidney or urinary tract stones.

Additionally, the compositions of the present invention may also be used in the treatment of cancer. Malignant ceiϊs have been shown to have altered fatty acid compositions. Addition of fatty acids has been shown to slow their growth, cause ceiS death and increase their susceptibility to chemotherapβutϊc agents. Moreover, the compositions of the present invention may also be useful for treating cachexia associated with cancer.

The compositions of the present invention may also be used to treat diabetes {see U.S. Patent No. 4,826,877 and Horrobin et a!., Am. J. Clin. NuIr. Vol. 57 (Suppf.) 732S-737S). Altered fatty acid metabolism and composition have been demonstrated in diabetic animals.

Furtherrpcre, λe compositions cf lie present invenjon comoπs.ng FlJFAs produced etiper directly or inάireciiy Through the use of the eiongase enzyme, may aiso be used <n the treatment of eczema, in the reduction of bteod pressure, and in the mprcwement of mathematics examination scores. Additionally, the compositions of the present invention may be used ir* inhibition of pϊatefet aggregation, induction of vasodilation, reduction in cholesterol levels, inhibition of proϊiferatϊon of vessel waff smooth muscle and fibrous tissue (Brenner et aL, Adv. Exp. Med. Biol Vol. 83, p.85- 101, 1976), reduction or prevention of gastrointestinal bleeding and other side effects of πoπ-steroida! anti-inflammatory drugs (see U.S. Patent No. 4.686,701). prevention or treatment of endometriosis and premenstrual syndrome (see U S. Patent No. 4,758,592), and treatment of myalgic encephalomyelitis and chronic fatigue after viraf infections (see U.S. Patent No. 5,116.871).

Further uses of the compositions of the present invention include use in the treatment of AfDS, multiple sciβrosss, and inflammatory sksn disorders, as weϊ! as for maintenance of genera! health,

Additionally, the composition of the present invention may be utilized for cosmetic purposes, it may be added to pre-existing cosmetic compositions such thai a mixture is formed or may be used as a soie composition.

Veterinary Applications

It should be noted that the above-described pharmaceutical and nutritional compositions may be utilized in connection with animals (i.e., domestic or non- domesttc), as well as humans, as animais experience many of the same needs and conditions as humans. For example, the oii or acids of the present invention may be utilized in animal or aquacuϊture feed supplements, animal feed substitutes, animal vitamins or in animal topical ointments.

The present invention may be illustrated by the use of the following πon-i ' mniting examples:

Example 1 Cloning of full length eloπqase like cDNA from Thraustochytrid sp. BlCC 7087

A cDNA library was constructed at lncyte Corporation (Wilmington, DE) from the fungus Thra ustoch vtrid sp. BlCC 7087 (BP0091 ). cDNA synthesis was initiated using an oiigo (dT) primer containing EcoRI restriction site in the first strand synthesis

reaction Fcf s o\v"ig ' h e second s'ranG s/π'nesis do J--'θ sif ST^βG cCNA v as z uTec isgateα Ό ι\oti aαapie^s αsgestec vvf ih Not! and Eco^ size se ecisd and c.oned .n;o the Noli (S of the cDNA ϊrsen) anc EcoR 1 (3 of the cDNA inserts sues of pBϊuescπpt (KS-r) vector The library ligation " x was diluted 10 fold and transformed ϊrao Escherichia col! DH1QB competent cells accorαng Ό ϊncyie's trans ' ormatson protocoϊ The tsbrary was stored in glycerol s*oek after recoveπng the uansfoimants The :κer appeared to be ! 14 milionε primary doπes/to*a[ ligation mix

Around 500G clones were then seαϋβπced using T3 pπrπer ard the sequence was ge^e r axeά from ihe 5 * end of each oone (EST) The sequenced r emplaτes thai oassed incyie s sequencing QC specification (overall success rate of 85% reads) were then processed through Incyie's BsosnformaHCS Sequence Editing PspeEiπe and assembled into eonugs Assemb ! ed sequences were then anno"a τ eσ usmg BLAST2 agasnst the appropriate GenBaπk dsvϊsions aπα using FASTX against GeπPept

A clone designated 660R1 ,' was obtained from sequencing of dones from Thraustochytπd so BiCC 7087 (T7087) cDNA library This molecule shared 30 6% amino aαd sequence idenifiy wsxh the mouse etøngase MELO4 and 35 3% ammo acid sequence identity w«ιh Thraustochytπd sp BlCC etoπgase TFLO1 (FlG ^)

To isoiaie the M-lengfb gene, ϊhe 660R1 EST clone was used as a template for PCR reaction with 10 pmo! of the 5' primer RO1206 (5"- AGA AGA CCA TGG GGG ACC TCG AAA GAT AC -3' SEQ ID NO3) and 10 pmoi of the 3' pπmer RCi 207 (5 " - AGA GCT AAG CW AGG CAG ATT TTG TCT TGG GC- 3 1 SEQ ID NO:4). RO1208 contains Nco! restriction site (CCATGG) and the start codon ATG (underisned) while RO1207 contains Hind! Il restriction site (AAGCTT) and the stop codon (underfined) PCR was earned out in a 50 μϊ volume containing: 1 μl of 660R1 cDNA, 0 2 μM CiNTP mix, 10 pmote of each pπmer, 5 μ! of 10 X buffer. 1 5 μl of 50 mM MgSO 15 , and 0 5 U of cDNA Polymerase Thermocycler conditions in Perkin Elmer 9600 were as follows- 94°C for 3 mSn, then 30 cycles of 94 0 C for 45 sec , 55 0 C for 30 sec , and 68°C for 2 mϊn. The PCR amplified mixture was run on a gel, an amplified molecule of approximately 810 bp was get purified, the termini of the molecule were digested with Wcol and HindlW, and the molecule was cloned into pYX242 (NcoUHinώW) {FIG 5). The recombinant plasmid was designated as pRAT-5A1 The nucleotide sequence (SEQ ID NO: 1) and the deduced amino acid sequence (SEQ ID NO: 2) are shown in Figure 2 and Figure 3, respectively

