EP1003882A1 - Proteines diacylglycerol acyltransferase - Google Patents

Proteines diacylglycerol acyltransferase

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Publication number
EP1003882A1
EP1003882A1 EP98926310A EP98926310A EP1003882A1 EP 1003882 A1 EP1003882 A1 EP 1003882A1 EP 98926310 A EP98926310 A EP 98926310A EP 98926310 A EP98926310 A EP 98926310A EP 1003882 A1 EP1003882 A1 EP 1003882A1
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EP
European Patent Office
Prior art keywords
dagat
activity
protein
plant
sequences
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EP98926310A
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German (de)
English (en)
Inventor
Kathryn Dennis Lardizabal
James George Metz
Michael W. Lassner
Gregory A. Thompson
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Monsanto Co
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Calgene LLC
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Publication of EP1003882A1 publication Critical patent/EP1003882A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • the present invention is directed to enzymes, methods to purify, and obtain such enzymes, amino acid and nucleic acid sequences related thereto, and methods of use for such compositions in genetic engineering applications.
  • Plant oils are used in a variety of industrial an edible uses. Novel vegetable oils compositions and/or improved means to obtain oils compositions, from biosynthetic or natural plant sources, are needed. Depending upon the intended oil use, various different fatty acid compositions are desired.
  • oilseed with a higher ratio of oil to seed meal would be useful to obtain a desired oil at lower cost. This would be typical of a high value oil product. Or such an oilseed might constitute a superior feed for animals . In some instances having an oilseed with a lower ratio of oil to seed meal would be useful to lower caloric content.
  • edible plant oils with a higher percentage of unsaturated fatty acids are desired for cardiovascular health reasons. And alternataively, temperate substitutes for high saturate tropical oils such as palm, coconut, or cocoa would also find uses in a variety of industrial and food applications .
  • One means postulated to obtain such oils and/or modified fatty acid compositions is through the genetic engineering of plants.
  • Fatty acids are made in plastids from acetyl-CoA through a series of reactions catalyzed by enzymes known collectively as Fatty Acid Synthetase (FAS) .
  • FAS Fatty Acid Synthetase
  • the fatty acids produced in plastids are exported to the cytosolic compartment of the cell, and are esterified to coenzyme A.
  • acyl-CoAs are the substrates for glycerolipid synthesis in the endoplasmic reticulum (ER) .
  • Glycerolipid synthesis itself is a series of reactions leading first to phosphatidic acid (PA) and diacylglycerol (DAG) .
  • PA phosphatidic acid
  • DAG diacylglycerol
  • Either or these metabolic intermediates may be directed to membrane phospholipids such as phosphatidylglycerol (PG) , phosphatidylethanolamine (PE) or phosphatidylcholine (PC) , or they may be directed on to form neutral triacylglycerol (TAG) the primary component of vegetable oil used by the seed as a stored form of energy to be used during seed germination.
  • PG phosphatidylglycerol
  • PE phosphatidylethanolamine
  • PC phosphatidylcholine
  • TAG neutral triacylglycerol
  • Diacylglycerol is synthesized from glycerol-3- phosphate and fatty acyl-CoAs in two steps catalyzed sequentially by glycerol-3 -phosphate acyltransferase (G3PAT) , lysophosphatidic acid acyltransferase (DAGAT) to make PA, and then an additional hydrolytic step catalyzed by phosphatidic acid phosphatase (PAP) to make DAG.
  • G3PAT glycerol-3 -phosphate acyltransferase
  • DAG lysophosphatidic acid acyltransferase
  • PAP phosphatidic acid phosphatase
  • DAG is used to make membrane phospholipids, the first step being the synthesis of PC catalyzed by CTP-phosphocholine cytidylyltransferase.
  • DAG is acylated with a third fatty acid in a reaction catalyzed by diacylglycerol acyltransferase (DAGAT) .
  • DAG diacylglycerol acyltransferase
  • acyltransferases from many temperate zone species of seeds to allow either a saturated or an unsaturated fatty acid at the sn-1 or the sn-3 position, but only an unsaturated fatty acid at the sn-2.
  • the absolute specificity for an unsaturated fatty acid at sn-2 is determined by the substrate preference of DAGAT enzyme.
  • TAG compositions suggest that this tendency is carried further in that there is an apparent preference for acylation of the sn-3 position with a saturated fatty acid, if the sn-1 position is esterified to a saturated fatty acid.
  • the reaction catalyzed by DAGAT is at a critical branchpoint in glycerolipid biosynthesis. Enzymes at such branchpoints are considered prime candidates for sites of metabolic regulation. Up through the synthesis of diacylglycerol, TAG and membrane lipid synthesis share in common G3PAT, DAGAT, and PAP. Since all cells have membranes, they must have these enzymes. What makes oil synthesis unique is the DAGAT reaction. The presence of DAGAT activity provides an alternative fate for DAG than going into membranes.
  • nucleic acid sequences capable of producing a phenotypic result in the incorporation of fatty acids into a glycerol backbone to produce an oil is subject to various obstacles including but not limited to the identification of metabolic factors of interest, choice and characterization of a protein source with useful kinetic properties, purification of the protein of interest to a level which will allow for its amino acid sequencing, utilizing amino acid sequence data to obtain a nucleic acid sequence capable of use as a probe to retrieve the desired DNA sequence, and the preparation of constructs, transformation and analysis of the resulting plants .
  • the identification of enzyme targets and useful tissue sources for nucleic acid sequences of such enzyme targets capapble of modifying oil structure and quantity are needed.
  • an enzyme target will be amenable to one or more applications alone or in combination with other nucleic acid sequences relating to increased/decreased oil production, TAG structure, the ratio of saturated to unsaturated fatty acids in the fatty acid pool, and/or to other novel oils compositions as a result of the modifications to the fatty acid pool.
  • enzyme targets (s) are identified and qualified, quantities of protein and purification protocols are needed for sequencing.
  • useful nucleic acid constructs having the necessary elements to provide a phenotypic modification and plants containing such constructs are needed.
  • Several putative isolation procedures have been published for DAGAT. Polokoff and Bell (1980) reported solubilization and partial purification of DAGAT from rat liver microsomes.
  • Solubilization of a multienzyme complex from Euglena gracilis having fatty acyl-SCoA transacylase activity is reported by Wildner and Hallick (Abstract from The Southwest
  • WO 93/10241 is directed to plant fatty acyl-CoA: fatty alcohol 0-acyltransferases .
  • a jojoba 57kD protein is identified as the jojoba fatty acyl-CoA: fatty alcohol 0- acyltransferase (wax synthase) .
  • the present inventors later reported that the 57kD protein from jojoba is a ⁇ -ketoacyl-CoA synthase involved in the biosynthesis of very long chain fatty acids (Lassner et al . ( The Plant Cell (1996) 8:281-292).
  • WO 93/10241 is directed to plant fatty acyl-CoA: fatty alcohol 0-acyltransferases .
  • a jojoba 57kD protein is identified as the jojoba fatty acyl-CoA: fatty alcohol 0- acyltransferase (wax synthase) .
  • the present inventors later reported that the 57kD protein from jojoba is a ⁇ -ketoacyl-CoA synthase involved in the biosynthesis of very long chain fatty acids (Lassner et al . ( The Plant Cell (1996) 8:281-292).
  • Figure 1 presents results of analysis of wax synthase activity in column fractions from a first wax synthase purification protocol.
  • Figure 1A provides results of Blue A agarose chromatography.
  • Figure IB provides results of ceramic hydroxyapatite chromatography.
  • Figure 1C provides results of sephracryl S-100 size exclusion chromatography.
  • Figure 1A provides results of hydroxyapatite chromatography.
  • Figure 2 presents results of analysis of wax synthase activity in column fractions from a second wax synthase purification protocol.
  • Figure 2A provides results of Blue A agarose chromatography.
  • Figure 2B provides results of hydroxyapatite chromatography.
  • Figure 2C provides results of Superdex 75 size exclusion chromatography.
  • Figure 3 presents results of wax synthase and DAGAT activity in fractions from a purified wax synthase preparation according to the wax synthase purification represented in Figure 1. Results are from the fractions obtained following the hydroxyapatite chromatography step.
  • Figure 4 presents results of analysis of wax synthase activity in column fractions from a DAGAT purification protocol utilizing Yellow 86-Agarose Chromotography .
  • Figure 5 presents results of analysis of DAGAT activity in column fractions from a second wax synthase purification protocol.
  • Figure 5A provides results of Heparin separose CL-6B chromatography.
  • Figure 5B provides results SDS-PAGE analysis of the peak fractions .
  • Figure 6A presents results of DAGAT purification from a yellow 86-Agarose chromotography.
  • Figure 6B presents the SDS- PAGE analysis of the peak fractions from the yellow 86-Agarose chromotography .
  • Figure 7 presents results of the purification of DAGAT from Mortierella ramanniana utilizing a yellow 86-Agarose chromotograph.
  • Figure 8 presents results of analysis of DAGAT activity in column fractions from a second wax synthase purification protocol.
  • Figure 8A provides results of Hydroxylapatite chromatography.
  • Figure 8B provides results SDS-PAGE analysis of the peak fractions .
  • Figure 9 presents results of analysis of DAGAT activity in column fractions from a DAGAT purification protocol.
  • Figure 9A provides results of tandem yellow 86-agarose/Hydroxylapatite chromatography.
  • Figure 9B provides results SDS-PAGE analysis of the peak fractions from the tandem chromotography.
  • compositions and methods of use related to diacylglycerol acyltransferase hereinafter also referred to as DAGAT
  • DAGAT diacylglycerol acyltransferase
  • methods and compositions of amino acid sequences related to biologically active DAGA are provided.
  • DAGAT protein preparations which have relatively high specific activity are of interest for use in a variety of applications, in vitro and in vivo.
  • protein preparations having DAGAT activities are contemplated hereunder .
  • the DAGAT obtainable from Mortierella rammaniana .
  • the jojoba wax synthase of the present invention also demonstrates diacylglycerol (DAGAT) activity.
  • DAG diacylglycerol
  • TAG is not naturally produced in jojoba and thus the activity of the wax synthase enzyme with DAG substrates suggests that the wax synthase is related to DAGAT, an enzyme responsible for production of TAG in most plant species, particularly in oilseed crop plants whose seeds contain high levels of storage TAG.
