MXPA00012868A - Diacylglycerol acyl transferase proteins - Google Patents

Abstract

The invention provides diacylglycerol acyl transferase (DAGAT) proteins, wherein said proteins are active in the formation of triacylglycerol from fatty acyl and diacylglycerol substrates. In one aspect, Mortierella ramanniana DAGAT proteins have been isolated and have molecular weights of between approximately 36 and 37kDa as measured by SDS-PAGE. The invention also provides novel DAGAT polynucleotide and polypeptide sequences and to methods of producing such polypeptides using recombinant techniques. In addition, methods are provided for using such sequences to alter triacylglycerol levels in plants and to treat diseases associated with altered DAGAT activity or expression.

Description

PROTEINS OF DIAZYLGLYCEROL ACIL TRANSFERASE This application claims the benefit of the provisional application of E.U.A. No. 60/091, 631 filed July 2, 1998, and provisional application 5 of E.U.A. No. 60 / 130,829, filed on April 23, 1999.

TECHNICAL FIELD The present invention is directed to enzymes, methods for purifying and obtaining said enzymes, amino acid and nucleic acid sequences related thereto, and methods of using said compositions in genetic engineering applications.

BACKGROUND OF THE INVENTION It is thought that triacylglycerol (TAG) is the most important energy reservoir for cells. Diacylglycerol acyl transferase (DAGAT) is an enzyme that is thought to regulate the structure of TAG and direct the synthesis of TAG. The reaction catalyzed by DAGAT is a critical branch point 20 in the biosynthesis of glycerolipids. It is considered that enzymes at said branch points are primary candidates for metabolic regulation sites. There are several enzymes that are common to the synthesis of ^ f ^^ M? LUÉ? a ^^ ?. . s > > r ** ,. > . . ... «- ''? ? -flli ^ | _! J_? JJfalttL¿ =? J diacylglycerol, TAG and membrane lipids; however, the DAGAT reaction is specific for the synthesis of oils. In plants, the TAG is the primary component of the vegetable oil that is used by the seed as a form of stored energy that will be used during the germination of the seed. The higher plants seem to synthesize oils through a common metabolic pathway. Fatty acids are synthesized in the plastids from acetyl-CoA through a series of reactions catalyzed by enzymes known collectively as fatty acid synthetase (FAS). The fatty acids produced in the plastids are translocated to the cytosolic compartment of the cell, and are esterified to coenzyme A. These acyl-CoAs are the substrates for the synthesis of glycerolipids in the endoplasmic reticulum (ER). The glycerolipid synthesis itself is a series of reactions that lead first to phosphatidic acid (PA) and diacylglycerol (DAG). Any of these metabolic intermediates can be directed towards membrane phospholipids such as phosphatidylglycerol (PG), phosphatidylethanolamine (PE) or phosphatidylcholine (PC), or they can be directed to form a neutral triacylglycerol (TAG). Diacylglycerol (DAG) is synthesized from glycerol-3-phosphate and acyl-CoAs fats in two sequentially catalyzed steps by glycerol-3-20 phosphate acyltransferase (G3PAT) and lysophosphatidic acid acyltransferase (LPAAT) to obtain PA, and then a additional hydrolytic step catalyzed by phosphatidic acid phosphotransferase (PAP) to obtain DAG. In most cells, DAG is used to synthesize membrane phospholipids, the first piiWllMIÉBIBii imilÉ i i i n i il il i i i P in ?? - M ^ M ^ MMM ^, ^ M ^ Mi ^. ^ M, ^ jM ^ MMMM ^^^^^^^^^ M ^^^^^ ia, ^ M ^^^^ JÉÍ | liÉi ||| step being the synthesis of PC catalyzed by CTP-phosphocholine citidyliltransferase. In cells that produce reserve oils, the DAG is acylated with a third fatty acid in a reaction catalyzed by diacylglycerol acyltransferase (DAGAT). Together, the reactions are part of what is commonly known as the Kennedy way. The structure of the TAG, in terms of the specific character of position of fatty acids, is determined by the specific character of each of the three acyltransferases for the acyl-CoA fat and the substrates of the glycerol base structure. In this way, for example, there is The tendency for acyltransferases of many temperate zone seed species to allow a saturated fatty acid or an unsaturated fatty acid in the sn-1 position or the sn-3 position, but only an unsaturated fatty acid in the sn-2 position. The absolute specific character for an unsaturated fatty acid in the sn-2 position is determined by preferencenzyme LPAAT by the substrate. In some species such as cocoa, the TAG compositions suggest that this trend is carried further, because there is an apparent preference for the acylation of the sn-3 position with a saturated fatty acid, if the sn-1 position is esterified to a saturated fatty acid. In this way, there is a higher percentage of TAG_20_structure of the SUS form (where S = saturated fatty acid and U = unsaturated fatty acid), which would be expected from the random distribution based on the general composition of fatty acids with the sn-2 position fixed with an acid unsaturated fat This suggests that DAGAT plays also an important function in the regulation of the TAG structure, as well as in the control of the TAG synthesis. Obtaining nucleic acid sequences capable of producing a phenotypic result in the incorporation of fatty acids into a glycerol base structure to produce an oil is subject to several obstacles including, but not limited to, the identification of factors metabolites of interest, choice and characterization of a protein source with useful kinetic properties, purification of the protein of interest up to a level allowing the sequencing of its amino acids using amino acid sequence data to obtain a nucleic acid sequence capable of being used as a probe to recover the desired DNA sequence, and the preparation of constructions, transformation and analysis of the resulting plants. In this way, the identification of enzyme targets and tissue sources useful for nucleic acid sequences of said enzyme targets capable of modifying the structure and amount of oils is required. Ideally, an enzyme target will be subject to one or more applications alone or in combination with other nucleic acid sequences related to the increased / decreased production of oils, TAG structure, the ratio of saturated fatty acids: unsaturated in the stock of fatty acids, and / or other novel oil compositions as a result of modifications to the fatty acid stock.

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For example, in some cases where you have an oilseed with a higher ratio of oil: seed meal, it would be useful to obtain a desired oil at a lower cost. This would be typical of a high-value oil product, or said oil seed could be a superior food for animals. In some cases where there is an oilseed with a lower ratio of oil: seed meal, a lower caloric content would be useful. In other uses, edible vegetable oils with a higher percentage of unsaturated fatty acids are desired for cardiovascular health reasons. And alternatively, tempered substitutes for highly saturated tropical oils such as palm oil, coconut and cocoa, would also find uses in a variety of industrial and food applications. In mammals, DAGAT plays an important role in the metabolism of cellular diacylglycerol, and is important in processes involving triacylglycerol metabolism that includes intestinal fat absorption, lipoprotein assembly, adipose tissue formation and lactation. As such, identification and isolation of the DAGAT protein, and of polynucleotide and polypeptide sequences are desired. Several putative procedures have been published for DAGAT. Polokoff and Bell (1980) reported the solubilization and partial purification of DAGAT from rat liver microsomes. This preparation was insufficiently pure to identify a specific protein factor responsible for the activity. Kwanyuen and Wilson (1986, 1990) reported the ^ + j m purification and characterization of the enzyme from soybean cotyledons. Nevertheless, the molecular mass (1843 Dka) suggests that this preparation was not exhaustively solubilized, and that the DAGAT protein contained therein was part of a large aggregate of many proteins. Little et al (1993) 5 reported the solubilization of DAGAT from rapeseed embryos derived from microspores, but also as with Kwanyuen and Wilson, the molecular mass of the material that was associated with the activity was so high that complete solubilization It was unlikely. Andersson et al (1994) reported the solubilization and 415 times the purification of DAGAT to from rat liver using immunoaffinity chromatography. However, there is no evidence that the antibodies they used recognized DAGAT epitopes, nor that the protein they purified was truly DAGAT. In addition, as in the case of Kwanyuen and Wilson, the DAGAT activity in their preparations exhibited a typical molecular mass of proteins membrane added. Finally, Kamisaka et al (1993, 1994, 1996, 1997) reported the solubilization of DAGAT from Mortierella rammaniana and its subsequent purification to homogeneity. They suggest that DAGAT solubilized from this fungus species has an apparent molecular mass of 53 kDa by SDS-PAGE. However, as shown in In Example 4 below, the fractions obtained using the protocol described by Kamisaka et al. they did not provide an abundant 53 kDa polypeptide that would correlate with the DAGAT activity.

BRIEF DESCRIPTION OF THE INVENTION The present invention is directed to diacylglycerol acyl transferase (DAGAT), and in particular to DAGAT polypeptides and polynucleotides. The polypeptides and polynucleotides of the present invention include those derived from plants, mammals including humans, and from nematodes and fungi. In another aspect, the invention provides DAGAT proteins having molecular weights between about 36 kDa and 37 kDa based on the SDS-PAGE analysis, and particularly molecular weights of 36 kDa and 36.5 kDa. The preferred DAGAT proteins of the invention are obtained from Mortierella rammaniana. In another aspect, the invention relates to oligonucleotides derived from DAGAT proteins and oligonucleotides that include partial or complete sequences encoding DAGAT. It is also an aspect of the present invention to provide recombinant DNA constructs that can be used for transcription or transcription and translation (expression) of DAGAT. In particular, constructs that are capable of transcription or transcription and translation in host cells of plants and mammals are provided. Particularly preferred constructs are those capable of transcription or transcription and translation in plant cells.

In another aspect of the present invention, methods are provided for the production of DAGAT in a host cell or progeny thereof. In particular, the host cells are transformed or transfected with a DNA construct that can be used for transcription or transcription and translation of DAGAT. Recombinant cells containing DAGAT are also part of the present invention. In another aspect, the present invention relates to methods for using polynucleotide and polypeptide sequences to modify the oil ratios relative to other constituents, as well as to modify the composition and / or structure of triglyceride molecules, particularly in oils of seeds of oil crops. Plant cells having said modified triglyceride are also contemplated in the present invention. Modified plants, seeds and oils obtained by the expression of DAGAT proteins are also considered as part of the invention. In another aspect, the invention relates to methods for using said polypeptides and polynucleotides in mammals. Such methods include treating or alleviating diseases associated with DAGAT activity, including diseases associated with the altered concentration of cellular diacylglycerol or the altered activity of the protein kinase. C including, but not limited to, cancer; diabetes; cardiopulmonary diseases that 1 mii ll I ni 1 1 tl ,, t ^ MM, M..MM- ^ | MM, ^^^^, ^^^^^^ | ^ M ^^^ i, | M ,, ^, ^ ^^ M ^ t ^^^^ | i | É < M ||| BM | include, but are not limited to, heart failure and atherosclerosis; adipocytosis; leukemia and skin carcinoma; fibroblastoma; metabolic disorders; obesity; diseases associated with abnormal lipid metabolism, diseases associated with abnormal absorption of fat, 5 secretion of lipoproteins and adipogenesis. In addition, methods are provided to alter the levels of DAGAT activity. In another aspect of the present invention, methods are provided for identifying DAGAT agonists and antagonists / inhibitors and treating conditions associated with DAGAT activity, or for altering the levels of DAGAT activity with said agonists or antagonists. It is also an aspect of the present invention to provide diagnostic tests to detect alterations in the level of DAGAT activity, and to diagnose conditions associated with DAGAT activity. 15 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the results of chromatography of the DAGAT activity of Mortierella rammaniana on a yellow agarose-86 column. Figure 2a shows the results of chromatography of the DAGAT activity of Mortierella rammaniana from the agarose column. yellow 86 on a column of Heparin Sepharose CL6B. The '"" • - • • -i »" - "» R - * »- Mta É Figure 2b shows the SDS-PAGE analysis of fractions of the Heparin Sepharose CL6B column. The protein bands are detected by silver staining. Figure 3A shows the results of chromatography of the DAGAT activity of Mortierella rammaniana from the second peak of activity of the Heparin Sepharose CL6B column chromatographed on a yellow agarose-86 column, where the protein was eluted during a gradient of KCl 75-150 mM. Figure 3B shows the SDS-PAGE analysis of fractions of the agarose-yellow column 86. The protein bands are detected by silver staining. Figure 4 shows the results of chromatography of the DAGAT activity of Mortierella rammaniana on a yellow agarose-86 column. Figure 5A shows the results of chromatography of the DAGAT activity of Mortierella rammaniana from the agarose column. yellow 86 on a column of hydroxylapatite (Bio-Gel HT). Figure 5B shows the SDS-PAGE analysis of fractions of the hydroxylapatite column. The protein bands are detected by silver staining. Figure 6 presents the results of the analysis of the DAGAT activity of Mortierella rammaniana in fractions of the column from a DAGAT purification protocol. Figure 6A provides the results of double yellow-agarose / hydroxylapatite chromatography. Figure 6B provides the results of the SDS-PAGE analysis of the peak fractions of the -tlimi, .tAj »a i 1.. «.. ^. ^ ^^ ^ j ^ jdj double chromatography. The protein bands are detected by silver staining. Figures 7A and 7B show the SDS-PAGE analyzes of preparations of the lipid body fraction of high salt concentration and low concentration of purified salts through agarose-yellow 86 / hydroxylapatite chromatography. The protein bands are detected by staining with Coomassie blue. Figure 8A shows the results of chromatography of the DAGAT activity of Mortierella rammaniana from the heparin column after chromatography on agarose-yellow 86 and hydroxylapatite (Bio-Gel HT). Figure 8B shows the SDS-PAGE analyzes of fractions of the heparin column. The protein bands are detected by silver staining. Figure 9 shows the results of chromatography of the DAGAT activity of Mortierella rammaniana on a yellow agarose-86 column. Figure 10A shows the results of chromatography of the DAGAT activity of Mortierella rammaniana grouped from 4 agarose columns. yellow 86 on a column of hydroxylapatite (Bio-Gel HT). Figure 10b shows the SDS-PAGE analyzes of fractions of the hydroxylapatite column. The protein bands are detected by silver staining.

. Figure 11A shows the results of chromatography of the DAGAT activity of Mortierella rammaniana from the hydroxylapatite column on a column of Heparin Sepharose CL6B. Figure 11B shows the SDS-PAGE analyzes of fractions of the Heparin Sepharose CL6B column. The protein bands are detected by staining with Coomassie blue. Figure 12A shows the chromatography results of the DAGAT activity of Mortierella rammaniana from the first activity peak of the Heparin Sepharose CL6B column chromatographed on a yellow agarose-86 column, where the protein was eluted during a gradient of KCl 75-150mM. Figure 12B shows the SDS-PAGE analyzes of fractions of the agarose-yellow column 86. The protein bands are detected by staining with Coomassie blue. Figure 13 shows the protein alienations of the two DAGAT proteins identified in Mortierella rammaniana. The total length protein sequence of the 36 kDa candidate is shown, as well as the partial sequence of the 36.5 kDa protein. Figure 14 shows data of DAGAT activity on membranes isolated from insect cells infected with an empty pFASTBAC vector or a pFASTBAC vector containing DNA sequences of the 36 kDa DAGAT sequence identified in Mortierella rammaniana. Figure 15 shows data of DAGAT activity on membranes isolated from insect cells infected with a vector tkMMtaMMM MM.a ^^ l empty pFASTBAC or a pFASTBAC vector containing DNA sequences from yeast DAGAT homologs and C. elegans. Figure 16 shows the relative content of triacylglycerol in insect cells infected with an empty pFASTBAC vector or a pFASTBAC vector containing DNA sequences of the 36 kDa DAGAT sequence identified in Mortierella rammaniana.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to diacylglycerol acyltransferase (hereinafter referred to as DAGAT), particularly the isolated DAGAT protein and the nucleic acid sequences encoding the DAGAT protein. A diacylglycerol acyltransferase of this invention includes any nucleic acid sequence encoding amino acids, such as a protein, polypeptide or peptide, that can be obtained from a cellular source, demonstrating the ability to catalyze the production of triacylglycerol to from 1, 2-diacylglycerol and fatty acid substrates under reaction conditions for enzyme. The term "reaction conditions for enzyme" means that any of the necessary conditions (i.e. factors such as temperature, pH, lack of inhibitory substances) that allow the enzyme to function are available in an environment.

MÉWHMIMÉLALÉMM «laH Isolated proteins, polypeptides and polynucleotides A first aspect of the present invention relates to isolated DAGAT proteins. As used in the present invention, "isolated" means that it is altered "by the hand of man" from its natural state. For example, if it occurs in nature, it has been changed or removed from its original environment or both. For example, a polynucleotide or a polypeptide that occurs in nature in a living organism is not "isolated", but the same polynucleotide or polypeptide when separated from the materials of its natural state is "isolated". In particular, DAGAT proteins were identified which have a molecular weight between about 36 kDa and about 37 kDa, in accordance with the SDS-PAGE analyzes. In particular, DAGAT proteins are provided which have molecular weights of approximately 36 kDa and 36.5 kDa and which can be obtained from Mortierella ramanniana. In addition, DAGAT proteins have been solubilized. "Solubilization" refers to the extraction of the DAGAT enzyme from the membranes in such a way that the latter then behaves in a manner typical of enzymes that are not membrane-associated. The DAGAT protein of the present invention can utilize a variety of acyl substrates in a host cell, including fatty acyl-CoA and fatty acyl-ACP molecules. In addition, the acyl substrates on which DAGAT acts may have varying carbon chain lengths and saturation degrees, although DAGAT may demonstrate preferential activity towards certain molecules. Another aspect of the present invention relates to DAGAT polypeptides. Such polypeptides include isolated polypeptides indicated in the Sequence Listing, as well as polypeptides and fragments thereof, particularly those polypeptides having DAGAT activity and also those polypeptides having at least 50%, 60% or 70% identity, preferably at least 80% identity, more preferred at least 90% identity, and even more preferred at least 95% identity with a polypeptide sequence that is selected from the group are sequences indicated in the Sequence Listing, and further includes portions of such polypeptides, in which such portions of the polypeptide preferably include at least 30 amino acids, more preferred are included by at least 50 amino acids. "Identity", as is well understood in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relationship between the polypeptide or polynucleotide sequences, as determined by the mating between the chains of such sequences. "Identity" can be easily calculated by known methods including, but not limited to, those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York (1988); Biocomputing: - ^ ..- »^ m. i. . ...... r ... . . . | _ ^ ¡L_Ul_ ^ l_ ^ Jl_l ___ j_ ^ JJJj¡¡jfl ($ ¡¡j¡, Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Griffin , AM and Griffin, HG, eds., Humana Press, New Jersey (1994), Sequence Aanalysis in Molecular Biology, von Heinje, G., Academic Press (1987), Sequence Analysis 5 Primer, Gribskov, M. and Devereux, J ., eds., Stockton Press, New York (1991), and Carillo, H., and Lipman, D., SIAM J. Applied Math, 48: 1073 (1988) .The methods for determining identity are designed to give the greater pairing between the sequences analyzed, and the methods to determine the identity are codified in programs available to the public.

