MXPA99011201A - Diacylglycerol acyl transferase proteins - Google Patents

Diacylglycerol acyl transferase proteins

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Publication number
MXPA99011201A
MXPA99011201A MXPA/A/1999/011201A MX9911201A MXPA99011201A MX PA99011201 A MXPA99011201 A MX PA99011201A MX 9911201 A MX9911201 A MX 9911201A MX PA99011201 A MXPA99011201 A MX PA99011201A
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dagat
activity
protein
sequences
column
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MXPA/A/1999/011201A
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Spanish (es)
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Dennis Lardizabal Kathryn
a thompson Gregory
George Metz James
W Lassner Michael
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Calgene Llc
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Abstract

By this invention, acyltransferase proteins are provided capable of catalyzing the production of triglycerides from 1,2-diacylglycerol and an acyl-CoA. The invention comprises a partially purified diacylglycerol acyltransferase (DAGAT), wherein said protein is active in the formation of triacylglycerol from fatty acyl-CoA and diacylglycerol substrates. Of special interest is a Mortierella ramanniana DAGAT having a molecular mass of approximately 40kD. Also considered are amino acid and nucleic acid sequences obtainable from DAGAT proteins and the use of such sequences to provide transgenic host cells with modified triacylglycerol levels.

Description

Diacylglycerol Acyl Transferase Proteins Technical Field The present invention is directed to enzymes, method for purifying and obtaining said enzymes, amino acid sequences and nucleic acids related thereto and methods for using said compositions in genetic engineering applications. INTRODUCTION Background Plant oils are used in a variety of industrial and edible uses. Novel vegetable oil compositions and / or improved means for obtaining oily compositions from biosynthetic or natural plant sources are necessary. Depending on the intended use of the oil, various different fatty acid compositions are desired. For example, in some cases, having an oily seed with a higher ratio of oil to seed, could be useful to obtain a desired oil at low cost. This could be normal for an oily product of superior value. Or said oil seed may constitute a superior food for animals. In some cases, an oily seed with a lower ratio of oil to seed could be useful for a lower caloric content. In other uses, oils from edible plants with a higher percentage of unsaturated fatty acids are desired for reasons of cardiovascular health. And alternatively, tempered substitutes for superior saturated tropical oils such as palm, coconut or cocoa could find use also in a variety of industrial and food applications. One postulated means to obtain said oils and / or modified fatty acid compositions is through plant genetic engineering. However, in order to genetically treat plants, one must have in place the means to transfer genetic material to the plant in a stable and hereditary manner. Additionally, one must have the nucleic acid sequences capable of producing the desired phenotypic result, the regulatory regions capable of targeting the correct application of said sequences, and the like. In addition, it should be appreciated that, in order to produce a desired phenotype, it is required that a desired phenotype that the Kennedy Route, so-called, for the synthesis of glycerolipids be modified to the extent that the ratios of the reactants and metabolic flux through of the route are modulated and changed. Higher plants seem to synthesize oils via a common metabolic pathway. Fatty acids are formed in acetyl-CoA plastids through serial catalytic reactions by enzymes collectively known as Fatty Acid Synthetase (SAG). The fatty acids produced in plastids are exported to coenzyme A. These acyl-CoA are the substrates for the synthesis of glycerolipids in the endoplasmic reticulum (ER). The synthesis of glycerolipids by itself is a series of reactions leading first to phosphatidic acid (PA) and diacylglycerol (DAG). These metabolic intermediates can be directed to membrane phospholipids such as phosphatidylglycerol (PG), phosphatidylethanolamine (PE) or phosphatidylcholine (PC) or that can be directed to form neutral triacylglycerol (TAG) in the main component of vegetable oils used by the seed as a form to store energy in order to be used during seed germination. Diacylglycerol (DAG) is synthesized from glycerol-3-phosphate and acyl-CoA fatty acids in two steps sequentially catalyzed by glycerol-3-phosphate acyltransferase (G3PAT), lysophosphatidic acid acyltransferase (DAGAT) to form PA and then a additional hydrolytic step catalyzed by phosphatidic acid phosphatase (PAP) to form DAG. In most cells, DAG is used to form the phospholipid membrane, the first step being PC synthesis catalyzed by CTP-phosphocholine cytidyltransferase. In cells that produce stored oils, DAG acylated with a third fatty acid in a reaction catalyzed by diacylglycerol acyltransferase (DAGAT). Collectively, the reactions are part of what is commonly referred to as the Kennedy Route. The structure of TAG, in terms of fatty acid position specificity, is determined by the specificity of each of the three acetyltransferases for acyl-CoA fatty acids and the substrates of the glycerol base structure. Thus, for example, there is a tendency for acyltransferases of many species in the main seed zone to give a saturated or unsaturated fatty acid