  • DAGAT diacylglycerol
  • the exemplified jojba and Mortierella rammaniana DAGATs are purified away from the membranes (i.e. solubilized), and the solubilized DAGAT preparation is subjected to various chromatographic analyses to identify a protein associated with the DAGAT activity. In this manner a protein having a molecular weight of approximately 40kDA is identified as associated with DAGAT activity. Further purification methods, such as column chromatography and polyacrylamide gel electrophoresis are utilized to obtain the DAGAT protein in sufficient purity for amino acid sequence analysis.
  • DAGAT proteins are used as a template in designing various synthetic oligonucleotides which may be used to obtain nucleic acid sequences encoding all or a portion of the DAGAT protein. Using the DAGAT encoding sequences so obtained, it is also possible to isolate other DAGAT genes which encode DAGAT proteins . Thus, this invention encompasses DAGAT peptides and the corresponding amino acid sequences of those peptides . Such sequences find particular use in the preparation of oligonucleotides containing DAGAT encoding sequences for analysis and recovery of DAGAT gene sequences .
  • the DAGAT encoding sequence may encode a complete or partial sequence depending upon the intended use. All or a portion of the genomic sequence, or cDNA sequence, is intended.
  • constructs which provide for transcription or transcription and translation (expression) of the DAGAT sequences.
  • constructs which are capable of transcription or transcription and translation in plant host cells are preferred.
  • Such constructs may contain a variety of regulatory regions including transcriptional initiation regions obtained from genes preferentially expressed in plant seed tissue.
  • this invention relates to a method for producing a DAGAT in a host cell or progeny thereof via the expression of a construct in the cell.
  • Cells containing a DAGAT as a result of the production of the DAGAT encoding sequence are also contemplated herein.
  • this invention relates to methods of using DNA sequences encoding DAGAT for the modification of triglyceride molecules, especially in the seed oil of plant oilseed crops .
  • Plant cells having such a modified triglyceride are also contemplated herein.
  • modified plants, seeds and oils obtained by expression of the plant LPAAT proteins of this invention are also considered in this invention.
  • a diacylglycerol acyltransferase (refered to herein as DAGAT) of this invention includes any sequence of amino acids, such as a protein, polypeptide or peptide, obtainable from a cell source, which demonstrates the ability to catalyze the production of triacylglycerol from 1, 2-diacylglycerol-3- phosphate and an acyl-CoA substrate under enzyme reactive conditions.
  • enzyme reactive conditions is meant that any necessary conditions are available in an environment (i.e., such factors as temperature, pH, lack of inhibiting substances) which will permit the enzyme to function.
  • solubilization refers to extraction of the DAGAT enzyme from the membranes in such a way that it then behaves in a manner typical of enzymes that are not membrane-associated. Because the membrane effectively links the DAGAT protein to other proteins which are also present therein, solubilization is an essential requirement for identification and purification of the DAGAT protein as described in the following examples. In testing for solubilization of DAGAT activity, three different indications of solubilization, as described in more detail in the following examples, are considered.
  • DAGAT activity is not sedimented by very high-speed centrifugation .
  • DAGAT activity migrates on a size-exclusion chromatography column as though it had a native molecular weight typical of enzymes which are not membrane-associated.
  • Proteins present in the DAGAT preparation are at least partially separable from each other by column chromatography.
  • the second criterion in which solubilized activity migrates more slowly through a size-exclusion column than the original membranes, may be compromised if the membranes themselves bind weakly to the column after exposure to detergent so that their migration through it is slowed.
  • the third criterion in which the solubilized proteins are chromatographically resolvable, is the least likely to be compromised by artifacts or unforeseen situations.
  • membranes could be partially dissociated by the solubilization procedure such that various aggregates of proteins are released. Such aggregates might then be resolved from each other chromatographically.
  • satisfaction of all three criteria is necessary to assure that DAGAT solubilization is achieved.
  • the jojoba wax synthase of this invention has a broad range of acyl substrates, including acyl-ACP and acyl-CoA molecules.
  • the acyl and fatty alcohol substrates may have a broad size range with respect to carbon chain length. For example, activity was tested using substrates having carbon chain lengths of from C12 to C24, and all were shown to be utilized by the enzyme. In addition, activity was shown with fatty acyl and fatty alcohols having varying degrees of unsaturation.
  • the purified jojoba wax synthase is also shown herein to have activity with diacylglycerol (DAG) and fatty acyl-CoA substrates to produce triacylglycerol (TAG) , even though TAG have not been reported to be exist in jojoba plant tissues.
  • DAG diacylglycerol
  • TAG triacylglycerol
  • the wax synthase has at least two acyltransferase activities, one in which the acceptor substrate for the acyl-CoA molecule is an alcohol (fatty alcohol acyltransferase) and another in which the acceptor subsrate is a diacylglycerol (DAG acyltransferase, or DAGAT) .
  • Solubilized preparations of Mortierella DAGAT are utilized in a variety of chromatographic experiments for identification and partial purification of the DAGAT protein.
  • a protein having a molecular weight of approximately 40kDa is identified as associated with DAGAT activity.
  • the 40kDa protein is partially purified by chromatography on yellow 86-agarose and hydroxyapatite columns. The protein is then obtained in substantially purified form by gel electrophoresis and blotting of the partially purified DAGAT preparation to nitrocellulose.
  • the 40kDA protein is recovered by cutting out that portion of the nitrocellulose filter containing the identified band.
  • the purified protein is then digested with various enzymes to generate peptides for use in determination of amino acid sequence.
  • the tryptic peptide of the 40kDa protein described herein represents a portion of a Mortierella ramanniana DAGAT.
  • Other Mortierella DAGAT peptides may be similarly obtained and the amino acid sequences determined.
  • amino acid sequences from DAGAT peptides to obtain nucleic acid sequences which encode DAGAT is described herein.
  • 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 partial DNA sequence of DAGAT genes .
  • the partial sequences so obtained are then used as probes to obtain DAGAT clones from a gene library prepared from Mortierella ramanniana tissue.
  • PCR polymerase chain reaction
  • probes may be used directly to screen gene libraries for DAGAT gene sequences.
  • a nucleic acid sequence of a DAGAT of this invention may be a DNA or RNA sequence, derived from genomic DNA, cDNA, mRNA, or may be synthesized in whole or in part.
  • the gene sequences may be cloned, for example, by isolating genomic DNA from an appropriate source, and amplifying and cloning the sequence of interest using a polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • the gene sequences may be synthesized, either completely or in part, especially where it is desirable to provide plant-preferred sequences.
  • all or a portion of the desired structural gene may be synthesized using codons preferred by a selected host.
  • Host-preferred codons may be determined, for example, from the codons used most frequently in the proteins expressed in a desired host species.
  • nucleic acid probes DNA and RNA
  • DNA and RNA DNA and RNA
  • homologous sequences are found when there is an identity of sequence, which may be determined upon comparison of sequence information, nucleic acid or amino acid, or through hybridization reactions between a known DAGAT and a candidate source.
  • Conservative changes such as Glu/Asp, Val/Ile, Ser/Thr, Arg/Lys and Gln/Asn may also be considered in determining sequence homology.
  • Amino acid sequences are considered homologous by as little as 25% sequence identity between the two complete mature proteins. (See generally, Doolittle, R.F., OF URFS and ORFS (University Science Books, CA, 1986.)
  • DAGATs may be obtained from the specific exemplified Mortierella protein preparations and sequences provided herein.
  • natural and synthetic DAGATs including modified amino acid sequences and starting materials for synthetic- protein modeling from the exemplified DAGATs and from DAGATs which are obtained through the use of such exemplified sequences .
  • Modified amino acid sequences include sequences which have been mutated, truncated, increased and the like, whether such sequences were partially or wholly synthesized. Sequences which are actually purified from plant preparations or are identical or encode identical proteins thereto, regardless of the method used to obtain the protein or sequence, are equally considered naturally derived.
  • a DAGAT sequence obtainable from the use of nucleic acid probes will show 60-70% sequence identity between the target DAGAT sequence and the encoding sequence used as a probe.
  • lengthy sequences with as little as 50-60% sequence identity may also be obtained.
  • the nucleic acid probes may be a lengthy fragment of the nucleic acid sequence, or may also be a shorter, oligonucleotide probe.
  • longer nucleic acid fragments are employed as probes (greater than about 100 bp) , one may screen at lower stringencies in order to obtain sequences from the target sample which have 20-50% deviation (i.e., 50-80% sequence homology) from the sequences used as probe.
  • Oligonucleotide probes can be considerably shorter than the entire nucleic acid sequence encoding an DAGAT enzyme, but should be at least about 10, preferably at least about 15, and more preferably at least about 20 nucleotides. A higher degree of sequence identity is desired when shorter regions are used as opposed to longer regions . It may thus be desirable to identify regions of highly conserved amino acid sequence to design oligonucleotide probes for detecting and recovering other related DAGAT genes. Shorter probes are often particularly useful for polymerase chain reactions (PCR) , especially when highly conserved sequences can be identified. (See, Gould, et al . , PNAS USA (1989) 85:1934-1938.)
  • genes for other related acyltransferase proteins may also be obtained using sequence information from the DAGAT and related nucleic acid sequences.
  • other acyltransferase enzymes are involved in plant lipid biosynthesis, including plastidial DAGAT, mitochondrial DAGAT, lysophosphosphatidylcholine acyltransferase (LPCAT) , lysophosphosphatidylserine acyltransferase (LPSAT) , lysophosphosphatidylethanolamine acyltransferase (LPEAT) , and lysophosphosphatidylinositol acyltransferase (LPIAT) .
  • acyltransferases including fatty acyl-CoA: fatty alcohol 0- acyltransferase (wax synthase) from jojoba may be related to diacylglycerol acylransferases .
  • DAGAT and wax synthase are members of a homologous family of proteins is supported by information obtained through the purification of DAGAT from a species of oleaginous fungus, Mortierella rammaniana (see Examples).
  • the fungal DAGAT activity is membrane bound, and may be solubilized only through the use of detergents.
  • solubilization it is necessary following solubilization to include a phospholipid (e.g., phosphatidic acid) in the assay mixture in order to restore enzyme activity of the fungal DAGAT.
  • a phospholipid e.g., phosphatidic acid
  • 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 Northern or Southern blots, or the filters containing cDNA or genomic clones to be screened.
  • Hybridization and washing conditions may be varied to optimize the hybridization of the probe to the sequences of interest. Lower temperatures and higher salt concentrations allow for hybridization of more distantly related sequences (low stringency). If background hybridization is a problem under low stringency conditions, the temperature can be raised either in the hybridization or washing steps and/or salt content lowered to improve detection of the specific hybridizing sequence.