Pfεsηd DNA pRA . -5A1 'vas Deposited w,τb Te Amencan I yoe Culitre Cclect.oi 1080' UnϊveiSity Bouievarα. Manassas Va 20110-2209. on Mov λ λ. 2006. under ihe terms of :he Budapest Treacy. and was accorded deposit number ATCC FTA- 8001

Exampfe 2 Expression of Thraυstochytήd sp BiCC 1 7087 E^ongase cDNA in Baker ' s Yeast

The construct pRAT-5A1 was transformed into Saccharomyces cerevisiae 334 (Hovefaπd et aL Gene. 1989: 83:57--S4) and screened for eloπgase activity. Saccharomyces cerevisiae 334 containing pYX242 vector afoπe was used as a control The cu l tures were grown for ^8 hours at 24 C C, in selective minimal media Sacking feucsne (Ausubef et a!.. Short Protocols in Molecular Biotogy, 1995. pp 4 8- 4.9), in the presence of 50 μM of LA. ALA or EPA For LA and ALA elongation. EDA and ETrA respectively were the predicted elongated products showing δ9-e!ongase activity. After 48 hours of incubation, cefϊs were harvested by centπfugation The cell pellet was washed once with sterile distϊf'ed/deϊonϊzed water. Total yeast lipids were then extracted and ihe fatty acid analysis was performed as described in Knutzon et ai. (J. Biol. Chem. 273:29360-29366 (1998)). Briefly, the rinsed ceil pellet was extracted with 30 ml of Ghloroform/methanoS (2:1 , v/v). Exaαϊy 17.216 μg of triheptadecaπoin was added to the lipid extract as the internal standard. The extracted lipids were derivatized with 2 m!_ 14% boron triflouride in methanol as described by Yamasaki et a! (JAOCS 76: 933-938, 1999). Fatty acid methyl esters were then analyzed by gas chromatography (GC) using a ftame-ionizatbn detector and a fused-sϊSϊca capillary column (Omegawax; 30 mm χ 0.32 mm, Ld., film thickness 0.25μm, Supeϊco, BeiSefonte, PA}. The identity of various metabolites was confirmed by gas chromatography using GLC reference standard # 461 (Nu Chek Prep). The enzyme activity was indicated by the conversion of substrates to products, based on the ratio of [product]/[producis + substrate] χ 1 G0%. The levels of substrate and its metabolic products taken up by the yeast were also calculated, based on-the fatty acid distribution as percentage of total fatty acids.

The lipid profiles of the recombinant yeast culture containing pRAT-5A-1 construct indicated that there was a 4.9% conversion of LA to EDA, 21% conversion of ALA to ETrA and 2.2% conversion of EPA to ω3-docosapentaenoic acid over pYX242 control (FiG. 6). Elongation of LA and ALA to EDA and EtrA, respectively, is by the action of δ9-elongase via the alternate delta 8 pathway (FIG. 1 ). From the results,

D R AT-5 Al appears tc oossess δQ-eSongafon activity preferπrg ALA ;21 % conversion) as a substrate over LA (5% conversion) under expenrn&ntai cond'taons ^n yeast Thus, this fungal sequence, pRAT-5A1 a"d rtε encoded protein, possesses δ9-efongase activity, in addition. ρRAT~5A1 -encoded protein was also found to elongate 18:1 rv-7 substrate to 18.1π-7 in yeast (data not shown).

Example 3 Sequence Comparison between pRAT~5A1 and o:her known elon-qases

The sequence analysis package of Vector MTi Suite 9 (invftrogen Corporation. Carfsbad, GA) was used to compare the pRAT-5A1 with known protein sequences. The nucleotide sequence of pRAT-5A1 open reading frame was first translated into amino acid sequence. This amino acid sequence of pRAT-5A1 was then used in the sequence homology comparison with other published δ9-eloπgase sequence from different organisms. Sequence alignment was performed using Vector NTI software and the percentile of positive alignment was determined. The amino add sequence of pRAT-5A1 had 32,0% identity with Danio rerio δ9-eloπgase (FlG. 7), 33.9% with isochrysϊs gaibana δ9-eϊongase (FiG. 8} and 35.4% with PavEova sauna δ9-efongase (FIG. 9). The functional activity of af! these δ9-eiongases have been established and published. The amino acid sequence of pRAT-5A1 was aiso used for performing BLAST searches on the NCBϊ-Genbank database to determine other sequence homologies. A sequence homology of 85.8% was found with a recently published sequence from Thraustochytrium sp. FJN-10 (Genbank Accession # ABC18314) (FIG.10), however no functional characterization of the protein encoded by this sequence has been demonstrated.

Exampie 4 identification of amino acid residues in pRAT-5A1 that are important determinants of eloπqase activity.

Amino acid sequence comparison of known fatty acid eloπgase proteins that are involved in PUFA biosynthesis has revealed certain shared structural characteristics. There is a highly conserved histidiπe-box motif, containing three histidine residues, HXXHjH, embedded m the fourth membrane spanning region, and the presence of

^ve hyd r ophobic stretches ρ r edjc τ ed τ o be iTieT-brsne-ςp^^πsng regions fie hssucne-box region is preαicteα >o be essenuas for eloπgase acxivi'y s.nce \ι is highly conserved across the PUFA-efoπgase family (Leonard ei ai {200^} Prog Lipia Res ^3(1}:38~54) pRAT-5A1 shares :hese same sequence characteristics wfth other PUFA-elongases, with ihe rπszsdhe box (HVLKH) focated oetwβen amino acid position H- r5λ and H t5S In addition, another motif that appears to be shared across PU FA-efongating enzymes in the 'HXYM Y " motif (FiG 11 ). St ;s not known if thrs HXYMY motif is essentrat for enzymatic activity.