The computer programs that can be used to determine the identity between two sequences include, but are not limited to, GCG (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984); five BLAST programs, three designed for investigations of nucleotide sequences (BLASTN, BLASTX and TBLASTX) and two designed for investigations on protein sequences (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology, 12: 76-80 (1994), Birren, et al., Genome Analysis, 1: 543-559 (1997)). The BLAST X program is available to the public at NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH, Bethesda, MD 20894: Altschul, S., et al., J. Mol. Biol. , 215: 403-410 (1990)). 0 The well-known Smith Waterman algorithm can also be used to determine identity. The parameters for polypeptide sequence comparison typically include the following: "Mu n - ->." "...." ... ".L¿t_ ^ _m Algorithm: Needleman and Wunsch J. Mol. Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62 by Hentikoff and Hentikoff, Pro. Nati, Acad Sci USA 89: 10915-10919 (1992) Punishment by space: 12 5 Punishment by space length 4 A program that can be used with these parameters is available to the public as the "gap" program of Genetics Computer Group, Madison Wisconsin The above parameters together with a "no endpoint penalty" are the pre-set parameters for the peptide comparisons. The parameters for the comparison of polynucleotide sequences include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970) Comparison matrix: pairs = + 10; no matings = 0 15 Punishment per space: 50 Punishment per length of space. 3 A program that can be used with these parameters is available to the public as the "gap" program of Genetics Computer Group, Madison Wisconsin. The above parameters are the pre-established parameters for nucleic acid comparisons. The invention also includes polypeptides of the formula: in which, at the amino terminus, X is hydrogen and at the carboxyl-terminal end, Y is hydrogen or a metal, Ri and R3 are any amino acid residue, n is an integer between 1 and 1000, and R2 is an amino acid sequence of the invention, particularly an amino acid sequence 5 which is selected from the group indicated in the Sequence Listing and preferably SEQ ID NO: 38 and 45. In the formula, R2 is oriented so that its amino terminal residue is on the left, bound to Ri and its carboxyterminal residue is on the right, linked to R3. Any stretch of amino acid residues indicated by any of the R groups, in which R is greater of 1, can be a heteropolymer or a homopolymer, preferably a heteropolymer. The polypeptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising a sequence that is selected from the group of a sequence contained in SEQ ID Nos: 37, 44 and 46-72. The polypeptides of the present invention have been shown to have DAGAT activity and are of interest because DAGAT is involved in the metabolism of cellular glycerolipids, and in particular catalyzes the formation of triacylglycerol from sn-1, 2-diacylglycerol and from the Acyl-CoA molecules fatty. DAGAT is the only enzyme unique to the triacylglycerol biosynthetic pathway (Coleman RA, (1992) Methods Enzymol 209: 98-104). The polypeptides of the present invention can be a mature protein or part of a fusion protein. i ». • I G IIII - III i? Íi] i a illeg ir ii "in mi iln liiii ^ - -MÉÉÉÍÉÍ- ^ MUafcMiM ^^^^ MI ^ JM ^^^ a ^^^^^^ It is also considered that the Fragments and variants of the polypeptides are part of the invention. A fragment is a polypeptide variant which has an amino acid sequence that is completely the same as part, but not all, of the amino acid sequence of the polypeptides previously described. The fragments can be "independent" or be comprised within a larger polypeptide of which the fragment forms a part or a region, more preferred as a continuous single region. Preferred fragments are biologically active fragments which are those fragments that mediate the activities of the polypeptides of the invention, including those with similar activity or improved activity or with reduced activity. Also included are those fragments that are antigenic or immunogenic in an animal, particularly a human. Polypeptide variants also include polypeptides that vary from the sequences indicated in the Sequence Listing by conservative amino acid substitutions, by substitution of one residue with another with similar characteristics. In general, such substitutions occur between Ala, Val, Leu and He, between Ser and Thr: between Asp and Glu; between Asn and Gln; between Lys and Arg, or between Phe and Tyr. Particularly preferred are variants in which 5 to 10 are substituted, deleted or added; 1 to 5; 1 to 3 or an amino acid or amino acids, in any combination. Variants that are fragments of the polypeptides of the invention can be used to produce the polypeptide with length »• • - - • • • complete corresponding by peptide synthesis. Therefore, these variants can be used as intermediates to produce the full-length polypeptides of the invention. Another aspect of the present invention relates to isolated DAGAT polynucleotides. The polynucleotide sequences of the present invention include isolated polynucleotides that encode the polypeptides of the invention having a deduced amino acid sequence that is selected from the group of sequences indicated in the Sequence Listing and for other polynucleotide sequences closely related to such sequences. sequences and variants thereof. The invention provides an identical polynucleotide sequence, along its entire length, to each of the coding sequences as indicated in the Sequence Listing. The invention also provides the coding sequence for the mature polypeptide or a fragment thereof, as well as the coding sequence for the mature polypeptide or a fragment thereof in a reading frame with other coding sequences, such as those encoding for a leader or secretion sequence, a pre-protein, pro-protein, or prepro-protein sequence. The polynucleotide can also include non-coding sequences, including but not limited to, for example, non-coding 5 'and 3' sequences, such as transcribed, untranslated sequences, termination signals, ribosome binding sites, sequences that stabilize to rRNA, introns, polyadenylation signals, and additional coding sequence that codes for • ^ - ^ J1 - - - '• "" - - ~ -' - - - - • - • '"" * • - - - - - - ^ - - - i - ^ - ^ Je¡¡¡ja¡ amino additional acids. For example, a marker sequence can be included to facilitate purification of the fused polypeptide. The polynucleotides of the present invention also include polynucleotides that comprise a structural gene and the naturally associated sequences that control the expression of the gene. The invention also includes polynucleotides of the formula: X- (R?) N- (R2) - (R3) n-Y in which, at the 5 'end, X is hydrogen, and at the 3' end, Y is hydrogen or a metal, Ri and R3 are any nucleic acid residue, n is an integer between 1 and 3000, preferably between 1 and 1000 and R2 is a nucleic acid sequence of the invention, particularly a nucleic acid sequence that is selected from the group indicated in the Sequence Listing and preferably SEQ ID Nos: 37, 44 and 46-72. In the formula, R2 is oriented so that its residue from the 5 'end is on the left, joined to R-i, and its residue at the 3' end is on the right, joined to R3. Any stretch of nucleic acid residues indicated by any of the R groups, in which R is greater than 1, can be a heteropolymer or a homopolymer, preferably a heteropolymer. The invention also relates to variants of the polynucleotides described in the present invention that code for variants of the polypeptides of the invention. The variants that are fragments of the polynucleotides of the invention can be used to synthesize full-length polynucleotides of the invention. Preferred embodiments are polypeptide variants that encode polynucleotides in which 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residue of a polypeptide sequence of the polypeptide are substituted, added or deleted. invention, in any combination. Particularly preferred are substitutions, additions, and deletions that are silent such that the properties or activities of the polynucleotide or polypeptide are not altered. Also preferred are embodiments of the invention that have at least 50%, 60%, or 70% identity throughout their entire length with a polynucleotide that encodes a polypeptide of the invention.10 invention, and the polynucleotides that are complementary to said polynucleotides. More preferred are polynucleotides that comprise a region that has at least 80% identity along its entire length with a polynucleotide that encodes a polypeptide of the invention and polynucleotides that are complementary thereto. In this In sense, polynucleotides having at least 90% identity throughout their entire length are particularly preferred, those having at least 95% identity are especially preferred. In addition, those who have at least 97% identity are quite preferred and those who have at least 98% and 99% identity are particularly preferred to a greater degree, being those that have at least 99% the most preferred. Preferred embodiments are polynucleotides that encode polypeptides that retain substantially the same function or activity The invention also relates to the biological activity of the mature polypeptides encoded by the polynucleotides indicated in the Sequence Listing. polynucleotides that hybridize with the sequences described above In particular, the invention relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described above As used in the present invention, the terms "astringent conditions" and "conditions" hybridization "means that hybridization will generally occur if there is at least 95% and preferably at least 97% identity between the sequences.An example of astringent hybridization conditions is incubation overnight at 42 ° C in a solution comprising 50% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate and 20 micrograms / milliliter of cut and denatured salmon sperm DNA followed by washing the hybridization support in OJx SSC at approximately 65 ° C. Other hybridization and washing conditions are well known and are exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor, NY (1989), particularly chapter 11. The invention also provides a polynucleotide consisting essentially of a polynucleotide sequence that can be obtained by selecting an appropriate library containing the complete gene for a polynucleotide sequence indicated in the Sequence Listing '' -ii 'irll-iii'ii l -nit ii?? fp II Mil ni iii -. i- ii, u l- * a? a, ^^?? ta ^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^ ßl ^^^^ under conditions of astringent hybridization with a probe having the sequence of said polynucleotide sequence or a fragment thereof; isolate said polynucleotide sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and primers such as those described in the present invention As discussed in the present invention with respect to the tests for polynucleotides of the invention , for example, the polynucleotides of the invention can be used as a hybridization probe for RNA, cDNA or genomic DNA to isolate full-length cDNA molecules or genomic clones that code for a polypeptide and to isolate cDNA or clones of other genes that have a high sequence similarity with a polynucleotide indicated in the Sequence Listing Such probes will usually comprise at least 15 bases. Preferably such probes will have at least 30 bases and can have at least 50 bases. Particularly preferred probes will have between 30 bases and 50 bases, inclusive. The coding region of each gene comprising or comprised by a polynucleotide sequence indicated in the Sequence Listing can be isolated by selection using a DNA sequence provided in the Sequence Listing to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of the gene of the invention is then used to select a library of cDNA, genomic DNA or mRNA to identify the elements of a | M | aitaHta || ^ M1 the library that hybridize with the probe. For example, synthetic oligonucleotides are prepared which correspond to the DAGAT peptide sequences. The oligonucleotides are used as primers in polymerase chain reaction (PCR) techniques to obtain the partial DNA sequence of the genes for DAGAT. The partial sequences obtained in this way are then used as probes to obtain the clones for DAGAT from a library that is prepared from Mortierella ramanniana tissue. Alternatively, where low degeneracy oligonucleotides can be prepared from particular DAGAT peptides, such probes can be used directly to select libraries for gene sequences for DAGAT. In particular, the selection of cDNA libraries in phage vectors is useful in such methods due to the low levels of background hybridization. Typically, a sequence for DAGAT that can be obtained from the use of nucleic acid probes will show 60-70% sequence identity between the DAGAT target sequence and the coding sequence used as a probe. However, elongated sequences can also be obtained with an amount as low as 50-60% sequence identity. The nucleic acid probes can be a long fragment of the nucleic acid sequence, or they can be a shorter oligonucleotide probe. When longer nucleic acid fragments are used as probes (greater than about 100 bp), the selection can be made with minor astringency in order to obtain jgggj ^^ iL sequences from the target sample that have 20-50% deviation (ie, 50-80% sequence homology) from the sequences used as a probe. The oligonucleotide probes may be considerably shorter than the entire nucleic acid sequence encoding a DAGAT enzyme, but must be at least about 10, preferably at least about 15, and more preferred at least about 20 nucleotides . A greater degree of sequence identity is desired when using shorter regions as opposed to longer regions. In this way it might be desirable to identify highly conserved amino acid sequence regions to design oligonucleotide probes to detect and recover other genes for related DAGATs. Frequently, shorter probes are particularly useful for polymerase chain reactions (PCR), especially when highly conserved sequences can be identified. (See, Gould, et al., PNAS USA (1989) 86: 1934-1938.). The polynucleotides and polypeptides of the invention can be used, for example, in the transformation of plants, as research reagents, and to discover treatments of, and for diagnostics of, diseases such as those discussed below in the present invention. The invention also provides polynucleotides that encode a polypeptide that is a mature protein plus the additional amino acids of the amino-terminal or carboxy-terminal, or amino acids within the A mature polypeptide (for example when the mature form of the protein has more than one polypeptide chain). Such sequences may, for example, play a role in the process of a protein from a precursor to a mature form, allow the transport of the protein, shorten or lengthen the life of the protein, or facilitate the manipulation of the protein. in tests or production. It is contemplated that cellular enzymes are used to remove any additional amino acid from the mature protein. A precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. Inactive precursors are usually activated when the prosecutions are removed. Some or all of the prosequences can be removed before activation. Said precursor protein is generally referred to as proproteins.

Plant constructions and methods of use Of particular interest is the use of nucleotide sequences in recombinant DNA constructs to direct the transcription or transcription and translation (expression) of the acyltransferase sequences of the present invention in a host plant cell. The constructions of The expression generally comprises a functional promoter in a host plant cell operatively linked to a nucleic acid sequence encoding a diacylglycerol acyltransferase of the present invention and a functional transcription termination region in a host plant cell. Those skilled in the art will recognize that there are a number of promoters that are functional in plant cells, and have been described in the literature. Also included are chloroplasts and promoters specific to plastids, chloroplasts or functional promoters of plastids, and chloroplast or promoters operable in plastids. A group of promoters are constitutive promoters such as CaMV35S or FMV35S promoters that produce high levels of expression in most organs in plants. Improved or duplicate versions of the CaMV35S and FMV35S promoters are useful in the practice of the invention (Odell, et al. (1985) Nature 313: 810-812; Rogers patent of US Patent No. 5,378,619). Furthermore, it would also be preferable to create the expression of the acyltransferase gene in specific tissues of the plant, such as leaf, stem, root, tuber, seed, fruit, etc., and the selected promoter should have the desired tissue and the specificity of development. Of particular interest is the expression of the nucleic acid sequences of the present invention from transcription initiation regions that are preferably expressed in a plant seed tissue. Examples of said preferred transcription initiation sequences in seeds include sequences derived from sequences encoding for plant storage protein genes or genes involved in the fatty acid biosynthesis in oilseeds. - | jrr - | f - í 'i lll i r. , 1 Mi I l.?,..l, ?? i, i. ?? i i ?? I ll llit mtalllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll Examples of such promoters include the 5'regional regulatory regions of these napin (Krid) et al., Seed Sci. Res. 1: 209: 219 (1991)), phaseolin, zein, soybean trypsin inhibitor, ACP, stearoyl-ACP desaturase, beta-subunit of β-conglycinin ( I am 7s, (Chen et al., Proc. Nati, Acad. Sci., 83: 8560-8564 (1986))) and oleosin. It would be advantageous to direct the location of the proteins by conferring DAGAT to a particular subcellular compartment, for example, to the mitochondria compartment, endoplasmic reticulum, vacuoles, chloroplasts or other plastid compartments. For example, wherein the genes of interest of the present invention will be directed to plastids, such as chloroplasts, for expression, the constructs would also employ the use of sequences to direct the gene to the plastid. Said sequences are referred to herein as chloroplast transit peptides (CTP) or plastid transit peptides (PTP). Thus, when the gene of interest is not inserted directly into the plastid, the expression construct will additionally comprise a gene encoding a transit peptide to direct the gene of interest to the plastid. Chloroplast transit peptides can be derived from the gene of interest, or they can be derived from a heterologous sequence having a CTP. Such transit peptides are well known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol. Chem. 264: 17544-17550; della-Cioppa eí al. (1987) Plant Physiol. 84: 965-968; Romer eí al. (1993) Biochem. Biophys, Res Commun. 196: 1414-1421; and Shah et al. (1986) Science 233: 478-481.

Depending on the determined use, the constructs may comprise the nucleic acid sequence encoding the complete DAGAT protein, or a portion thereof. For example, when antisense inhibition of a determined DAGAT protein is desired, the complete DAGAT sequence is not required. Furthermore, when the DAGAT sequences used in constructions are intended to be used as probes, it would be advantageous to prepare constructs containing only a particular portion of the DAGAT coding sequence, for example a sequence that is discovered, encodes a highly conserved DAGAT region. The person skilled in the art will recognize that there are different methods for the inhibition of expression of endogenous sequences in a host cell. Such methods include, but are not limited to antisense suppression (Smith, et al. (1988) Nature 334: 724-726), co-suppression (Napoli, et al. (1989) Plant Cell 2: 279-289), ribozymes (PCT publication WO 97/10328), and Waterhouse sense and antisense combinations, ei al. (1998) Proc. Nati Acad. Sci. E.U.A. 95: 13959-13964. Methods for the suppression of endogenous sequences in a host cell typically employ the transcription or transcription and translation of at least a portion of the sequence to be deleted. Said sequences can be homologous to the coding as well as non-coding regions of the endogenous sequence. The regulatory regions of transcription termination can be provided in plant expression constructs of this invention. The terminating regions of the transcript may be provided by the DNA sequence encoding the diacylglycerol acyltransferase or a convenient transcription terminus region derived from a different gene source, eg, the transcription terminus region that is associated naturally with the region of initiation of transcription. One skilled in the art will recognize that any convenient transcription terminus region that is capable of terminating transcription in a plant cell can be employed in the constructions of the present invention. Alternatively, the constructs can be prepared to direct expression of the DAGAT sequences directly from the plastid of the host plant cell. Such constructions and methods are known in the art and are generally described, for example, in Svab. i to the. (1990) Proc. Nati Acad. Sci. E.U.A. 87: 8526-8530 and Svab and Maliga (1993) Proc. Nati Acad. Sci. E.U.A. 90: 913-917 and US patent. No. 5,693,507. A plant cell, tissue, organ or plant into which the recombinant DNA constructs containing the expression constructs have been introduced are considered transformed, transfected or transgenic. A transgenic or transformed cell or plant also includes progeny of the cell or plant and progeny produced from a breeding program employing said transgenic plant as a parent in a cross and presenting an altered phenotype resulting in the presence of an acid sequence. DAGAT nucleic acid ^ * Plant expression or transcription constructs having a plant DAGAT as the DNA sequence of interest for increased or decreased expression thereof can be employed with a wide variety of plant life, particularly, plant life involved in the production of vegetable oils for edible and industrial uses. Most preferred are the transformed oilseed crops. Plants of interest include, but are not limited to, rapeseed (tall varieties in erucic acid and cañola), sunflower, safflower, cotton, soybean, peanut, coconut and palm oil, and corn. Depending on the method for introducing the recombinant constructs into the host cell, other DNA sequences may be required. It should be noted that this invention is applicable to similar dicotyledonous and monocotyledonous species and will be readily applicable to new and / or improved transformation and regulation techniques. Of particular interest, is the use of plant DAGAT constructs in plants that have been genetically engineered to produce a particular fatty acid in the plant seed oil, where TAG in the seeds of unmanaged plants of the manipulated species, does not they naturally contain said particular fatty acid. Therefore, the expression of novel DAGAT in plants may be desirable for the incorporation of unique fatty acyl groups in the sn-3 position. Other genetic engineering applications in plants for DAGAT proteins of this invention include their use in the preparation of structured plant lipids containing TAG molecules that have ^ MH lMi. desirable fatty acyl groups incorporated at particular positions on the TAG molecules. It is contemplated that gene sequences may be synthesized, either completely or in part, especially when it is desired to provide preferred plant sequences. Therefore, all or a portion of the desired structural gene (the portion of the gene encoding the DAGAT protein) can be synthesized using codons preferred by a selected host. Preferred host codons can be determined, for example, from codons most frequently used in proteins expressed in a desired host species. One skilled in the art will readily recognize that antibody preparations, nucleic acid probes (DNA and RNA) and the like can be prepared and used to select and recover "homologous" or "related" DAGATs from a variety of plant sources. Homologous sequences are found when there is a sequence identity, which can be determined by comparing the sequence information, the nucleic acid or the amino acid, or through hybridization reactions between a known DAGAT and a candidate source. Conservative changes, such as Glu / Asp, Val / lie, Ser / Thr, Arg / Lys and Gln / Asn can also be considered to determine sequence homology. The amino acid sequences are considered homologous for as little as 25% sequence identity between the two complete mature proteins. (See, uutf ^^ «ÉÉÉiA? iífea? taÉiÉlife - - • * - * - < Generally, Doolittle, RF, OF URFS and ORFS (University Sciencie Books, CA, 1986.) Therefore, other DAGATs can be obtained from the specific exemplified Mortierella protein preparations and sequences provided herein. , it will be evident that natural and synthetic DAGATs can be obtained, including modified amino acid sequences and starting materials for synthetic protein modeling from exemplified DAGATs and from DAGATs that are obtained through the use of said exemplified sequences. modified include sequences that have been mutated, truncated, increased and the like, where said sequences were partially or totally synthesized, sequences that are currently purified from plant preparations or are identical or encode proteins identical thereto., regardless of the method used to obtain the protein or sequence, they are also considered naturally derived. For immunological selection, antibodies to the DAGAT protein can be prepared by injecting rabbits or mice with the purified protein or portion thereof, said methods for preparing antibodies are well known to those skilled in the art. Monoclonal or polyclonal antibodies can be produced, although polyclonal antibodies are typically more useful for the isolation of genes. Western analysis can be performed to determine that a related protein is present in a crude extract of the plant species ^^^ ^ ¿^? Jm ¡? desired, as determined by cross-reaction with antibodies to the DAGAT of Mortierella ramanniana. When cross-reactivity is observed, the genes that code for the related proteins are isolated by selecting expression libraries that represent the desired 5-plant species. Expression libraries can be constructed in a variety of commercially available vectors, including lambda gt11, as described in Sambrook et al., (Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). 10 Many plants use DAGAT proteins in the production of TAG from storage in seeds, and therefore any plant species can be considered as a source of additional DAGAT proteins. Plants that have high amounts of TAG with acyl palmitate or stearate groups that are found in positions sn-1 and sn-3 with oleate or linoleate in sn-2 are preferred candidates for obtaining DAGATs capable of incorporating saturated fatty acids in the sn-3 position of TAG that shows special selectivity for synthesis of structured TAG of the SUS form, where S represents a saturated fatty acid and U represents an unsaturated fatty acid. For example, oils from several tropical plants including cocoa, illipe, salt, shea and Garcinia species such as kokum have been shown to accumulate high amounts of TAG in this form.

U?? ~ ~ ¡^. .r. ^. . r, .. «. Ll £ __l | ^ ^ JT * Plants that have important medium chain fatty acids in their seed oils are preferred as candidates to obtain vegetable DAGATs capable of incorporating medium chain fatty acids in the sn-3 position of TAG. Several species in the genus Cuphea 5 accumulate triglycerides containing medium chain fatty acids in their seeds, for example, procumbens, lute, hookeriana, hyssopifolia, wrightii and inflata. Another source of natural plant of medium chain fatty acids are seeds of the Lauraceae family. In addition to California Bay (Umbellularia californica), Pisa (Actinodophne hookeri), Sweet Bay (Laurus nobilis) and 10 Cinnamomum camphora (camphor) accumulate medium chain fatty acids. Other plant sources include Ulmaceae (elm), Palmae, Myristicaceae, Simarubaceae, Vochysiaceae and Salvadoraceae. Also of particular interest are DAGATs of plant species that incorporate unusual long chain fatty acids in the storage of TAG. For example, nasturtium and meadowfoam contain 22: 1 acyl groups in the seed. It should be noted that plant DAGATs from a variety of sources can be used to investigate the TAG biosynthesis events of plant lipid biosynthesis in a wide variety of applications in 20 v / Vo. Because plants appear to synthesize lipids through the common metabolic pathway, the study and / or application of a plant DAGAT to a heterologous plant host can easily be achieved in a variety of species. In other applications, a vegetable DAGAT can «*» > < «» * «.». ,. . »-. . . . ,. . * ..,. tK I_L_Í_Í_ A 3T L ^ - It should be used outside the native plant source of DAGAT to increase the production and / or modify the modification of the TAG produced or synthesized in vitro. In addition to the isolation of other DAGATs, it is considered that genes for other related acyltransferase proteins can be obtained using sequence information from DAGAT and related nucleic acid sequences. For example, other acyltransferase enzymes are involved in the biosynthesis of plant lipids, including DAGAT plastidial, mitochondrial DAGAT, lysophosphatidylcholine acyltransferase (LPCAT), lysophosphatidylserine acyltransferase (LPSAT), lysophosphatidylethanolamine acyltransferase (LPEAT), phosphatidylcholine diacylglycerol acyltransferase (PDAT), and lysophosphatidylinositol. acyltransferase (LPIAT). Although many of these enzymes catalyze acyltransferase reactions involving the sn-2 position of lysophospholipids, the genes encoding these sequences may also be related to the acyl-CoA plant DAGAT sequences of the present invention and obtainable forms thereof. To determine whether a related gene can be isolated by hybridization with a given sequence, the sequence is labeled to allow detection, typically using radioactivity, although other methods are available. The labeled probe is added to a hybridization solution, and incubated with filters containing the desired nucleic acids, such as Northerm or Southerm blots, or the filters containing cDNA or genomic clones to be selected. Hybridization and washing conditions may vary to optimize hybridization of the probe to the sequences of interest. Low temperatures and high salt concentrations allow the hybridization of more related sequences (low astringency). If the background hybridization is a problem under low stringency conditions, the temperature may be increased in the steps of hybridization or washing and / or decrease in salt content to improve the detection of the specific hybridization sequence. Hybridization and washing temperatures can be adjusted based on the estimated melting temperature of the probe as presented in Beltz, et al. (Methods in Enzymology (1983) 100: 266-285). A useful probe and appropriate hybridization as well as the washing conditions as described above, cDNA or genomic libraries have been identified using the labeled sequences and the optimized conditions. The nucleic acid sequences associated with the vegetable DAGAT proteins will find various uses. For example, recombinant constructs can be prepared which can be used as probes, or which will confer for the expression of the DAGAT protein in host cells to produce an accessible source of the enzyme and / or modify the triglyceride composition found therein. Other useful applications may be found when the host cell is a plant host cell, either in vitro or in vivo. For example, by increasing the amount of a respective medium chain preferred DAGAT available for the vegetable TAG biosynthesis pathway, an increased percentage of medium chain fatty acids in the TAG can be obtained. Similarly, for some applications it would be desirable to decrease the amount of DAGAT endogenously expressed in a plant cell by antisense technology. For example, to allow more opportunity for a foreign inserted DAGAT to transfer saturated acyl groups, or unusual long chain or medium chain fatty acyl groups to the sn-3 position, a decreased expression of a native long Brassica long chain DAGAT would be desirable. . As presented above, the nucleic acid sequence encoding a plant DAGAT of this invention can include genomic sequence, cDNA or mRNA. "Coding" means that the sequence corresponds to a particular amino acid sequence in either a sense or antisense orientation. "Extrachromosomal" means that the sequence is outside the genome of the plant to which it is naturally associated. By "recombinant" is meant that the sequence contains a genetically engineered modification through manipulation via mutagenesis, restriction enzymes and the like. Once the nucleic acid sequence of vegetable DAGAT is obtained, it can be manipulated in different ways. When the sequence involves non-coding flanking regions, the flanking regions can be subjected to reception, mutagenesis, etc. Therefore, transitions, transversions, deletions and insertions can be made in a naturally occurring sequence. In addition, all or part of the sequence can be synthesized. In the structural gene, one or more codons can be modified to provide a modified amino acid sequence, or one or more codon mutations may be introduced to provide a convenient restriction site or other purpose involved with the construction or expression. The structural gene can be further modified using synthetic adapters, linkers to introduce one or more convenient restriction sites, or the like. The nucleic acid or amino acid sequences encoding a plant DAGAT of this invention can be combined with other sequences that are not native, or "heterologous," in various forms. "Heterologous" sequences means any sequence that is not naturally found bound to the plant DAGAT, including, for example, combinations of nucleic acid sequences from the same plant that are not naturally linked together. The DNA sequence encoding a plant DAGAT of this invention can be used together with all or part of the sequences normally associated with DAGAT. In its component parts, a DNA sequence encoding DAGAT is combined in a DNA construct having, in the direction of the 5 'end to the 3' end of transcription, a transcription initiation control region with the capacity of promote transcription and translation in a host cell, the DNA sequence encoding the plant DAGAT and a transcription and translation determination region.

Potential host cells include both prokaryotic and eukaryotic cells. A host cell can be unicellular or is located in a differentiated or undifferentiated multicellular organism depending on the intention of use. The cells of this invention can be distinguished by having DAGAT foreign to the wild-type cell present therein, for example, having a recombinant nucleic acid construct encoding a plant DAGAT therein. Depending on the host, the regulatory regions will vary, including regions of viral, plasmid or chromosomal genes, and the like. For expression in prokaryotic or eukaryotic microorganisms, particularly unicellular hosts, a wide variety of constitutive or regulatory promoters may be employed. Expression in a microorganism can provide an easy source of the plant enzyme. Among the regions of transcriptional initiation that have been described, there are regions of bacterial and yeast hosts, such as E. coli. B. subtilis. Saccharomyces cerevisiae, including genes such as beta-galactosidase, T7 polymerase, tryptophan E and the like. The methods used for the transformation of the host plant cell are not so important for the present invention. The transformation of the plant is preferably permanent, that is, by integration of the expression constructions introduced into the host plant genome, so that the introduced constructions pass over successive plant generations. Those skilled in the art will recognize that a wide variety of transformation techniques exist in the art, and that new techniques are continuously available. Any technique that is suitable for the target host plant may be employed within the scope of the present invention. For example, constructs can be introduced in a variety of ways including, but not limited to, a DNA strand, a plasmid, or an artificial chromosome. The introduction of the constructs into the target plant cells can be achieved by a variety of techniques, including, but not limited to calcium phosphate-DNA coprecipitation, electrophoration, microinjection, Agrobacterium infection, transformation of liposomes or microprojectiles. Those skilled in the art can refer to the literature for more details and to select suitable techniques for use in the methods of the present invention. Normally, included within the DNA construct will be a structural gene that has the regulatory regions necessary for expression in a host and provide for the selection of transforming cells. The gene can confer resistance to a cytotoxic agent, for example, antibiotic, heavy metal, toxin, etc., and a complementation that confers prototrophy to an auxotrophic host, viral immunity or the like. Depending on the number of different host species, constructs or expression components are introduced therein, one or more markers that can be used, where the different conditions for selection for different hosts are used.