  • Hybridization and washing temperatures can be adjusted based on the estimated melting temperature of the probe as discussed in Beltz, et al .
  • genes encoding the related proteins are isolated by screening expression libraries representing the desired plant species.
  • Expression libraries can be constructed in a variety of commercially available vectors, including lambda gtll, as described in Maniatis, et al . (Molecular Cloning: A Laboratory Manual , Second Edition (1989) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) .
  • All plants utilize DAGAT proteins in production of membrane phospholipids, and thus any given plant species can be considered as a source of additional DAGAT proteins.
  • Plants having significant medium-chain fatty acids in their seed oils are preferred candidates to obtain plant DAGATs capable of incorporating medium-chain fatty acids into the sn- 2 position of TAG.
  • Several species in the genus Cuphea accumulate triglycerides containing medium-chain fatty acids in their seeds, e.g., procumbens , lutea, hookeriana, hyssopifolia, wrightii and inflata .
  • Another natural plant source of medium-chain fatty acids are seeds of the Lauraceae family. In addition to the exemplified California Bay
  • Umbel 1 ul aria californica Pisa (Actinodophne hooker i ) , Sweet Bay (Laurus nobilis) and Cinnamomum camphor a (camphor) accumulate medium-chain fatty acids .
  • Other plant sources include Ulmaceae (elm) , Palmae, Myristicaceae , Simarubaceae , Vochysiaceae, and Salvadoraceae .
  • DAGATs from plant species which incorporate unusual long-chain fatty acids in the storage TAG.
  • nasturtium and meadowfoam contain 22:1 acyl groups in the seed TAG, and meadowfoam has been shown to contain an DAGAT capable of incorporating 22:1
  • (erucic) fatty acyl groups into the sn-2 position.
  • An DAGAT having such activity may find use in production of "tri- erucic" Brassica oil, which to date is not found due to the selectivity of Brassica seed DAGAT towards unsaturated fatty acids, such as 18:1 and 18:2.
  • plant DAGATs from a variety of sources can be used to investigate TAG biosynthesis events of plant lipid biosynthesis in a wide variety of in vivo applications. Because all plants appear to synthesize lipids via a common metabolic pathway, the study and/or application of one plant DAGAT to a heterologous plant host may be readily achieved in a variety of species. In other applications, a plant DAGAT can be used outside the native plant source of the DAGAT to enhance the production and/or modify the composition of the TAG produced or synthesized in vi tro .
  • nucleic acid sequences associated with plant DAGAT proteins will find many uses. For example, recombinant constructs can be prepared which can be used as probes, or which will provide for expression of the DAGAT protein in host cells to produce a ready source of the enzyme and/or to modify the composition of triglycerides found therein. Other useful applications may be found when the host cell is a plant host cell, either in vi tro or in vivo . For example, by increasing the amount of a respective medium-chain preferring DAGAT available to the plant TAG biosynthesis pathway, an increased percentage of medium-chain fatty acids may be obtained in the TAG.
  • DAGAT endogenously expressed in a plant cell by anti-sense technology.
  • DAGAT decreased expression of a native Brassica long-chain preferring DAGAT may be desired.
  • the constructs may contain the sequence which encodes the entire DAGAT protein, or a portion thereof. For example, where antisense inhibition of a given DAGAT protein is desired, the entire DAGAT sequence is not required. Furthermore, where DAGAT constructs are intended for use as probes, it may be advantageous to prepare constructs containing only a particular portion of an DAGAT encoding sequence, for example a sequence which is discovered to encode a highly conserved DAGAT region.
  • nucleic acid sequence encoding a plant DAGAT of this invention may include genomic, cDNA or mRNA sequence.
  • encoding is meant that the sequence corresponds to a particular amino acid sequence either in a sense or anti-sense orientation.
  • extrachromosomal is meant that the sequence is outside of the plant genome of which it is naturally associated.
  • recombinant is meant that the sequence contains a genetically engineered modification through manipulation via mutagenesis, restriction enzymes, and the like.
  • a cDNA sequence may or may not contain pre-processing sequences, such as transit peptide sequences or targetting sequences to facilitate delivery of the DAGAT protein (such as mitochondrial DAGAT) to a given organelle or membrane location.
  • pre-processing sequences such as transit peptide sequences or targetting sequences to facilitate delivery of the DAGAT protein (such as mitochondrial DAGAT) to a given organelle or membrane location.
  • the use of any such precursor DAGAT DNA sequences is preferred for uses in plant cell expression.
  • a genomic DAGAT sequence may contain the transcription and translation initiation regions, introns , and/or transcript termination regions of the plant DAGAT, which sequences may be used in a variety of DNA constructs, with or without the DAGAT structural gene.
  • nucleic acid sequences corresponding to the plant DAGAT of this invention may also provide signal sequences useful to direct protein delevery into a particular organellar or membrane location, 5' upstream non-coding regulatory regions (promoters) having useful tissue and timing profiles, 3' downstream non-coding regulatory region useful as transcriptional and translational regulatory regions and may lend insight into other features of the gene.
  • the desired plant DAGAT nucleic acid sequence may be manipulated in a variety of ways. Where the sequence involves non-coding flanking regions, the flanking regions may be subjected to resection, mutagenesis, etc. Thus, transitions, transversions , deletions, and insertions may be performed on the naturally occurring sequence. In addition, all or part of the sequence may be synthesized.
  • one or more codons may be modified to provide for a modified amino acid sequence, or one or more codon mutations may be introduced to provide for a convenient restriction site or other purpose involved with construction or expression.
  • the structural gene may be further modified by employing synthetic adapters, linkers to introduce one or more convenient restriction sites, or the like.
  • nucleic acid or amino acid sequences encoding a plant DAGAT of this invention may be combined with other non-native, or “heterologous", sequences in a variety of ways.
  • heterologous sequences is meant any sequence which is not naturally found joined to the plant DAGAT, including, for example, combinations of nucleic acid sequences from the same plant which are not naturally found joined together.
  • the DNA sequence encoding a plant DAGAT of this invention may be employed in conjunction with all or part of the gene sequences normally associated with the DAGAT.
  • a DNA sequence encoding DAGAT is combined in a DNA construct having, in the 5' to 3 ' direction of transcription, a transcription initiation control region capable of promoting transcription and translation in a host cell, the DNA sequence encoding plant DAGAT and a transcription and translation termination region.
  • Potential host cells include both prokaryotic and eukaryotic cells.
  • a host cell may be unicellular or found in a multicellar differentiated or undifferentiated organism depending upon the intended use.
  • Cells of this invention may be distinguished by having a plant DAGAT foreign to the wild- type cell present therein, for example, by having a recombinant nucleic acid construct encoding a plant DAGAT therein.
  • the regulatory regions will vary, including regions from viral, plasmid or chromosomal genes, or the like.
  • a wide variety of constitutive or regulatable promoters may be employed. Expression in a microorganism can provide a ready source of the plant enzyme.
  • transcriptional initiation regions which have been described are regions from bacterial and yeast hosts, such as E. coli , B . subtilis, Sacchromyces cerevisiae, including genes such as beta-galactosidase, T7 polymerase, tryptophan E and the like.
  • the constructs will involve regulatory regions functional in plants which provide for modified production of plant DAGAT, and possibly, modification of the fatty acid composition.
  • the open reading frame, coding for the plant DAGAT or functional fragment thereof will be joined at its 5' end to a transcription initiation regulatory region.
  • the use of all or part of the complete plant DAGAT gene is desired; namely all or part of the 5' upstream non-coding regions (promoter) together with the structural gene sequence and 3 ' downstream non-coding regions may be employed.
  • a different promoter such as a promoter native to the plant host of interest or a modified promoter, i.e., having transcription initiation regions derived from one gene source and translation initiation regions derived from a different gene source
  • numerous transcription initiation regions are available which provide for a wide variety of constitutive or regulatable, e.g., inducible, transcription of the structural gene functions.
  • the transcription/translation initiation regions corresponding to such structural genes are found immediately 5' upstream to the respective start codons.
  • transcriptional initiation regions used for plants are such regions associated with the T-DNA structural genes such as for nopaline and mannopine synthases, the 19S and 35S promoters from CaMV, and the 5' upstream regions from other plant genes such as napin, ACP, SSU, PG, zein, phaseolin E, and the like.
  • Enhanced promoters, such as double 35S, are also available for expression of DAGAT sequences.
  • 5' upstream non-coding regions are obtained from other genes regulated during seed maturation, those preferentially expressed in plant embryo tissue, such as ACP and napin-derived transcription initiation control regions, are desired.
  • seed-specific promoters may be obtained and used in accordance with the teachings of U.S.
  • Transcript termination regions may be provided by the DNA sequence encoding the plant DAGAT or a convenient transcription termination region derived from a different gene source, for example, the transcript termination region which is naturally associated with the transcript initiation region. Where the transcript termination region is from a different gene source, it will contain at least about 0.5 kb, preferably about 1-3 kb of sequence 3 ' to the structural gene from which the termination region is derived.
  • Plant expression or transcription constructs having a plant DAGAT as the DNA sequence of interest for increased or decreased expression thereof may 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 especially preferred are temperate oilseed crops. Plants of interest include, but are not limited to, rapeseed
  • DAGAT proteins of this invention include their use in preparation of structured plant lipids which contain TAG molecules having desirable fatty acyl groups incorporated into particular positions on the TAG molecules.
  • the method of transformation in obtaining such transgenic plants is not critical to the instant invention, and various methods of plant transformation are currently available. Furthermore, as newer methods become available to transform crops, they may also be directly applied hereunder . For example, many plant species naturally susceptible to Agrojbacteriii infection may be successfully transformed via tripartite or binary vector methods of Agrobacterium mediated transformation. In many instances, it will be desirable to have the construct bordered on one or both sides by T-DNA, particularly having the left and right borders, more particularly the right border. This is particularly useful when the construct uses A . tumefaciens or A . rhizogenes as a mode for transformation, although the T-DNA borders may find use with other modes of transformation. In addition, techniques of microinjection, DNA particle bombardment, and electroporation have been developed which allow for the transformation of various monocot and dicot plant species.
  • included with the DNA construct will be a structural gene having the necessary regulatory regions for expression in a host and providing for selection of transformant cells.