τ o determine rf ihe Hisiidsπe-box (including some flanking conserved regions) and ihe 'HXYMY' motifs are impoπaπt determinants for etongase enzymatic activity, site- directed mutagenesis was earned out on the pRAT-5A1 gene. Two separate sets of mutatfons were generated wrthϊn the ρRAT-5A1 sequence, one within the Hϊsiϊdsne- box mow (including some conserved flanking regions) (pRAT-5A1 mutant 1 }, and one at the HXYMY motif (pRAϊ-5A1 mutant 2). A comparison of the proteins encoded by [ pRAT-5A1 mutant V (SEQ !D NO:5) and 'pRAT-δA- 5 mutant 2 (SEQ ID NO:6) is depicted ϊrt FiG 11 ρRAT-5A1 mutant 1 shares 98 5% amino add sequence identity with pRAT-5A1, and pRAϊ-5A1 mutant 2 shares 98.9% amino acid sequence identity with pRAT-5A1. The resulting changes in the encoded amino acid sequence of the mutants are as indicated below:

To generate the 'pRAT-5A1 mutant 1, the pRAT-5A1 gene (cloned into pYX242) was used as a template for PCR amplification . A 623 bp sequence {Fragment A) containing the 5' end of the gene aiong with part of the 5' flanking vector sequence was PCR amplified used the following primers:

pYX242 vector FP (SEQ SD NO:7) 5'-AGT GAA CTT GCA ACA TTT AC-3' PRAT5A1 mutant 1 RP (SEQ ID NO:8)

" GC G T C GTC CAA GAC ATG CGG CGC ACG CAA CTT GTT GCC CTT G-3 "

The remaining 803 bp {Fragment B) of the pRA7-5A1 gene along with some of the 3 " flanking vector was PCR amplified used the following primers:

pYX242 vector RP (SEQ ID NO:9)

5'-CGA CGG CCA GTG AAT TGϊ-3' pRAT5A1 mutant 1 FP (SEQ D HO: 10)

5'-CAA GGG CAA CAA GTT GGG TGC GCC GCA TGT CTT GGA CGA CGC AAC

CAC CTT TTG GCT T~3 ;

The PCR reaction mix consisted of 200 ng template DMA, 200 μM (final concentration} dNTPs, 1X PWO polymerase buffer (10 mM Tris-CE pH 8.8, 25 mM KCI 1 5 mM {WH4) 2 SG 4 and 2mM Mg 2 SO 4 ), 100 pmoles each of forward and reverse primers and 1 unit of PWO DNA polymerase (Roche). PCR amplification conditions were as follows: An initial denaturation at 94°C/2 mϊn followed by 30 cycϊes of [Denaturation at 94°C/30 sec: Annealing at 58 e C/30 sec; Extension at 72°C/30 secj. This was followed by extension at 72°C/2 msn and finally held at 4 0 C indefinitely.

To generate the fυϊi-Iength "pRAT-5A1 mutant 1'. overlapping PCR was carried out using Fragment A and Fragment B as a template along with vector primers 'pYX242 vector FP' (SEQ ID NO: 7) and pYX242 vector RP (SEQ ID NO:9). PCR amplification conditions were as described above. This generated a 1.2 kb DNA fragment containing the 'pRAT-5A1 mutant 1' gene and vector flanking regions with the Ncoϊ/Hindfii restriction sites. The fuil-ϊength 'pRAT~5A1 mutant 1' gene was then cloned into pYX242 vector at the Nco!/Hind!il site.

Similarly, to generate the 'pRAT-5A1 mutant 2' gene, the pRAT-5A1 gene (cloned into pYX242) was used as a template for PCR amplification. A 720 bp sequence (Fragment A) containing the 5' end of the gene along with part of the 5' flanking vector sequence was PCR amplified used the following primers:

pYX242 vector FP (SEQ ID NO:7) 5'-AGT GAA CTT GCA ACA TTT AC-3'

pR&T5A-i minant 2 RP (S≡Q 1 D NO I ^

5 -AGG GAC GAA AGT AGT GCG CCG CCG CGA CCG ~GT CAA TAA AAG CAT

TCA CGG GG-3

The remaining 502 bp (Fragment B) of the pRAT-5A1 gene aiong with some of ihe 3 " flanking vector was PCR amplified used the following primers:

pYX242 veαor RP (SEQ ID NO:9) 5 ' CGA CGG CCA GTG AAT TGT 3 '

pRAT5A1 mutant 2 FP (SEQ ID hiθ:12)

5' CGC CGT GAA TGC TTT TAT TGA CAC GGT CGC GGC GGC GCA CTA CTT

TCG TCC CT 3 "

PCR amplification and generation of the fuif-iength "pRAT-5A1 mutant 2' gene was earned out using the same technique described for the isolation of pRAT-5A1 mutant 1.

The two mutant clones thus obtained were sequenced to verify that the appropriate mutations were created in the pRAT-5A1 sequence. The two mutant clones were then transformed into Saccharomyces cerevisiae SC 334 and screened for δ9- eiongase activity as described in Example 2. Fatty acid substrates tested were iinoSeϊc acid (LA : 18:2 n-6) and afpha-lϊnoienic acid (ALA, 18:3 n-3). Fatty acid extraction and analysis was carried out as described in Example 2.

The lipid profiles of the recombinant yeast culture expressing the 'pRAT-5A1 mutant 1' or 'pRAT-5A1 mutant 2' indicated that there was a considerable decrease in activity as compared to the activity of the original pRAT-5A1 done (FiG 12). With both mutants, the elongation activity decreased by greater than 50% {F!G 12). 'pRAT-5A1 mutant 2' appeared to !ose greater activity in comparison to that of * pRAT-5A1 mutant 1" (FIG 12). These studies confirm the importance of the Histidine-box region for enzyme functionality. In addition, here it has been demonstrated that the region flanking the Histidine-box (FLHXXHH) may also be important for enzyme functionality. These studies demonstrate, for the first time, the importance of the 'HXYMY' motif in elongation activity. AH these regions may be directly involved in the catalytic activity of the enzyme and/or may play an essential role in stabilizing the protein structure.