When Agrobacterium is used for the transformation of a plant cell, a vector can be used that can be introduced into an Agrobacterium host for homologous recombination with T-DNA or the Ti-o Ri-plasmid present in the host Agrobacterium. The Ti- or R-plasmid that contains the T-DNA for recombination, can be assembled (with the capacity to provoke a gill formation) or disarm (which do not have the capacity to cause the formation of gall), the last one being permissible, as long as the vir genes are present in the transformed host Agrobacterium. The armed plasmid can give a mixture of normal plant cells and galls. In some cases, when Agrobacterium is used as the vehicle to transform the host plant cells, the expression or transcription construct constructed limited by the T-DNA border region (s) in a vector with a wide host range will be inserted. with the capacity of replication in E.coli and Agrobacterium; there are vectors with a wide range of hosts that are described in the literature. PRK2 is commonly used, as well as derivatives thereof. See, for example Ditta, et al., (Nat. Acad. Sc / E. (JA (1980) 77: 7347-7351) and EPA 0 120 515, which are incorporated herein by reference. , sequences to be expressed in plant cells can be inserted into a vector containing separate replication sequences, one of which is stabilized in the vector in E. coli and the other in Agrobacterium.See, for example McBride and Summerfelt (Plant Mol. Biol. (1990) 74: 269-276), where the origin of replication of pRiHRI (Jouanin, et al., Mol.Gennet.Genet. (1985) 201: 370-374) is used and provides an aggregate stability of the plant expression vectors in Agrobacterium host cells, including one or more markers, which will allow the selection of transformed Agrobacterium plant cells and transformed plant cells, will be included with the expression construct and the T-DNA. large number of markers for use with plant cells, ta such as resistance to chloramphenicol, kanamycin, aminoglycoside G418, hygromycin, or the like. The marker used in particular is not essential for this invention, one or the other marker is preferred depending on the particular host and the manner of construction. For the transformation of plant cells using Agrobacterium, explantations can be combined and incubated with the transformed Agrobacterium to allow sufficient time for transformation, for the bacteria to be killed, and for the plant cells to be cultured in an appropriate selective medium. Once calluses are formed, shoot formation can be encouraged by using the appropriate plant hormones according to known methods and the shoots can be transferred to a root formation medium for regeneration of the plants. Then the plants can be grown to produce seeds and the seed can be used to establish repetitive generations and for the isolation of vegetable oils.

There are several possible ways to obtain plant cells of this invention that contain multiple expression constructs. Any means for producing a plant comprises a construct having a DNA sequence encoding diacylglycerol acyltransferase of the present invention, and at least one different construct having another DNA sequence encoding an enzyme and which are included or contemplated by the invention. present invention. For example, the expression construct can be used to transform a plant at the same time as the second construct either by inclusion of both expression constructs in a simple transformation vector or by using separate vectors, each of which expresses the desired genes . The second construction can be introduced in a plant that has already been transformed with the DAGAT expression construction, or alternatively, transformed plants, one expressing the DAGAT construction and the other expressing the second construction, and can be crossed to include the constructions together in the same plant.

Other constructions and methods of use The invention also relates to vectors that include a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombination techniques. Cell-free translation systems can be used to produce said protein using RNAs derived from DNA constructs of the invention. For recombinant production, the host cells can be genetically engineered to incorporate expression systems of portions thereof or polynucleotides of the present invention. The introduction of a polynucleotide into a host cell can be effected by methods that are described in various standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology, (1986) and Sambrook et al, Molecular Cloning: A Laboratory Manual 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989). Such methods include, but are not limited to, calcium phosphate transfection, DEAE dextran-mediated transfection, transvection, microinjection, lipid-mediated cationic transfection, electrophoration, transduction, ballistic introduction, scratch loading and infection. Representative examples of appropriate hosts include bacterial cells, such as streptococcal cells, staphylococci, enterococci, E. coli, Streptomyces, and Bacillus subtilis; fungal cells, such as yeast cells and Aspergillus cells; insect cells, such as Drosophiila S2 and Spodoptera S19 cells; animal cells such as CHO, COS, HeLa. C127, 3T3, BHK, 293 and Bowes melanoma cells; as well as plant cells as described above.

A variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, but are not limited to, chromosomal vectors, episomal vectors and virus-derived vectors, eg, vectors of bacterial plasmids, bacteriophages, transposons, yeast episomes, insert elements, yeast chromosomal elements, viruses such as baculovirus, viruses of papova such as SB40, vaccinia virus, adenovirus, nodular rash virus, seudorabies virus and retroviruses, and vectors derived from combinations of said viruses, such as those derived from bacteriophage and plasmid genetic elements, such as cosmids and phagemids . The constructions of the expression system may contain control regions that regulate and procreate expression. Generally, any system or vector that is suitable for maintaining, propagating or expressing polynucleotides and / or expressing a polypeptide in a host can be used for expression. The appropriate DNA sequence can be inserted into the chosen expression by any variety of well-known and routine techniques, such as, for example, those established in Sambrook et al., Molecular Cloning. A Laboratory Manual, (supra). Appropriate secretion signals, whether homologous or heterologous, can be incorporated into the expressed polypeptide to allow secretion of the protein within the lumen of the endoplasmic reticulum, periplasmic space or extracellular medium.The polypeptides of the present invention can be recovered and purified from recombinant cell cultures by any number of well-known methods, including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anionic or cation exchange chromatography, chromatography. of phosphocellulose, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. It is preferred to use high performance liquid chromatography (HPLC) for purification. Any of the well known techniques for protein folding can be used to regenerate an active confirmation if the polypeptide is denatured during isolation and / or purification. This invention also relates to the use of polynucleotides of the invention as diagnostic reagents. The detection of a mutated form of a gene can be used as a diagnostic tool that is used to assist in the diagnosis of a disease or susceptibility to a disease that results from underexpression, overexpression or altered expression of the gene. A variety of highly known techniques can be used to detect, at the DNA level, an individual that has a mutation in the gene. Nucleic acids can be obtained for the diagnosis of individual infected cells and tissues, such as bone, blood, muscle, cartilage and skin. Genomic DNA can be used directly for detection or can be amplified prior to analysis using PCR or other amplification techniques. RNA or cDNA can also be used in the same way. Deletions and infections can be detected by a change in the size of the purified product compared to the genotype of a reference sequence. Point mutations can be identified by hybridizing amplified DNA to labeled polynucleotide sequences of the invention. Perfectly pairing sequences can be distinguished from unpaired duplexes by RNA-loop digestion or by differences in melting temperature. Differences can also be detected in the sequence, at the DNA level, by comparing the electrophoretic mobility of the DNA fragments in gels, with or without denaturing agents; or by direct DNA sequencing (see, for example, Myers et al., Science 230: 1242 (1985)). A change in sequence at a particular site can also be detected using nuclease protection tests, such as RNA-ase and SI protection or by the chemical cut-off method (see, for example, Cotton et al., Proc. Nati, Acad. Sci., USA 85: 4397-4401 (1985) It is anticipated that an array of oligonucleotide probes including a DAGAT nucleotide sequence or fragments thereof may be used for selection, particularly for mutations. The methods of disposition technology are well known and are useful in gene expression, genetic linkage and analysis of genetic variation capacity (eg see, M. Chee et al., Science, 27: 610- 613 (1996)).

The invention further provides a method for diagnosis or determination of susceptibility to a disease associated with DAGAT activity, particularly diseases associated with altered diacylglycerol cell concentration or protein kinase C activity, including, but not limited to, cancer.; diabetes; cardiopulmonary diseases including, but not limited to heart failure, and atherosclerosis, adipocytosis; leukemia and carcinoma of the skin; fibroblastoma; metabolic disorders; obesity; diseases associated with abnormal lipid metabolism; diseases associated with abnormal absorption of fats; secretion of lipoproteins and adipogenesis by determining an abnormally altered level of polypeptide or mRNA from a sample. The altered expression can be measured at the RNA level by any of the techniques already known in the field of polynucleotide quantification, including, but not limited to, amplification, PCR, RT-PCR, and RNA-loop protection. Northern blotting and other hybridization methods.

Diagnostic tests that detect levels of protein expression including, but not limited to, radioimmunoassays, competitive binding assays, Western Blot analysis and ELISA tests are also contemplated. The nucleotide sequences of the present invention can also be used in the identification of the chromosome. The polypeptides of the invention or variants thereof, or cells expressing them, can be used as an immunogen to produce antibodies that are immunospecific for polypeptides of the present invention. "Immunospecific" means that the antibodies have a substantially higher affinity for the polypeptides of the present invention, compared to the affinity of the antibodies for other related polypeptides. The term "Antibodies" includes monoclonal and polyclonal antibodies, including chimeric, single chain, simionized, humanized, surface renewed, and other types of antibodies replaced in the complementarity determining region (CDR); as well as Fab fragments, including products from a Fab immunoglobulin expression library. Antibodies can be obtained by administering polypeptides or epitopes that carry fragments, analogs or cells to an animal, preferably non-human, using routine protocols. Any of the highly known techniques among continuous cell culture methods can be used to prepare monoclonal antibodies including hybridoma technology (see, for example, Kohier, G. and Milstein, C, Nature 256: 495-497 (1975)); trioma technology; Human B-cell hybridoma technology (Kozbor et al., Immunology Today 4:72 (1983)); and Hybridoma-EBV technology (Colé et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 77-96, (1985)). Single chain, humanized, surface renewed, simionized antibodies and other types of antibodies replaced in CDR can be produced according to methods that are well known in the art.

The antibodies already described can be used to isolate or identify clones that express for the polypeptide or purify polypeptides by affinity chromatography. Antibodies can also be used to treat diseases associated with DAGAT activity, particularly diseases associated with altered diacylglycerol cell concentration or protein kinase C activity including, but not limited to cancer; diabetes; Cardiopulmonary disease is including, but not limited to heart failure, and atherosclerosis; adipocytosis; leukemia and carcinoma of the skin; fibroblastoma; metabolic disorders; obesity; diseases associated with abnormal lipid metabolism; diseases associated with abnormal absorption of fats, lipoprotein secretion and adipogenesis. The present invention also relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, fused to portions of the constant regions of the heavy or light chains of immunoglobulins of the various subclasses (IgG, IgM, IgA and IgE). Preferably, the constant portion of the heavy chain of human IgG, particularly IgG1, is used with fusion in the hinge region. The use of the Fc portion is particularly preferred. (See, for example, WO 94/29458 and WO 94/22914.) The polypeptides of the present invention can also be used to identify compounds that bind to the polypeptide, and in particular, to inhibit or stimulate the activity of the polypeptide by binding. The binding of substrates and ligands of a molecule of reduced size can be evaluated for example in cells, cell-free preparations, chemical libraries, as well as mixtures of natural products.Agonists or antagonists / inhibitors can be natural substrates or ligands or can be a functional or structural mimicry of them., for example, Coligan et al., Current Protocol, Immuno, 1 (2): Chapter 5 (1991). The invention also provides a method for selecting compounds to identify those compounds that bind to the polypeptides or polynucleotides of the present invention and particularly those compounds that increase (agonists) or inhibit (antagonists) the action of polypeptides or polynucleotides of the invention. High-performance selection techniques can be used. As an example, to select antagonists or agonists, a synthetic reaction mixture, a cellular compartment, such as a membrane, cell membrane or cell wall, or a preparation of any of these, comprising the polypeptide of the invention and a substrate is incubated or labeled ligand of said polypeptide in the absence or presence of a candidate compound that has been selected. The ability of the candidate compound to agonize or antagonize a polypeptide of the invention is detected by a reduction in the binding of the labeled ligand or by a decrease in the production of the product coming from the substrate. Candidate compounds that bind freely, without inducing the effects of a polypeptide of the invention, are more susceptible to being good antagonists. On the other hand, compounds that bind adequately and increase the speed of production of the substrate product are considered agonists. The detection of the speed or level of product production of the substrate can be increased using a reporter system such as, but not limited to, colorimetric labeling, inclusion of a reporter gene that is responsible for changes in the activity of the polynucleotide or polypeptide and of binding tests known in the art. Competitive tests that combine a polypeptide of the invention and a potential antagonist with a compound that binds to the polypeptide, ligands or natural substrates, or substrate or ligand mimetics to select antagonist compounds can also be used. The polypeptide of the invention can be labeled, by radioactivity or colorimetric compound, so that the number of such polypeptide molecules that bind to the binding molecule or that are converted to product can be determined to evaluate the efficacy of the potential antagonist. Potential antagonists may include, but are not limited to, small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide or polypeptide of the invention and thereby, inhibit or partially or completely block its activity. The antagonists may also include small organic molecules, peptides, polypeptides and antibodies that bind to the same site in a binding molecule without inducing the activities that are induced by a polypeptide of the invention, thus preventing the action of the polypeptide by preventing it from binding. . Potential antagonists also include small molecules that bind to and occupy the polypeptide binding site, thereby preventing the polypeptide from binding to cell binding molecules, so as to avoid or reduce the normal biological activity of the polypeptide. Examples of such small molecules include, but are not limited to, small organic molecules, peptides and peptide-like molecules. Other potential antagonists include antisense molecules (see, for example, Okano, J. Neurochem, 56: 560 (1991)).; Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Ratón. FL (1988). Antagonists and agonists of DAGAT activity are particularly useful, since DAGAT is important in the formation of chylomicra in the small intestine, VLDL in the liver and for energy storage as triacylglycerol in adipose tissue. In this way, inhibiting the activity of DAGAT in the small intestine, liver and adipose tissues, will reduce the absorption of lipids and triglyceride levels in plasma and decrease adipogenesis. In addition, hypertriglyceridemia has been shown to be an independent risk factor for atherosclerosis (Kugiyama, K., et al., (1998) Circulation 97: 2519-2526.) And is an increased risk marker for coronary artery disease and may serve as a marker for several atherogenic factors. (Grundy, S.M., (1998) Am. J. Cardiol, 81: 18B-25B). Compounds that inhibit DAGAT activity are also useful for controlling intestinal fat absorption, altering the secretion of TAG-rich lipoproteins and controlling serum TAG and reducing adipogenesis (Owen MR, et al. (1997) Biochem J 323: 17-21, Jamdar SC and Cao WF (1995) Biochim Biophys Acta 1255: 237-243). In addition, the diacylglycerol substrate of DAGAT is a signal transducing molecule within the cell and is a known modulator of protein kinase C activity. The altered concentration of cellular diacylglycerol and protein kinase C activity has been associated with cancer (da Costa et al., (1993) J. Biol. Chem. 268: 2100-2105), diabetes (Koya D and King GL (1998) Diabetes 47: 859-866), cardiac deficiency (Okumura, et al., (1991) J. Mol. Cell, Cardiol 23: 409-416), adipocytes (Baldo et al., 81995) J. Lipid Res., 36: 1415-1426), leukemia and skin carcinoma cells (Goldokorn T., and Ding .T. (1997) Adv. Exp. Med. Biol., 400A.461-472), and rat fibroblasts (Pai et al., (1991) Proc. Nati. Acad. Sci., 88: 598-602 ). As such, the agonists and antagonists of the invention are particularly useful for treating or ameliorating diseases associated with DAGAT activity, including diseases associated with altered concentration of cellular diacylglycerol or protein kinase C activity, including but not limited to cancer, diabetes , cardiopulmonary diseases, which include but are not limited to cardiac deficiency and atherosclerosis; adipositosis; leukemia and skin carcinoma; fibroblastoma, metabolic disorders; obesity, diseases associated with abnormal lipid metabolism; diseases associated with abnormal fat absorption, lipoprotein secretion and adipogenesis. The invention also relates to compositions comprising the polynucleotide or the polypeptide, or variants, agonists or antagonists thereof. The polypeptides of the invention can be used in combination with a sterile or non-sterile vehicle or vehicles for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a subject. Such compositions comprise, for example, a therapeutically effective amount of a polypeptide or other compound of the invention and a pharmaceutically acceptable carrier or excipient. Such vehicles include but are not limited to saline, pH regulated saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation must be consistent with the mode of administration. The invention further relates to pharmaceutical and diagnostic packages or equipment comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. The polypeptides and other compounds of the invention can be administered alone or in combination with other compounds. The pharmaceutical compositions can be administered in any effective and convenient manner, including but not limited to topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular routes., subcutaneous, intranasal and intradermal. The dose scale required will depend on the peptide or other compound of the present invention that is used, the route of administration, the nature of the formulation, the nature of the condition of the subject and the judgment of the physician. Generally, adequate doses will be on the scale from about OJ to 100μg / kg. A large dose variation is expected due to the variety of compounds and differences in administration efficiency. As an example, it is expected that oral administration requires more cans than intravenous administration. The expert doctor can determine the appropriate dose using standard empirical methods. The polypeptides can also be generated endogenously in the subject, which is generally referred to as "gene therapy". For example, cells from a subject can be designed with a polynucleotide, such as a DNA or RNA, to encode a polypeptide, ex vivo, and by the use of a retroviral plasmid vector. The cells are then introduced into the subject. The polynucleotide and polypeptide sequences can also be used to identify additional sequences which are homologous to the sequences of the present invention. The most convenient and preferred method is to store the sequence in a computer-readable medium, for example, floppy disk, CD ROM, hard disk drives, external disk drives, and DVD and then use the stored sequence to search a database of sequence with well-known search tools. Examples of public databases include the DNA Database of Japan (DDB) (http://www.ddbj.nig.ac.jp/): Genebank; (http://www.ncbi.nim.nih.gov/web/Genbank/lndex.htlm); and the Nucleic Acids Sequence Database of the Molecular Biology Laboratory of Europe (EMBL) (http://www.ebi.ac.uk/ebi docs / embl db.html). The person skilled in the art has a number of different search algorithms, one example of which is the series of programs referred to as BLAST programs. There are five implementations of BLAST, three designed for nucleotide sequence issues (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence issues (BLAST and TBLASTN) (Coulson, Trends in Biotechnology, 12: 76-80 (1994)). ); Birren, et al., Genome Analysis, /: 543-559 (1997)). Additional programs for the analysis of identified sequences are available in the art, such as sequence alignment programs, programs for the identification of related sequences more distantly and the like, and which are known to the person skilled in the art. The invention now being generally described, it will be more readily understood with reference to the following examples, which are included only for purposes of illustration and are not intended to limit the present invention.

EXAMPLES EXAMPLE 1 Tests of diacylglycerol acyltransferase (DAGAT) Methods for testing DAGAT activity in preparations of non-solubilized or solubilized proteins are described for Mortierella ramanniana.

A. Non-solubilized samples The activity of DAGAT is tested with 3.67 μM 1-14C-18: 1 -coenzyme A (53.5-54.5 Ci / mol, New England Nuclear, Boston, MA) and 1.5 mM of 1, 2- 18: 1 diacylglycerol (DAG) (Sigma D-0.138, prepared as a 150 mM supply material in 2-methoxyethanol) a pH regulator containing 10 mM potassium phosphate (pH 7.0), 100-150 mM KCl , and 0.1% TX-100 (w / v) in a total volume of 100 μl as described in a similar manner by Kamisaka et al. (1993) supra and Kamisaka et al. (1994) supra. The tests are carried out at 30 ° C for 5 minutes and are terminated with the addition of 1.5 ml of heptane: isopropanol: 0.5M H2S04 (10:40: 1, v / v / v). If necessary, the samples can be diluted with pH regulator before being tested in order to maintain a linear rate of product formation during the test.

B. Solubilized Samples The sample is made as described for unsolubilized samples with the following changes: the amount of 1, 2-18: 1 DAG is reduced to 0.5 mM, the amount of Triton X-100 is increased by 0.2%, and the KCl concentration is maintained between 100-125 mM. It is also necessary to include La-phosphatidic acid (Sigma P-9511, prepared as a 50 mM supply material in 1% Triton x-100 (w / v) to recover activity followed by solubilization with detergent as described in Kamisaka et al. al. (1996 and 1997) supra, with the following protocol modifications: The use of 300 μM of phosphatidic acid instead of 500 μM gives a greater stimulation of DAGAT activity following the treatment by Triton X-100. DAGAT is sensitive to the amount of KCl introduced in the test with the optimal level between 100-125 mM.The tests are performed at 30 ° C for 5 to 30 minutes and are terminated as described for unsolubilized samples.

C. Sample Test Processing After the tests are completed, the samples can be stored at 4 ° C for processing at a later date or they can be processed immediately by the addition of 0J ml 1 M NaHC03 followed by 1 ml of heptane that contains 15 nmoles / ml of triolein as a vehicle for extraction. The samples are subjected to swirling action and after separation of the aqueous and organic phases, the upper organic phase is removed to a new glass flask and washed with 1 ml 1 M NaCl. 40% of the final organic phase is removed for counting by liquid scintillation and the remaining organic phase is transferred to a clean bottle and evaporated to dry under nitrogen gas. The residue is resuspended in 45 μl of hexane and placed on a plate for thin layer chromatography, silica gel glass G (TLC) with a preabsorbent loading zone (Analtech # 31011, Newark, Delaware). The TLC plate is developed in hexane: diethyl ether.acetic acid (50: 50: 1 v / v / v) to the top then dried and scanned by a radioimaging analyzer (AMBIS 3000. San Diego, CA) to determine the portion of radioactivity incorporated in triacylglycerol. The activity is reported in units such as pmol min.

EXAMPLE 2 Culture conditions of Mortierella ramanniana Mortierella ramanniana is grown by inoculating 1 liter of defined glucose media (30 g of glucose, 1.5 g of (NH4) 2S04, 3 g of K2HP04, 0.3 g of MgSO4 »7H20, 0.1 g of NaCl, 5 g of CH3C00Na.3H20 , 10 mg of FeS04 «7H20, 1.2 mg CaCI2» 2H20, 0.2 g of CuS04 «5H20, 1.0 mg of ZnS04« 7H20, 1.0 mg of MnCl2 «4H20, 2 mg of thiamine-HCI and 0.02 mg of biotin in 1 L of water purified by reverse osmosis (pH 5.7) with spores of 1.5-3 x 106 and incubating at 30 ° C with agitation at 200 rpm for 9-11 days.The cultures are harvested by filtration through a Miracloth layer (Calbiochem, La Jolla, CA) The surplus liquid is removed by manual compression.The average yield of packed cells per cultivated liter is 22.5 g.

EXAMPLE 3 Analysis of SDS-PAGE The samples of the column fractions are diluted in SDS-PAGE sample pH buffer (1 x pH regulator = 2% SDS w / v, 250 mM of β-mercaptoethanol, 0.0025% of bromophenol blue) and analyzed by electrophoresis The polyacrylamide gradient gel electrophoresis (10-13%) is performed according to the method of Laemmli ((1970) Nature 227: 680-685) with some of the modifications of Delepelaire (1979) Proc. Nati Acad. Sci. USA 76: 111-115. Sodium dodecylsulfate is used in the reservoir pH regulator above 0.1% but is omitted from the lower reservoir pH regulator, and stacking and resolution gels. The stacking gel contains 5% of a supply material of 30% acrylamide (acrylamide.N.N'-methylene acrylamide, 37.5: 1 Bio-Rad, Hercules, CA), 0.06% ammonium persulfate and 0.1% TEMED (v / v). The resolution gel contains a linear gradient of 10-13% acrylamide supply material stabilized by a linear gradient of 0-10% sucrose. Electrophoresis is performed at room temperature at 150V, constant voltage, for 7-9 hours. Proteins are visualized by staining with silver Aüfr-to j -jo. according to the method of Blum et al. (1987) Electrophoresis 8: 93-99, or with Coomassic blue (0.1% coomassie blue R-250, 50% methanol (v / v), 10% acetic acid (v / v).