  • the gene may provide for resistance to a cytotoxic agent, e.g. antibiotic, heavy metal, toxin, etc., complementation providing prototrophy to an auxotrophic host, viral immunity or the like.
  • a cytotoxic agent e.g. antibiotic, heavy metal, toxin, etc.
  • complementation providing prototrophy to an auxotrophic host, viral immunity or the like.
  • one or more markers may be employed, where different conditions for selection are used for the different hosts.
  • a vector may be used which may be introduced into the Agrobacterium host for homologous recombination with T-DNA or the Ti- or Ri-plasmid present in the Agrobacterium host.
  • the Ti- or Ri-plasmid containing the T-DNA for recombination may be armed (capable of causing gall formation) or disarmed (incapable of causing gall formation), the latter being permissible, so long as the vir genes are present in the transformed Agrobacterium host.
  • the armed plasmid can give a mixture of normal plant cells and gall.
  • the expression or transcription construct bordered by the T-DNA border region (s) will be inserted into a broad host range vector capable of replication in E. coli and Agrobacterium, there being broad host range vectors described in the literature. Commonly used is pRK2 or derivatives thereof. See, for example, Ditta, et al . , ( Proc . Na t . Acad . Sci . , U. S. A . (1980) 77:7347-7351) and EPA 0 120 515, which are incorporated herein by reference.
  • a vector containing separate replication sequences one of which stabilizes the vector in E. coli , and the other in Agrobacterium.
  • McBride and Summerfelt Plant Mol . Biol . (1990) 24:269-276
  • the pRiHRI Jaanin, et al . , Mol . Gen . Genet . (1985) 201:370-374
  • origin of replication is utilized and provides for added stability of the plant expression vectors in host Agrobacterium cells .
  • markers which allow for selection of transformed Agrobacterium and transformed plant cells.
  • a number of markers have been developed for use with plant cells, such as resistance to chloramphenicol, kanamycin, the aminoglycoside G418, hygromycin, or the like.
  • the particular marker employed is not essential to this invention, one or another marker being preferred depending on the particular host and the manner of construction.
  • explants For transformation of plant cells using Agrobacterium, explants may be combined and incubated with the transformed Agrobacterium for sufficient time for transformation, the bacteria killed, and the plant cells cultured in an appropriate selective medium. Once callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants. The plants may then be grown to seed and the seed used to establish repetitive generations and for isolation of vegetable oils.
  • the substrate generally used in the wax synthase assays [l- 14 C]palmitoyl-CoA, is purchased from Amersham (Arlington Heights, IL) .
  • Other chain length substrates were synthesized in order to perform chain length specification studies.
  • Long chain [1- 1 C] fatty acids (specific activity 51-56 Ci/mole) , namely 11-cis-eicosenoic acid, 13-cis-docosenoic acid and 15- cis-tetracosenoic acid are prepared by the reaction of potassium [ 14 C] cyanide with the corresponding alcohol mesylate, followed by the base hydrolysis of the alcohol nitrile to the free fatty acid.
  • the free fatty acids are converted to their methyl esters with ethereal diazomethane, and purified by preparative silver nitrate thin layer chromatography (TLC) .
  • TLC thin layer chromatography
  • the fatty acid methyl esters are hydrolyzed back to the free fatty acids .
  • Radiochemical purity is assessed by three TLC methods: normal phase silica TLC, silver nitrate TLC, and C18 reversed phase TLC. Radiochemical purity as measured by these methods was 92-98%.
  • Long chain [1- 14 C] acyl-CoAs are prepared from the corresponding [1- 1 C] free fatty acids by the method of Young and Lynen ( J. Bio . Chem. (1969) 244:377), to a specific activity of lOCi/mole.
  • [1- 14 C] hexadecanal is prepared by the dichromate oxidation of [l- 1 C]hexadecan-l-ol , according to a micro-scale modification of the method of Pletcher and Tate (Tet. Lett. (1978) 1601-1602) .
  • the product is purified by preparative silica TLC, and stored as a hexane solution at -70°C until use.
  • Wax synthase activity in a microsomal membrane preparation is measured by incubation of 40 ⁇ M [1- 1 C] acyl-CoA (usually palmitoyl-CoA, sp. act. 5.1-5.6 mCi/mmol) and 200mM oleyl alcohol with the sample to be assayed in a total volume of 0.25ml.
  • the incubation mixture also contains either 25 mM HEPES (4- [2-hydroxyethyl] -1-piperazineethane-sulfonic acid), pH 7.5, as the buffering agent with 20% w/v glycerol, ImM DTT, 0.5M NaCl or 25 mM Tricine-NaOH, pH 7.8 , as the buffering agent with 0.28M NaCl, 10% glycerol, and 2mM ⁇ - mercaptoethanol .
  • Initial studies were performed with the first buffer system, when the pH was chosen to accomodate the preference of the acyl-CoA reductase enzyme. Membrane preparations were later changed to the second buffer system to accomodate the higher pH optimum of wax synthase.
  • a substrate mixture is prepared in a glass vial, with oleyl alcohol being added immediately before use, and is added to samples. Incubation is carried out at 30°C for up to one hour. The assay is terminated by placing the assay tube on ice and immediately adding 0.25ml isopropanol : acetic acid (4:1 v/v) . Unlabeled wax esters (O.lmg) and oleyl alcohol (O.lmg) are added as carriers. The [ 1 C] lipids are extracted by the scaled-down protocol of Hara and Radin (Anal. Biochem . (1978) 50:420) .
  • Solubilized wax synthase is assayed using up to 50 ⁇ l sample in a 250 ⁇ l assay that contains 40 ⁇ M 1- 14 C-16:0 CoA (5 Ci/mol) , 200 ⁇ M 18:1-0H, 0.07% soybean phospholipid (Sigma, P- 3644), 0.2 %CHAPS, 280 mM NaCl, 25 mM Tricine-NaOH, pH 7.8, 2mM ⁇ -ME and 5.6% glycerol.
  • Phospholipid 50mg/ml in 0.5% CHAPS
  • a cocktail containing the remaining assay components is added directly to the sample, which is in 1% CHAPS, then diluted by a cocktail containing the remaining assay components.
  • Wax synthase is sensitive to detergent and requires the amount of phospholipid (PL) and detergent (CHAPS) to be balanced at 2.8/1 (CHAPS/PL, w/w) in the assay for maximal activity.
  • Assays for wax synthase activity in samples concentrated by ultra-filtration require a readjustment of the sample volume assayed because of the concentration of CHAPS. Introducing too much CHAPS into the assay results in inhibition of activity.
  • the sample is applied to a silica TLC plate, and the plate is developed in hexane/diethy1 ether/acetic acid (80:20:1 or 70:30:2 v/v/v) .
  • the distribution of radioactivity between the lipid classes is measured using an AMBIS radioanalytic imaging system (AMBIS Systems Inc., San Diego, CA) . If necessary the individual lipid classes can be recovered from the TLC plate for further analysis. Reversed- phase TLC systems using C18 plates developed in methanol have also been used for the analysis.
  • Embryo development was tracked over two summers on five plants in Davis, CA. Embryo fresh and dry weights were found to increase at a fairly steady rate from about day 80 to about day 130. Lipid extractions reveal that when the embryo fresh weight reaches about 300mg (about day 80) , the ratio of lipid weight to dry weight reaches the maximum level of 50%.
  • Wax synthase activity was measured in developing embryos as described in Example IB. As the jojoba seed coats were determined to be the source of an inhibiting factor (s), the seed coats were removed prior to freezing the embryos in liquid nitrogen for storage at -70°C.
  • s inhibiting factor
  • Jojoba embryos are harvested at approximately 90-110 days after flowering, as estimated by measuring water content of the embryos (45-70%) .
  • the outer shells and seed coats are removed and the cotyledons quickly frozen in liquid nitrogen and stored at -70°C for future use.
  • frozen embryos are powdered by pounding in a steel mortar and pestle at liquid nitrogen temperature. In a typical experiment, 70g of embryos are processed.
  • the powder is added, at a ratio of 280ml of solution per 70g of embryos, to the following high salt solution: 3M NaCl, 0.3M sucrose, lOOmM HEPES, 2mM DTT, and the protease inhibitors, ImM EDTA, 0.7mg/ml leupeptin, 0.5mg/ml pepstatin and 17mg/ml PMSF.
  • a cell free homogenate (CFH) is formed by dispersing the powdered embryos in the buffer with a tissue homogenizer (Kinematica, Switzerland; model PT10/35) for approximately 30 sec. and then filtering through three layers of Miracloth (CalBioChem, LaJolla, CA) . The filtrate is centrifuged at 100,000 x g for one hour.
  • the resulting sample consists of a pellet, supernatant and a floating fat pad.
  • the fat pad is removed and the supernatant fraction is collected and dialyzed overnight (with three changes of the buffering solution) versus a solution containing 1M NaCl, lOOmM HEPES, 2mM DTT and 0.5M EDTA.
  • the dialyzate is centrifuged at 200,000 x g for 1 1/2 hour to yield a pellet, DP2.
  • the pellet is suspended in 25mM HEPES and 10% glycerol, at 1/20 of the original CFH volume, to yield the microsomal membrane preparation.
  • Example 3 Purification of Jojoba Wax Synthase Methods are described which may be used for isolation of a jojoba membrane preparation having wax synthase activity, solubilization of wax synthase activity, and further purification of the wax synthase protein.
  • Example 2 The following modification of the method described in Example 2 is employed and provides an improved membrane fraction useful for purification of wax synthase from solubilized membranes .
  • 100 g of jojoba embryos are added to 400 ml of extraction buffer (40 mM Tricine-NaOH, pH 7.8, 200 mM KC1, 10 mM EDTA, 5 mM ⁇ -mercaptoethanol) , ground in a blender, and homogenized with a Polytron tissue disrupter. All subsequent steps are performed at 4°C.
  • the blended material is filtered through Miracloth (CalBioChem) . Centrifugation (20,000 x g; 20 min.
  • the filtrate yielded a floating wax layer, a turbid supernatant fraction and a dark green pellet.
  • the supernatant fraction is collected and centrifuged (100,000 x g; 2 h) to obtain membrane pellets which are then resuspended in 40 ml of Buffer A (25 mM Tricine-NaOH, pH 7.8, 200 mM KC1, 5 mM EDTA, 5 mM ⁇ -mercaptoethanol) containing 50% (w/v) sucrose.