! wo additional sequence varans cf pRAT-5A1 were ^πa-yzed for ^-e'oπgase activity. These sequence variants, designated pRAT-5Ai 1 and pR£T-5B6 were isolated during the process of PCR amplification of the fuSS-ϊength gene of pRAT~5A1 The random mutations in these sequences were probably PCR-ϊnduced errors caused by certain DNA polymerases that have low proof-reading activity (enzyme specificity). The encoded amino acid sequences of ρRAT-5A11 (SEQ [D 15), pRATSBδ (SEQ ID 16) anά pRAT-5A1 (SEQ ID 2) are depicted in FiG 13. Amino add residues that differ from the pRAT-5A1 encoded protein are underlined and highlighted in FIG 13. pRAT-5A11 shares 98.5% amino add sequence identity wrih pRAT-5A1. and ρRAT-5B6 shares 99.6% amino acid sequence identity with pRAT- 5A1.

Yeast expression studies with the pRAT-5A11 -encoded protein revealed that pRAT- 5A11 had much tower λ9-elongase activity than pRAT-5A1 {60% decrease in enzymatic activity), in the conversion of LA to EDA and ALA to ETrA (data not shown}. This indicates that the four amino add residues that differ between pRAT- 5A11 and pRAT-5A1 are important determinants for enzymatic activity. These four amino add residues include S 33 , V^ 9 , L 72 and Pus as seen in pRAT-5A1 (Figure 13).

Expression of ρRAT-5B6 in yeast did not reveal any differences in enzymatic activity as compared to expression of pRAT-5A1. This indicates that this single amino add mutation (D101 → N1G1) can be accommodated without much change to the enzyme activity of pRAT-5A1.

Example 5

Seed-Specific Expression of pRAT-5A1 in Arabidopsis

The pRAT-5A1 gene cloned into a plant expression vector p 0308-Ds Red, to test for activity in pfants. For generation of the construct, pRAT-5A1 was PCR amplified with the Phusion polymerase (New England Bioϊabs) according to conditions specified by the manufacturer. The primers used for this PCR included:

Sense primer (SEQ ID NO:13) S'-TATGAATTCAAAATGGGGGACCTCGAAAGATAC-S'

Aniisense primer (SEO !D NO,14) 5 -T ATACCGAGTTAGGCAG ATTTTGT CTTGGGC-3

The PCR amplified gene was :hers digested with restriction enzymes EcoRl and Xho\. and the resulting product was linked on its 5 ' -eπd to the seed-specffϊc giydnin-1 promoter from soybean and on its 3 ' -end to the giydnin-1 3 " untranslated region (n the binary vector pO3O8-DsRed to generate pϊasmtd pRS1 (FIG 14). The giycinin-1 regulatory elements have been previously described by Nieϊsen et af., (1989) Plant Cell 1:313-328. This vector also contains a Ds-Red transgeπe under control of the cassava mosaic virus promoter for selection of transgenic seeds by fluorescence anά a kanamycin resistance marker for bacterial selection. The eϊorsgase gene was sequenced in the binary vector to confirm the absence of any mutations resulting from PCR amplification.

pRS1 was introduced into Agrohacteπum tumefadens strain C58 MP90 by electroporafϊon. Kaπamycin-reststant agrσbacterium was then used for transformation of Arabidopsis thaliana ecoiype CoS-O by the floral dip method (Cloυgh et at., (1998) Plant J 16:735-743). For these experiments, a fad3/fae1 mutant of Arabidopsis was used that contains iow ieveϊs of α-linofenic acid and very-long chain fatty acids (≥C20) but elevated ieveϊs of ϋnoteic acid in its seed oiS (Cahoon et aL, (2006) Phyiochemistry 67:1168-1176). This genetic background approximates the fatty acid profile of seed oils from crops such as safflower and iow linolersic acid soybean. Transgenic seeds obtained from the agro bacterium-dipped Arabidopsis plants were identified by fluorescence of the DsRed marker protein using the methodology described by Pidkowich et af. {Pidkowtch et a!., (2007) Proc Natl Acad Set U S A 104:4742-4747). Single transgenic and nontransgenic control seeds were subjected to direct transesterication of the constituent lipids, including triacySglyceroIs, by use of the protocoi described by Cahoon and Shankliπ (Cartoon et ai., (2000) Proc Natl Acad Sci U S A 97:12350-12355). Fatty acid methyl esters obtained from the single seeds were analyzed by gas chromatography with flame ionization detection by use of an Agilent 6890 gas chromatograph fitted with an iNNOWax column (30 m length x 0.25 mm inner diameter) and oven temperature programming from 185°C (1 min hold) to 230 0 C (2 min hold) at 7°C/min. Component fatty acid methyl esters were identified based on their retention times relative to fatty acid methyl esters of known identity from seeds of wiid-type Arabidopsis thaliana CoI- 0 and by structural analysis using gas chromatography-mass spectrometry.

Shovvrs n F'guje " 15 are íne "3:ty acid compositions c f s'ngte seeds vo r v- five mdependβπi transformaiϊcn events λ'ϊih the pRS J construct λføch contains the PRAT-5A1 gene Afso snown are the fai'y aαd compositions of norr-iransgenic coπtroϊ seeds As seei in FIG 15. ihe transgenic lines that express " .he ρRAT-5A1- encoded enzyme actively elongate tmoϊeic acid (LA 1 18:2n-6} substrate to produce ωβ-eicosadienoic add (EDA. 20:2n-6) (FlG 15} Line 3 shows ϊhe highesi activity with a 26% conversion of 18:2n-6 substrate to 20:2π-β product. This activity is not defected in the non-transgerac fines thai do not express pRA7-5A1 Thus the pRAT- 5A1 -encoded enzyme wiil be useful for the production of transgenic σiis containing aracbidonic aαd (ARA 1 2(M π-6), eicosapeπfaeπotc acid (EPA. 20:5 n-3) and or docosahexaenosc acid (DHA, 20:6 n-3), by virtue of its functionality sπ the alternate pathway (δ9-elongase-δ8 desaturase pathway) teadtng to biosynthesis of ARA. EPA and DHA (FiG I ).