in the sn-1 or sn-3 position, but only an unsaturated fatty acid in the position sn-2. The absolute specificity for an unsaturated fatty acid at the sn-2 position is determined by the substrate preference of the DAGAT enzyme. In some species such as cocoa compositions, TAG suggests that this trend is carried out since there is an obvious preference for acylation of the sn-3 position with a saturated fatty acid, if the sn-1 position is esterified to an acid saturated fat. Therefore, there is a higher percentage of structured TAG of the SUS form (where S = saturated fatty acid and U = unsaturated fatty acid), which could be expected from a random distribution based on the overall fatty acid composition with the sn-2 position fixed with an unsaturated fatty acid. This suggests that DAGAT also plays an important role in the regulation of the TAG structure, if not also in the control of the TAG synthesis. The reaction catalyzed by DAGAT is at a critical branching point in glycerolipid biosynthesis. Enzymes at said branch points are considered prime candidates for metabolic regulatory sites. Through the synthesis of diacylglycerol, TAG and synthesis of membrane lipids share in common G3PAT, DAGAT and PAP. Because all cells have membranes, they must have these enzymes. The single oil synthesis formed is the DAGAT reaction. The presence of DAGAT activity provides an alternative destination for DAG that goes inside the membranes. It is logical to think that what drives the synthesis of TAG is the presence of the DAGAT enzyme, and that either directly or indirectly through the regulatory cascade, the activity of DAGAT and / or concentrations of diacylglycerol, can play a role in controlling the flow in glycerolipids. The obtained nucleic acid sequences capable of producing a phenotypic result in the incorporation of fatty acids into a glycerol base structure in order to produce an oil that is subjected to various obstacles including, but not limited to, the identification of factors metabolites of interest, choice and characterization of a protein source with useful kinetic properties, purification of the protein of interest at a level that can allow its sequencing, using the amino acid sequence data to obtain a nucleic acid sequence capable of being used as a probe to restrict the desired DNA sequence, and the preparation of construction, transformation and analysis of the resulting pfantas. Therefore, it is necessary to identify target enzymes and tissue sources useful for nucleic acid sequences of the target enzymes capable of modifying the oil structure and the desired amount. Ideally, a white enzyme could be susceptible to one or more applications alone or in combination with other related nucleic acid sequences for increased / decreased oil production, the structure of TAG, the ratio of saturated to unsaturated fatty acids in the fatty acid poor and / or in other novel oily compositions, as a result of the fatty acid combinations. Once the target enzymes were identified and scored, the amounts of protein and purification protocols are required for sequencing. Finally, useful nucleic acid constructs that have the necessary elements to provide a phenotypic modification and quantification of plants of said constructions are necessary. Several putative isolation procedures have been published for DAGAT. Polokoff and Bell (1980) reported the solubilization and partial purification of DAGAT from rat liver microsomes. This preparation was insufficiently pure to identify a specific protein factor responsible for the activity. Kwanyuen and Wilson (1986, 1990) report the purification and characterization of the soybean cotyledon enzyme. However, the molecular mass (1843 kDa) suggests that this preparation is not extensively solubilized and any DAGAT protein contained therein was part of a larger aggregate of the major proteins. Little and others (1993) reported the solubilization of DAGAT from embryos derived from rapeseed microspores, but as with Kwanyuen and Wilson, the molecular mass of the material that was associated with the activity was so high that complete solubilization is not likely. . Andersson and others (1949) reported the solubilization and 415-fold purification of DAGAT from "liver" rats using immunoaffinity chromatography, however, there is no evidence that the antibodies can be used to recognize DAGAT epitopes, nor that the protein that Purified to be truly DAGAT In addition, as with Kwanyuen and Wilson, the DAGAT activity in their preparations exhibits a molecular mass usually of aggregated membrane proteins.Finally, Kamisaka et al. (1993, 1994, 1996, 1997) report the solubilization of DAGAT. of Mortierella rammaniana and subsequent purification for homogeneity This shows the evidence that DAGAT of these fungal species has a molecular mass of 53 kDa, which is the first published report of a DAGAT that has really been solubilized. our laboratory (see Example 4), but we could not obtain a homogenous preparation, or associate the enzyme activity with a n 53 kDa polypeptide. In addition, we were able to show that a 53 kDa polypeptide tends to be the one claimed by Kamisaka and others, it does not correlate with the DAGAT activity in the fractions obtained using its protocol. Relevant Literature It was reported that cell-free homogenates of developing jojoba embryos have acyl-transferase activity of acyl-CoA fatty alcohol. The activity was associated with a floating wax pad that was formed under differential centrifugation (Poollard et al. (1979) supra).; Wu et al., (1981) supra).