EXAMPLE 4 Evaluation of the chromatography used by Kamisaka et al. (1997) in the purification of DAGAT A. Preparation of the fraction of lipidium bodies The following steps were carried out at 4 ° C. Normally, 70-75 g of moist-packed Mortierella ramanniana cells (stored at -70 ° C) are used for each preparation of lipid bodies. Just before use, the cells are thawed on ice and resuspended in 150 ml of buffer pH A (10 mM potassium phosphate (pH 7.0), 0.15 M KCl, 0.5 M sucrose, and 1 mM EDTA). The following protease inhibitors are added to reduce proteolysis: 0.1 μM of aprotinin, 1 μM of leupeptin, and 100 μM of Pefabloc (all from Boehringer Mannheim, Germany). The cells are divided into 5 50 ml tubes and lysed with a Polytron tissue homogenizer (Kinematic GmbH, Brinkman Instruments, Switzerland) in position # 7 with a 1 cm diameter probe for 7 x 1 min. The resulting suspension is transferred to centrifuge tubes (29 x 104 mm) and solid waste is made to pellet by centrifugation at 1500 xg (Beckman Instrumets, J2-21, JA-20 rotor, 3500 rpm) for 10 minutes at 4 minutes. ° C. The supernatant is removed and the pellets are washed with another 5 ml of pH regulator A. After centrifugation, the supernatant volumes are combined. This fraction is referred to as 'S1'. S1 is divided into 6 ultracentrifuge tubes (25 x 89 mm, Beckman Instruments, Fullerton, CA) and each is covered with 5 ml of pH regulator B (10 mM potassium phosphate, pH 7.0, 0J5 M KCl, 0.3 M sucrose, and 1 mM EDTA). Samples are centrifuged at 100,000 x g (Beckman Instruments, L8-M, SW-28 rotor, 21000 rpm) at 4 ° C for 3 hours. The fraction of lipid bodies (LBF), which floats on the upper part of the cover, is recovered with a spatula and transferred to a glass homogenizer (Potter-Elvehjem). The small amounts of LBF remaining in the centrifuge tube are recovered with a pipette by removing 4 ml of the cover of the pH regulator B and combining it with the LBF in the homogenizer. The final LBF is homogenized in 40 rnl of the pH regulator B. The remaining fractions are collected as follows: interface fraction (the interface between pH regulators of 0.3 and 0.5 M sucrose), soluble fraction (the volume of liquid underneath of the interface), and the membrane fraction (a roasted / brown pellet at the bottom of each tube). All are frozen and stored at -70 ° C for solubilization and subsequent purification.

B. Solubilization of DAGAT activity The LBF is thawed on ice and the solubilization is achieved by the addition of Triton X-100 (Boehringer, Mannheim, Germany) from a supply material of 10% (w / v) to a final concentration of 1.3% (w / v). Solid sucrose (Mallinckrodt, Paris, Kentucky) is added to achieve a final concentration of 0.5M. The sample treated with detergent is shaken at 4 ° C for one hour and then divided into six centrifuge tubes (25 x 89 mm Beckman Instruments). Each tube is covered with 5 ml of pH regulator B. Samples are centrifuged at 100,000 x g (Beckman Instruments, L8-M, SW-28 rotor, 21000 rpm) at 4 ° C for 3 hours. The solubilized material, referred to as the "Triton X-100 extract" is recovered by inserting a thin tube through the cover in 1 cm from the bottom of each ultracentrifuge tube and removing the lower layer of 0.5M sucrose with moderate suction leaving behind the top cover of 0.3M sucrose (including a floating fat layer) and the pellet. In the protocol described by Kamisaka et al. (1997) supra, the Lipid Body Fraction was solubilized with 0.1% (w / v) Triton X-100 and subsequently centrifuged at 100,000 x g or filtered through a 0.2 μm filter. As described in Kamisaka et al. (1997) supra, it was necessary to increase the concentration of Triton X-100 to 1.5% for DAGAT activity to join the first column.

C. Chromatography used in the purification of DAGAT Regulator depH C, used for chromatography, contains 10mM of potassium phosphate (pH 7.0), 0.1% of Triton X-100 (w / v) (Boehringer, Mannheim, Germany), 10% of glycerol (w / v), 0J μM of Aprotinin, 1 μM of Leupeptin, 100 μM of Pefabloc (all from Boehringer, Mannheim, Germany) and variable amounts of potassium chloride (75-500 mM). This pH regulator differs from the corresponding column pH regulator used by Kamisaka et al. (1997) supra, in which glycerol is substituted by ethylene glycol and EDTA, DTT and PMSF are omitted while Aprotinin, Leupeptin and Pefabloc are included. Following the protocol of Kamisaka et al. (1997) supra, a column (1.5 cm x 5.8 cm) of Rose-Yellow Aga 86 (Sigma R-8504, St. Louis, MO) is prepared and equilibrated with 150 mM KCl in pH C regulator. Part of the DAGAT activity present in the extract of Triton X-100 did not bind to the agarose-yellow column 86. However, a significant portion of the DAGAT activity was bound to the column by diluting the KCl concentration of the sample applied at 75 mM with an equal volume of pH C regulator (without KCl). In accordance, the column of yellow 86-agarose in KCl at 75 mM is also equilibrated in pH regulator C. Following the application of the sample at 0.56 ml / min, the column is washed with 4 column volumes of pH regulator. balance The activity of DAGAT and the proteins bound to the column are eluted with KCl at 500 mM in pH C regulator (FIG. 1). The activity of DAGAT eluted from the yellow 86-agarose column (fractions 17-20), 1: 3.33 with pH C regulator is diluted to reduce the concentration of KCl to 150 mM. The diluted stock (103 ml) is applied to a heparin-Sepharose CL-6B column (Pharmacia, Uppsala, Sweden, 0.5 cm x 4. 8 cm) equilibrated with KCl at 150 mM in pH C regulator at 0.2 ml / min. The column is washed with 5 volumes in equilibrium pH regulator and the activity of DAGAT and protein is eluted in a linear gradient of 15 ml of KCl at 150-500 mM in pH C regulator. DAGAT activity is eluted in two overlapping peaks. The first peak elutes during the gradient, as discovered by Kamisaka et al. (1997) supra. and a second peak, not discovered by Kamisaka et al., elutes at the end of the gradient with much less protein (Figure 2A). A portion (250 μl) of the two peak fractions of the heparin column is further purified by size exclusion chromatography on a Superdex-200 column (1 x 30 cm, Bio-Rad, Hercules, CA) at 0.2 ml. / min equilibrated with KCl at 150 mM in pH regulator C. By calibration alone, the column is equilibrated with KCl at 150 mM in a modified pH C regulator in which Triton X-100 is replaced with Triton X-100 R ( Calbiochem, La Jolla, CA). The column is calibrated using the filtration standards of Bio-Rad Gel. The DAGAT activity of each of the two peaks of heparin-sepharose CL-6B is eluted to an estimated molecular one of 99 kDa. Additional chromatography is performed on the peak that is eluted later from the heparin column, which contained DAGAT at a higher specific activity. In this case, the second peak of the heparin column (fractions 36-41) is diluted to 1: 6.6 with pH C regulator at a volume of 46.7 ml. The sample is applied to a yellow 86-agarose column (1.0 cm x 6.4 cm) equilibrated with KCl at 75 mM in pH C regulator at 0.5 ml / min.

After washing with 5 column volumes in equilibrium pH regulator, bound proteins and all DAGAT activity are eluted in a linear gradient of 40 ml of KCl at 75-500 mM in pH C regulator. DAGAT activity it elutes as a single peak (Figure 3A). The DAGAT activity is eluted as a single peak (Fig. 3A). The protein composition of the fractions containing DAGAT activity of the heparin and second yellow 86 columns is analyzed by gradient SDS-PAGE according to the protocol in Example 3. Protein bands are detected by silver staining. The band pattern eluting from these columns, fraction by fraction, is compared with the respective DAGAT activity profile. Many protein candidates are present that correlate with the presence of DAGAT activity. This purification protocol is insufficient to identify a particular protein candidate associated with the DAGAT activity (Figures 2B, 3B).

EXAMPLE 5 New purification protocol to identify DAGAT protein candidates A. Preparation of the lipid body fraction The following steps are carried out at 4 ° C. Typically, it is used at 70-75 g of wet-packed Mortierella ramanniana cells (stored at -70 ° C) for each lipid body preparation. Shortly before use, the cells are thawed on ice and 350 ml of pH A buffer (10 mM dipotassium phosphate (pH 7.0), 0.15 M KCl, 0.5 M sucrose, 1 mM EDTA) is resuspended. The following protease inhibitors are added to reduce to proteolysis: 0.1 μM aprotinin, 1 μM leupeptin and 100 μM pefabloc (all from Bead-Beater, Ciospec Products, Germany). The samples are lysed with a disgregator using glass globules of 0.5 mm. The sample chamber is filled with 180 ml of glass globules. The wet packed cells are thawed on ice and resuspended in 150 ml of pH regulator A. The diluted mixture of cells is poured onto the glass beads. In general, an additional 40-50 ml of pH regulator A is needed. To fill the chamber for proper operation. This volume is used to rinse the traces of the diluted mixture of cells from their original container, so that it can be combined with the rest of the sample. The cells are ground (set to "homogenize") for 45-90 seconds depending on the viscosity of the sample. Divide the diluted mixture of the cells containing glass globules in tubes (29 x 104 mm) and centrifuge at 500 x g (Beckman Instruments, GP centrifuge, horizontal rotor GH 3.7 at 1500 rpm) and at 4 ° C. The supernatant is removed and the pellets are washed with another 5 ml of pH regulator A. After centrifugation, the supernatant volumes are combined. This reaction is referred to as the "S1". The S1 is divided into six ultracentrifuge tubes (25 x 89 mm, Beckman Instruments) and each is covered with 5 ml with modified B-regulator (dipotassium phosphate at 10 mM, pH 7.0, KCl at 0J5M and sucrose at 0.3 M). The EDTA of pH regulator B is omitted (see example 4) since it interferes with hydroxylapatite chromatography. The samples are centrifuged at 100,000 x g (Beckman Instruments, L8-M, SW-28 rotor, 21000 rpm) at 4 ° C for 3 hours. The fraction of the lipid body (LBF) that floats on top of the layer with a spatula is recovered and transferred to a glass homogenizer. Small amounts of LBF remaining in the centrifuge tube are recovered with a pipette, removing 4 ml of the buffer layer of pH B by combining it with the LBF in the homogenizer. The final LBF is homogenized in 40 ml pH regulator B. The remaining fractions are pooled as follows: the interface fraction (the interface between the sucrose pH regulators from 0.3 to 0.5 M), the soluble fraction (the liquid volume) below the interface) and the membrane fraction (a cinnamon / coffee pellet at the bottom of each tube). Freeze all and store at -70 ° C for further solubilization and purification.

B. Solubilization of the DAGAT activity of the lipid body fraction Prior to solubilization, a protein determination is made with an aliquot of the lipid body fraction by the Bradford method (Bio-Rad Reagent, Hercules, CA ) using bovine serum alum as a standard. The LBF is frozen on ice, then diluted to a concentration of 1 mg protein / ml and treated with Triton X-00 in a ratio of detergent to protein of 15: 1 (w / w, equivalent to 1.3% Triton X-100). Solid sucrose (Mallinckrodt, Paris, Kentucky) is added to achieve the final concentration of 0.5M. The treated sample was rocked with detergent at 4 ° C for 1 h and then divided into 6 ultracentrifuge tubes (25 x 89 mm, Beckman Instruments). Each tube is covered with 5 ml of modified pH B regulator. The samples are centrifuged at 100,000 x g (Beckman Instruments, L-8M, broken SW-28, 21,000 rpm) at 4 ° C for 3 hours. The solubilized material is recovered, which is referred to as the "Triton X-100 extract, inserting a thin tube through a cap up to 1 cm from the bottom of each ultracentrifuge tube, removing the lower layer, sucrose at 0.5M, with gentle suction, while leaving the top layer of sucrose at 0.3M (including a flat floating layer) and the pellet behind.

C. DAGAT column chromatography A method of purification of yellow 86-agarose followed by hydroxylapatite chromatography is used to further purify the protein. The method is performed in two ways. In protocol A, the activity is linked to the first column and, after elution, the fractions are assayed for activity. The active fractions are then combined and applied to the second column (which is also referred to as the sequential cycle). In protocol B, the activity is attached to the first column, then eluted and flows directly over the second column without joining and testing in the meantime (which is also referred to as the tandem cycle). In protocol A, Triton X-100 extract is applied to a yellow 86-agarose column (2.5 cm x 6.4 cm) equilibrated with KCl at 75 mM in buffer pH C (example 4.C) at 2 ml / min. The column is washed with 5 column volumes of the equilibration pH regulator, then eluted with KCl at 500 mM in pH C regulator at 0.5 ml / min (FIG. 4). The other more active fractions (64 and 65), which contains 93% of the eluted activity, are put together and loaded onto a hydroxylapatite column (Bio-Gel HT, Bio-Rad, 1 cm x 25.5 cm) equilibrated with KCl a 500 mM in pH regulator C at 0.5 ml / min. The DAGAT activity flows through the column, while most of the proteins bind to the column. The column is washed with three volumes of equilibration pH regulator. The bound proteins are eluted with dipotassium phosphate at 100 mM and KCl at 500 mM the pH regulator C at 0.5 ml / min (FIG. 5A). A portion of the fractions containing the peak of DAGAT activity on SDS-PAGE gradient gel is handled as described in example 9. The proteins are stained with silver and the pattern of the bands is compared, fraction by fraction, with the activity profile (Figure 5B). Several protein DAGAT candidates correlate with activity. In particular, attention is drawn to the bands that move in positions corresponding to approximately 43 kDa, 36.5 kDa, 33 kDa, 29 kDa, 29 kDa and 27 kDa. There does not seem to be a candidate protein in the 53 kDa region that correlates with the activity. In protocol B, Triton X-100 extract is applied to a yellow 86-agarose column (1.5 cm x 5.8 cm) equilibrated with 75 mM KCl in pH C regulator at 1 ml / min. The column is washed with 5 column volumes of equilibration pH regulator. Then, the output of the yellow 86-agarose column is connected to the inlet of a hydroxylapatite column (1.0 cm x 26.2 cm, Bio-Gel HT, Bio-Rad, Hercules, CA) equilibrated with KCl at 500 mM of regulator of pH C. The DAGAT activity bound to the yellow 86 column is eluted with 110 ml of pH C buffer containing 500 mM KCl and passed directly through the hydroxylapatite column at 0.2 ml / min. Finally, the hydroxylapatite column is disconnected from the yellow 86-agarose column and the proteins bound to the hydroxylapatite column are eluted with dipotassium phosphate at 100 mM and KCl at 500 mM in pH-C buffer. DAGAT activity is found in fractions of the hydroxylapatite column during a 110 ml wash with pH C buffer containing 500 mM KCl. Most of the protein in the Triton X-100 extract does not bind to the 86-agarose yellow column and is discarded. A small subset of proteins, including DAGAT, if bound to the yellow 86-agarose column and diluted with KCl at 500 mM in pH C regulator. When this eluate product is applied to the hydroxylapatite column, the DAGAT activity it flows through, whereas most of the remaining proteins bind to the column and separate (Figure 6A). A portion of the fractions containing the peak of DAGAT activity on SDS-PAGE of the gradient gel is handled and stained with silver. The pattern of bands eluting from these columns is compared, fraction by fraction, with the respective activity profile of DAGAT. Examination of stained protein bands indicates that a protein at approximately 33 kDa correlates better with DAGAT activity (Figure 6B). The 36.5 kDa candidate protein sequence seen in Figure 5B and the 33 kDa candidate seen in Figure 6B is obtained as described in Examples 8 and 9 and the peptides are used to search the databases. Peptides generated by the 36.5 kDa candidate were paired with glyceraldehyde-3-phosphate (GAP) dehydrogenase. The best match for the 33 kDa candidate peptides is RNA helicase.

EXAMPLE 6 Protocol modified to identify DAGAT A. Preparation of the lipid body fraction The following steps were carried out at 4 ° C. Typically, 72-75 g of moist Mortierella ramanniana packed cells (stored at -70 ° C) are used for each lipid body preparation. Shortly before use, the cells are thawed on ice and resuspended in 150 ml of pH A buffer (10 mM dipotassium phosphate (pH 7.0), 1 M KCl, 0.5 M sucrose, 1 mM EDTA). The KCl concentration is increased from 0.15 M to 1 M in order to reduce the non-specific binding of soluble proteins with the lipid body fraction. The following protease inhibitors are added to reduce proteolysis: aprotinin at 0.1 μM, leupeptin at 1 μM and pefabloc at 100 μM (all from Boehringer Mannheim, Germany). The samples are lysed with a cell disrupter (globule beater, Biospec Products, Bartlesville, OK) using 0.5 mm glass beads. The sample chamber was filled with 180 ml of glass globules. The wet packed cells are thawed on ice and resuspended in 150 ml of pH regulator A. The diluted mixture of cells is poured onto the glass beads. In general, an additional 40-50 ml of pH regulator A is needed to fill the chamber for proper operation. This volume is used to rinse the traces of the diluted mixture of cells from their original container, so that it is combined with the rest of the sample. The ice chamber is surrounded in order to keep the sample cold during the lysis. The cells are ground (set to "homogenize") for 15 seconds, then cooled for one minute and the procedure repeated 2 times. The gaseous mixture of cells containing glass globules is divided into tubes (29 x 104 mm) and centrifuged at 1500 x g (Beckman Instruments, GP centrifuge, horizontal rotor GH 3.7 at 2460 rpm) for 10 minutes at 4 ° C. The supernatant is removed and the pellets are washed with another 5 ml of pH regulator A. After centrifugation, the supernatants are combined. This fraction is referred to as "S1". The S1 is divided into 6 ultracentrifuge tubes (25 x 89 mm, Beckman Instruments) and each is covered with 5 ml of modified pH B regulator (dipotassium phosphate 10 mm, pH 7.0, KCl at 1 M and sucrose at 0.3 M) The EDTA of pH regulator B is omitted (see example 4) since it interferes with hydroxylapatite chromatography. The samples are centrifuged at 100,000 x g (Beckman Instruments, L8-M, SW-28 rotor, 21000 rpm) at 4'C for 3 hours. The lipid body fraction (LBF), which floats on top of the layers, is recovered with a spatula and transferred to a glass homogenizer for solubilization. The remaining fractions are pooled as follows: the soluble fraction (the liquid volume below the lipid body fraction) and the membrane fraction (the tan / brown pellet at the bottom of each tube) of each tube are combined and he recovers for his trial. The membrane fraction is resuspended in 3.8-4 ml of modified pH A regulator (in which the concentration of KCl to KCl has been reduced to 75 mm).

B. Solubilization of the DAGAT activity of the lipid body fraction. The same day the final LBF is homogenized in 50 ml the pH regulator of solubilization (dipotassium phosphate at 10 mM (pH 7.0), KCl at 75 mM, sucrose a 0.5 M, Triton X-100 at 1.5%) and the homogenized product is centrifuged at 90,000 xg for 1.8 hours in SW-28 at 27 K rpm). Following the centrifugation, the floating lipid layer is discarded and the solubilized layer (Triton X-100 extract) is combined and stored at -70 ° C pending further purification. The extract of Triton X-100 is ready to be loaded onto the first column without further dilution.

C. DAGAT column chromatography using yellow 86-agarose and HA in tandem mode (protocol B) To compare with the protocol described in example 5, a lipid body fraction is prepared as described in example B (low salt ) and another lipid body fraction is prepared as described in example 6B (high salt). Each preparation is solubilized with Triton X-100. The extracts of Triton X-100 are chromatographed through yellow 86-agarose and hydroxylapatite as described in example 5 C, protocol B. The amount of protein recovered in the high salt preparation is greater than that recovered in the low salt preparation as shown in Figure 7A (high salt) and 7B (low salt).

All subsequent preparations are made using the high salt protocol described in Example 6A-B. These two comparative preparations also reveal additional DAGAT protein candidates after SDS-PAGE analysis that are not previously seen, specifically using the high-salt protocol. Active fractions of the two purifications are prepared for gel digestion by precipitating fractions from the HA column as described in example 8B and separating by gel SDS-PAGE from the gradient as described in example 8C. Coomassie-stained proteins of approximate sizes of 55, 50, 39, 36.5, 36.33.32.5, 32.39 and 27 kDa are cleaved, the gel made from the high salt preparation (Figure 7A). Coomassie stained proteins of approximate sizes of 39, 36.5, 36, 35, 32, 31, 29 and 27 are cleaved from the gel made from the low salt preparation (Figure 7B). These candidates are stored at -70 ° C for later use in protein sequencing. The kDa band of the high salt preparation was named Mr18. The 36 kDa band of the low salt preparation was named Mr19.

D. DAGAT column chromatography using Agarose-yellow 86, hydroxylapatite and Heparin. The extract of Triton X-100 described in Example 6B is melted and applied to a column of Agarose-yellow 86 (2.5 cm x 6.4 cm) balanced with 75 mM KCl in a pH regulator of pH C (10 mM phosphate) of potassium (pH 7.0), 0J% by weight of Tx-100, 10% by weight of glycerol) at 2 ml / min. most of the protein does not bind the column, but part of the protein and DAGAT activity bind the column. The column is washed with volumes of 5 buffer pH columns of equilibration pH, then the bound protein and the DAGAT activity are eluted on a linear gradient of 120 ml of 75 to 500 nM KCl in a pH buffer pH C at 2 ml / min. The fractions are analyzed immediately and the active fractions are mixed and concentrated 8 times by ultrafiltration using a stirred pressurized cell (Amicon) mounted with a YM-30 membrane. The concentrate is placed in a hydroxylapatite column (approximately 1.0 cm x 26 cm, Bio-Rad, Hercules, CA) equilibrated with 500 mM KCl in a buffer pH of pH C at 0.5 ml / min. and the column is washed with 40 ml of equilibrium pH regulator. Since the DAGAT activity is in the direct and washed fluid, the combined proteins are not eluted in this experiment. The active fractions are mixed and diluted in a 1: 3.3 ratio to reduce the KCl concentration from 500 to 150 mM. The diluted sample is applied to a column of Heparin (0.55 x 4.7 cm) equilibrated with 150 mM KCl in a pH regulator of pH C at 0.5 ml / min. The column is washed with 5 volumes of buffer of pH of equilibration and the bound protein is eluted in a linear gradient of 10 ml of 150 to 500 mM of KCl in a pH regulator of pH C at 0.25 ml / min. After the gradient the column is washed with 15 volumes of 500 mM KCl in a pH regulator of pH C at 0.25 ml / min. The activity of DAGAT is eluted in two peaks. One during the gradient and another during the laying of 500 mM KCl after the gradient. The fractions on the profile of the column, including those containing DAGAT activity, are concentrated by precipitation as in example 8. The precipitated samples are separated by SDS-PAGE gradient gel and the gel is stained with silver as in the example 3. The pattern of bands eluting from the column are compared, fraction by fraction, with the respective DAGAT activity profile (Figure 8A). Examination of the stained protein bands indicates that a protein in the size range from about 36 kDa to about 37 kDa has better correlation with the DAGAT activity found in the peak that elutes during the washing of 500 mM KCl (Figure 8B). ). Based on this information, protein bands of about 36 to about 37 kDa removed from the two gels described in Example 6C are sent to gel digestion and protein sequencing.

EXAMPLE 7 Progressive increase of the purification protocol to identify DAGAT protein candidates from Mortierella ramanniana The purification protocol described in example 6D indicates that two possible forms of DAGAT may be present in this preparation, however, there is not enough protein in the final purification step to proceed with protein sequencing, therefore, a Progressive increase of the protocol.

A. Progressive Increase by Agarose- yellow 86 The extract of Triton X-100 described in Example 6A and 6B is melted and applied to a column of Agarose-yellow 86 (2.5 cm x 6.4 cm) equilibrated with 75 mM KCl in a pH regulator of pH C (10 mM potassium phosphate (pH 7.0), 0.1% by weight of Tx-100, 10% by weight of glycerol at 2 ml / min.The majority of the protein does not bind the column but a part of the protein and DAGAT activity bind the column.The column is washed with 5 column volumes of equilibration pH regulator, then the bound protein and the DAGAT activity are eluted with 500 mM KCl in a pH regulator from pH C to 2 pnl / min (Figure 9) DAGAT activity is stable to melt / freeze in this purification step so the eluted fractions are typically stored at -70 ° C at this stage. The eluted fractions are also analyzed to find DAGAT activity in accordance with Example 1 B.