  • Buffer A 25 mM Tricine-NaOH, pH 7.8, 200 mM KC1, 5 mM EDTA, 5 mM ⁇ -mercaptoethanol
  • This homogenate is distributed into four SW28 centrifuge tubes (Beckman) and each is overlaid with 10 ml Buffer A containing 20% sucrose and then with 13 ml Buffer A.
  • a membrane fraction is collected from the 20%/50% sucrose interface, diluted with four volumes Buffer A and collected by centrifugation (200,000 x g; 1 h) .
  • the membranes are then homogenized in 10 ml storage buffer [25 mM Tricine-NaOH, pH 7.8 , 1 M NaCl, 10% (w/v) glycerol, 5 mM ⁇ -mercaptoethanol)].
  • the protein concentration of membranes prepared via this protocol is typically between 7 and 9 mg/ml. Protein concentrations are estimated as described (Bradford, 1976) using BSA as the protein standard.
  • the membrane suspension is adjusted to approximately 0.83mg of protein per ml by dilution with storage buffer (25mM Tricine-NaOH, pH 7.8 , 1M NaCl, 10% glycerol, 5 mM ⁇ - mercaptoethanol) .
  • Solid 3- ( [3-cholamidopropyl] dimethyl- " ammonio) -1-propanesulfate (CHAPS) is added to achieve a final concentration of 2% (w/v) and a detergent to protein ratio of 24:1. After incubation on ice for 1 hr, the sample is centrifuged (200,000g for 1 hr) , and the supernatant fraction collected.
  • the 200,000g supernatant fraction is diluted (with 0.57% CHAPS, 25 mM Tricine-NaOH, pH 7.8, 20% glycerol) to yield final concentrations of NaCl and CHAPS of 0.3M and 1%, respectively.
  • the sample is loaded onto a Blue A-agarose (Amicon, Inc., Beverly, MA) column that has been equilibrated with buffer B (25 mM Tricine-NaOH, pH 7.8, 1% CHAPS, 20% glycerol,) containing 0.3M NaCl. After washing with equilibration buffer, wax synthase activity is eluted with buffer B containing 2M NaCl. Active fractions eluted from the Blue A column are pooled (Blue Pool) and used for further chromatography.
  • Protocol 1 Figure 1
  • the Blue Pool was concentrated 5.4 fold by ultrafiltration in a pressure cell fitted with a YM 30 membrane (Amicon, Inc., Beverly, MA) .
  • YM 30 membrane Amicon, Inc., Beverly, MA
  • One- half of the concentrate was applied to a Ceramic Hydroxyapatite (CHT) column (Bio-Scale CHT-2 ; Bio-Rad,
  • the stacking gel contained 5% of a 30% acrylamide stock (29.2% acrylamide, 0.8% ⁇ , ⁇ ' -bis-methyleneacrylamide, w/v), 0.06% ammonium persulfate (w/v) and 0.1% TEMED (v/v).
  • the resolving gel contained a 10-13% linear gradient of acrylamide stock stabilized by a 0-10% linear gradient of sucrose. Electrophoresis was carried out at room temperature at 150V, constant voltage, for 9-10 hours. Proteins were visualized by staining with silver according to the method of Blum et al .
  • Example 3D 10-13% acrylamide gradient gel
  • proteins were resolved by electrophoresis at 150V, constant voltage, for 9.5 hours.
  • the gel was stained with 0.1% Coomassie Blue in 50% methanol, 10% acetic acid for 15 minutes then destained in 50% methanol, 10% acetic acid for 2 x 20 minutes.
  • the 33 kD Wax Synthase band was excised from the gel and destained in 50% ethanol for 3 x 20 minutes. One lane contained a streak of protein and was not used in the final digestion.
  • the final three pellets for each pooled set of samples were resuspended with the same 50 ⁇ l of SDS sample buffer by transfering the buffer from one vial to the next.
  • the emptied vials, that had already been resuspended, were washed with lO ⁇ l of sample buffer for a total resuspended volume of 60 ⁇ l for each pooled sample.
  • the samples were applied to a 12% acrylamide Tris/Glycine mini-gel (Novex, San Diego, CA, 1.5mm x 10 well) and proteins were resolved by electrophoresis at 150 V, constant voltage, for 20 minutes beyond the elution of dye from the foot of the gel.
  • the gel was stained with Coomassie Blue and destained using Gel-Clear (Novex, San Diego, CA) . Wax Synthase was excised from three non-equivalent lanes on the gel representing the peak and tailing fractions from the column.
  • the gel slices were placed in 1.5 ml vials and destained with 1 ml of 50% methanol, 10% acetic acid for 2 hours. The destain solution was removed and the gel slices were frozen in liquid nitrogen and sent on dry ice, overnight, to the W M Keck Foundation Biotechnology Resource Laboratory at Yale University for in-gel-digestion.
  • One gel slice from the sample concentrated by ultrafiltration and three gel slices from the samples concentrated by precipitation were pooled for in-gel tryptic digestion.
  • Protein sequencing was performed at the W.M. Keck Foundation Biotechnology Resource Laboratory, Yale University. Procedures include amino acid analysis of a portion (10-15%) of the gel slice for quantitation and amino acid composition, digestion of the protein with one of the proteolytic enzymes (trypsin or lysyl endopeptidase) , and fractionation of the products by reverse phase HPLC . Absorbance peaks are selected from the HPLC run and subjected to laser desorption mass spectrometry to determine the presence, amount, and mass of the peptide prior to protein sequencing. The longest peptides are selected for microsequencing.
  • proteolytic enzymes trypsin or lysyl endopeptidase
  • RNA is isolated from jojoba embryos collected at 80-90 days post-anthesis using a polyribosome isolation method, initially described by Jackson and Larkins ( Plant Physiol .
  • extraction buffer 200mM Tris pH 9.0, 160mM KC1, 25mM EGTA, 70mM MgC12 , 1% Triton X-100, 05% sodium deoxycholate, ImM spermidine, lOmM ⁇ -mercaptoethanol, and 500mM sucrose
  • extraction buffer 200mM Tris pH 9.0, 160mM KC1, 25mM EGTA, 70mM MgC12 , 1% Triton X-100, 05% sodium deoxycholate, ImM spermidine, lOmM ⁇ -mercaptoethanol, and 500mM sucrose
  • the supernatant is decanted into a 500ml sterile flask, and 1/19 volume of a 20% detergent solution (20% Brij 35, 20% Tween 40, 20% Noidet p-40 w/v) is added at room temperature. The solution is stirred at 4°C for 30 minutes at a moderate speed and the supernatant is then centrifuged at 12,000 x g for 30 minutes .
  • a 20% detergent solution (20% Brij 35, 20% Tween 40, 20% Noidet p-40 w/v
  • resuspension buffer 40mM Tris pH 9.0, 5mM EGTA, 200mM KC1, 30mM MgCl 2 , 5mM ⁇ - mercaptoethanol
  • the tubes are placed on ice for 10 minutes, after which the pellets are thoroughly resuspended and pooled. The supernatant is then centrifuged at 120 x g for 10 minutes to remove insoluble material.
  • RNA is precipitated by adding 1/10 volume of sodium acetate and 2 volumes of ethanol. After several hours at 20°C RNA is pelleted by centrifugation at 12,000 x g at 4°C for 30 minutes. The pellet is resuspended in 10ml of TE buffer (lOmM Tris, ImM EDTA) and extracted with an equal volume of Tris pH 7.5 saturated phenol. The phases are separated by centrifuging at 10,000 x g for 20 minutes at 4°C. The aqueous phase is removed and the organic phase is re- extracted with one volume of TE buffer. The aqueous phases are then pooled and extracted with one volume of chloroform. The phases are again separated by centrifugation and the aqueous phase ethanol precipitated as previously described, to yield the polyribosomal RNA.
  • TE buffer lOmM Tris, ImM EDTA
  • Polysaccharide contaminants in the polyribosomal RNA preparation are removed by running the RNA over a cellulose column (Sigma-cell 50) in high salt buffer (0.5M NaCl, 20mM Tris pH 7.5, ImM EDTA, 0.1% SDS). The contaminant binds to the column and the RNA is collected in the eluant . The eluant fractions are pooled and the RNA is ethanol precipitated. The precipitated total RNA is then resuspended in a smaller volume and applied to an oligo d(T) cellulose column to isolate the polyadenylated RNA.
  • Polyadenylated RNA is used to construct a cDNA library in the plasmid cloning vector pCGN1703, derived from the commercial cloning vector Bluescribe M13- (Stratagene Cloning
  • the polylinker of Bluescribe M13- is altered by digestion with BamHI, treatment with mung bean endonuclease, and blunt-end ligation to create a BamHI-deleted plasmid, pCGNl700.
  • pCGNl700 is digested with EcoRI and SstI (adjacent restriction sites) and annealed with a synthetic linker having restriction sites for BamHI, PstI, Zbal, Apal and Smal, a 5' overhang of AATT, and a 3 ' overhang of TCGA.
  • the insertion of the linker into PCGN1700 eliminates the EcoRI site, recreates the S ⁇ rfcl (also, sometimes referred to as "Sad” herein) site found in Bluescribe, and adds the new restriction sites contained on the linker.
  • the resulting plasmid pCGN1702 is digested with HindiII and blunt-ended with Klenow enzyme; the linear DNA is partially digested with PvuII and ligated with T4 DNA wax synthase in dilute solution.
  • a transformant having the lac promoter region deleted is selected (pCGN1703) and is used as the plasmid cloning vector.
  • the cloning method for cDNA synthesis is as follows.
  • the plasmid cloning vector is digested with SstI and homopolymer T-tails are generated on the resulting 3 ' -overhang stick-ends using terminal deoxynucleotidyl transferase.
  • the tailed plasmid is separated from undigested or un-tailed plasmid by oligo (dA) -cellulose chromatography.
  • the resultant vector serves as the primer for synthesis of cDNA first strands covalently attached to either end of the vector plasmid.
  • the cDNA-mRNA-vector complexes are treated with terminal transferase in the presence of deoxyguanosine triphosphate, generating G-tails at the ends of the cDNA strands.
  • the extra cDNA-mRNA complex, adjacent to the BamHI site, is removed by BamHI digestion, leaving a cDNA-mRNA- vector complex with a BamHI stick-end at one end and a G-tail at the other.
  • This complex is cyclized using an annealed synthetic cyclizing linker which has a 5' BamHI sticky-end, recognition sequences for restriction enzymes NotI , EcoRI and SstI , and a 3' C-tail end.