The solubilization of a methylenezyme complex of Euglena gracilis having acyl transacelase activity-fatty SCoA, is reported by Wildner and Hallick (Summary of The Southwest Consortium Fifth Annual Meeting, April 22-24, 1990, Las Cruces, NM. ). The ten-fold acyl-CoA purification of jojoba: alcohol transacylase protein is reported by Pushnik et al. (Summary of The Southwest Consortium Fourth Annual Meeting, February 7, 1989, Riverside, Ca.). An analysis for the acyl-CoA transacilase activity of jojoba: alcohol was reported by Garver et al. (Analytical Biochemistry (1992) 207: 335-340). WO 93/10241 is directed to a fatty acyl-CoA 0-acyltransferase plant: fatty alcohol. A 57 kD jojoba protein is identified as the acyl-CoA acyltransferase jojoba fat: fatty alcohol (wax synthase). The present inventors reported to the latter that the 57 kD jojoba protein is a β-ketoacyl-CoA synthase implicated in the biosynthesis of very short chain fatty acids (Lassner et al. {The Plant Cell (1996) 8: 281 -292). The photoaffinity label of a postulated 47 kD jojoba seed polypeptide can be an acyl-CoA acyltransferase: fatty alcohol reported also by Shockey et al. (Plant Phys. (1995) 107: 155-160). "Kamisaka and Nakahara," Characterization of the Diacylglycerol Acyltransferase Activity from the Lipid Body Fraction from an Oleaginous Fungus ", J. Biochem. (1994) 116: 1295-1301.
Kamisaka et al., "Purification and Characterization of Diacylglycerol Acyltransferase Activity from the Lipid Body Fraction from an Oleaginous Fungus", J. Biochem. (1997) 121: 1107-1114. WO 93/10241 is described for acyl-CoA O-acyltransferases: fatty alcohol plants. A 57 kDa jojoba protein is identified as an acyl-CoA O-acyltransferase: fatty alcohol (wax synthase). The present inventors reported finally that the 57 kD jojoba protein is a β-ketoacyl-CoA synthase implicated in the biosynthesis of very long chain fatty acids (Lassner et al. (The Plant Cell (1996) 8: 281-292 ) BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the results of wax synthase activity analysis in the column fractions of a first wax synthase purification protocol, Figure 1A provides the results of Blue A agarose chromatography. Figure 1B provides the results of ceramic hydroxyapatite chromatography.
Figure 1C provides the results of S-100 fractionation size chromatography. Figure 1A provides the results of hydroxyapatite chromatography. Figure 2 presents the results of analysis of the wax synthase activity in the column fractions of a second wax synthase purification protocol. Figure 2A provides the results of Blue A agarose chromatography. Figure 2B provides the results of hydroxyapatite chromatography. Figure 2C provides the results of size exclusion chromatography 75 of Superdex. Figure 3 presents the results of wax synthase and DAGAT activity in fractions of a purified wax synthase preparation according to the wax synthase purification depicted in Figure 1. The results from the obtained fractions followed by the step of hydroxyapatite chromatography. Figure 4 presents the results of the wax synthase activity analysis in the column fractions of a DAGAT purification protocol using Yellow Agarose Chromatography 86. Figure 5 presents the results of analysis of the DAGAT activity in the fractions of column of a second synthase purification protocol. Figure 4a provides the results of Heparin Sepharose CL-6B chromatography. Figure 5B provides the results of the SDS-PAGE analyzes of the itch fractions. Figure 6A shows the results of the DAGAT purification of a Yellow Agarose 86 chromatography. Figure 6B repressed the SDS-PAGE analysis of the peak fractions. of the Yellow Agarose 86 chromatography. Figure 7 shows the results of the DAGAT purification of Mortierella rammaniana using a Yellow Agarose 86 chromatograph.