B. Hydroxylapatite Chromatography After four preparations are purified by Agarose-yellow 86, the most active fractions are mixed, concentrated 12 to 14 times by ultrafiltration (Amicon stirred cell, YM-30 membrane) and applied (0.5 ml). / min) to a column of hydroxylapatite (Bio-Gel HT, Bio-Rad, 1 cm x 25.5 cm) equilibrated with 500 mM KCl in a pH regulator of pH C. The concentration of the sample is carried out before HA chromatography in order to reduce the time required to load the sample. The DAGAT activity flows through the column while most of the remaining proteins bind the column and separate. The column is washed with three pH buffer volumes of equilibration pH. The combined proteins are eluted with 100 mM dipotassium phosphate and 500 mM KCl in a buffer pH of pH C at 0.5 ml / min (Figure 10A). A portion of the fractions containing the DAGAT activity peak are performed in SDS-PAGE gradient gel as described in example 3. The proteins are stained with silver and the pattern of the bands is compared, fraction by fraction, with the activity profile (figure 10B). Several DAGAT protein candidates keep correlation with the activity. In particular, the bands that migrate to positions that correspond approximately to 36. KD, 36 kD, 35 kDa, 34 kD, 33kD and 31 kD draw attention. Again, there does not appear to be a candidate protein in the previously described 53 kD region that retains correlation with activity.

C. Chromatography in Heparin Following hydroxylapatite chromatography, the DAGAT activity is not stable to freeze / melt so that the fractions are analyzed immediately and the active fractions are mixed for another chromatography.

The pool is diluted with a pH C pH regulator to lower the KCl concentration from 500 mM to 150 mM KCl. The diluted pool is loaded on a Heparin column (0.55 x 4.7 cm) equilibrated with 150 mM KCl in a pH C regulator. The protein and DAGAT activity are eluted during a gradient of 10 ml of 150 to 500 mM. KCl in a pH C regulator followed by a 10 ml wash with 500 mM KCl in a pH C regulator. The DAGAT activity is eluted in two peaks, an acute peak is found during the KCl gradient and another peak wide during washing (Figure 11A). A portion of the fractions containing the peak DAGAT activity are performed on a gradient SDS-PAGE gel and stained with silver. The pattern of the bands that are eluted from the column is compared, fraction by fraction, to the respective DAGAT activity profile. Examination of the stained protein bands indicates that a protein at 36 kDa has a better correlation with the DAGAT activity found at the larger peak (FIG. 11B). Several proteins (approximately 36.5 kDa, 35 kDa, 34 kDa) are associated with the activity found in the most acute peak. Candidates of approximately 33 kDa and approximately 31 kDa do not appear to be correlated with DAGAT activity. Table 1 shows the number of times of the purification from the 1500 x g fraction by Heparin.

TABLE 1 The four identified candidates (at approximately 36.5 kDa, 36 kDa, 35 kDa and 34 kDa) are prepared for gel digestion by fractions that are precipitated from the Heparin column as described in Example 8B and separated by SDS-gradient gel. PAGE as described in example 8C. In this way, the peptide maps of each of the DAGAT candidates are obtained and individual peptides are selected for protein sequencing.

D. Yellow Agarose-86 Chromatography with gradient elution In order to examine another purification protocol, DAGAT is purified by hydroxylapatite as described in example 6A, diluted in 75 mM KCl and applied to a yellow agarose-86 column ( 1.3 x 6.3 cm) equilibrated with 75 mM KCl in pH C regulator. The column is washed with 25 ml of equilibration pH regulator and the combined proteins are eluted on a gradient of 40 ml of 75 to 500 mM KCl in PH regulator C. The fractions are analyzed to find DAGAT activity as in example 1 B. The activity of DAGAT appears as a simple peak in the middle of the gradient. Fractions containing DAGAT activity are concentrated by precipitation as in example 8B and separated by SDS-PAGE analysis as in example 8C. The pattern of bands eluting from the column, fraction by fraction, is compared to the respective DAGAT activity profile (Figure 12A). The 34 kDa protein candidate elutes early in the gradient and does not appear to correlate with the DAGAT activity (Figure 12B). Three remaining protein candidates (approximately 36.5 kDa, 36 kDa and 35 kDa, designated Mr21, Mr22, Mr23, respectively) correlate with DAGAT activity. EXAMPLE 8 Preparation of protein for gel digestion After a protein candidate has been identified, it is necessary to prepare sufficient quantities for sequencing. Protein sequencing can be performed using a wide variety of methods known in the art. Another technique involves the digestion of the protein, using enzymes such as trypsin, while it is in a gel.

^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ SDS. Several commercial companies have established protocols to obtain peptides in this way. Following the generation of peptides, standard techniques are used to separate and sequence them. In order to gel purify a protein candidate, it is often necessary to first concentrate the liquid sample so that it can be gel loaded. Samples containing large amounts of detergent can pose special problems. Depending on the micelle size of the detergent, it can concentrate during ultrafiltration and pose problems during electrophoresis. An alternative method for concentrating the protein sample must then be employed.

A. Sample preparation for SDS-PAGE analysis by concentration Fractions can be concentrated in an adapted pressure cell with a membrane of the appropriate limit of molecular weight retention. Alternatively, the sample can be concentrated using filtration by centrifugation in individual units, for example a product such as Centricon-30 (Amicon, Inc., Beverly, MA), at volumes of approximately 50μl. After concentration, the samples can be treated with charge buffer, for example Laemmli.

B. Preparation of samples for SDS-PAGE analysis by precipitation Sometimes it is convenient to concentrate the samples by precipitation. This can be achieved by using an acid and / or ketone. A typical protocol would be to add trichloroacetic acid (TCA) from a concentrated stock (40% to 50% (w / v) to a final concentration of 7 to 10% (w / v). After about 10 minutes on ice the samples will be centrifuge (12,000 xg, 15 minutes at 4 C) to pellet the precipitated protein.The supernatants are removed and the precipitated detergent is removed, the pellets are washed with ice-cold acetone and centrifuged again. The precipitates can be resuspended with a sample charge pH regulator (ie, Laemmli sample pH regulator or SDS-PAGE analysis as in example 3). The SDS-PAGE analysis can be performed using melted gels in the laboratory, as described in example 3 or gels prepared by commercial sources.

C. SDS-PAGE Analysis Samples may be heated or not heated before loading the gel. It has been observed that some membrane proteins have the tendency to aggregate after being heated. In this case, the samples are generally applied to the gel after standing at room temperature for 15 minutes. Acrylamide gels can be purchased commercially or prepared in the laboratory. A protocol for preparing 10 to 13% (w / v) of acrylamide gel is described in Example 3. After electrophoresis, the gel can be stained with 0.1% (w / v) of Coomassie Blue in 50% ( v / v) of methanol, 10% (v / v) acetic acid and then fading. The deinking can be achieved with the use of a commercial product, such as Gel-Clear (Novex, San Diego, Ca) or in 50% (v / v) methanol, 10% (v / v) acetic acid. Protein candidates can be extracted from the gel and sent to gel digestion with or without further deinking.

EXAMPLE 9 Determination of amino acid sequence Some commercial companies have been established that provide protein sequencing as a service. Among the available techniques, the generation of peptides by gel digestion using an endopeptidase, such as trypsin, followed by purification by CLAR, has proven to be the most useful. N-terminal sequencing in PVDF, and to a lesser degree the generation of peptides by limited treatment of cyanogen bromide from PVDF proteins, has also proven successful. Processes for gel digestion include the analysis of amino acids from one part (10 to 15%) of the gel sample for quantification and amino acid composition, digestion of the protein with one of the proteolytic enzymes (trypsin or lysyl endopeptidase) , and fractionation of the products by inverted phase CLAR. The absorption peaks can be selected by HPLC and subjected to a laser desorption mass spectrometry to determine the presence, amount and mass of the peptide before protein sequencing. The longest peptides are selected for microsequencing. In particular, DA05AT candidates are gel purified and sent to Argo Bioanalytica (a commercial service) for gel digestion and microsequencing.

EXAMPLE 10 Amino acid sequence of peptides generated from Tripsin The amino acid sequence of peptides generated from the approximately 36 kDa protein, also called MR1, (see examples 6C and 6D) by trypsin digestion as described in example 9, is as shown below (the first two digits of the number of sequence designate the Mr bands described in Examples 6C and 7C): # sequence Amino acid sequence No. Id. Sec 19-138 ELHDSYMHAV 1 19-169 KlqHALgFTMplFhgr 2 19-181 HPIYTiv 3 18-146 NAAwpk 4 18-151 VKELEFVE 5 18-159-1 FGF 6 18-159-2 yxhDayphave 7 18 -164 ELHDSYMHAVQDLYDR 8 18-208-1 GVFNYDFGLLPHR 9 18-208-2 xlagifpa 10 18-219-1 IAVQTGAGLVPTLSF 11 18-219-2 slAIWgSASEsINA 12 18-219-3 gffNYDFxxl 13 22-158 ELHDSYMHAV 14 The amino acid sequence of peptides generated from the approximately 36.5 kDa protein, also called MR2, (see Example 7B) by trypsin digestion as described in Example 9, is as shown below: # sequence Sequence of amino acids No. Id. Sec, 21-134 VHWAPLR 15 21-149-1 KLPLFk 16 21-149-2 VDIDxAPpR 17 21-160-1 ITGFTVPHAH 18 21-160-2 ELHDSHMLxV 19 21-218 GIFNYNAGFIPFR 20 21-178 hPIYTIVGKpipv 21 21-101 gsCEAILR 22 21- 221 hPIVTWGKPIAVpLLAegeteppse 23 21-197 sRDsTPVITEHKQPMeQvqvtalldhipv 24 The amino acid sequence is represented using the one letter code. The amino acids represented by lowercase letters represent the residues that were identified with a lower degree of reliability. The peptide map of the 35 kDa candidate, Mr23 in Example 7C, is substantially similar to the candidate peptide map of 36.5, MNr21 in Example 7C. The amino acid sequences in the above peptides are compared to the known protein sequences in public and private databases. No significant homology was found between the DAGAT peptides and any sequence encoding an enzyme of known function including any part of glyceraldehyde 3-phosphate hydrogenase (GAP) that is known to migrate at 36kDa by SDS-PAGE analysis.

EXAMPLE 11 Identification of Nuclear Acid Sequences of DAGAT of Mortierella ramanniana In general, oligonucleotides containing the sense orientation sequence corresponding to the coding sequences are prepared to be used as polymerase chain reaction (PCR) primers of a single-stranded DNA model transcribed in reverse form from mRNA. of DAGAT peptide. For the "reverse" reaction of the amplification of the DNA coding arm, an oligonucleotide containing a sequence complementary to the coding sequence of the DAGAT peptide must be designed.

Alternatively, an oligonucleotide can be designed to be identical to a part of a bait used to prepare a DNA model for PCR. This oligonucleotide can be used either as the bait that goes "forward" or "reverse" as described above. Where the DAGAT peptide sequences contain amino acids that can be encoded by a number of different codons, forward or reverse primers can be "degenerate" oligonucleotides, that is, they contain a mixture of all or some of the possible coding sequences for a particular peptide region. To reduce the number of different oligonucleotides present in said mixture, it is preferable to select regions of peptides having the least number of possible coding sequences when preparing the synthetic oligonucleotide for the PCR primers.

A. Identification of DAGAT MR1 To identify the nucleic acid sequence for DAGAT MR1 from Mortierella ramanniana, peptide 18-151 is used to design the degenerate primer 5'-CACTGCAGACRAAYTCNARYTCYTTNAC-3 '(SEQ ID NO: 25), the peptide 18-208-1 to design primers 5'-CCAAGCTTGGNGTNTTYAAYTAYGAYTTYG-3 '(SEQ ID NO: 26) and 5'-CACTGCAGCRAARTCRTARTTRAANACNCC-3' (SEQ ID NO: 27), peptide 18-164 is used to design the 5'-initiator CACTGCAGCYTGNACNGCNGCRTGCATRTA-3 '(SEQ ID NO: 28), peptide 18-129-1 is used to design the 5'-CCAAGCTTATHGCNGTNCARACNGGNGC3' primer (SEQ ID NO: 29), peptide 19-181 is used to design the primers 5'-CCAAGCTTAARCAYCCNATHTAYACNAT-3 '(SEQ ID NO: 30) and 5'-CACTGCAGACDATNGTRTADATNGGRTG-3' (SEQ ID NO: 31), the peptide 19-169 is used to design the primers 5'- CCAAGCTTGCNYTNGGNTTYACNATGCC-3 '(SEQ ID NO: 32), 5'-CCAAGCTTTTYACNATGCCNYTNTTYCA-3' (SEQ ID NO: 33) and 5'-CACTGCAGAARTGRAANARNGGCATNGT-3 '(SEQ ID NO: 34). DNA fragments obtained by PCR are analyzed for nucleic acid sequence encoding the amino acid sequence found in the peptides in example 10. To obtain the entire coding region corresponding to the DAGAT MR1 protein of Mortierella ramanniana, oligonucleotide primers are designed to amplify the 5 'and 3' ends of partial cDNA clones containing MR1 sequences. Initiators are designed according to the DAGAT MR1 sequence of Mortierella ramanniana and are used in Rapid Amplification of cDNA end reactions (RACE) (Frohman et al. (1988) Proc. Nati. Acad. Sci. USA 85: 8998- 9002). The amplification of flanking sequences of cDNA clones is performed using the Marathon cDNA Amplification kit (Clontech, CA). For example, PCR reactions can be performed with 3 'RACE primer 5'-GGTTTTGCTCCCCCATCGCCATCCTATC-3' (SEQ ID NO: 35) and 5 'RACE primer 5'-GATAGGATGGCGATGGGGGAGCAAACC-3' (SEQ ID NO: 36). In this way the complete MR1 coding sequence of 1065 nucleotides is determined (SEQ ID NO: 37). The protein sequence predicted for MR1 DAGAT is also determined (SEQ ID NO: 38). DAGAT nucleic acid sequences are obtained which can be analyzed for nucleic acid sequence and can be used for DAGAT expression in several hosts, both prokaryotic and eukaryotic. The primers 5- AATTCGCGGCCGCATGGCCAGCAAGGATCAACATTTACAGC-3 '(SEQ ID NO: 39) and 5'-TGCTGCAGCTATTCGACGAATTCTAGTTCTTTTACCCGATCC-3' (SEQ ID NO: 40) are used to amplify the open reading frame (ORF) of the Marathon cDNA library by PCR of Mortierella ramanniana made in accordance with the manufacturer's protocol (Clonetech). These primers introduce Notl and Pst1 restriction sites at the 5 'and 3' ends of ORF, respectively. The PCR product is cloned into plasmid pCR2J according to the manufacturer's protocol (Invitrogen) to produce the plasmid pCGN8707. The double-stranded DNA sequence is obtained to verify that no errors are introduced by PCR amplification. For expression of the M. ramanniana DAGAT MR1 protein in insect cells using a baculovirus expression system, the Notl-Pst1 fragment of pCGN8707 is cloned into the pFASTBACI plasmid digested with Notl-PstI (Gibco), and the resulting plasmid, pCGN8708, is transformed into E. coli DH10BAC (Gibco). Bacmid DNA is used to transfect insect cells. For expression of the DAGAT MR1 sequence of Mortierella ramanniana in plants, the Notl-Pstl fragment of pCGN8708 is cloned into the binary vector pCGN8622 digested with Notl-Pstl to produce the plasmid pCGN8709 under the control of a napin promoter. Plasmid pCGN8709 is introduced into Agrobacierium tumefaciens EHA105.

B. Identification of DAGAT MR-2 To identify the nucleic acid sequence for DAGAT MR2 from Montierella ramanniana, peptide 21-221 is used to design the degenerate primer 5'-GGCACNGCDATNGGYTTNCCNAC-3 '(SEQ ID NO: 41) and use peptide 21-218 to design the primer 5'-CCNGCRTTRTARTTRAADATNCC-3 '(SEQ ID NO: 42). These are used in a nested PCR as antisense primers in Rapid Amplification of cDNA End Reactions (RACE) (Frohman et al. (1988) Proc. Nati. Acad. Sci. USA 85: 8998-9002) using a cDNA library built with Marathon's cDNA Amplification equipment (Clontech) according to the manufacturer's instructions. The RACE amplification of the 5 'region corresponding to the DAGAT MR2 protein of Mortierella ramanniana is carried out with the primer 5'-TGCCTAGTGACATCATGAAATCTCG-3' (SEQ ID NO: 43) using a cDNA library constructed with the Amplification cDNA equipment of Marathon (Clontech) according to the instructions of the manufacturers. In this way the partial nucleotide coding sequence is determined (SEQ ID NO: 44). A partial amino acid sequence for the MR2 protein is also predicted (SEQ ID NO: 45). Those skilled in the art will recognize that additional RACE reactions will lead to the cloning of the complete nucleic acid sequence that can be used for DAGAT expression in various hosts, both prokaryotic and eukaryotic.

C. MR1 and MR2 sequence comparison The analysis of protein sequence alignments between the protein sequences of the DAGAT sequences of Mortierella ramanniana MR1 (SEQ ID NO: 38) and MR2 (SEQ ID NO: 45) (FIG. 13) ) shows that they share a 55% similarity.

EXAMPLE 12 Identification of sequences related to DAGAT Because DAGAT in plants are unknown in the art, the nucleic acid and DAGAT protein sequences of Mortierella ramanniana are used to search public and patented EST databases as well as public genomic databases to identify other DAGAT-like sequences.

Three EST sequences can be identified by tblastn in the patented corn database, which are assembled into two contigs using the GCG assembly program (SEQ ID NO: 46-47). An EST can be identified in each of the patented databases of Brassica napus (SEQ ID NO: 48) and soy (SEQ ID NO: 49). Two EST sequences can be identified in patented Arabidopsis thaliana databases (SEQ ID NO: 50-51), and a patented genomic sequence (SEQ ID NO: 52). The MR1 protein sequence is used to search for patented mouse and human databases. The results of this search identified approximately 45 human EST sequences, which are assembled into 5 contigs using the GCG assembly program (SEQ ID NO: 53-57) and 12 mouse assemblies, which are assembled into 3 contigs using the assembly program. GCG (SEQ ID NO: 58-60). Searches of Aspergillus fumigatus (SEQ ID NO: 61 and 62), Aspergillus oraceus (SEQ ID NO: 63), Candida albicans (SEQ ID NO: 64), Fusarium graminearum (SEQ ID NO: 65), Mortierella alpina (SEQ ID NO : 66), and Schizochytrium aggregatum (SEQ ID NO: 67) patented, produce additional EST sequences. Together with these EST sequences, database searches of the predicted public proteins of the genomic and amino acid sequence databases of C. elegans yield four similar sequences, W01A11.2 (SEQ ID NO: 68), K07B1. 4 (SEQ ID NO: 69), F59A1.10 (SEQ ID NO.70), as well as the protein sequence y53G8B_93.B (SEQ ID NO: 71). Similar searches of the predicted protein database of S. cerevisae public produce a sequence, YOR245c (SEQ ID NO: 72). Total RNA was collected from these two organisms, a first-strand cDNA library was created using the Marathon cDNA library kit (Clontech.) The primers 5'-GCGCGGCCGCCTGCAGTCACTGGAAGATGAG-3 '(SEQ ID NO: 73) and 5' -GCGCGGCCGCATGAGACTCCGGCTGAGCTCG-3 ' (SEQ ID NO: 74) are used to PCR amplify W01A11.2 of the C. elegans cDNA library. The primers 5'-GAGCGGCCGCATGCCACATCTACTAGGAGTTGA-3 '(SEQ ID NO: 75) and 5'-CGGCGGCCGCCTGCAGTTAATTGATAACAAGTTGT-3' (SEQ ID NO: 76) are used to PCR amplify the CEK07B1.42 from the C. elegans cDNA library. . 5'-GCGCGGCCGCATGCTAAACTACCAAATTCACA-3 '(SEQ ID NO: 77) and 5'-TGGCGGCCGCCTGCAGTCACTGAAAAACGAGCC-3' (SEQ ID NO: 78) are used to PCR amplify CEF59A1 J0 2 from the C. elegans cDNA library. The primers 5'-CAGCGGCCGCATGTCAGGAACATTC-3 '(SEQ ID NO: 79) and 5'-CACTGCAGTTACCCAACTATCTTCAA-3' (SEQ ID NO: 80) are used to PCR amplify the YOR245C of the cDNA library of S. cerevisae. The PCR products were cloned into pCR2J TOPO according to the manufacturer's protocol (Invitrogen), and these sequences were verified.

EXAMPLE 13 Sequence comparisons Sequence alignments between DAGAT-like sequences from several different sources are compared to identify the similarity between the sequences. The longest sequences are aligned using the Clustal Algorithm in DNASTAR. The following similarity values in percent are obtained compared to the MR1 sequence: ATgC-A1X01ds10429d10a1 19.8% ATLIB22-029-Q1-E1-G7 19.0% ATLIB24-124-Q1-E1-E2 16.8% BNLIB3034-036-Q1-E1-C3 18.2% CEF59A1.10 37.1% CEK07B1.4 36.3% CEW01A11. 2 39.0% HS4371967H1CON 42.0% HS4818474H1 25.9% MALIB26-037-Q1-E1-D8 41.6% MMg2813274 32.4% MMg2892216 30.2% MMg2989686 38.7% MR2 53.9% ZMLIB3136-059-Q1-K1-F10 14.6% GM701121562H1 15.2% The sequences of proteins containing a conserved region corresponding to bases 355 to 796 of MR1 are aligned and truncated to this region, the following percentage of similarity is achieved.

AF804547551 F1 35.1% ATgC-A1X01ds10429d10a1 22.3% ATLIB22-029-Q1-E1-G7 20.0% ATLIB24-124-Q1-E1-E2 18.8% BNLIB3034-036-Q1-E1-C3 19.0% CA803535474F1 33.6% CEF59A1.10 44.9% CEK07B1.4 46.3% CEW01A11.2 50.3% GM701121562H1 25.4% HS4371967H1CON 52.4% MALIB26-037-Q1-E1-D8 55.6% MMg2989686 49.7% MR2 60.3% SCYOR245C 42.4% ZMLIB3136-059-Q1-K1-F10 26.3% EXAMPLE 14 Expression constructions A. Baculovirus expression constructions Constructs are prepared to direct the expression of the DAGAT protein of M. ramanniana in cultured insect cells. The Notl-Pst 1 fragment from pCGN8707 is cloned into the plasmid pFASTBACI digested with Notl-Pstl (Gibco), and the resulting plasmid, pCGN8708, is transformed into E. coli DH10BAC (Gibco). Bacmid DNA is used to transfect insect cells.