  • the circular complexes are transformed into E. coli strain DH5a (BRL, Gaithersburg, MD) to generate the cD ⁇ A library.
  • the jojoba embryo cD ⁇ A bank contains between approximately 1.5xl0 6 clones with an average cD ⁇ A insert size of approximately 500 base pairs.
  • jojoba polyadenylated R ⁇ A is also used to construct a cD ⁇ A library in the cloning vector 1ZAPII/EcoRI (Stratagene, San Diego, CA) .
  • the library is constructed using protocols, D ⁇ A and bacterial strains as supplied by the manufacturer.
  • Clones are packaged using Gigapack Gold packaging extracts (Stratagene) , also according to manufacturer's recommendations.
  • the cDNA library constructed in this manner contains approximately 1 x 10 6 clones with an average cDNA insert size of approximately 400 base pairs.
  • Synthetic Oligonucleotides In general, for use as PCR primers from single stranded DNA template reverse-transcribed from mRNA, oligonucleotides containing the sense orientation sequence corresponding to wax synthase peptide encoding sequences are prepared. These oligonucleotides are used as primers for the "forward" amplification reaction to produce sense strand DNA.
  • an oligonucleotide may be designed to be identical to a portion of a primer used to prepare DNA template for PCR.
  • oligonucleotides which contain sequence complementary to wax synthase peptide encoding sequences may be used in combination with a "forward" wax synthase oligonucleotide primer as described above.
  • the forward or reverse primers may be "degenerate" oligonucleotides, i.e. containing a mixture of all or some of the possible encoding sequences for a particular peptide region. To reduce the number of different oligonucleotides present in such a mixture, it is preferable to select peptide regions which have the least number of possible encoding sequences when preparing the synthetic oligonucleotide for PCR primers. Similarly, where the synthetic oligonucleotide is to be used to directly screen a library for wax synthase sequences, lower degeneracy oligonucleotides are preferred.
  • oligonucleotide names reflect the particular wax synthase peptide fragment numbers as listed in Example 6.
  • the letter “F” in the oligonucleotide name designates a PCR forward reaction primer.
  • the letter “R” designates a PCR reverse reaction primer.
  • oligonucleotide is used for reverse transcription from poly (A) + or total RNA to prepare single- stranded DNA for use as a PCR template.
  • the oligonucleotide contains restriction digestion sequences for Hindlll, PstI and Sstl .
  • the sequence of TSYN is as follows:
  • TAMP An oligonucleotide, is useful in the reverse reaction of PCR for amplification of the antisense strand of a wax synthase encoding sequence. It is noted that where the template for PCR is single stranded DNA reverse-transcribed from mRNA, the reverse reaction will not occur until completion of the first forward reaction. The first strand reaction results in production of a sense strand template which may then be used in amplification of the antisense DNA strand from the reverse primer.
  • the sequence of TAMP is as follows :
  • the nucleotide base codes for the above oligonucleotides are according to the IUPAC standard as follows:
  • Y cytosine or thymine
  • H adenine, cytosine or thymine
  • N adenine, cytosine, guanine or thymine
  • W adenine or thymine
  • K guanine or thymine
  • M adenine or cytosine
  • Poly (A) + RNA is isolated from total RNA prepared from jojoba tissue as described above.
  • Single-stranded cDNA is prepared from poly (A) + or total RNA by reverse transcription using Superscript reverse transcriptase (BRL) and TSYN as the oligonucleotide primer. The reaction is conducted according to manufacturer's directions, except that the reaction is run at 45_C rather than 37_C.
  • the jojoba single-stranded cDNA is used in PCR reactions 1-12 as set forth below.
  • PCR is conducted in a Perkin Elmer Cetus GeneAmp PCR System 9600 PCR machine using reverse transcribed single- stranded cDNA as template.
  • Commercially available PCR reaction and optimization reagents are used according to manufacturer's specifications.
  • the portion of the cDNA located 3 ' of the primers above can be amplified in the following reactions: Reaction Forward Primer Reverse Primer
  • the PCR products of reactions containing primers designated -FI can be used as a template for a second round of PCR reactions using the primers designated -F2.
  • the PCR products of reactions containing primers designated -Rl can be used as a template for a second round of PCR reactions using the primers designated -R2.
  • the entire cDNA can be amplified using 5' and 3' RACE (Frohman et al . , 1988) using the Marathon cDNA Amplification Kit (Clontech Laboraties Inc.) according to the manufacturers instructions.
  • Primers WSpep29-Fl, WSpep29-Fl, WSpep33-Fl and WSpep33-F2 are used for the 3 ' RACE reactions.
  • Primers WSpep29-Rl, WSpep29-Rl, WSpep33-Rl and WSpep33-R2 are used for the 5 ' RACE reactions.
  • DNA fragments generated in PCR reactions are cloned into pCR2.1 according to the manufacturers protocol (Invitrogen Corp.) .
  • the DNA sequence of the cloned fragments are determined to confirm that the cloned fragments encode wax synthase peptides .
  • Wax synthase DNA fragments obtained by PCR are labeled and used as a probe to screen clones from the cDNA libraries described above.
  • DNA library screening techniques are known to those in the art and described, for example in Maniatis et al . (Molecular Cloning: A Laboratory Manual , Second Edi tion (1989) Cold Spring Harbor Laboratory Press) . In this manner, wax synthase nucleic acid sequences are obtained which may be analyzed for nucleic acid sequence and used for expression of wax synthase in various hosts, both procaryotic and eucaryotic .
  • Constructs which provide for expression of wax synthase and reductase sequences in plant cells may be prepared as follows .
  • Expression cassettes which contain 5' and 3' regulatory regions from genes expressed preferentially in seed tissues may be prepared from napin, Bce4 and ACP genes as described, for example in WO 92/03564.
  • a napin expression cassette, pCGN1808, which may be used for expression of wax synthase or reductase gene constructs is described in Kridl et al . ( Seed Science Research (1991) 2:209-219).
  • An additional napin expression cassette, pCGN3223 contains an ampicillin resistance background, and essentially identical 1.725 napin 5' and 1.265 3' regulatory sequences as found in pCGN1808. The regulatory regions are flanked with Hindlll, NotI and Kpnl restriction sites and unique Sail, Bglll, Pstl , and Xhol cloning sites are located between the 5 ' and 3 ' noncoding regions .
  • a cassette for cloning of sequences for transcriptional regulation under the control of 5 ' and 3 ' regions from an oleosin gene may also be used. Sequence of a Brassica napus oleosin gene was reported by Lee and Huang ( Plant Phys . (1991) 55:1395-1397). Sequence of an oleosin cassette, pCG ⁇ 7636, is provided in Figure 4 of USPN 5,445,947.
  • the oleosin cassette is flanked by BssHII, Kpnl and Xbal restriction sites, and contains Sail, BamHI and Pstl sites for insertion of wax synthase, reductase, or other DNA sequences of interest between the 5' and 3' oleosin regions.
  • Wax synthase and reductase gene sequences may be inserted into such cassettes to provide expression constructs for plant transformation methods.
  • a construct for expression of reductase in plant cells using 5' and 3' regulatory regions from a napin gene is described in USPN 5,445,947.
  • Binary vector constructs are transformed into Agrobacterium cells, such as of strain EHA101 (Hood et al . , J. Bacteriol (1986) 158:1291-1301), by the method of Holsters et al . (Mol . Gen . Genet . (1978) 153:181-187) and used in plant transformation methods as described below.
  • DAGAT activity is assayed with 3.67 ⁇ M 1- 14 C-18 : 1-Coenzyme A (53.5-54.5 Ci/mole, New England Nuclear, Boston, MA) and 1.5 mM 1,2-18:1 diacylglycerol (DAG) (Sigma D-0138, prepared as a 150 mM stock in 2-methoxyethanol) in a buffer containing 10 mM potassium phosphate (pH 7.0), 100-150 mM KC1, and 0.1 % TX-100 (w/v) in a total volume of 100 ⁇ l as similarly described by Kamisaka et al . (1993, 1994).
  • DAG diacylglycerol
  • Assays are performed at 30 °C for 5 min and terminated with the addition of 1.5 ml of heptane: isopropanol : 0.5M H 2 S0 4 (10:40:1, v/v/v) . If necessary, samples may be diluted with buffer prior to assay in order to maintain a linear rate of product formation during the assay.
  • the assay is performed as described for non-solubilized 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 to 0.2%, and the KC1 concentration is maintained between 100-125 mM. It is also necessary to include L- - phosphatidic acid (Sigma P-9511, prepared as a 50 mM stock in 1% Triton X-100 (w/v) ) to recover activity following solubilization with detergent as described by Kamisaka et al . (1996, 1997), with slight modification of the protocol.
  • L- - phosphatidic acid Sigma P-9511, prepared as a 50 mM stock in 1% Triton X-100 (w/v)
  • assays After assays are stopped, they can be stored at 4 °C for processing at a later date or immediately processed by addition of 0.1 ml 1 M NaHC0 3 followed by 1 ml of heptane containing 15 nmoles/ml triolein as a carrier for extraction.
  • the contents are vortexed and, after separation of aqueous and organic phases, the upper organic phase is removed to a new glass vial and washed with 1 ml IM NaCl. Forty percent of the final organic phase is removed for liquid scintillation counting and the remaining organic phase is transferred to a clean vial and evaporated to dryness under nitrogen gas .
  • the residue is resuspended in 45 ⁇ l hexane and spotted onto a silica gel-G, glass, thin-layer chromatography (TLC) plate with a preadsorbent loading zone (Analtech #31011, Newark, Delaware).
  • TLC thin-layer chromatography
  • 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 radio-image analyzer (AMBIS 3000, San Diego, CA) to determine the portion of radioactivity incorporated into triacylglycerol. Activity is reported in units as pmole/min.
  • Example 8 Growth and Harvesting of Mortierella ramanniana cultures .
  • Mortierella ramanniana is cultured by inoculating 1 liter of Defined Glucose Media (30 g glucose, 1.5 g (NH 4 ) 2 S0 4 , 3 g K 2 HP0 4 , 0.3 g MgS0 4 »7H20, 0.1 g NaCl, 5g CH 3 C00Na»3H 2 0, 10 mg FeS0 4 »7H 2 0, 1.2 mg CaCl 2 »2H 2 0, 0.2 mg CuS0 4 »5H 2 0, 1.0 mg ZnS0 4 »7H 2 0, 1.0 mg MnCl 2 »4H 2 0, 2 mg thiamine-HCl and 0.02 mg biotin in 1 L of water purified by reverse osmosis (pH 5.7)) with 1.5-3 x 10 6 spores and incubating at 30 °C with shaking at 200 rpm for 9-11 days. Cultures are harvested by filtration through one layer of Miracloth (Calbiochem, La Jolla, CA) . Excess liquid
  • Polyacrylamide gradient gel electrophoresis (10-13%) is carried out according to the method of Laemmli (1970) with some of the modifications of Delepelaire (1979).