Figure 8 presents the results of analysis of the DAGAT activity in the column fractions of a second synthase purification protocol. Figure 8A provides the results of the hydroxylapatite chromatography. Figure 8B provides the results of the SDS-PAGE analysis of the peak fractions. Figure 9 presents the results of the DAGAT activity in the column fractions of a DAGAT purification protocol. Figure 9A provides the results of yellow 86 agarose / hydroxylapatite tandem chromatography. Figure 9B provides the results of the SDS-PAGE analysis of the peak fractions of the random chromatography. SUMMARY OF THE INVENTION By means of this invention, the compositions and methods for use in connection with the diacylglycerol acyltransferase, hereinafter referred to as DAGAT, are provided. Also of interest are the methods and compositions of the amino acid sequences related to the biologically active DAGATs. In particular, DAGAT protein preparations having the relatively high specific activity of interest for use in a variety of applications, in vitro and in vivo. Specifically, protein preparations having DAGAT activities are contemplated below. Of special interest is that DAGAT obtained from Mortierella rammaniana.
Also of particular interest is the discovery that the jojoba wax synthase of the present invention also demonstrates the activity of diacylglycerol (DAGAT). TAG does not occur naturally in jojoba and therefore the activity of the wax synthase enzyme with DAG substrates suggests that wax synthase is related to DAGAT, an enzyme responsible for the production of TAG in most species of plants, particularly in oily seed crop plants, whose seeds contain high levels of stored TAG. Therefore, the use of the jojoba wax synthase protein and / or its coding sequence for the isolation of plant genes encoding DAGAT are considered in the present invention. The illustrative jojoba and the DAGATs of Mortierella rammaniana are purified from the membranes (ie, they are solubilized) and the preparation of solubilized DAGAT is subjected to various chromatographic analyzes to identify a protein associated with the DAGAT activity. In this form, a protein having a molecular weight of approximately 40 kDa is identified as being associated with DAGAT activity. In addition, purification methods, such as column chromatography and polyacrylamide gel electrophoresis, are used to obtain the DAGAT protein in sufficient purity for amino acid sequence analysis. Peptide fragments of DAGAT proteins are used as a template in the design of several synthetic oligonucleotides that can be used to obtain the nucleic acid sequences encoding all or a portion of the DAGAT protein. Using DAGAT encoding the sequences thus obtained, it is also possible to isolate other DAGAT agents that encode the DAGAT proteins. Therefore, this invention encompasses the DAGAT peptides and the corresponding amino acid sequences of those peptides. Said sequences found in particular are used in the preparation of oligonucleotides containing the DAGAT coding sequences for the analysis and recovery of the DAGAT gene sequences. The DAGAT coding sequence can encode a complete or partial sequence depending on the intended use. All or a portion of the genomic sequence or a cDNA sequence is intended. Of special interest are the recombinant DNA constructs that provide the transcription or transcription and translation (expression) of the DAGAT sequences. Particularly preferred are constructs that are capable of transcription or transcription and translation in host cells of plants. Such constructs may contain a variety of regulatory regions including transcriptional initiation regions obtained from genes preferentially expressed in the tissue of plant seeds. In still a different aspect, 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 DAGAT encoding the sequence are also contemplated herein. Furthermore, this invention relates to methods for using the DNA sequences encoding DAGAT for the modification of triglyceride molecules, especially in the seed oil of oilseed crop plant. Plant cells having said modified triglycerides are also contemplated herein. Also modified in this invention are the modified plants, seeds and oils obtained by the expression of the LPAAT proteins of plants of this invention. DETAILED DESCRIPTION OF THE INVENTION A diacylglycerol acyltransferase (herein referred to as DAGAT) of this invention includes any amino acid sequence, such as a protein, polypeptide or peptide, obtained from a cellular source, which demonstrates the ability to catalyze the production of triacylglycerol of 1,2-diacylglycerol-3-phosphate and an acyl-CoA substrate under the reactive conditions of enzyme. By "reactive enzyme conditions" it is meant that any necessary condition is available in an environment (i.e., such factors as temperature, pH, lack of inhibitory substances) that allow the enzyme to function.