Nutritional Compositions

The PUFAs described in the Detailed Description may be utilized in vanous nutritional supplements, ϊnfarrf formulations, nutritional substitutes and other nuiritional solutions.

1. INFANT FORMULATSONS

A. ϊsomiϊ® Soy Formuia with Iron:

Usage: As a beverage for infants, children and adults with an aliergy or sensitivity to cows milk. A feeding for patients with disorders for which lactose should be avoided: including lactase deficiency, lactose iπtoferaπce and galactosemia.

Features:

-Soy protein isolate to avoid symptoms of cow's-mϋk-proteiπ aflergy or sensitivity.

-Lactose-free formulation to avoid lactose-associated diarrhea.

-Low osmolality (200 mθs/kg water).

-Dual carbohydrates (corn syrup and sucrose) designed to maximize absorption and minimize risk of malabsorption.

Ingredients: 43.2% Corn Syrup Solids, 14.6% Soy Protein Isolate, 11.5% High

Oleic Safflower Oil, 10.3% Sugar (Sucrose), 8.4% Soy Oil, 8.1% Coconut Oil:

Less Than 2% Of: Calcium Phosphate, Potassium Citrate, Potassium

Chloπde sv!agressurn Cfilcnce Sodium Ch'onae. AscorbfG Ac s d. Choline Chloride L-Meihiortfne. " "aunne. Ascorbyi Palmita:e, Ferrous Sulfate, m- ! nositoS, W>xeα Tocopherols. Zinc Sulfate, d-Aϊpha-Tocophery! Acexate. L- Camttine. Niacinamide, Calcium Pantothenate, Cupπc Sυϊfaie. Thiamine Chloride Hydrochloride, Vitamin A Palmftate. Riboflavin. Pyπdoxine Hydrochloride. FoSic Add. Potassium iocfde, Potassium Hydroxide. Phyiioquinone, Biotin, Sodium Setenate, Beta-Carotene, Vitamin D3 and Cyaπocobaiamϊn.

B Isømii® DF Soy Formuϊa For Diarrhea:

Lfsage: For the dietary management of diarrhea in infants and ioddSers.

Features.

-First infant formula to contain added dietary fiber from soy fiber specifically for diarrhea management

-Cfrnscalfy shown to reduce ihe duration of toose, watery stools during msfd to severe diarrhea in infants

-Lactose-free formulation to avoid lactose-associated diarrhea.

-Low osmolality (240 mOsm/kg water) to reduce the risk of osmotic diarrhea. ingredients: 85.7% water, 4,8% com syrup, 2.6% sugar (sucrose), 2.1% soy off, 2.0% soy protein isolate, 1.4% coconut oil, 0.77% soy fiber, cafcium citrate, potassium citrate, calcium phosphate, potassium phosphate, potassium chEoride, mono and dϊg{ycerides, soy Secithin, magnesium chloride, carrageenan, ascorbic acid, t-methjomne, sodium chloride, choline chloride, taurine, ferrous sulfate, m-ϊπositol, d-alpha-tocopheryf acetate, zinc sulfate, L- camitine, niacinamide, caicϊum pantothenate, cupric sulfate, vitamin A paϊmitate, thiamine chloride hydrochloride, riboflavin, pyridoxine hydrochloride, folic acid, manganese sulfate, potassium iodide, phylloquϊnorte, btotin, sodium seienite, vitamin D3 and cyanocobalamin.

C. Isomil® Advance® Soy Formula with Iron:

Usage: As a beverage for infants, children and aduits with an allergy or sensitivity to cows mfik. A feeding for patients with disorders for which lactose should be avoided: including iactase deficiency, lactose intolerance and galactosemia. Features:

- Coπiams DHA and ARA :vo nutπenrs fo ^ nd sπ creai rni ' k irrnorarF for mental and visual develooment

-Soy protein isolate to avosd symptoms of cow's-Tisik-protein allergy or sensitivity.

-Lactose-free forrπciaiiαn to avoid Factose-associated diarrhea

-Low osmolality (200 mOs/kg water).

-DuaS carbohydrates (com syrup and sucrose) designed >o maximize absorption and minimize πsk of malabsorpxiαn.

Eπgredients: 43.2% Com Syrup Solids. 14 6% Soy Protein Isolate, 11.5% High

OEeic Safflower Oil. 10.3% Sugar (Sucrose), 8A% Soy Oil. 7.7% Coconut OEL

C- Cohnii Oi. M. Afpϊπa Oil Caictum Phosphate, Potassium Citrate,

Potassium Chfonde. Magnesium Chloride. Sodium Chloride, Ascorbϊc Acid,

Choϋne Chloride. [.-Methionine, Taurine, Ascort>yi Paϊmϊtate, Ferrous Sulfate. m-lnositol. Mixed Tocopherols, Zinc Sulfate, d-Aϊpha-Tocopheryl Acetate. L-

Camitine, Nfacinamrde, Caidum Pantothenate. Cupric Suifate, Thiamine

ChJoride Hydroch Sonde, Vitamin A Pa imitate. Riboflavin, Pyridoxtne

Hydrochloride. Folic Acid. Potassium iodide, Potassium Hydroxide,

Phyfioquinone, Biotin. Sodium Sefenate, Beta-Carotene, Vitamin D3 and

Cyartccobaiamϊn.