  • Sodium dodecyl sulfate is used in the upper reservoir buffer at 0.1% but is omitted from the lower reservoir buffer, stacking and resolving gels.
  • the stacking gel contains 5% of a 30% acrylamide stock (acrylamid:N, N' -Methylenacrylamid, 37.5:1, Bio-Rad, Hercules, CA) , 0.06% ammonium persulfate and 0.1% TEMED (v/v) .
  • the resolving gel contains a 10-13% linear gradient of acrylamide stock stabilized by a 0-10% linear gradient of sucrose. Electrophoresis is carried out at room temperature at 150V, constant voltage, for 7-9 hours.
  • Proteins are visualized by staining with silver according to the method of Blum et al . (1987) or with Coomassie Blue (0.1% Coomassie Blue R-250, 50% methanol (v/v), 10% acetic acid (v/v) ) .
  • wet packed cells typically, 70-75 g of wet packed cells (stored at -70 °C) are used for each lipid body preparation. Just prior to use, cells are thawed on ice and resuspended in 150 ml of Buffer A (10 mM potassium phosphate (pH 7.0), 0.15 M KC1, 0.5 M sucrose, and 1 mM EDTA) .
  • Buffer A 10 mM potassium phosphate (pH 7.0), 0.15 M KC1, 0.5 M sucrose, and 1 mM EDTA
  • the following protease inhibitors are added to reduce proteolysis: 0.1 ⁇ M Aprotinin, 1 ⁇ M Leupeptin, and 100 ⁇ M Pefabloc (all from Boehringer Mannheim, Germany) .
  • Cells are divided into five, 50-ml tubes and lysed with a Polytron Tissue Homogenizer (Kinematic GmbH, Brinkman Insruments, Switzerland) on setting #7 with a 1 cm diameter probe for 7 x 1 min.
  • the resulting slurry is transferred to centrifuge tubes (29 x 104 mm) and solid debris made to pellet by spinning at 1500 x g (Beckman Instruments, J2-21, JA-20 rotor, 3500 rpm) for 10 min at 4 °C .
  • the supernatant is removed and the pellets washed with another 5 ml of Buffer A. Following centrifugation, the supernatant volumes are combined. This fraction is referred to as the 'SI'.
  • the SI is divided into six ultracentrifuge tubes (25 x 89 mm, Beckman Instruments, Fullerton, CA) and each is overlayed with 5 ml of Buffer B (10 mM potassium phosphate pH, 7.0, 0.15 M KC1, 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 Lipid Body Fraction (LBF) , floating on top of the overlay, is recovered with a spatula and transferred to a glass homogenizer (Potter-Elvehjem) .
  • Buffer B 10 mM potassium phosphate pH, 7.0, 0.15 M KC1, 0.3 M sucrose, and 1 mM EDTA .
  • the LBF is thawed on ice and solubilization is achieved by addition of Triton X-100 (Boehringer Mannheim, Mannheim, Germany) from a 10 % (w/v) stock 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 detergent-treated sample is rocked at 4 °C for one hour then divided into six ultracentrifuge tubes (25 x 89 mm, Beckman Instruments) . Each tube is overlayed with 5 ml of Buffer 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 overlay to within 1 cm of the bottom of each ultracentrifuge tube and removing the lower, 0.5M sucrose, layer with gentle suction while leaving the upper 0.3M sucrose overlay (including a floating fat layer) and the pellet behind.
  • Kamisaka et al the protocol described by Kamisaka et al .
  • Buffer C used for chromatography, contains 10 mM potassium phosphate (pH 7.0), 0.1% Triton X-100 (w/v)
  • This buffer differs from the corresponding column buffer used by Kamisaka et al . (1997) in that glycerol is substituted for ethylene glycol and EDTA, DTT, and PMSF are omitted while Aprotinin, Leupeptin and Pefabloc are included.
  • a Yellow 86-Agarose Sigma R-8504, St.
  • DAGAT activity and proteins bound to the column are eluted with 500 mM KCl in Buffer C ( Figure 4) .
  • DAGAT activity eluted from the Yellow 86-Agarose column (fractions 17-20) is diluted 1:3.33 with Buffer C to reduce the KCl concentration to 150 mM.
  • the diluted pool (103 ml) is applied to a Heparin-Sepharose CL-6B column (Pharmacia, Uppsala, Sweden, 0.5 cm x 4.8 cm) equilibrated with 150 mM KCl in Buffer C at 0.2 ml/min.
  • the column is washed with 5 volumes of equilibration buffer and DAGAT activity and protein are eluted in a 15 ml linear gradient of 150-500 mM KCl in Buffer C.
  • DAGAT activity elutes in two overlapping peaks. The first peak elutes during the gradient, as found by Kamisaka et al . (1997) and a second peak, not found by
  • a portion (250 ⁇ l) of the two peak fractions from the Heparin column are 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 150 mM KCl in Buffer C.
  • the column is equilibrated with 150 mM KCl in a Modified Buffer C in which Triton X-100 is replaced with Triton X-100 R (Calbiochem, La Jolla, CA) .
  • the column is calibrated using Bio-Rad Gel Filtration
  • the second peak from the Heparin column (fractions 36-41) is diluted 1:6.6 with Buffer C to 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 75 mM KCl in Buffer C at 0.5 ml/min.
  • bound proteins and all of the DAGAT activity elute in a 40 ml linear gradient of 75-500 mM KCl in Buffer C.
  • DAGAT activity elutes as a single peak ( Figure 6A) .
  • the protein composition of the fractions containing DAGAT activity from the Heparin and second Yellow 86 columns are analyzed by gradient SDS-PAGE according to the protocol in Example 3. Protein bands are detected by silver-staining. The pattern of bands eluting from these columns is compared, fraction by fraction, to the respective DAGAT activity profile. Many protein candidates are present that correlate with the presence of DAGAT activity. It is our opinion that the purification protocol is insufficient to identify a particular protein candidate associated with DAGAT activity ( Figure 5B, 6B) .
  • Example 11 New purification protocol for identifying DAGAT protein candidates purified from Mortierella ramanniana
  • wet packed cells typically, 70-75 g of wet packed cells (stored at -70 °C) are used for each lipid body preparation. Just prior to use, cells are thawed on ice and resuspended in 150 ml of Buffer A (10 mM potassium phosphate (pH 7.0), 0.15 M KCl, 0.5 M sucrose, 1 mM EDTA) .
  • Buffer A 10 mM potassium phosphate (pH 7.0), 0.15 M KCl, 0.5 M sucrose, 1 mM EDTA
  • the following protease inhibitors are added to reduce proteolysis: 0.1 ⁇ M Aprotinin, 1 ⁇ M Leupeptin, and 100 ⁇ M Pefabloc (all from Boehringer Mannheim, Germany) .
  • Samples are lysed with a cell disrupter (Bead-Beater, Biospec Products, Bartlesville, OK) using 0.5 mm glass beads.
  • the sample chamber is filled with 180 ml of glass beads.
  • Wet- packed cells are thawed on ice and resuspended in 150 ml of Buffer A.
  • the cell slurry is poured over the glass beads.
  • an additional 40-50 ml of Buffer A are needed to fill the chamber for proper functioning. This volume is used to rinse the remains of the cell slurry from its original container so that it can be combined with the rest of the sample.
  • Cells are ground ('Homogenize' setting) for 45-90 seconds depending on the viscosity of the sample.
  • the cell slurry containing glass beads is divided into tubes (29 x 104 mm) and centrifuged at 500 x g (Beckman Instruments, GP centrifuge, GH 3.7 Horizontal rotor at 1500 rpm) and 4 ⁇ C . The supernatant is removed and the pellets washed with another 5 ml of Buffer A. Following centrifugation the supernatant volumes are combined. This fraction is referred to as the 'SI'.
  • the SI is divided into six ultracentrifuge tubes (25 x 89 mm, Beckman Instruments) and each is overlayed with 5 ml of Modified Buffer B (10 mM potassium phosphate pH, 7.0, 0.15 M KCl, and 0.3 M sucrose). EDTA is omitted from Buffer B (see Example 4) since it interferes with hydroxylapatite chromatography. Samples are centrifuged at 100,000 x g
  • LBF Lipid Body Fraction
  • Interface fraction the interface between the 0.3 and 0.5 M sucrose buffers
  • Soluble fraction the liquid volume beneath the interface
  • Membrane fraction a tan/brown pellet at the bottom of each tube
  • a protein determination is made with an aliquot of the Lipid Body Fraction by the method of Bradford (Bio-Rad Reagent, Hercules, CA) using bovine serum albumin as a standard.
  • the LBF is thawed on ice, then diluted to a concentration of 1 mg protein/ml and treated with Triton X-100 at a detergent to protein ratio of 15:1 (w/w, equivalent to 1.3% Triton X-100).
  • Solid sucrose (Mallinckrodt , Paris, Kentucky) is added to achieve a final concentration of 0.5M.
  • the detergent-treated sample is rocked at 4 °C for one hour then divided into six ultracentrifuge tubes (25 x 89 mm, Beckman Instruments) . Each tube is overlayed with 5 ml of Modified Buffer B. Samples are centrifuged at 100,000 x g (Beckman Instruments, L-8M, SW-28 rotor, 21000 rpm) at 4 "C tor 3 hours.
  • the solubilized material referred to as the 'Triton X-100 extract', is recovered by inserting a thin tube through the overlay to within 1 cm of the bottom of each ultracentrifuge tube and removing the lower, 0.5M sucrose, layer with gentle suction while leaving the upper 0.3M sucrose overlay (including a floating fat layer) and the pellet behind.
  • Protocol A activity is bound to the first column and after elution, fractions are assayed for activity. The active fractions are then pooled and applied to the second column. We refer to this as a sequential run.
  • Protocol B activity is bound to the first column then elutes and flows directly onto the second column without pooling and assaying in between. We refer to this as a tandem run.