"Solubilization" refers to the extraction of the DAGAT enzyme from the membranes in such a way that it then behaves in a normal form of enzymes that are not associated with the membrane, because the membrane effectively lacks the DAGAT protein for other proteins that are also present therein, solubilization is an essential requirement for the identification and purification of the DAGAT protein as described in the following examples. In the test for the solubilization of DAGAT activity, three different indications of solubilization are considered, as described in more detail in the following examples. 1) The DAGAT activity is not sedimented by centrifugation at very high speed. 2) The DAGAT activity migrates in a size exclusion chromatography column if it has a native molecular weight of enzymes that do not associate with the membrane. 3) The proteins present in the preparation of DAGAT are at least partially separable with each other by column chromatography. Due to the potential alternative interpretations that can be applied to any of the above criteria individually, it is necessary to confirm that all three criteria have been met to confirm the solubilization of DAGAT. For example, the first criterion, of failure for sediment at very high g-forces, could be altered if the density of the solution used for the solubilization is similar to the non-solubilized membranes so that they settle very slowly. This situation is illustrated in the following examples, in which a published solubilization procedure is shown, which is inadequate to obtain DAGAT substantially separated from the cytoplasmic membranes. The second criterion, in which the solubilized activity migrates more slowly through the size exclusion column than the original membranes, can be compromised if the membranes by themselves bind weakly to the column after exposure to the detergent so that Your migration is very slow. The third criterion, in which the solubilized proteins are revealed chromatographically, is at least less likely to be compromised by artifacts or unforeseen situations. However, it is possible that the membranes may be partially associated by the solubilization process so that several aggregates of proteins are released. Said aggregates could then be revealed with each other chromatographically. Therefore, the satisfaction of the three criteria is necessary to ensure that solubilization of DAGAT is achieved. Having obtained the solubilized jojoba wax synthase protein, it can be seen that additional experiments can be carried out to characterize the enzyme in terms of substrate specificity, cofactor requirements and possible activity inhibiting agents. For example, it has been found that the jojoba wax synthase of this invention has a wide range of acyl substrates, including acyl-ACP and acyl-CoA molecules. In addition, the acyl and fatty alcohol substrates can have a scale of broad size with respect to the carbon chain length. For example, the activity was tested using substrates having carbon chain lengths of C12 to C24 and all show that they can be used by the enzyme. In addition, the activity is shown with fatty acyl and fatty alcohols having varying degrees of unsaturation. Surprisingly, the purified jojoba wax synthase is also shown herein to have diacylglycerol (DAG) activity and acyl-CoA fatty substrates to produce triacylglycerol (TAG), even through TAG that has not been reported leaving from tissues of jojoba plants, therefore, wax synthase has at least two acyltransferase activities, one in which the acceptor substrate for the acyl-CoA molecule is an alcohol (acyltransferase of fatty alcohol) and another in which the acceptor substrate is a diacylglycerol (acyltransferase DAG or DAGAT). The presence of the DAGAT activity of the wax synthase enzyme suggests that the wax synthase protein is closely related to the DAGAT proteins in other plant species. The solubilization of DAGAT from Mortierella is described in the following examples. The solubilization of DAGAT is confirmed by the demonstration of each of the above solubilization criteria. The solubilized preparations of DAGAT from Mortierella are used in a variety of chromatographic experiments for the identification and partial purification of the DAGAT protein. In this way, a protein having a molecular weight of approximately 40kDa were identified as being associated with DAGAT activity. As described in more detail in the following examples, the 40 kDa protein is partially purified by chromatography on yellow 86-agarose and hydroxyapatite columns. The protein is then obtained in the substantially purified form by gel electrophoresis and spots of the DAGAT preparation partially purified to nitrocellulose. The 40 kDa protein is recovered by cutting the portion of the nitrocellulose filter containing the identified band. The purified protein is then digested with various enzymes to generate