D. fsomii® Advance® 20 Soy Formula With Iron Ready To Feed, 20 Cal/fi oz.:

Usage: When a soy feeding is desired. fngredients: 85.9% water, 6.7% corn syrup, 1.9% soy protein isolate, 1.4% high oieic safflower oil, 1.3% sugar{sucrose), 1.1 % soy oiϊ, 1.0% coconut oil, C. cohnii oil, m. aSpfna oϊi, calcium citrate, calcium phosphate, potassium citrate, potassium chloride, mono- and diglycerides, soy lecithin, carrageenan, abscorbic acid, L-methioπine, magnesium chloride, potassium phosphate, sodium chloride, choline chloride, taurine, ferrous sulfate, m-iπositol, d-a!pha- tocopheryi acetate, zinc suifate, L-camitine, niacinamide, calcium pantothenate, cupric sulfate, vitamin A paimϊtate, thiamine chloride hydrochloride, riboflavin, pyridoxine hydrochloride, folic acid, manganese sulfate, potassium iodide, phylloquinone, biotin, sodium seϊenite, vitamin D3 and cyanocobalamin.

E. Simϊiac® Infant Formula:

Usage' When an miar>ι forrru^ is needed f ire oecssOn >s nade to discontinue breastfeeding before age 1 year ή a supplement TO breastfeeding is needed or as a routine feeding if breastfeeding is not adopted Powder, Concentrated Liquid and Ready τ o Feed forms. Ingredients: Water, nonfat miik. (actose. high oleic safflcwer oil, soy oil, coconut oil whey protein concentrate, potassium cftrate. calcium carbonate abscorbic add. soy lecithin, moriog Syce rides, carrageenan, potassium chforide. magnesium chloride, ferrous sulfate, choline chloride, choline bϊtartrate, taurine, m-inosrtol, zinc sulfate, niacinamide, d-alpha-tocophery! acetate, cafcrurn pantothenate , [-carnitine, vitamin A pafmitate, riboflavin, cupric sulfate, thiamine chloride hydrochloride, pyrϊdoxϊrte hydrochloride, folic acid, manganese sulfate, phylloquiπorse. bsotin, beta-carotene, sodium seSerate, vitamin D3, cyanocobaϊamin, calcium phosphate, potassium phosphaie, sodium chfoπde. potassium hydroxide and nucleotides (adenosine 5 " -rnonophosphaie, cytidine 5'-monoposphate. disodium guanosiπe 5'- monophosphate, disodium undine 5'-monophsphate}.

F Sirnilac® Advance® infant Formula with Iron:

Usage: For use as a supplement of alternative io breastfeeding. Powder, Concentrated Liquid and Ready To Feed forms. Ingredients: Water, nonfat milk, lactose, high oleic saffiower oil, soy oil, coconut oil, whey protein concentrate. C. cohniϊ oil. M. afpina oil, potassium citrate, caicium carbonate, abscorbic acid, soy lecithin, monogfycerides, carrageenan, potassium chforide, magnesium chloride, ferrous sulfate. choline chloride, choline bitartrate, taurine, nvϊnositoS, zinc sulfate, niacinamide, d-aipha-tocopheryj acetate, cafcϊum pantothenate, i-camitine, vitamin A palmitate, riboflavin, cupric sulfate, thiamine chloride hydrochloride, pyridoxine hydrochloride, folic acid, manganese sulfate, phylSoquinone. biotin, beta-carotene, sodium sefenitβ, vitamin D3, cyanocobaiamiπ, calcium phosphate, potassium phosphate, sodium chloride, potassium hydroxide and nucleotides (adenosine 5'-monophosphate, cytidine S'-monophosphate, disodium guanosine 5'-monophosphate, disodium uridine 5'-monophsphate).

G. Similac® NeoSure® Advance® Infant Formula With iron:

Usage: A special formula for conditions such as prematurity.

Features

-Weil absorbed fat Diend that contains 25% added medium-chain ϊrigfycerides(fv!CTs).

-Higher levels of protein, vitamins and minerals per 100 CaS than standard term formula

-More calcium and phosphorus than standard term formula.

Ingredients: nonfat milk, corn syrup solids, lactose, soy oil. high oleic safflower oii, whey protein concentrate, medium chain triglycerides, cocorsui oil, c. cohnii oil, m. aipϊπa oil, potassium citrate, calcium phosphate, m- inositot, ascorbic acid, magnesium chforide, calcium carbonate, taurine. ferrous suifate, choline bitartrate, choline chforide, ascorbyϊ paimitate, L- camitine, potassium chioride, sodium chloride, zinc sulfate, mixed tocopherols. d-alpha-!ocopheryl acetate, sodium citrate, niacinamide, potassium phosphate, calcium pantothenate, cupric sulfate, vitamin A paimitate, thiamine chloride hydrochloride, riboflavin, pyridoxine hydrochloride, beta carotene, folic acid, manganese suifate, phyitoquϊnone, biotin, sodium sefeπite, vitamin D3. cyanocobalamiπ and nucleotides

(adenosine S'-monophosphate, cytidine 5"-monophosphate. disodium guanoslne S'-monophosphate. disαdium uridine S'-monophsphate).

H.Similac Natural Care Advance Low-iron Human Miik Fortifier Ready To Use, 24 CaM oz.;

Usage: Designed to be mixed with human milk or to be fed alternatively with human milk to tow-bsrth-weight infants.

Ingredients: Water, nonfat milk, corn syrup solids, lactose, medium-chain triglycerides, whey protein concentrate, soy oil, coconut oil, C. cohnϋ oil, M. aipina oil, caicϊum phosphate, potassium citrate, ascorbic acid, calcium carbonate, magnesium chloride, soy lecithin, mono and diglycerides, m- inositol, sodium citrate, carrageenan, choline bϊtartrate, taurine, choline chloride, niacinamide, d-alpha tocopheryi acetate, L-camitine, zinc suifate, potassium chloride, potassium phoshphate dibasic, calcium pantothenate, ferrous suifate, cupric sulfate, riboflavin, vitamin A paimitate, thiamine chloride hydrochloride, pyridoxine hydrochloride, biotin, folic acid, beta carotene, manganese suifate, phyiloquϊnone, vitamin D3, sodium selenite, cyanocobalamin and nucleotides {adenosine 5'-moπophosphate, cytidine 5'-

monophosphate dssodiu^ gυanos.ne 5 -rnonconcsoh-a'e oisodmn undine o - monopnsphaie)

Various PUFAs of this invention can be substituted and/or added to ihe infant formulae described above and to other infant formulae known to those in the art.