B. Preparation of Plant Expression Construction Constructs that provide expression of DAGAT sequences in plant cells can be prepared in the following manner. A plasmid containing the napin cassette derived from pCGN3223 (described in U.S. Patent No. 5,639,790, which is incorporated herein by reference in its entirety) is modified to make it more useful for cloning large DNA fragments containing sites of multiple restriction, and to allow the cloning of multiple napin fusion genes into binary transformation vectors of plants. An adapter comprising the self-priming oligonucleotide of sequence 5'- CGCGATTTAAATGGCGCGCCCTGCAGGCGGCCGCCTGCAGGGCGCGCC ATTTAAAT-3 * (SEQ ID NO: 81) is ligated into the cloning vector pBC SK + (Estratagen) after digestion with the restriction endonuclease BssHIl to construct the vector pCGN7765. Plasmids pCGN3223 and pCGN7765 are digested with Notl and ligated together. The resulting vector, pCGN7770, contains the base structure of pCGN7765 with the napin seed-specific expression cassette of pCGN3223. The plasmid pCGN8618 is constructed by ligating the oligonucleotides 5, -TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGG-3, (SEQ ID NO: 82) and 5'-TCGACCTGCAGGAAGCTTGCGGCCGCGGATCC-3 '(SEQ ID NO: 83) in pCGN7770 digested with Sall / Xhol. A fragment containing the napin promoter, polylinker and 3 'napin region is cut from pCGN8618 by digestion with Asp 7181; the fragment is shaved at its ends by filling at the 5 'protruding ends with Klenow fragment and then it is ligated into pCGN5139 which has been digested with Asp718l and Hindlll and shaved at its ends by filling at the 5' protruding ends with Klenow fragment. A plasmid containing the insert oriented so that the napin promoter is closer to the shaved Asp718l site of pCGN5139 and the 3 'napin is closest to the shaved Hindlll site is subjected to sequence analysis to confirm both insert orientation and integrity. of cloning unions. The resulting plasmid is designated pCGN8622. The Notl / Pst1 fragment of pCGN8708 containing the entire DAGAT coding region is ligated into pCGN8622 directed with Notl / Pst 1 to provide the expression construct pCGN8709 having the DAGAT coding sequence of Mortierella ramanniana placed for transcription of the sense sequence under the regulation of the napin promoter. further, the MR1 nucleic acid sequence is re synthesized (SEQ ID NO: 84) for use as the plant's preferred codon and used to produce expression constructs for transformation into host plant cells. The binary vector constructs are transformed into Agrobacterium cells, such as strain EHA105 (Hood et al., Transgenic Research (1993) 2: 208-218), by the method of Holsters et al. (Mol. Gen. Genet. (1978) 163: 181-187) and used in plant transformation methods as described below.

EXAMPLE 15 Expression of DAGAT in insect cell culture A baculovirus expression system is used to express the full length 36 kDa Mortierella ramanniana cDNA encoding a putative DAGAT in cultured insect cells. The baculovirus expression construct pCGN8708 (see example 14A) is transformed and expressed using the BAC-to-BAC Baculovirus Expression System (Gibco-BRL, Gaithersburg, MD) according to the manufacturer's instructions, except that the harvest of recombinant virus was performed 5 days after transfection. The supernatant of the transfection mixture was used to generate a virus supply which in turn is used to infect Sf9 cells for use in the test.

A. DAGAT Enzyme Activity Testing in Insect Cell Culture Membranes The transformed insect cells can be tested for DAGAT or other acyltransferase activities using the methods described herein. The insect cells are centrifuged and the resulting tablet cells can be used immediately or stored at -70C for further analysis. Cells are resuspended in Medium I (100 mM Tricine / NaOH, pH 7.8, 10% (w / v) glycerol, 280 mM NaCl with: 0.1 μM Aprotinin, 1 μM Leupeptin, and 100 μM from Pefabloc (all from Boehringer Mannheim, Germany) and are lysed by sound treatment (2 x 10 sec) Tablets from cell walls and other residues are formed by centrifugation (14,000 xg, 10 min, 4 ° C). it is transferred to a new bottle and membranes are compressed by centrifugation (100,000 xg, Ti 70.1 rotor, 46,000 rpm for 1 hour at 4 ° C) .The total membranes are resuspended in Medium I. The activity of DAGAT is tested in a 0J ml reaction mixture containing 30 mM Tricine / NaOH, pH 7.8, 56 mM NaCl, 10 mM MgCl 2, 0.2 mM 1,2-diolein in 2-methoxyethanol, 25 mM of 1- 14 C-palmitoyl-CoA (17,600dpm / nmoles), and 0.2-30 rng of membrane protein The 5 minute reaction is terminated by addition of a 1.5 ml solution of and isopropanoheptane: 0.5M sulfuric acid (80: 20: 2, v / v / v). The reaction mixture can be stored at 4 ° C or can be processed immediately as described in example 1C. The 36 kDa Mortierella candidate, when expressed in insect cells, demonstrates 94 times greater DAGAT activity than the control membranes isolated from insect cells infected with an empty vector (Figure 14). The result of the DAGAT activity test demonstrates that this DNA sequence of Mortierella romanniana encodes a protein with DAGAT activity. Similarly, DAGAT homologs identified from yeast (SCYOR245c) and C. elegans (CEK07B1.4, CEF59A1 J 0, and CEWOLA11.2) were also cloned into the pFASTBACI vector (Gibco) to create baculoviral expression constructs pCGN8821 , pCGN8822, pCGN8823, and pCGN8824, respectively. The results of DAGAT enzyme activity tests show significant increases in DAGAT enzyme activity on control vectors when expressed in insect cells (FIG. 15). For example, membranes isolated from insect cells infected with a vector for the expression of the sequence of yeast homologs have a greater than 95 fold increase in DAGAT enzyme activity compared to control membranes isolated from insect cells infected with an empty vector (figure 15). Also, membranes isolated from insect cells infected with a vector for the expression of the C. elegans homologue sequence (pCGN8823) have an approximately 15-fold increase in DAGAT enzyme activity (FIG. 15). Therefore, additional DAGAT coding sequences can now be easily identified using the sequences of the present invention.

B. Production of triacylglycerol in insect cell culture Transformed insect cells can be tested for triacylglycerol, phosphatidylcholine or other lipid classes by methods described herein. A culture cell suspension of the insect is diluted to a standard optical density of 0.3 to 0.6 at an absorbance of 600 nm with the culture medium. To a 4.5 ml sample of culture suspension in the culture medium is added 200 μl of glacial acetic acid, internal standards consisting of 12.5 ug c17: 0 TAG and 25 ug c15: 0 PC, and 10 ml of chloroform: methanol ( 1: 1, v / v). After subjecting to swirling action, the phases are separated by centrifugation (approximately 500 x g, 5 min.). The lower organic phase (OP1) is stored and the upper aqueous phase is extracted again with the lower organic phase of a mixture of 200 μl of glacial acetic acid, 10 ml of chloroform: methanol (1: 1, v / v), and 4.5 ml of water. The samples are re-swirled and centrifuged to separate the phases. The lower organic phase is stored (OP2). The OP1 is filtered through a 0.45 μm filter and the filter is rinsed with OP2. The filtered products are combined and concentrated under nitrogen gas at a final volume of 0.4 ml. Twenty-five percent of the final volume is placed on a GHL TLC plate of hard-shell silica gel with inorganic binder (Alltech Associates, Inc., Newart, Delaware). The TLC plate is developed for 30 minutes in hexane: diethyl ether: acetic acid (80: 20: 2, v / v / v) containing 20 mg / 100 ml of propyl gallate as an antioxidant. After the plate is dried, it is sprayed with 0.001% primulin in 80% acetone and the lipid bands are identified under ultraviolet light. TAG and phospholipid bands are scraped off the TLC plate in glass jars. The samples are methanolized in 2 ml of 5% H2SO4 in methanol at 90 ° C for 2 hours. After the samples have cooled, add 2 ml of 0.9% NaCl and 0.50 ml of hexane. After the sample is subjected to swirling action, it is centrifuged to separate the phases, and the upper hexane layer is taken up for analysis of fatty acid methyl esters (FAME) by gas chromatography using methods well known in the art. The Mortierella candidate of 36 kDa when expressed in insect cells demonstrates a 3J 5 fold increase in triacylglycerol content compared to the control culture of insect cells infected with an empty vector (Figure 16). For comparison, tests were normalized to find the phosolipid content of the cell. The result of the triacylglycerol analysis showed that this DNA sequence of Mortierella ramanniana codes for a protein that leads to the production of triacylglycerol.

EXAMPLE 16 Transformation of plants A variety of methods have been developed to insert a DNA sequence of interest into the genome of a host plant to obtain transcription or transcription and translation of the sequence to effect phenotypic changes. Brassica transgenic plants are obtained by Agrobacterium-mediated transformation as described by Radke et al., (Theor.Appl. Genet. (1988) 75: 685-694, Plant Cell Reports (1992) 11: 499-505). Arabidopsis thaliana transgenic plants can be obtained by Agrobacterium-mediated transformation as described by Valverkens et al., (Proc. Nat. Acad. Sc /. (1988) 85: 5536-5540), or as described by Bent et al. ((1994), Science 265: 1856-1860). Bechtold et al ((1993), C.R. Acad. Sci, Life Sciences 316: 1194-1199). Other plant species can be transformed in a similar way using related techniques. Alternatively, microprojectile bombardment methods, such as those described by Klein et al. (Bio / Technology 10: 286-291) can also be used to obtain nuclear transformed plants. The DAGAT activity can be analyzed in other seeds or other transformed plant materials using the DAGAT test methods described in Examples 1 to 7. The above results demonstrate the ability to obtain partially purified DAGAT proteins that are active in the formation of triacylglycerols. from fatty acyl and diacylglycerol substrates. Methods are provided to obtain the DAGAT proteins and amino acid sequences thereof. In addition, DAGAT nucleic acid sequences can also be obtained from the amino acid sequences using PCR and library selection methods provided herein. Such nucleic acid sequences can be engineered to provide transcription of sequences and / or expression of DAGAT proteins in host cells, whose proteins can be used for a variety of applications. Such applications include the modification of triacrylglycerol levels and compositions in host cells.

All publications and patent applications cited in the present detailed description are incorporated by reference as if each publication or individual patent application will be specifically and individually indicated to be incorporated by reference. Although the invention has been described in detail by way of illustration and example for purposes of clarity and understanding, it will be readily apparent to those skilled in the art by virtue of the teachings of the present invention that certain changes and modifications can be made thereto without Separate from the spirit or scope of the appended claims.