peptides for use in the determination of the amino acid sequence. Therefore, the tryptic peptide of the 40 kDa protein described herein represents a portion of a DAGAT of Mortierella rammaniana. Other DAGAT peptides from Mortierella can be obtained in a similar manner and the determined amino acid sequences. For the use of amino acid sequences of the DAGAT peptides to obtain nucleic acid sequences encoding DAGAT are described herein. For example, synthetic oligonucleotides will be prepared that correspond to the DAGAT peptide sequences. The oligonucleotides used as primers in the polymerase chain reaction (PCR) techniques to obtain partial DNA sequence of the DAGAT genes. The partial sequences thus obtained are then used as probes to obtain DAGAT clones from a gene bank prepared from Mortierella rammaniana tissue. Alternatively, where the low degeneracy oligonucleotides can be prepared from particular DAGAT peptides, said probes can be used directly in the gene bank screening for the DAGAT gene sequences. In particular, screening of cDNA libraries in phage vectors are useful in such methods due to the lower levels of the background hybridization. A nucleic acid sequence of a DAGAT of this invention may be a DNA or RNA sequence, derived from DNA, cDNA, mRNA or may be synthesized in whole or in part. These gene sequences can be cloned, for example, by isolating the genomic DNA from an appropriate source and amplifying and cloning the sequence of interest using a polymerase chain reaction (PCR). Alternatively, the gene sequences can be synthesized, either completely or in part, especially where it is convenient to provide sequences of preferred plants. Therefore, all or a portion of the desired structural gene (the portion of the gene encoding the DAGAT protein) can be synthesized using preferred codons for a selected host. The preferred host codons can be determined, for example, from the codons most frequently used in the proteins expressed in a desired host species. - One skilled in the art should readily recognize that antibody preparations, nucleic acid probes (DNA and RNA) and the like can be prepared and used to screen and recover "homologues" or "related" DAGATs from a variety of plant sources. . The homologous sequences are found in a sequence identity, which can be determined in comparison to the sequence information, the nucleic acid, the amino acid or through the hybridization reactions between a known DAGAT and a candidate source. Conservative changes, such as Glu / Asp, Val / Me, Ser / Thr, Arg / Lys and Gln / Asn can also be considered in the determinant sequence homology. The amino acid sequences are considered homologous by a sequence identity with less than 25% between the two complete mature proteins. (See generally Doolittle, R.F., OF URFS and ORFS (University Science Books, CA, 1986.) Therefore, other DAGATs may be obtained from the specific exemplified Mortierella protein preparations and sequences provided herein., it will be apparent that natural and synthetic DAGATs can be obtained, including modified amino acid sequences and starting materials for synthetic protein models of the exemplified DAGATs and DAGATs that are obtained through the use of said exemplified sequences. Modified amino acid sequences include sequences that have been mutated, truncated, increased, and the like, so that said sequences are partially or completely synthesized. The sequences that are currently purified from plant preparations or are identical or identical proteins encoded by themselves despite the method used to obtain the protein or sequences, are naturally derived as well considered. Typically, a DAGAT sequence obtainable from the use of nucleic acid probes that show 60-70% sequence identity between the target DAGAT sequence and the coding sequence used as a probe. However, length sequences with at least 50-60% sequence identity can also be obtained. The nucleic acid probes can have a length fragment of the nucleic acid sequence or can also be a short oligonucleotide probe. When the larger nucleic acid fragments are used as probes (greater than about 100 bp), one can be screened at lower resistances in order to obtain sequences from the blank sample that have 20-50% deviation (ie, homology). of 50-80% sequence) of the sequences used as probes. The oligonucleotide probes can be considerably shorter than the total nucleic acid sequence encoding a DAGAT enzyme, but could be at least 10, preferably at least about 15 and more preferably at least about 20 nucleotides. The high degree of sequence identity as desired when the short regions are used as opposed to larger regions. Therefore it may be convenient for regions of highly conserved amino acid sequence identity to designate oligonucleotide probes to detect and recover other related DAGAT genes. Short 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) 96: 1934-1938).