Sl. NUTRlT[QMAt FORMULATIONS

A. ENSURE®

Usage: RlCn, creamy-tasting ENSURE provides a source of complete, balanced nutrition for suppϊementaf use between or with meals and for interim sole-source feeding. ENSURE can benefit peopfe who are at nutrition πsk. experiencing invofuntary weight loss, recovering from ineεs or surgery, or on modified or low-residue diets. For oral feeding. For interim sole-source feeding. Retail product for supplemental ora! nutrition Ingredients: Water. Sugar (Sucrose), Corn Maltodextrin. Milk Protein isolate, Soy Oil, Corn OiL Canola Oil, Soy Protein Concentrate, Potassium Citrate, Natural & Artificial Flavor. Magnesium Phosphate, Sodium Citrate, Soy Lecithin, Calcium Phosphate, Magnesium Chloride, Salt (Sodium Chioride), Choline Chloride, Carrageenaπ, Ascorbic Acid, di-Aϊpha-Tocopheryl Acetate, Ferrous Sulfate, Zinc Sulfate, Niacinamide, Cafcium Pantothenate, Manganese Sulfate, Cupric Sulfate, Vitamin A Paimitate, Thiamine Chioride Hydrochloride, Pyrϊdoxine Hydrochloride, Riboflavin, Folic Acid, Chromium Chioride, Bϊotfn, Sodium Mofybdate, Sodium Selenate, Phyifoquirtone, Potassium Iodide, Vitamin D3 and Cyanocobalamin.

B. ENSURE® HSGH PROTEIN:

Usage: ENSURE HIGH PROTEfN is useful for people who need extra protein and nutrition in their diet ENSURE HIGH PROTEIN is suitable for use by people recovering from general surgery or hip or other bone fractures, and is a good source of nutrition for those who have or are at risk for pressure ulcers. For supplemental oral nutrition. ingredients: Water, Sugar (Sucrose), Com Maitodextriπ, Calcium and Sodium Caseinates, Soy Oil, Soy Protein Isolate, Corn Oii, Potassium Citrate, Canola

OiS, Cafeium Phosphate Soαsur" Curate, I Phosphate, Artificial Flavor, Sah (Sodium Chloride}, Soy Lecsihip Choline Chloride. Ascorbic Acid. Carrageenaπ Zinc Sulfate. dl-ASpha-Tocopheryf Acetate, Ferrous Suffaie. Geliaπ Gum, Niacinamide, Calcium Pantothenate, Manganese Sulfate. Cupπc Sulfate, Vitamin A Palmitate, Thiamine Chionde Hydrochloride, Pyridoxiπe Hydrochloride, Riboflavin. Folic Add, Chromium Chloride. Bioirn, Sodium Molybdate. Potassium Iodide, Sodium Seienate, Phylioqulnone. Vitamin D3 and Cyanocobafamsn.

C. ENSURE PLUS®

Usage: ENSURE PLUS is a source of complete, balanced nutrition thai provides concentrated caloπes and protein to help patients gain or maintain healthy weight. It can oe used with or between meals or as a meaS replacement For oral feeding. For Interim sofe- source feeding. For patients with fuid restrictions or require volume-limited feedings

Features:

- 650 mg omega-3 fatty acid ALA (40% of 1.6 g RDi) to support heart health.

Excellent source of 24 esseπtiaf vitamins and minerals.

Source of antioxidants selenium and vitamins C and E to strengthen the immune system.

Low in cholesterol.

Kosher.

G!uten-free.

Lactose-free.

Ingredients: VaniSia: Water Com Syrup, Mattodextrin (Corn), Corn Oil, Sodium and Calcium Caseinates, Sugar (Sucrose), Soy Protein isolate, Magnesium Chloride, Potassium Citrate, Caicium Phosphate Tribasic, Soy Lecithin, Natural and Artificial Flavor, Sodium Citrate, Potassium Chloride, Choline Chloride, Ascorbic Acid, Carrageenan, Zinc Sulfate, Ferrous Sulfate, Alpha-Tocopheryi Acetate, Niacinamide, Calcium Pantothenate, Manganese Sulfate, Cupric Sulfate, Thiamine Chloride Hydrochloride, Pyridoxine Hydrochloride, Riboflavin, Vitamin A Paimitate, Folic Acid, Biotin, Chromium

Cbioπde. Sod:um Mo ! ybαaιe Potassium Odide Sodium gelemte. Phylioquinone. Cy8TOCob8'aτwn and Vitamn D3

D ENSURE© POWDER:

Usage: ENSURE© POWDER (recoπsϊϊtuted with water) is complete, balanced nutrition for supplemental use with or between meais. I! may bercefrc people who are on modified diets, at nutrition risk, experiencing involuntary weight loss, recovering from illness or surgery, or on low-residue diets

Features:

-Convenient, easy to mix

-Low residue

-Lactose and gSυfeπ free ingredients: Com Syrup, Com Maitodextrin. Sugar (Sucrose). Corn OsI.

Sodfum and Calcium Casemates. Soy Protein isolate, Artificial Flavor,

Polassium Citrate, Magnesium ChSoride, Sodium Citrate, Calcium Phosphate.

Potassium Chloride, Soy Lecithin, Ascorbic Add, Choline Chloride, Zinc

Sulfate, dl-ASpha-Tocopheryi Acetate. Niacinamide. Ferrous Sulfate, Caϊcium

Pantothenate, Manganese Sulfate, Cuprϊc Sulfate, Thiamine CbEoride

Hydrochforide, Pyridoxine Hydrochloride, Riboflavin, Vitamin A Paimitale,

Folic Acid, Biotin, Chromium Chloride, Sodium Molybdate, Potassium fodide,

Sodium Selenate, Phylloquinone, Cyanocobatamln and Vitamin D3.