^ MÜU & LIST OF SEQUENCES < 110 > Lardizabal, Kathryn D Hawkins, Deborah J Thompson, Gregory A < 120 > Diacylglycerol Acyl Transferase Proteins < 130 > 17253/00 / WO < 140 > < 141 > < 160 > 84 < 170 > Patentln Ver. 2.0 < 210 > 1 < 211 > 10 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 1 Glu Leu His Asp Ser Tyr Met His Ala Val 1 5 10 < 210 > 2 < 2"11> 16 <212> PRT <213> Mortierella ramanniana <400> 2 Lys lie Gln His Wing Leu Gly Phe Thr Met Pro Leu Phe His Gly Arg 1 5? O 15 < 210 > 3 < 211 > 7 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 3 His Pro He Tyr Thr He Val 1 5 < 210 > 4 < 211 > 6 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 4 Asn Ala Ala Trp Pro Lys < 210 > 5 < 211 > 8 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 5 Val Lys Glu Leu Glu Phe Val Glu 1 5 10 < 210 > 6 < 211 > 3 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 6 Phe Gly Phe 1 < 210 > 7 15 < 211 > 11 < 212 > PRT < 213 > Mortierella ramanniana < 220 > < 221 > INSECURE < 222 > (2) < 400 > 7 Tyr Xaa His Asp Ala Tyr Pro His Ala Val Glu 20 1 5 10 < 210 > 8 < 211 > 16 > 212 > PRT < 213 > Mortierella ramanniana < 400 > 8 Glu Leu His Asp Ser Tyr Met His Wing Val Gln Asp Leu Tyr Asp Arg, 5 10 15 ÜHMifeMa itMM ^? J? ß ^ ^^^^^^ a? ^ ". i .i. i, ",.,!,. my IÉ i, a?. ",« i, .. ^ MfcJ ^ __ ttM, mlM «< 210 > 9 < 211 > 13 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 9 Gly Val Phe Asn Tyr Asp Phe Gly Leu Leu Pro His .Arg 1 5 10 < 210 > 10 < 211 > 8 < 212 > PRT < 213 > Mortierella ramanniana < 220 > < 221 > INSECURE < 222 > (1) < 400 > 10 10 Xaa Leu Ala Gly He Phe Pro Ala 1 5 < 210 > 11 < 211 > 15 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 11 -i c He Wing Val Gln Thr Gly Wing Gly Leu Val Pro Thr Leu Ser Phe 1 5 10 '- 5 < 210 > 12 < 211 > 15 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 12 Ser He Ala He Val Val Gly Ser Ala Ser Glu Ser He Asn Ala 1 5 10 15 < 210 > 13 < 211 > 10 < 212 > PRT < 213 > Mortierella ramanniana < 220 > < 221 > INSECURE < 222 > (8) . (9) _ ^ _ ^^ j_m_ ^ ¡í < 400 > 13 Gly Phe Phe Asn Tyr Asp Phe Xaa Xaa Leu 1 5 10 < 210 > 14 < 211 > 10 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 14 Glu Leu HIS Asp Ser Tyr Met His Ala Val 1 5 10 < 210 > 15 < 211 > 7 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 15 Val His Trp Ala Pro Leu Arg i 1 5 < 210 > 16 < 211 > 6 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 16 Lys Leu Pro Leu Phe Lys < 210 > 17 < 211 > 9 > 212 > PRT < 213 > Mortierella ramanniana < 200 > < 221 > INSECURE < 222 > (5) < 400 > 17 Val Asp Leu Asp Xaa Ala Pro Pro Arg 1 5 < 210 > 18 < 211 > 10 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 18 He Thr Gly Phe Thr Val Pro His Ala His 1 5 lo < 210 > 19 < 211 > 10 < 212 > PRT < 213 > Mortierella ramanniana < 220 > < 221 > INSECURE < 222 > (9) < 400 > 19 Glu Leu His Asp Ser His Met Leu Xaa Val 1 5 10 < 210 > 20 < 211 > 13 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 20 Gly He Phe Asn Tyr Asn Wing Gly Phe He Pro Phe Arg 1 5 10 < 210 > 21 < 211 > 13 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 21 His Pro He Tyr Thr He Val Gly Lys Pro Ha Pro Val 1 5 10 < 210 > 22 < 211 > 8 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 22 Gly Ser Cys Glu Ala He Leu Arg 1 5 < 210 > 23 < 211 > 26 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 23 His Pro He Val Val Val Vai Gly Lys Pro He Ala Val Pro Leu Leu 1 5 10 15 Ala Glu Gly Glu Thr Glu Pro Pro Ser Glu 20 25 < 210 > 24 < 211 > 29 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 24 Ser Arg Asp Ser Thr Pro Val He Thr Glu His Lys Gln Pro Me t Glu 1 5 10 15 Gln Val Gln Val Thr Ala Leu Leu Asp Hi s He Pro Va l 20 2 5 < 210 > 25 < 211 > 28 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: Degenerate primer for peptide in SEQ ID NO: 5 < 400 > 25 cac tgcagac raaytcnary tcyt tnac " < 210 > 26 < 211 > 30 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: Degenerate initiator for SEQ ID NO: 9 < 400 > 26 ccaagcttgg ngtnttyaay taygayttyg 30 < 210 > 27 < 211 > 30 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: Degenerate primer for SEQ ID NO: 9 < 400 > 27 10 cactgcagcr aartcrtart traanacncc 30 < 210 > 28 < 211 > 30 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: Degenerate primer for 15 SEQ ID NO: 8 < 400 > 28 cactgcagcy tgnacngcng crtgcatrta ^ Q < 210 > 29 < 211 > 28 > 212 > DNA < 213 > Artificial sequence 20 < 220 > < 223 > Artificial sequence description: Degenerate primer for SEQ ID NO: 11 < 400 > 29 ccaagcttat hgcngtncar acnggngc 23 Ma ^ MáWÉMta ^^ _ MiÉÍáÉato.Ml > MMÉ «MÉM ..._ MM _áiK.ata« M_UM > > MB > MHWto ^^ WÉM < 210 > 30 < 211 > 28 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: Degenerate initiator for SEQ ID NO: 3 < 400 > 30 ccaagcttaa rcayccnath tayacnat < 210 > 31 < 211 > 28 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: Degenerate primer for SEQ ID NO: 3 < 400 > 31 cactgcagac datngtrtad atnggrtg < 210 > 32 < 211 > 28 15 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: Degenerate primer for SEQ ID NO: 2 < 400 > 32 ccaagc ctgc nytnggntty acnacgcc ¡20 < 210 > 33 < 211 > 28 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: Degenerate initiator for SEQ ID NO: 2 MtM fiMÉÜiu Míttl. < 400 > 33 ccaagctttt yacnatgccn ytnttyca; < 210 > 34 < 211 > 28 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: Degenerate primer for SEQ ID NO: 3 < 400 > 34 caccgcagaa rtgraanarn ggcatngt < 210 > 35 < 211 > 27 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: RACE PCR Initiator for MR1 < 400 > 35 ggtttgctcc cccatcgcca tcctatc ' < 210 > 36 < 211 > 27 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: RACE PCR Initiator for MR1 < 400 > 36 gataggatgg cgatggggga gcaaacc < 210 > 37 < 211 > 1065 < 212 > DNA < 213 > Mortierella ramanniana < 400 > 37 atggccagca aggatcaaca ttcacagcag aaggtcaagc atacgctaga agctatccca 60 tcccctcgct atgctccatt gcgagtgcca ttaagacgga gattacaaac attggcagta 120 ttcatcatgg tgttccatga tgtcaatatg catgtcatat tcttcttttt atgctcatcc 180 ctgttctcct ttggttccca ttatctttat ttgacctgga tcttggtgtg ggataaggcg 240 ccagagaacg gtggaagacc tatccgctgc ctgcggaatg ctgcctggtg gaagctgctt 300 gcagggtatt ttcccgcaca tatcatcaag gaagccgatt tagatccatc caagaacaca 350 atcaccccca atccttggtt tggaatcata tccatgggct cgttctgtac ttctaagtcc 120 aatgctactg gcttcgatga ggcaeccggc cttgtcccca gacaccaaca catcgctttt 480 tctaatetta atatcccact ttatcgtgat cattt gatgg cgtgcggact ttgctccgCc 540 tccaaaacac cctgtcaaaa tattttaacc aaaggtggtc cgggccgttc cactgccatt 500 gtcgtgggag gtgcttccga gtctctcaat gctagacccg gtgtcatgga ccttgtgctg 560 aagagacgct ttggttttat caagattgct gttcaaaccg gtgcaagtcc agtgcccaot 720 gtgaaaatga atcagttttg gctgtacgaa cagattgaaa gcaatgaaaa ctcaaagctg 780 catagacggc aaaagaagat tcaacatgce cttggtttta ctatgccgct ctttcatgga 840 cgcggt gtat tcaattatga ctbtggtttg ctcccccatc gccatcctat ctacacgatt 900 gtcggaaagc ccatccccgt ccccagcatc aagtatggac agacaaagga Cgagattata 960 agagaactac atgactcgta catgcatgcc gtgcaggatc tctatgatcg tbacaaggat L020 abcbabgcaa aggatcgggt aaaagaacta gaattcgCcg aatag 1065 < 210 > 38 < 211 > 354 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 38 Met Ala Ser Lys Asp Gln His Leu Gln Gln Lys Val Lys His Thr Leu 1 5 10 15 Glu Ala He Pro Pro Pro Arg Tyr Ala Pro Leu Arg Val Pro Leu Arg 20 25 30 Arg Arg Leu Gln Thr Leu Ala Val Phe He Met Val Phe His Asp Val 35 40 45 Asn Met His Val He Phe Phe Phe Leu Cys Ser Phe Leu Phe Ser Phe 50 55 60 Gly Ser His Tyr Leu Tyr Leu Thr Trp He Leu Val Trp? Sp Lys Wing 65 70 75 80 Pro Glu Asn Gly Gly Arg Pro He Arg Cys Leu Arg Asn? The Wing Trp 85 90 95 Trp Lys Leu Phe Wing Gly Tyr Phe Pro Wing His He He Lys Glu Wing 100 105 110 Asp Leu Asp Pro Ser Lys Asn Thr He Phe Gly Tyr His Pro His Gly and ^ j ^^^ 115 120 125 He He Is Being Met Gly Being Phe Cys Thr Phe Lys Being Asn Wing Thr Gly 130 135 140 Phe Asp Asp Leu Phe Pro Gly He Arg Pro Be Leu Leu Thr Leu Thr 145 150 155 160 Be Asn Phe Asn He Pro Leu Tyr Arg Asp Tyr Leu Met Wing Cys Gly 165 170 175 Leu Cys Ser Val Ser Lys Thr Ser Cys Gln Asn He Leu Thr Lys Gly 180 185 190 Gly Pro Gly Arg Ser He Ala He Val Val Gly Gly Ala Ser Glu Ser 195 200 205 Leu Asn Ala Arg Pro Gly Val Met Asp Leu Val Leu Lys Arg Arg Phe 210 215 220 Gly Phe He Lys lie Wing Val Gln Thr Gly Wing Ser Leu Val Pro Thr 225 230 235 240 I Have To Be Phe Gly Glu Asn Glu Leu Tyr Glu I Gln He Glu To Be Asn Glu 245 250 255 Asn Ser Lys Leu His Arg:? Gln Lys Lys He Gln His Wing Leu Gly 260 265 270 Phe Thr Met Pro Leu Phe His Gly Arg Gly Val Phe Asn Tyr Asp Phe 275 280 285 Gly Lau Leu Pro His Arg His Pro He Tyr Thr He Val Gly Lys Pro 290 295 300 He Pro Val Pro Be He Lys Tyr Gly Gln Thr Lys Asp Glu He He 305 310 315 320 Arg Glu Leu Kis Asp Ser Tyr Met Kis Ala Val Gln Asp Lau Tyr Asp 325 330 335 Arg Tyr Lys Asp He Tyr A.la Lys Asp Arg Val Lys Glu Leu Glu Phe 340 345 350 Val Glu < 210 > 39 < 21 1 > 41 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: Initiator for sequence amplification coding for MR1 < 400 > 39 41 aat tcgcggc cgcatggcca gcaaggatca acatt tacag c < 210 > 40 < 211 > 42 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: Initiator for sequence amplification coding for MR1 < 400 > 40 tgccgcagct attcgacgaa tt? Tagttct tttacccgat cc 42 < 210 > 41 < 211 > 23 15 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: Degenerate primer for SEQ ID NO: 23 < 400 > 41 ggcacngcda tnggyt tncc nac. < 210 > 42 < 211 > 23 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: Degenerate initiator for SEQ ID NO: 20 ? ^^ * tKM ?? * ^ H * ^ m < 400 > 42 -1 3 ccngcrttrt arctraadat r ce < 210 > 43 < 211 > 25 > 212 > DNA < 213 > Artificial sequence 5 < 220 > < 223 > Artificial sequence description: Initiator for RACE amplification at the 5 'end of MR2 < 400 > 43 tgcctagtga catcatgaaa teteg < 210 > 44 10 < 211 > 845 > 212 > DNA < 213 > Mortierella ramanniana < 400 > 44 atggaacaag tccaagtcac tgcattgctc gaccacattc ccaaagtcca ttgggcaccg 60 tccctttgaa ctccgcggga gcgtcgctta caaacgtcgg ctatcgtcac atggctggct 120 tcgcttccta tctgtc? Cat tatataectg tacetattea ccattccctt attatggccc 180 ateetcatta tgtatacgat atggctgttt ttcgacaaag cccctgaaaa cggaggcaga 240 cgaatttcgc tggtgaggaa attgccgctg tggaagcact ttgecaacta tttcccagcc 300 aggaaggaga actttgatca cctcgacccc aagggaaact acatcatgtc atatcatceg 360 catggaataa tatccatggc ggcttttgcc aattttgcga ctgaggcgac tgggtttccc 420 gagcaatatc cgggtattgt tccttcatta ctgacgctag catccaattt tcggttgcca 480 ttgtaccgag atttcatgat gtcactaggc atgtgcccgg tategegaca ctcctgtgaa 540 gccacccttc gttcggggcc cggtcgatcc attgtgatcg ttacaggcgg agcttcagaa 600 tcccttagcg cacgaccagg caccaacgac ctcaccctca agaaaegatt gggtttcatc 660 cgactagcca ttcgaaatgg tgccagttta gtgcctatct tttcgttcgg agagaaegac 720 atetaegage aataegacaa caaaaagggc agtctgatat ggcggtacca aaaatggttc 780 caaaaaatta caggattcac ggtccctttg gctcatgccc gtggaacytt caactacaac 840 gcggg 345 twenty ^ ^ i ^ áá itít < 210 > 45 < 211 > 281 < 212 > PRT < 213 > Mortierella ramanniana < 400 > 45 Me t Glu G ln Val Gln Val Thr Ala Leu Leu Asp His He Pro Lys Val 1 5 10 15 His Trp Wing Pro Leu Arg Gly He Pro Leu Lys Arg Arg Leu Gln Thr 20 25 30 Ser Ala He Val Thr Tr? Leu Ala Leu Leu Pro He Cys Leu He He 35 40 45 Tyr Leu Tyr Leu Phe Thr He Pro Leu Leu Trp Pro He Leu He Met 50 '55 60 Tyr Thr He Tro Leu Phe Phe Asp Lys Wing Pro Glu A.sn Gly Gly .Arg 65 70 75 30 Arg He Ser Leu Val Arg Lys Leu Pro Leu Trp Lys His. Phe Ala Asn 85 90 95 Tyr Phe Pro Val Thr Leu He Lys Glu Gly Asp Leu Asp Pro Lys Gly 100 105 110 Asn Tyr He Met Being Tyr His Pro His Gly He He Being Mfet Ala Wing 115 120 125 Phe Ala Asn Phe Ala Thr Glu Ala Thr Gly Phe Ser Glu Gln Tyr Pro 130 135 140 Gly He Val Pro Ser Leu Leu Thr Leu Ala Ser Asn Phe Arg Leu Pro 145 150 155 160 Leu Tyr Arg Asp Phe Met Met Ser Leu Gly Met Cys Ser Val Ser Arg 165 170 175 His Ser Cys Glu Wing He Leu Arg Ser Gly Pro Gly Arg Ser He Val 180 185 190 lie Val Thr Gly Gly Wing Ser Glu Ser Leu Ser Wing Arg Pro Gly Thr 195 200 205 ^ Wa - ^ ___ ^^^^^ _ ^^^^^^ __ ^^^^ a As Asp Leu Thr Leu Lys Arg Leu Gly Phe He Arg Leu Ala Ha 210 215 220 Arg Asn Gly Ala Ser Leu Val Pro He Phe Ser Phe Gly Glu Asn Asp 225 230 235 240 He Tyr Glu Gln Tyr Asp Asn Lys Lys Gly Ser Leu He Trp? Rg Tyr 245 250 255 Gln Lys Trp Phe Glr. Lys He Thr Gly Phe Thr Val Pro Leu Wing His 260 265 270 A. Arg Gly He Phe Asn Tyr Asn Ala 275 280 < 210 > 46 < 211 > 355 < 212 > DNA < 213 > Zea mays < 400 > 46 CCGT taagac TGGT tgcttt tcttaaatca agaaaaggt tt tgtcaagat agc tatacag 60 tctggatgtc ct t tagtccc agttttc tgc tt tgggcaga gc ta tgcata caagtggtgg 120 aggcctggtg gtaaattgtt tatcaagatc gc tagagcag t TAAAT t tac tcctattatc 180 t tctgggata GAIT tggcac ACCAT ccccc t tcccaaaac ccatgcatgt ggtcgcgggt 240 aaaccaattg aagtcaataa gat tccccat cc tacaattg acgagattaa tgaagtccac 300 ggacagttca tcat tgccat gcgggacc tc tt tgagagct gtaccatcag tgtcc 355 < 210 > 47 < 211 > 375 < 212 > DNA < 213 > Zea mays < 400 > 47 cccacgcgtc cgcgagctta tgtttttgct tatgaaccgc attcggtgct gcctattggc 60 gtttgtgcgc ttgcggatca tacaggtttt tcgcccctgc cgaagattaa ggctcttgcg 120 agtaccgcgg tctCcta.tgt gccgtttgcg aggcagatat ggacatggct ggggcttgcc 180 cctgcgtcga gaaggaattc ttacgagtac ttggcggctg ggtatagttg catcatagtg 240 ccgggcggtg tgcaggagtt gttgtatatg gaatgcgatt cggaggtcgc Ctttcttaaa 300 tcaaggaaag gatttgtaaa gatagccatg gagatgggcc aaccCccCgc acctgtattc 360 tgcttcggcc AGAGT 375 < 210 > 48 < 211 > 477 < 212 > DNA < 213 > Brassica napus < 400 > 48 aacnttactt gccaggcacc ggtcaagaan tcccgggtcg acccacgcgt ccgcaaatac 60 ggtcgaatgc tcgctaggta catatgtaaa cacgcgtgta gttatttccc cgttactctc 120 catgtcgagg attacgaagc ttccagcct actcgtgcct atgctttcgg ttatgaacca 180 cattcggtgt ggcctattgg agctgctgca cccgctgacc ctacggggtt catgcctctt 240 cctaacatca aagttcttgc gtttcctaca tagtactgct caccctcict gaggcaaata 300 tggacgtggt tagggctcgc ccctgcttct aggaagaatt ccgcttccta tttggactct 360 ggccatagtt gtatcctcgc acccggCggC gtccaggaga caccccacat gaaacacgat 420 gttgagaact tattcccttc atccgagaan ggggtttgtg cgcaccgcca tgggagc 477 < 210 > 49 < 211 > 498 > 212 > DNA < 213 > Glycine max < 400 > 49 nngctcccca tcaacgtgca gtgggatctg gccgaagatt gtcgaggctc atatgcaagc oO acgcagtgca attactctcc gatcacgctc cacgtagagg atatgaaagc ctccga cct 120 aaccgcgcct acgcttttgg gtatgaacca cattcagcct tgccaattgg caCacgtnnt 130 gcaccggccg accacacagg cttcatgcct cttccaaaag ttaaagttct tgctagcagc 240 acggtgcccc acacaccatt ttacacacca tttttgagac acatatggac atggttgggc 300 ctaacgccag cgacaaagaa aaggtttacc tcgctgttgg atgctggcta tagttgtacc 350 ttgatacctg gtggagtgca agaagcatt cccatcgagc atggttctga gattgccoct 420 ctcaaaccaa ggagaggacc cgtccgcata gcaatggaga agggaaaacc cctggttcca 430 gttccctgct ttggtcag 'i9s < 210 > 50 < 211 > 393 > 212 > DNA < 213 > Arabidopsis thaliana < 400 > 50 gcgtccgtcg ccatggccat ctggctcggc gccattcacc tcaacgtcgc ccctgttctc 60 tgttctctca tt tccttcc tccttctcta tctctcatgg tcttgggctt gctctctctg 120 tCtatctCCa Ccccaatcga tcatcgtagc aaacatggtc gtaagctcgc caggcacata 130 tgcaagcacg cgtgtaatta tttccccgtc tccccg acg Ccgaggatta cgaanctttc 240 cagcctaatc gCgccCatgt ccttggttat gaaccacact cggtgctacc gattggantt 300 gtngctcttt gtgatctcac anggcttatg cctaatccca acaccaaagt tcttgcaant 360 agtgctaaac tcaaaattcc ctttcaaagg ata 393 ^^ MatatB a? B < 210 > 51 < 211 > 344 < 212 > DNA < 213 > Arabidopsis thaliana < 400 > 51 cttgccgtgc cggtccgaaa taacgggCcg acncacgcgt ccgtgtacgt cgaggattac 60 gaagctttcc agcctaatcg tgcctatgcc cttggttatg aaccacattc ggtgccaccg 120 aCtggagttg ttgctctttg tgatctcaca gggtttatgc cnaCtcctaa cactaaagtt 130 cttgcaagta gtgctatacc ccacaccccc cccccaaggc acatatggac atggtcaggg 240 cCcaccgctg cctctaggaa naatttcacc Ccccttttgg attctggcta cagttgcgCt 300 cttgtacctg gtgggtgtgc aggagacttt tcanatgcan catg 344 < 210 > 52 < 211 > 462 > 212 > DNA < 213 > Arabidopsis thaliana < 400 > 52 cgagagaagc acatttctgg gtacgcacca cacggtatgt tcccgatggg cgcctctcac 50 ccccacaaca cctcgacgtg gacggaactc cccccaaaca Ccgcgcctta tacactcaca 120 gcgacggcga cccaccCggc tccgCCtcta agagaagtga ctcagtataa cggaggtgtt 130 gaagtcagtc aaagcagctt tgcaaacgcg tcgatgaaat tcaaaaacgt tctgccggcc 240 cccggaggac aacacgaaac gctacccatc agcgacgacc acaacgaagC gcctttatcc 300 gccaaacaca agggatccat tcgattagcc tCgcaatcgg cagcagaaaa cccagatgaa 360 gCcaCcaacc tcgtcccggt gtacgctt: t ggagaaaaag acaaaatgta taacgcattc 420 cctgcgagtc tctctctgca gcgatatctg gcggccaagc tg 462 < 210 > 53 < 211 > 489 > 212 > DNA < 213 > Human < 400 > 53 gcctcgggca cccagcccca ggccgtggtc atcatggtgg ggggtgcgca cgaggccctg 60 cattcagtcc ccggggagca ctgccttacg ctccagaagc gcaaaggctt cgtgcgcctg 120 gcgctgaggc acggggcgcn cnCggcgccc gCgcactcct CCggggagaa CgacaCcCtt 180 agacttaagg cttttgccac aggnncctgg cagnattggc gccagctcac cttcaagaag 240 ctcatgggct tntcnccttg catnttctgg ggtngcggtn tcttctcagc cacntcntgg 300 ggcctgctgn nctttgctgt gcccatcacn actgtggtgg nnngnacnat nnccntnaan 360 cagaaccncc acccnaccga ggaggaaacn aatnactatn acgnnnccta catgacggnc 420 ntggagcagn tcttcgagga gnanaaggaa agntgtgggg acccngcCtc cacccgcntn 480 accttnaCc 489 < 210 > 54 < 211 > 333 < 212 > DNA < 213 > Human < 400 > 54 tggccctccc cCgtttttta cttggtgtgg ctctatgtgg actgggacac acccaaccaa 60 ggggttggggaaaaggggcc ggttttccggggaaggttgg ggaattaaaaggggaaaacc ccggggggccaaaattttcc ggggaaggaaccaaaaccCC aaaaggggggaaCCCCaaCC 112200 CCaaCCccccCCggCtccaa aaggccctggggctggaaaa aaaaccaaggccaaggaagg ccttggccccccccccgggg aattccggggaaaaccttaa ccggttggccttggggggcc 113300 aarcsggsgsaactccaatt gsCtgscpaaccaagsgscc ttttcccctctgta atCCcCccac cgagagcaat 240 ggcttctccc agctcttccc ggggctccgg CCCT tacctcccgg tctatcgcga ctacatcatg tec 333 < 210 > 55 < 211 > 222 > 212 > DNA < 213 > Human < 400 > 55 ateat egeag ggggcgccca ggaggccc cg tgccaggc ga t c Cgga TCCT cacgctgc ca 60 c tgcggaacc gaaagggctt cgtcaggctc gccctgacac acggggcacc cctggCgcca 120 cc aka Acc c t e gaccaga ccggggagaa tgaectat t t I ccaac CCCCC tggcccc tgg 180 ttacgc CACA tecagaateg gttgcagaag atcacgggca 222 cc < 210 > 56 < 211 > 827 > 212 > DNA < 213 > Human < 400 > 56 cCccagCggg Ccccgtcctt ccttgtactg ggagtggcct gcagcgccat cctcatgtac 60 atattctgca ctgattgctg gctcatcgct gtgctctact tcactcggct ggtgtttgac 120 tggaacacac ccaagaaagg tggcaggagg tcacagtggg tccgaaactg ggctgtgtgg 180 cgctactttc gagactactt tcccatccag ctggtgaaga cacacaacc gctgaccacc 240 tctttggata aggaactata ccacccccat ggtatcatgg gcctgggtgc cttctgcaac 300 ttcagcacag aggccacaga agtgagcaag aagtt ccag gcatacggcc ttacctggct 360 acactggcag gcaacttccg aatgcctgtg ttgagggagt acctgatgtc tggagg atc 420 tgccctgtca gccgggacac catagaccat ctgcccccaa agaacgggag cggcaaCgcc 480 accatcatcg tggtcggggg tgcggctgag tctctgagct ccatgcctgg caagaatgca 540 gtcaccctgc ggaaccgcaa gggctttgcg aaactggccc tgcgtcatgg agctgacctg 600 gttcccatct actcctttgg agagaatgaa gcgcacaagc aggCgatctt cgaggagggc 660 gatgggtcca tcctggggcc gaagaagttc cagaaataca ccggcttcgc cccacgcatc 720 ttccatggtc gaggcctctt ctcctccgac acctgggggc CggCgcccCa ctccaagccc 0.780 atcaccactg ttgtgggaga gcccatcacc atccccaagc tggagca. 827 < 210 > 57 < 211 > 265 > 212 > DNA < 213 > Human < 400 > 57 agcgaCtatt tccctctcaa gcttctgaag actcatgaca tctgccccag ccgcaactac 60 atcctcgtct gccaccctca tgggctccCC gcccacggac ggcccg? RCCA ccttgccaca 120 gaggcctcag gcttctccaa gatattcccn ggcaccaccc cCCacat.acC cacaccggga 180 gccttCCCcC ggacgccccc ccccagagaa cacgcaatgC cCacaggggc ccgctccgtg 240 agtcgatcct ccattgacct tctgc 265 < 210 > 58 < 211 > 342 > 212 > DNA < 213 > Murino < 400 > 58 ctmgtgcagg tgtgcattgg aattatggtg atgctggtcc tgtacaacta ttggttcctt 60 tacatcccat atctggtctg gttttactat gactggagaa ccccagagca aggaggcaga 120 agatggaact gggtccaaag ccggcctgtg tggaagtaCt ttaaggagta ttttccaatc 130 aaacgcagga tgtcttgtca tttggatccg ggccacaatt atatatttgg gtttcaccct 240 catggaatat tcgtgcctgg agccttcgga aaccCccgca caaaacacCc ggacttcaag 300 aagctatttc ctggctttac atcgtatccc cacg ggcca 342 ag < 210 > 59 < 211 > 375 > 212 > DNA < 213 > Murino < 400 > 59 nctacctccc tcagggtccc gggcatcats tctcgcCcCa Cgaagactga acacttacag ° 0 agcctgagcc ttctgcagtg gcccttgagc tacgctgcca cgccttggat tgtgcagcca 120 ttgtcaacct gcctattgtt cacacccttg tggccgctac caacagttta ctttgtctgg 180 ttacttctcg actggaagac tccagataaa ggtggcaggc gttcagactg ggtacggaac 240 tggaatgtct ggaaccacat cagggaccat ttccccatca gactaaggac caaccctgaa 300 ctgtcacctt cagagaacta catcatgggg gtccacccca tnggtctcct gaccctcggc 360 ACTTC gccttctgca. ^ 75 < 210 > 60 < 211 > 885 < 212 > DNA < 213 > Murino < 400 > 60 Q gtactacaat gggtcctatc cttcctggtg ccaggagcgg cctgcagtgt catcctcatg 60 tacaccttct gcacagactg ctggctgata gctgtgctct actccacccg gccggcactc 120 cgcccaagaa gaccggaaca aggcggcagg agatcgcagt gggtgcgaaa ctgggccgtg 130 tggcgctact tccgagacta ctttcccacc cagctggtga agacacacaa cctgctgacc 240 accaggaact atatctttgg ataccacccc catggcatca tgggcctggg tgccttctgt 300 aacctcagca cagaggcCac Cgaagccagc aagaagcccc ctggcataag gccctatttg 360 gctacgttgg ctggtaactt ccggatgcct gtgcttcgcg agtacctgat gtctggaggc 420 atctggcctg tcaaccgaga caccatagac tacttgctct ccaagaatgg gagtggcaac 480 gctatcatca Ccgtggtggg aggtgcagct gagtccctga gctccatgcc tggcaagaac 540 gcagtcaccc tgaagaaccg caaaggcttt gtgaankyyg gatccmtgcg ccatggagct 600 gatctggttc ccacttattc ctttggagag aatgaggtat acaagcaggt gatctttgag 660 gagggttcct ggggccgatg ggtccagaag aagttccaga agtatatcgg tttcgccccc 720 tgcatcttcc atggccgagg cctcttctcc tctgacacct gggggctggt gcccacCcca 780 agcccaCcac caccgtcgtg ggggagccca tcactgtccc caagctggag cacccgaccc 840 agaaagacat cgacctgtac catgccatgc acatggaggc CCTGG 885 < 210 > 61 < 211 > 809 > 212 > DNA < 213 > Aspergillus fumigatus 20 < 400 > 61 acatcctcta gtcctcctcn cgtcaaatat ctcgccaaag cacacaaaac cggcacttta 60 gctctccgca acgaccgcct ccgcacgtcc tggatctgga aagcctacgc ctcccacttc 120 cccctccgcc tctaccgctc ggtgcccntc tccccccgca aaaagcacaC c tcggctac 130 catccccacg gcatcgcccc ccgaggagca tagccgccga ctcgggaccc cgccgccgca 240 ttctccgatc tcttccccgg cgttacgaac acgctcctga tgaaagacga ggcgttctac 300 cagcccatat atagggagta acgggggtga ccttctctct gcggcgtgtc ccactcgtcg 360 jl_ _t_? _ l_? _¡ tgtatccgac acctgacccg cgcaggacat gatgggcagg gtatgggccg ggcgactacc 420 atcaccgttg gcggaagtcg cgagtataac attgcgcggc cggggacgat gtgcgcggtc 430 gtccgcatcc gcaanggctt tgtgcgggtg gcggttgaga cgggggcgga tctcgttcct 540 gttattgcct tcggggagaa tgagctcttt gattgtgtga atgtgtcctc gCcgactgtg 500 ccgggggttg tggccagggt acgggagcgg gccgccggcc acaaggtggc gtttccgatc 660 gccggcccaa cacccccgcc cgcaccgcgg ccggcgaatg ttgccgcngg gganccgact 720 cctgcgacgc ancancggcg ggatccgatc aagcgtatan tgaccattgc atggcatata 780 tccanggcac cggaanaacc tcnggaaCg 309 < 210 > 62 < 211 > 668 > 212 > DNA < 213 > Aspergillus fumigatus < 400 > 62 nctgcatttg ctactgaagc actcggattt tcgaggttgt ccccgggaac tacaaacacc 60 ttacttaccc ttgattcgaa ttttcgaatt ccgttctaca gagaatatgc tcttgccatg 120 ggactcgcca gtgCttcccg ggagtcctgt gaaaacctgc tatctaaagg tggtgctgat 130 ggggaaggca tgggccgcgc gattacaatt gtcatcggcg gggctcgtga gtccctgcac 240 gctttacctc actctctgcg aaatgccgca ccttgtttta aaggctagca aaggattcat 300 attcgcaccg gtgctgatct tgtgccagta cttgctttcg gcgaaaacga tctctatgag 360 cagatcagca caggtgcgat tcccattata cacaagcttc aaatgctcat taagcgtacg 420 atggggttca cagttccgct ctttcatgct cgtggggttt tcaattatga cgtgggaccg 430 atgccccatc gacgtccgtt gaatattgtc gttggcagac ctatacaagc cgttcaacag 540 cgtgacagag acaagattga cgaaacgtac 'attgacgacc ttcatgccaa gtacaCacaa 600 ttcga GAAC gancaataca cgcttrtngg aaagatgtct tttgcggaag gacccgaatc ctcccgga 660 668 < 210 > 63 < 211 > 435 > 212 > DNA < 213 > Aspergillus ochraceus < 400 > 63 atcaccatgc tcattacgtc ttgcttgaag cgacgtatgg ggttcataaa gctagccatc 60 cgcactggtg ctgaccctgc accagtcttg gcttttggag aaaatgatct atacgaacag 120 gtccgt cag atagccatcc ccttattcac aagttccaaa tgttggtgaa acagacactg 180 ggattcacca ttccgctgct ccatgcacgc ggCgcCcCca attacgatgt tggcttgatg 240 gcccgctgaa ccgtaccgcc CaCCgccgcc ggccggccaa CCcaCgcggt Ccagcaacag 300 gacagaaaca aaaCcaaCga cgacCatatt gatcaactcc attcagagta cgtgagagaa 360 cccgagaggc Cgcgggaaga gtggaaggac gcctacgcca aagaccgggc ccccgaaatc 420 gaaatagtgg cctag 435 < 210 > 64 < 211 > 597 < 212 > DNA < 213 > Candida albicans < 400 > 64 atgaaaaatt tcatcacctg ggattggttt gtcagatatt tccctataaa ggtttataag 60 tctgccgaat tggaaccaac attcaaagaa gttttggtag aggagactga aagttcagaa 120 gatgatgatg agcaagattt agtgtctgaa cggagcagaa cgttagttga taaagttttc 180 aaatcttttg ggttgaaaaa acgtttgaat gacacttctc tggggaagtc agaaacctac 240 aagacagtgt ctactggtcc caggtatatt tttggatacc atcctcatgg agttatttca 300 atgggcgggg ttggtttatt tgctactaat tcattacgca acgagccata tacgccattt 360 ctaaaatttt tgaaaccatt cttccatgac agttccaaag gtgaacgitt atttcctggt ' 420 cttggaaata ttttcttgtt gacaattacc acacaatttg ccataccatt ttatcgtgat 480 tatttaatgg gattgggggt- tactagtgca tcagcaaaga atattagaag tttgattagc 540 aacggcgaCa aCCcCgCctg tattgtagcc ggtggggcag aagagtcttt gttaaac 597 < 210 > 65 < 211 > 362 > 212 > DNA < 213 > Fusarium graminearum < 400 > 65 atgtctattg ccacattggt ttcggccttt tggttgattt gcgccaaccc acttgcctgg 60 cctattatta tcccttattt aattcatctt gctctatcaa ctgccggCac taatggcaac 120 ttgacaCacc gcCcagaatg ggtccgaagc ctgaagttgt ggaaactt t cgctggatat 180 agttgcacaa ttccccatga aacgcacgat ctgcccaccg aCagaaa? RCa catttttgga 240 caccatcccc acggtatcat ttcccatggt gcctttgccg cttttggtac caatgccctt 300 ggattccgcg agctcttccc tgggatcaca aacacgttac gggggatcca ttactctaga 360 362 ct < 210 > 66 < 211 > 431 > 212 > DNA < 213 > Mortierella alpina < 400 > 66 cccctgatca tcatgtacct gctccgggcc tcatactgg accgaggccc agagcgtggt 60 gcacgcccag tgcagtggta tcgtaactgg atcggatgga aacaccCCgc CcaaCacCtC 120 cccaCgacCc CCgCcaagga aggagaaccg gaCccgCcca agaaccacaC ccctggctac 180 cacccgcacg gcatcatctc cttgggcgcg ttctgcacct tcgggaccga nggccttcat 240 ttctcaaaac gctttcnagg catcaagccg cagctgttga ccctgcatgc caactttcan 300 gttccgctct accgcgaaat ggtcatggcc cacggctgtg cttcggtctc tagagcctct 360 tgtgaacaca ttctgcggcc cggtgaagga cgctcgg cg tgaccgtcnt tggggggcgc 420 tcaaganant t 431 < 210 > 67 < 211 > 285 < 212 > DNA < 213 > Schizochytrium aggregatum < 4QO > 67 cccatctcan nggcccatct gggaaatccg cgcatcanng gcanacggcg cttgggaccc 60 cggatattcc nttttcgcat tgttgaagac catttcagcc tctcgatggt gcgcacgtct 120 gaagagcctt gggacccgga gcacgagtac atttgtggcC atcaccctca cggnctcgcg 130 cccttgggng ccgcttacat gaaaatgacc ccacaatggt cggagctcct ccccaatatt 240 3cgcccntta ctctcagcgc angcatt cs cntcangtac dwarf -535 < 210 > 68 < 211 > 1080 > 212 > DNA < 213 > Caenorhabditis elegans < 400 > 68 atgagactcc ggctgagctc gatatctgga aaggcgaagc ttcccgataa agaaatatgc 60 tcaccagctt cgagaatatt ggcaccatcg cttgttccat ggaagcgacg actegagact 120 ctcgccgtga tgggtttcat tttcatgtgg gtaatectac cctctgggta caatcatgga 130 ccattccacg tcttgttcaa tactegatgg tggttccccg ttccactcta cgctgtctgg 240 tt tactatg attttgatac accgaaaaaa gcttcaagaa gatggaattg ggecagaaga 300 cacgtagcct ggaagtactt tgccagctac ttcccattga gattgatcaa gactgctgac 360 cttccggcgg atcgtaatta catcattggc tctcacccCc aCggaatgtt ctcggttggt 420 ggttttactg caatgagcac caacgcgacc ggattcgaag acaagctccc gggaaCaaaa 430 tgacgctaaa tcccacatca tgggcaattt atttcccat attcggaata ttcgtcgaga 540 atgctcggtg gaatcgaagt ttcgaaagaa tcacttgaat acactcCaac taaatgtgga 600 aaaggacgag caCgcgcaaC tgtcacCggc ggagcctcgg aggctcttga agctcatccc 660 aaCaaaaaca cattgacgtt gatcaatcga cgtggtttcC gcaaatatgc tccgaagttc 720 ggggcagacc CcgCaccaaC gtacaatttc ggagagaatg atttatacga gcagtatgaa 780 aacccgaagg gatctagatt gcgagaagtt caggagaaaa tcaaggacat gttcggattg 840 tgtcccccat t gctccgcgg tcgatcgttg ttcaaccaat accttatcgg attgctgccg 900 ttccgaaaac cagttacaac agtcatggga aggccaattc gggtcaccca aaccgacgag 960 ccaaccgttg agcagattga tgagctgcat gcaaaatatt gtgatgctct ctacaaCctg 1020 ttcgaggagt acaagcatct tcaccccatt cctcccgaca ctcatctcat cttccagtga 1080 < 210 > 69 < 211 > 1020 > 212 > DNA < 213 > Caenorhabditis elegans 10 < 400 > 69 atgccacatc tactaggagt tgagtgggct ccgctcaata ttccgttggc tcggcgtctt 60 caaactttgg gagcacttca tttcttcttc atcacccCcC CcacaccagC acCcgCCcCc 120 accgCtccat tctacatgtt atataccgta ctctggcctt tgatctctct gtatgggctt 180 tggatgattt acgattggaa ttcaccaaag aagggagccc aCaCgajcaa CCggcCccag 240 agacaaagaa ttcattcgtg gtatgccaac tatttcccag CcaaaCCgca cacaacatct 300 gacatgccag aagaacataa ctatttgatt gggtaccatc cgcatggaat aatttcaatg 360 tcaactttgc gccgcattca aacaaatgga actggaatCc CcgaCacCcC Cccacgaact 420 cgtttccatt tgtgcacact tgtcggtcaa ttctggactc cgtggagacg tgagtgggga 480 ttgttgcacg gaaCgataga ctgcagtcga gaaagcatca ggagcatgaa agcacgtttt 540 aagaaaggaa aagcagttgt attggtggtt ggtggagctg aagaagcact tgatgcacat 600 ccaggacgcc atattttgac tttgaaaaaa aggaaaggat tcgtgaaaat tgccctgcaa 660 actggagctc aactggttcc atgccactca ttcggcgaaa atgatatttt caatcaagcc 720 gaaaatccaa agggatcaac aattcgacag ttccaaacga taatgaaaag agccccggga 780 cagcattcta tccccccctc tgggagagga gtattcaact atacatttgg tcttcttcca 340 ttcaggaaac ctatcaacac tgttctcggc gctccaacct cagtgacaaa gacagtgaat 900 aacaaatcga ccaacccaag cacacttcat cagacataca tggaccgtct tcatgagcct 950 ctcgaggagc acaagacaaa atacgacgtc tctccaacta cacaacttgt tatcaaccaa 1020 MMküákíÜ? IÉ. < 210 > 70 < 211 > 1038 > 212 > DNA < 213 > Caenorhabditis elegans < 400 > 70 atgctaaact accaaattca caaaaagccc accgacatca agtgggtgaa catcttctcc 60 ccatgggatc gccagcgtgc ctacttcgcc ttggtcgtct ggttcgggct catctaccca 120 ttctgctgcc tgtgccaggt ggctccgttt gtgctctttt tcaccggcca gtggattatt 180 ttgggtctct acgcagtttg gtaccttcac gatcgagaat ctccgagaag aggaggatat 240 cgggataacc ggttcagaaa tttgtcgctg cacaagtggt tcgccgagta ttttcctgtt 300 aaactccaca aaactgcgga gttggatcca aaccaaaact atttactcgg atatcatcct 360 catggaattc tcggtgtcgg agcgtggtct tgctttggat ttgatgcgtg caatgtgaag 420 caagtgttca aaggcatccg cttcaacatc tgcaccttgc ccggcaactt caccgcaatg 480 ttccgccgcg agatcctcct cagcatcggt atgatcgaga gctccaaaga atccatcgag 540 actccgagga cacgtgctca aaagggccgt gccgttgtaa ttgtcgtggg tggagccgct 600 gaagctcttg aagctcaccc agggaagcat actctaacac tggcaaatcg caaaggtttc 650 gtgagagaag ccgtgaagac cggagctcat cCggcgccag cctatgcgtt tggagagaat 720 agcaaattga gacatatata caacccggaa ggctcgaaat tacggaaaat tcaagaatgg 780 ggaaagaaga aaatgggaat ttcactgcca ctaatctacg gaagagsata ttttcaaatg 840 gctcttgggc Ctcttc caat gagccgagct gtgaatgtag tcgtcggagc gcctattcaa 900 gtggaaaaag agctcgatcc ttctaaggaa gtcattgatg aaattcatgg agtttatatg 960 gaaaagcccg ccgagttatt tgaagagcac aaggcaaagt tcggagtttc caaggacact 1020 cggctcgttt ttcagtga 1033 < 210 > 71 < 211 > 315 < 212 > PRT < 213 > Caenorhabditis elegans < 400 > 71 Met Ala Glu Thr His Arg Ala Gly Xaa Ser Ser Pro Kis Arg Val Ser 1 5 10 15 Phe Pro Leu Lys Met Pro Gln Phe Leu Gly He Glu Trp Val Asp Leu 20 25 30 Phe Ser Ser He Gln Arg Lys Lys Thr Tyr Leu Gly Val Val Tyr His 35 40 45 Phe Met Leu Thr Tyr Pro Leu Ala Leu Phe Val Thr He Leu Pro Phe 50 55 60 10 Phe Leu Leu Phe Thr Phe Gin Trp His Ha Leu Ala Leu Tyr Ala Cys 65 70 75 80 '< aa Val Leu Leu Arg Tyr Gly Phe Ser Glu Xaa Glu Val Asp He Pro 35 90 95 muml Édßfa ^ * ^ h_á Xaa Asp Trp Met Wing Cys Gln Arg Leu Gly Ser Pro Asn Thr Ser Gln 100 105 110 Be Thr Cys Thr Lys Leu Pro Asn Ser Pro Arg Thr Arg Thr He Trp 115 120 125 Leu Glu Be He Xaa His Gly He He Be Met Ala Ala Wing Trp Ser Asn 130 135 140 Phe Ala Thr Asn Gly Thr Gly He Tyr Glu Lys Phe Pro Gly He Arg 145 150 155 160 Trp Asn Leu Cys Thr Leu Wing Leu Gln Phe Arg Mee Wing He Arg Arg 165 170 175 Glu Leu Leu Leu Leu Thr Gly Leu He Asp Cys Ser Arg Glu Ser He 180 185 190 10 Glu Tyr Val Leu Asp Lys Cys Gly Gln Lys Gly Arg Ala Val Val Leu 195 200 205 Val He Gly Gly Ala Glu Glu Ala Leu Asp Ala His Pro Gly Tyr His 210 215 220 Thr Leu Thr Leu Wing Ser Arg Lys Gly Phe Val Arg Glu Wing Leu He 225 230 235 240 Thr Gly Wing Tyr Leu Val Pro Val Tyr Ser Phe Gly Glu Asn Asp Val 245 250 255 Phe Glu Gln MeC Glu Asn Pro Val Gly Ser Arg Leu Arg Asn Phe Gln 260 255 270 Glu Trp Cys Lys Ser He Phe Gly He Ser Tyr Pro He Phe His Gly 275 280 285 Arg Gly Phe Phe Gln Leu Thr Phe Gly Tyr Leu Pro Phe Arg Lys Pro 290 295 300 He Asp Thr Val Xaa Arg Ser Pro Asn Ser Arg 305 310 315 EÍÉaüai &AdíliM- < 210 > 72 < 211 > 1257 < 212 > DNA < 213 > Saccharomyces cerevisiae < 400 > -at 72 ACCG aaaggcatga gaacaagtct tcgtcaagca tcgataaaag agaacagact 120: aajccac aactagagtc atgcCgtcca tCggcgaccc cctttgaaag aaggttacaa 180 -ctggctg tagcatggca cacttctcca tttgtactct tctccatatt tacgttactc 240 atrccga caccagcacc gtgggttcct gctattccat atatgatcta ttccttttcc 300 caggtctc ctgcaactgg cgaagtggta aatcgatact ctcttcgatt tcgttcattg 360 acttgga agcggtattg tgattatttc cctataagtt tgattaaaac tgtcaattta 420, accaactt ccacgctctc aaaaaataag agagtcaacg aaaaaaatta caagactaga 480 gtggccaa ctaagtattc cattaatctc aaaagcaact ctactattga ctatcgcaac 540 ggaatgca cagggccaac gtacttatct ggtcaccatc cacacggcat aggagcactt 600 cgcgtctg gagcgtttgc aacagaaggt tgtaactatt ccaagatttt cccaggtatt 660 cattcctc tgatgacact ggtcacacaa tttcatatcc cattgCaCag agaccaccta 720 ggcgCCag gtaüttcttc agtatctcgg aaaaacgctt taaggactct aagcaaaaat 780 gccgacct gcattgttgc tggtggcgct agggaatctt tattaagttc aacaaatggt 840 ttttaaacaa acaactga aagaaagggt tttatcaaac tggccattca aacggggaat 900 taacctag tgcctgtgtt Cgcatttgga gaggtggact gttataatgc tctgagcaca 960 aaaagatt cagtcctggg taaaatgcaa ctatggttca aagaaaactc tggttttacc 1020, tcccatcc tccacgcaag aggattattc aattacgatt tcggtttgtc gccatttaga 1080 gcctatca atgttgttgt tggaaggcct atatacgttg aaaagaaaac aacaaatccg 1140 agatgatg ttgttaatca tttccatgat ttgtatattg cggagttgaa aagactatat 1200 gagaaaaata cgaaaaca tggggtaccg gacgcagaat tgaagatagt tgggtaa 1257 < 210 > 73 < 211 > 31 15 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: PCR primer for SEQ ID NO: 68 < 400 > 73 gcgcggccgc ctgcagtcac tggaagacga g 31 < 210 > 74 < 211 > 31 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: PCR Initiator for SEQ ID NO: 68 duí ^ a? 400 > 74 gcgcggccgc atgagac tcc ggc tgagc tc g 3. < 210 > 75 < 211 > 33 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: PCR primer for SEQ ID NO: 69 < 400 > 75 gagcggccgc atgccacatc tactaggagt tga 33 < 210 > 76 < 211 > 35 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: PCR primer for SEQ ID NO: 69 < 400 > 76 cggcggccgc ctgcagttaa ttgataacaa gttgt 35 < 210 > 77 < 211 > 32 < 213 > DNA < 213 > Artificial sequence ^^^^ < 220 > < 223 > Artificial sequence description: PCR primer for SEQ ID NO: 70 < 400 > 77 gcgcggccgc atgccaaacc accaaattca ca 32 < 210 > 78 < 211 > 33 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial sequence: PCR primer for SEQ ID NO: 70 < 400 > 78 «tggcggccgc ctgcagtcac tgaaaaacga gcc 33 < 210 > 79 < 211 > 25 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: PCR primer for SEQ ID NO: 71 < 400 > 79 cagcggccgc atgtcaggaa catee 25 < 210 > 80 < 211 > 26 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: PCR primer for SEQ ID NO: 71 < 400 > 80 cactgcagt t acccaactat c cccaa 26 , ^ - Má ^ ááMill < 210 > 81 < 211 > 55 > 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: Cloning adapter < 400 > 81 cgcgacttaa atggcgcgcc ctgcaggcgg ccgcccgcag ggcgcgccat tcaat 55 < 210 > 82 < 211 > 32 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: Cloning adapter < 400 > 82 tcgaggaccc gcggccgcaa gctccctgca gg 32 < 210 > 83 < 211 > 32 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: Cloning adapter < 400 > 83 CcgaccCgca ggaagcCCgc ggccgcggac cc .. < 210 > 84 < 211 > 1068 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: Nucleic acid sequence for resinsized MR1 < 400 > 84 aggaccagca atggctagca cctccaacag aaggtgaagc ggccatccca acacccctga 60 tcccctaggt atgctccact cagggtccca cttaggagaa ggctccaaac ccttgcCgtt 120 ctcctctggt gctccatgae gagcacc gc to gttcatct tcttcttcct ctgcagcatc 180 cctgtgctcc tttggtcccc aactaCcctc tacttgacct ggatcttggt gtgggataag 240 gcccctgaga acggaggcag acccatcagg cggcccagga acgcagcCtg gtggaagcCc 300 cttgctggat acttcccagc tcatgttaCc aaggaggc g accttgaccc atccaagaac 360 tacatctttg gccaccaccc acaCggCatc atcagcacgg gCagctCctg caccttctcc 420 accaacgcta ctggttCcga CgaccCcttc ccaggaatca ggccttcctt gctcaccctc 480 accagcaacC Ccaacatccc actctacagg gattacccca tggcctgtgg actccgctca 540 gtgtctaaga cctcctgcca gaacatcctc accaagggcg gCccaggaag gtccatcgct 600 attgtggtgg gaggtgcctc tgagCccctg aacgccagac caggagtgat ggaccttgtg 550 ttgaagagga ggcttggatt catcaagatt gctgtgcaga ctggtgctag ccttgtccct 720 accacccccc ccggtgagaa cgagcttcac gagcagactg agagcaacga gaaccctaag 780 cttcacaggt ggcagaagaa gaCccagcaC gcCcCcggtt tcaccat? rcc actcttccat 840 ggaaggggtg egttcaacta cgactccggt cccctcccac acaggcaccc aatctacacc 900 attgtgggCa agccaaCccc agccccaCcC atcaagtacg gCcagaccaa ggatgagatc 960 atcagggagc tccatgactc ttacatgcac gctgtgcagg acctctatga caggtacaag 1020 gacatctacg ccaaggacag ggtcaaggag cttgagcttg tggagcga 1068