In addition to isolating the other DAGATs, it is convenient that the genes for the different related acetyltransferase proteins can also be obtained using the sequence information of the DAGATs and the related nucleic acid sequences. For example, other acyltransferase enzymes are involved in plant lipid biosynthesis, including plastic DAGAT, DAGAT mitochondrial acyltransferase liposofosfofatidilcolina (LPCAT), liposofosfofatidilserina acyltransferase (LPSAT), lysophosphatidylethanolamine acetyltransferase (LPEAT) and lysophosphatidylinositol acyltransferase (LPIAT). These enzymes catalyze the acyltransferase reactions involved in the sn-2 position of lysophospholipids and the genes encoding these sequences can also be related to the plant acyl-CoA DAGAT sequences of the present invention and obtainable therefrom. Therefore, as demonstrated herein, other acyltransferases include fatty acyl-CoA O-acyltransferase: fatty alcohol (wax synthase) of jojoba may be related to diacylglycerol acyltransferases. DAGAT and wax synthase are members of a homologous protein family supported by the information obtained through the DAGAT purification of an oleaginous fungus species, Mortierella rammaniana (See Examples). Firstly, the similar jojoba wax synthase, DAGAT fungal activity is membrane binding and can only be solubilized through the use of detergents. Second, as with jojoba wax synthase, if necessary following solubilization to include a phospholipid (e.g., phosphatidic acid) in the analysis mixture in order to restore the activity of the DAGAT fungal enzyme, third, fungal DAGAT works very similarly with jojoba wax synthase during purification chromatography. Specifically, both the washing of enzyme species through a hydroxyapatite column with only light delay, while another species of protein in the membrane preparations binds to the column matrix (see Examples). Indeed, experience with the jojoba enzyme allows the prediction that hydroxyapatite chromatography can be a key step in the purification of DAGAT from Mortierella rammaniana. The addition of this step provided essential for the purification successively of DAGAT fungal, and reading the conclusion of the Kamisaka et al. Protocol (1997) was not sufficient to identify the correct protein species associated with DAGAT activity. Finally, the apparent molecular weight of a fungal DAGAT polypeptide (33 kDa) was determined by SDS-PAGE is identical to that observed for jojoba wax synthase in SDS-PAGE (see Examples). To determine whether a released gene can be isolated by hybridization with the given sequence, the sequence is labeled to allow detection, usually using radioactivity, although other methods are available. The labeled probe is added to the hybridization solution, and incubated with filters containing the desired nucleic acids, such as Northern or Southern blot analysis or filters containing the cDNA or genomic clones to be screened. Hybridization and washing conditions may vary to optimize the hybridization of the probe to the sequences of interest. Lower temperatures and higher salt concentrations allow the hybridization of more distantly related sequences (low resistance). If the antecedent hybridization is a problem under conditions of low resistance to temperature that can be raised in the hybridization or wash steps and / or lower salt content to provide detection of the specific hybridization sequence. Hybridization and wash temperatures can be adjusted based on the estimated melting temperature of the probe as described in Beltz, et al. (Methods in Enzymology (1983) 700: 266-285). A probe useful in appropriate hybridization and washing conditions that have been identified as described above, the cDNA or genomic libraries are screened using the labeled sequences and optimal conditions. For immunological screening, antibodies of the coconut DAGAT protein can be prepared injected into rabbits or mice with the purified protein, said methods of antibody preparation are well known to those skilled in the art. Monoclonal or polyclonal antibodies can be produced, through normally polyclonal antibodies are most useful for the isolation of genes. Western analysis can be carried out to determine that a released protein is present in a crude extract of the desired plant species, as determined by the cross-reaction with the coconut DAGAT antibodies. When cross-reactivity is observed, the coding genes of the related proteins are isolated by the screening expression banks representing the desired plant species. Expression banks can be constructed in a variety of commercially available vectors, including gt 11 lambda, as described in Maniatis, and others (Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). All plants used in DAGAT proteins in the production of membrane phospholipids and therefore any given plant species can be considered as a source of additional DAGAT proteins. Plants having significant medium chain fatty acids in their oil oils are preferred candidates for obtaining DAGAT from plants capable of incorporating the chain fatty acids measured at the sn-2 position of TAG. Several species in the Cuphea genus accumulate triglycerides that contain the medium chain fatty acids in their seeds, eg, procumbenss, luteal, hookerian, hyssopifolia, wrightii and inflata. Another source of natural plant of medium chain acids are seeds of the Lauranceae family. In addition to the California laurel (Umberllularia califonrica); Pisa (Actinodophe hookeri), Laurel Dulce (Laurus nobilus) and Cinnamomun camphora (camphor) accumulate medium chain fatty acids. Other plant sources include Ulmaceae (elm), Palmae, Myristicaceae, Simarubaceae, Vochysiaceae and Salvadoraceae. Also of particular interest are DAGATs of plant species that incorporate long-chain fatty acids unusual in stored TAG. For example nastrutium and meadowoam contain 22: 1 acyl groups in the seed TAG and meadowfoam have been shown to contain a DAGAT capable of incorporating 22: 1 fatty acyl groups (erucic) at the sn-2 position. A DAGAT having said activity can find use in the production of "tri-erucic" Brassica oil, whose damages are not due to the selectivity of DAGAT from Brassica seed to unsaturated fatty acids, such as 18: 1 and 18 :2.