E. ENSURE® PUDDING

Usage: ENSURE PUDDING is a nutritious alternative to other snacks or desserts. It provides complete, balanced nutrition in a delicious easy-to-eat form, it is appropriate for those who are underweight or undernourished, or are on a fluid-restricted or voluroe-iimsted diet For people on consistency- modified diets (eg, soft, pureed, or fuiϊ liquid). For people with swallowing impairments. For supplemental oral nutrition Features:

-Good source of 24 essential vitamins and minerals. -Convenient-needs no refrigeration. -Gluten-free.

-Includes 1 g or FOS per serving (FOS are prebiotics that stimulate the growth of beneficial bacterial in the colon).

Jngred'eπts

Vaπsϊϊa * Water, Sugar (Sucrose). Moameύ Corn Siarch, Partially Hydrogeπated Soybean Oil. Milk Protein Concentrate. Nonfat Milk, Fπjctooiigosaccharides. Magnesium Sulfate, Potassium Phosphate. Sodium Phosphate. Sodium Stearoyi Lactyfate, Artificial Fiavor. Sodium Ascrobate. Zrπc Sulfate, d[-A!pha-Tocophery! Acetate. Ferrous Sulfate, Niacinamide. Manganese Sulfate, Cateiυrn Pantothenate. FD&G Yei!ow #5 & #6, Cupric Sulfate, Thiamine Chloride Hydrochloride, Pyridoxine Hydrochloride. Vitamin A Pafmϊiate, Riboflavin. Folic Acid, Chromium Chloride, Biotiπ, Sodium MoSybdate, Potassturn iodide. Sodium Sefenate, Phylloquinone, Vitamin D3 and Cyanocobaiamin,

F. ENSURE© WiTH FiBER:

Usage: ENSURE FIBER is a source of complete, bafanced nutrition for people who can benefit from increased dietary fiber and nutrients. The fiber bSend with FOS. a prebiotlc, heips maintain digestive-tract health. ENSURE

FIBER SS suitable for peopie who do not require a low-residue diet, ft can be fed orally or by tube. ENSURE FfBER can benefit people who are on modified diets, are at nutritional risk, are experiencing involuntary weight foss, or are recovering from iifπess or surgery. For oral feeding. For interim sote-source feeding.

Features:

-includes 1 g of FOS/ 8 fl 02. FOS fiber (nondtgetifafe carbohydrate) heϊps promote natural defenses in the colon.

-Excellent source of 24 essential vitamins and minerals.

-Provides 2.8 g total dietary fiber per 8-fl-oz serving.

-Lactose and gluten-free. ingredients:

Vaniϊfa: Water; Corn Maltodextrin, Sugar (Sucrose), Sodium and Calcium

Caseinates, Soy Oi!, Soy Protein Isolate, Corn Oil, Oat Fiber,

Fructooiigosacchardϊes, Canoia Oil, Soy Fiber, Calcium Phosphate,

Magnesium Chloride, Potassium Citrate, Cellulose GeI 1 Soy Lecithin,

Potassium Phosphate, Sodium Citrate. Natural and Artificial Flavors, Choline

Chloride, Magnesium Phosphate, Ascorbic Acid, Cellulose Gum, Potassium

Chloride, Carrageenan, Ferrous Sulfate, dl-Alpha-Tocophery! Acetate, Zinc

Sulfate, Niacinamide, Manganese Sulfate, Calcium Pantothenate, Cupric

Sulfate. Vitarπn A Paimitate i hiamine Chioride Hydrochloride, Pynύoxtne Hydrochloride, Riboflavin. FoUc Acid. Chromium Chfoπde. Bioiin. Sodium Wofybdafe. Poiassium iodide. Sodium Sefenate. Phylloquinone. Vitamin D3 and CyaπccobaSamsn.

The various nufrϊtionaf supplements described above and known to others of skis! in the art can be substituted and/or supplemented with the PUFAs produced in accordance with the present invention.

G. Oxepa™ Nutritional Product

OXEPA is cϋnicaiiy shown Io modulate the inflammatory response in critically ilL mechanically ventilated patients, it is appropriate for patients who have sepsis, SIRS (systemic inflammatory response syndrome), ALI (acute iuπg injury), or ARDS (acute respiratory distress syndrome). For tube feeding. For sole-source nutrition

Caloric Distribution:

The distribution of Calories in Oxepa is shown in Table A.

Table A. Caloric Distribution of Qxepa per 8 fl oz. per liter % of CaI

Calories 355 1,500 —

Fat (g) 22.2 93.8 55.2

Carbohydrate(g) 25 105.3 28.1 Protein (g) 14.8 62.5 16.7

Water (g) 186 785

ingredients: Water, Calcium and Sodium Caseiπates, Sugar (Sucrose), Canoia Oil. Medium Chain Triglycerides, Sardine Oil, Borage Oil, Magnesium Chloride, Calcium Phosphate, Soy Lecithin, Potassium Citrate, Sodium Citrate, Ascorbic Acid, Potassium Phosphate, Natural and Artificiaf Flavor, Choline Chloride, Taurine, d-A!pha-Tocophery! Acetate, L-Carnitine, Salt {Sodium Chloride), Geilan Gum, Zinc Sulfate, Ferrous Suffate, Niacinamide, Calcium Pantothenate, Manganese Sulfate, Cupric Sulfate, Thiamine Chloride Hydrochloride, Pyridoxine Hydrochloride, Riboflavin, Beta-Carotene, Vitamin A Palmitate, Folic Acid, Chromium Chloride, Biotin, Sodium

Wciybdaie Potass^u^ f odiG'e Sodium Setensie D 'ny'!ccu]pore, V ' τamiπ D3 and CyanocoDafamin

The vaπ ' ous fatty add components of Oxepa ^ " nutritsona! product can be substituted and/or supplemented with the PUFAs produced in accordance with this invention