  • Protocol A the Triton X-100 extract is applied to a Yellow 86-Agarose column (2.5 cm x 6.4 cm) equilibrated with 75 mM KCl in Buffer C (Example 4.C) at 2 ml/min.
  • the column is washed with 5 column volumes of equilibration buffer then eluted with 500 mM KCl in Buffer C at 0.5 ml/min ( Figure 7) .
  • the two most active fractions (64 and 65), containing 93% of the eluted activity, are pooled and loaded onto a hydroxylapatite column (Bio-Gel HT, Bio-Rad, 1 cm x 25.5 cm) equilibrated with 500 mM KCl in Buffer C at 0.5 ml/min. DAGAT activity flows through the column whereas the majority of the proteins bind the column. The column is washed with 3 volumes of equilibration buffer. Bound proteins are eluted with 100 mM dipotassium phosphate and 500 mM KCl in Buffer C at 0.5 ml/min ( Figure 8A) .
  • a portion of the fractions containing the DAGAT activity peak are run on gradient gel SDS-PAGE as described in Example 3.
  • the proteins are stained with silver and the pattern of the bands are compared, fraction by fraction, to the activity profile ( Figure 8B) .
  • Several DAGAT protein candidates correlate with activity.
  • attention is called to bands migrating at positions corresponding to 43 kD, 36.5 kD, 33 kDa, 29 kD, 28 kD and 27 kD. There does not appear to be a candidate protein in the region of 53 kD that correlates with activity.
  • Protocol B the 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 Buffer C at 1 ml/min. The column is washed with 5 column volumes of equilibration buffer. Then, the outlet from the Yellow 86-Agarose column is connected to the inlet of a hydroxylapatite column (1.0 cm x 26.2 cm, Bio-Rad, Hercules, CA) equilibrated with 500 mM KCl in Buffer C.
  • a hydroxylapatite column 1.0 cm x 26.2 cm, Bio-Rad, Hercules, CA
  • DAGAT activity bound to the Yellow 86 column is eluted with 110 ml of Buffer C containing 500 mM KCl and passes directly through the hydroxylapatite column at 0.2 ml/min. Finally, the hydroxylapatite column is disconnected from the Yellow 86- Agarose column and proteins bound to the hydroxylapatite column are eluted with 100 mM dipotassium phosphate and 500 mM KCl in Buffer C. DAGAT activity is found in fractions from the hydroxylapatite column collected during the 110-ml wash with Buffer C containing 500 mM KCl.
  • Triton X-100 extract does not bind the Yellow 86-Agarose column and is discarded.
  • Buffer C When this eluate is applied to the hydroxylapatite column, DAGAT activity flows through while most of the remaining proteins bind the column and are separated ( Figure 9A) . A portion of the fractions containing the DAGAT activity -peak are run on gradient gel SDS-PAGE and are silver-stained. The pattern of bands eluting from these columns is compared, fraction by fraction, to the respective DAGAT activity profile. Examination of the stained protein bands indicate a protein at 33 kDa correlates best with DAGAT activity ( Figure 9B) .
  • Example 12 Preparation of Protein for In-Gel Digestion A. Preparation of Samples for SDS-PAGE by Concentration
  • Odd numbered fractions from the flow through/wash of the final HA column were pooled and concentrated three fold by ultrafiltration in a pressure cell fitted with a YM 30 membrane (Amicon, Inc., Beverly, MA) . The sample was further concentrated using two Centricon-30 units (Amicon,
  • Tris/Glycine mini-gel (Novex, San Diego, CA, 1.5mm x 10 well) and proteins were resolved by electrophoresis at 150 V, constant voltage, for 20 minutes beyond the elution of dye from the foot of the gel.
  • the gel was stained with Coomassie Blue and destained using Gel-Clear (Novex, San Diego, CA) .
  • Wax Synthase was excised from three non-equivalent lanes on the gel representing the peak and tailing fractions from the column.
  • the gel slices were placed in 1.5 ml vials and destained with 1 ml of 50% methanol, 10% acetic acid for 2 hours.
  • the destain solution was removed and the gel slices were frozen in liquid nitrogen and sent on dry ice, overnight, to the W M Keck Foundation Biotechnology Resource Laboratory at Yale University for in-gel-digestion.
  • One gel slice from the sample concentrated by ultrafiltration and three gel slices from the samples concentrated by precipitation were pooled for in-gel tryptic digestion.
  • Protein sequencing was performed at the W.M. Keck Foundation Biotechnology Resource Laboratory, Yale University. Procedures include amino acid analysis of a portion (10-15%) of the gel slice for quantitation and amino acid composition, digestion of the protein with one of the proteolytic enzymes (trypsin or lysyl endopeptidase) , and fractionation of the products by reverse phase HPLC. Absorbance peaks are selected from the HPLC run and subjected to laser desorption mass spectrometry to determine the presence, amount, and mass of the peptide prior to protein sequencing. The longest peptides are selected for microsequencing .
  • proteolytic enzymes trypsin or lysyl endopeptidase
  • Example 14 Isolation of Mortierella ramanniana DAGAT Nucleic Acid Sequences
  • oligonucleotides containing the sense orientation sequence corresponding to DAGAT peptide encoding sequences are prepared. These oligonucleotides are used as primers for the "forward" amplification reaction to produce sense strand DNA.
  • an oligonucleotide may be designed to be identical to a portion of a primer used to prepare DNA template for PCR.
  • oligonucleotides which contain sequence complementary to DAGAT peptide encoding sequences may be used in combination with a "forward" DAGAT oligonucleotide primer as described above.
  • the forward or reverse primers may be "degenerate" oligonucleotides, i.e. containing a mixture of all or some of the possible encoding sequences for a particular peptide region. To reduce the number of different oligonucleotides present in such a mixture, it is preferable to select peptide regions which have the least number of possible encoding sequences when preparing the synthetic oligonucleotide for PCR primers. Similarly, where the synthetic oligonucleotide is to be used to directly screen a library for DAGAT sequences, lower degeneracy oligonucleotides are preferred.
  • DAGAT DNA fragments obtained by PCR are labeled and used as a probe to screen clones from cDNA libraries.
  • Complementary DNA and DNA constructioin and library screening techniques are known to those in the art and described, for example in Maniatis et al . (Molecular Cloning: A Laboratory Manual , Second Edi tion (1989) Cold Spring Harbor Laboratory Press) .
  • DAGAT nucleic acid sequences are obtained which may be analyzed for nucleic acid sequence and used for expression of DAGAT in various hosts, both procaryotic and eucaryotic .
  • Example 15 Mortierella ramanniana DAGAT Constructs for Plant Expression Constructs which provide for expression of DAGAT sequences in plant cells may be prepared as follows.
  • Expression cassettes which contain 5' and 3' regulatory regions from genes expressed preferentially in seed tissues may be prepared from napin, Bce4 and ACP genes as described, for example in WO 92/03564.
  • a napin expression cassette, pCGN1808, which may be used for expression of wax synthase or reductase gene constructs is described in Kridl et al . ( Seed Science Research (1991) 1:209-219) .
  • An additional napin expression cassette, pCGN3223 contains an ampicillin resistance background, and essentially identical 1.725 napin 5' and 1.265 3' regulatory sequences as found in pCGNl808. The regulatory regions are flanked with Hindlll, No tl and Kpnl restriction sites and unique Sail, Bglll, Pstl, and Xhol cloning sites are located between the 5 ' and 3 ' noncoding regions .
  • a cassette for cloning of sequences for transcriptional regulation under the control of 5' and 3' regions from an oleosin gene may also be used. Sequence of a Brassica napu ⁇ oleosin gene was reported by Lee and Huang ( Plant Phys . (1991) 95:1395-1397). Sequence of an oleosin cassette, pCGN7636, is provided in Figure 4 of USPN 5,445,947.
  • the oleosin cassette is flanked by BssHII, Kpnl and Xbal restriction sites, and contains Sail, BamHI and Pstl sites for insertion of wax synthase, reductase, or other DNA sequences of interest between the 5' and 3' oleosin regions.
  • DAGAT gene sequences may be inserted into such cassettes to provide expression constructs for plant transformation methods.
  • a construct for expression of reductase in plant cells using 5' and 3' regulatory regions from a napin gene is described in USPN 5,445,947.
  • Binary vector constructs are transformed into Agrobacterium cells, such as of strain EHA101 (Hood et al . , J. Bacteriol (1986) 158:1291-1301), by the method of Holsters et al . (Mol . Gen . Genet. (1978) 253:181-187) and used in plant transformation methods as described below.
  • Example 16 Plant Transformation Methods and Analyses
  • a variety of methods have been developed to insert a DNA sequence of interest into the genome of a plant host to obtain the transcription or transcription and translation of the sequence to effect phenotypic changes.
  • High erucic acid varieties such as cultivar Reston, or Canola-type varieties of Brassica napus may be transformed using Agrobacterium mediated transformation methods as described by Radke et al . ( Theor. Appl . Genet . (1988) 75:685- 694; Plant Cell Reports (1992) 12:499-505).
  • Transgenic Arabidopsis thaliana plants may be obtained by Agrobacterium- mediated transformation as described by Valverkens et al . , ( Proc . Nat . Acad.
  • Seeds or other plant material from transformed plants may be analyzed for DAGAT activity using the DAGAT assay methods described in Example 1.
  • DAGAT nucleic acid sequences may also be obtained from the amino acid sequences using PCR and library screening methods provided herein. Such nucleic acid sequences may be manipulated to provide for transcription of the sequences and/or expression of DAGAT proteins in host cells, which proteins may be used for a variety of applications. Such applications include the modification of triacylglycerols levels and compositions in host cells. All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Abstract

La présente invention se rapporte à des protéines acyltransférase capables de catalyser la production des triglycérides à partir de 1,2-diacylglycérol et d'un acyl-coA. L'invention comprend un diacylglycérol acyltransférase (DAGAT) partiellement purifié, ladite protéine étant active dans la formation du triacylglycérol à partir d'un acyl-CoA gras et de substrats de diacylglycérol. Un élément particulièrement intéressant est une DAGAT Mortierella ramanniana dont la masse moléculaire est d'environ 40KD. L'invention se rapporte également à des séquences d'acides aminés et d'acides nucléiques pouvant être obtenues à partir des protéines DAGAT, ainsi qu'à l'utilisation de telles séquences pour obtenir des cellules hôtes transgéniques avec des taux de triacylglygérol modifiés.
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JP2002504819A (ja) 2002-02-12
CA2292766A1 (fr) 1998-12-10

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