Claims (46)

NOVELTY OF THE INVENTION CLAIMS

1. - An isolated DNA sequence that codes for an active enzyme in the formation of triacylglycerol from diacylglycerol and fatty acyl substrates.

2. The isolated DNA sequence according to claim 1, wherein said nucleic acid sequence encodes diacylglycerol acyl transferase.

3. The isolated DNA sequence according to claim 1, wherein said nucleic acid sequence is isolated from a source of eukaryotic cells.

4. The isolated DNA sequence according to claim 3, wherein said source of eukaryotic cells is selected from the group consisting of mammalian cells, nematodes, fungi and plants.

5. The sequence coding for DNA according to claim 4, wherein said diacylglycerol acyl transferase protein is from Mortierella ramanniana.

6. The sequence coding for DNA according to claim 4, wherein said source of cells is selected from the group consisting of soybean, Brassica napus, Arabidopsis thaliana, corn, human, murine, Mortierella alpina, Aspergillus fumigatus, Aspergillus oraceus, Fuserium graminearum, Schzichytrium aggregatum, Caenorhabditis elegans, and Sacchromyces cervaisae.

7 '.- The sequence coding for DNA according to claim 4, wherein said enzyme is selective for the synthesis of a structured triacylglycerol.

8. The sequence encoding DNA according to claim 7, wherein said structured triacylglycerol has a structure of formula: S-U-S, wherein S represents a saturated fatty acid and U represents an unsaturated fatty acid.

9. The sequence encoding DNA according to claim 4, wherein said diacylglycerol acyl transferase protein is encoded by a sequence including a nucleotide sequence selected from the group consisting of SEQ ID Nos: 46 to 72, inclusive .

10. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 38.

11- An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 45.

12. An isolated polypeptide encoded by the nucleotide sequence as was established in SEQ ID NO: 37.

13. An isolated polypeptide encoded by the nucleotide sequence as set forth in SEQ ID NO: 44.

14. - An isolated polypeptide encoded by the nucleotide sequence as set forth in SEQ ID NO: 84.

15. An isolated polynucleotide selected from the group consisting of: a) an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 38; b) an isolated polynucleotide comprising SEQ ID NO: 37; c) an isolated polynucleotide comprising a nucleotide sequence having at least 70% identity compared to SEQ ID NO: 37 in the total length of SEQ ID NO: 37; d) an isolated polynucleotide comprising a nucleotide sequence having at least 80% identity compared to SEQ ID NO: 37 in the total length of SEQ ID NO: 37; e) an isolated polynucleotide comprising a nucleotide sequence having at least 90% identity as compared to SEQ ID NO: 37 in the total length of SEQ ID NO: 37; f) an isolated polynucleotide comprising a nucleotide sequence having at least 95% identity as compared to SEQ ID NO: 37 in the total length of SEQ ID NO: 37; g) an isolated polynucleotide that hybridizes, under stringent conditions to SEQ ID NO: 37 or a fragment thereof; and h) an isolated polynucleotide complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), (f) or (g).

16. An isolated polynucleotide selected from the group consisting of: a) an isolated polynucleotide comprising SEQ ID NO: 84; b) an isolated polynucleotide comprising a nucleotide sequence having at least 70% identity compared to SEQ ID NO: 84 in the total length of SEQ ID NO: 84; c) an isolated polynucleotide comprising a nucleotide sequence having at least 80% identity as compared to SEQ ID NO: 84 in the total length of SEQ ID NO: 84; d) an isolated polynucleotide comprising a nucleotide sequence having at least 90% identity as compared to SEQ ID NO: 84 in the total length of SEQ ID NO: 84; e) an isolated polynucleotide comprising a nucleotide sequence having at least 95% identity as compared to SEQ ID NO: 84 in the total length of SEQ ID NO: 84; f) the isolated polynucleotide that hybridizes, under stringent conditions, to SEQ ID NO: 84 or a fragment thereof; and g) an isolated polynucleotide complementary to the polynucleotide sequence of (a), (b), (c), (d), (e) or (f).

17. An isolated polynucleotide selected from the group consisting of: a) an isolated polynucleotide comprising a nucleotide sequence coding for the polypeptide of SEQ ID NO: 45; b) an isolated polynucleotide comprising SEQ ID NO: 44; c) an isolated polynucleotide comprising a nucleotide sequence having at least 70% identity compared to SEQ ID NO: 44 in the total length of SEQ ID NO: 44; d) an isolated polynucleotide comprising a nucleotide sequence having at least 80% identity compared to SEQ ID NO: 44 in the total length of SEQ ID NO: 44; e) an isolated polynucleotide comprising a nucleotide sequence having at least 90% identity compared to SEQ ID NO: 44 over the total length of SEQ ID NO: 44; f) an isolated polynucleotide comprising a sequence ^ Mift ^ ftttMM ^ Üti of nucleotide having at least 95% identity as that of SEQ ID NO: 44 in the total length of SEQ ID NO: 44; g) the isolated polynucleotide that hybridizes, under stringent conditions, to SEQ ID NO: 44 or a fragment thereof; and h) an isolated polynucleotide complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), (f) or (g).

18. An isolated polynucleotide selected from the group consisting of SEQ ID Nos: 46 to 67, inclusive.

19. An acyl transferase protein of Mortierella, wherein said acyl transferase is active in the formation of triacylglycerol from diacylglycerol and fatty acyl substrates, and wherein said acyl transferase is isolated from other native proteins of Mortierella.

20. An acyl transferase protein, wherein said protein has an apparent molecular mass of about 36 kD in SDS-PAGE, said protein being substantially purified from membranes and other native cell proteins and capable of catalyzing the production of triglycerides from substrates of diacylglycerol and fatty acyl.

21. An acyl transferase protein, wherein said protein has an apparent molecular mass of about 36.5 kD in SDS-PAGE, said protein being substantially purified from membranes and other native cell proteins and capable of catalyzing the production of triglycerides from substrates of diacylglycerol and fatty acyl.

22. A nucleic acid construct comprising as components operably linked in the direction of the 5 'end to the 3' end of the transcript: a transcription initiation region; and a polynucleotide sequence encoding an active enzyme in the formation of triacylglycerol from diacylglycerol and fatty acyl substrates.

23. The nucleic acid construct according to claim 22, wherein said enzyme is diacylglycerol acyl transferase.

24. A host cell comprising a DNA construct according to claim 22.

25.- The host cell according to claim 24, wherein said host cell is selected from the group consisting of bacteria cells, insects, fungi, mammals and plants.

26. A plant comprising a cell according to claim 25.

27.- A method for producing a recombinant host cell comprising: transforming or transfecting a cell with a nucleic acid construct comprising a transcription initiation region and a polynucleotide sequence encoding an active enzyme in the formation of triacylglycerol from diacylglycerol and fatty acyl substrates, such that said host cell under suitable culture conditions produces an acyl transferase protein.

28. The method according to claim 27 wherein said host cell is a plant cell.

29. A method for producing a recombinant host cell comprising: transforming or transfecting a cell with a nucleic acid construct comprising a transcription initiation region and a polynucleotide sequence selected from the group consisting of a polynucleotide according to claim 12 and a polynucleotide according to claim 13, so that said host cell under suitable culture conditions produces an acyl transferase protein.

30. The method according to claim 29, wherein said polynucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 37.

31.- The method according to claim 29, wherein said host cell is a plant cell.

32.- A method for modifying the composition of triacylglycerol in a plant cell said method comprises: transforming a plant cell with a nucleic acid construct comprising a transcription start region and a polynucleotide sequence that encodes an active enzyme in the formation of triacylglycerol from diacylglycerol and fatty acyl substrates, or fragments or variants thereof.

33. The method according to claim 32, wherein said polynucleotide sequence is in an antisense orientation.

34. The method according to claim 32, wherein said polynucleotide sequence is in a sense orientation.

35. The method according to claim 32, wherein the activity of the diacylglycerol acyl transferase protein is suppressed.

36. - The method according to claim 32, wherein the activity of the diacylglycerol acyl transferase protein is improved.

37.- A method for modifying the composition of lipids in a plant cell, said method comprises: transforming a plant cell with a nucleic acid construct comprising a transcription start region and a polynucleotide sequence selected from the group consisting of a polynucleotide according to claim 12 and a polynucleotide according to claim 13, so that said plant cell under suitable culture conditions produces an acyl transferase protein.

38. The method according to claim 37, wherein said polynucleotide sequence is in an antisense orientation, wherein the mRNA transcribed from said sequence is complementary to the transcribed mRNA equivalent from the endogenous gene and the activity of said diacylglycerol protein. acyl transferase in said plant cell is deleted.

39. The method according to claim 37, wherein said polynucleotide sequence is in a sense orientation.

40.- An immunospecific antibody for the polypeptide of claim 10.

41.- An immunospecific antiquase for the polypeptide of claim 11.

42.- The use of an agonist of DAGAT activity for the preparation of a medicament for treating or alleviating an illness or ^ t_a_ß¡ _ _tl_f_? condition associated with the altered activity of diacylglycerol acyl transferase (DAGAT) in a subject.

43.- The use of a DAGAT activity agonist for the preparation of a medicament for treating or improving a disease or condition associated with the altered activity of diacylglycerol acyl transferase (DAGAT) in a subject.

44.- A method to diagnose a disease or susceptibility to a disease associated with the expression or activity of DAGAT in a patient, said method comprises the step of: determining the presence or absence of a mutation in the nucleotide sequence encoding DAGAT in the genome of said patient.

45.- A method to diagnose a disease or susceptibility to a disease associated with the expression or activity of DAGAT in a patient, said method comprises the step of: detecting the presence or amount of DAGAT or an indicator of the same in a sample obtained from said patient.

46. A method for identifying a compound that stimulates or inhibits the expression or activity of DAGAT comprising: making a composition comprising a polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 38 and a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 45 makes contact with a candidate compound; detecting an interaction between said polypeptide and said candidate compound. 47.- An agonist of a polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 38 and a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 45. 48.- A antagonist of a polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 38 and a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 45.