It should also be noted that DAGAT plants from a variety of sources can be used to investigate the TAG biosynthesis events of plant lipid biosynthesis in a wide variety of in vivo applications. Because plants for synthesizing lipids via a common metabolic pathway, the study and / or application of a DAGAT plant for a host heterologous plant can be easily achieved in a variety of species. In other applications, a DAGAT plant can be used outside the native plant source of DAGAT to increase production and / or modify the composition of the TAGs produced or synthesized in vitro. Nucleic acid sequences associated with DAGAT plant proteins could find many uses, for example, recombinant constructs can be prepared that can use probes, or that could be provided for the expression of 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 can be found when the host cell is a host cell of plants, either in vitro or in vivo. For example, increasing the amount of a preferred medium chain DAGAT available from the plant TAG biosynthesis pathway, an increased percentage of medium chain fatty acids after being obtained in the TAG. In a similar way, for some applications it may be convenient to increase the amount of DAGAT endogenously expressed in plant cells by the counter-sense technology. For example, to more conveniently allow a foreign inserted DAGAT to transfer medium chain or longer chain fatty acyl groups to the sn-2 position, it may be desirable to decrease the expression of a preferably long-chain Brassica DAGAT. Therefore, depending on the intended use, the constructs may contain the sequence encoding the total DAGAT protein or a portion thereof. For example, when the inhibition of the sense of a given DAGAT protein is desired, the total DAGAT sequence is not required. In addition, where DAGAT constructs are intended to be used as probes, it may be advantageous to prepare constructs that contain only a particular portion of a sequence encoding DAGAT, for example a sequence that is described to encode a highly conserved DAGAT region. A nucleic acid sequence described above that encodes a plant DAGAT of this invention may include genomic cDNA or a mRNA sequence. By "coding" it is meant that the sequence corresponds to a particular amino acid sequence in a direction of sense and nonsense. By "extrachromosomal" is meant that the sequence is outside the genome of the plant to which it is naturally associated. By "recombinant" is meant that the sequence contains a genetically modified modification through manipulation via mutagenesis, restriction, enzymes and the like.
A cDNA sequence may or may not contain pre-processing sequences, such as temporal peptide sequences or target sequences to facilitate delivery of the DAGAT protein (such as mitochondrial DAGAT) to a given organelle or membrane location. The use of any precursor DAGAT DNA sequence is preferred for uses in the expression of plant cells. A genomic DAGAT sequence can contain the transcriptional and translational initiation regions, introns, and / or transcriptional termination regions of DAGAT from plants, whose sequences can be used in a variety of DNA constructs, with or without the DAGAT structural gene. . Therefore, the nucleic acid sequences corresponding to DAGAT of plants of this invention also provide useful signal sequences to direct delivery of the protein at a particular organelle location or membrane, the regulatory regions without upstream coding of the protein. '(promoters) having useful tissue profiles and time control, the regulatory region without downstream coding 3' useful as transcriptional and translational regulatory regions, and can lead to penetrate other aspects of the gene. Once the nucleic acid sequence is obtained from DAGAT of desired plants, can be manipulated in a variety of ways. Where the sequence involves the flanking regions without coding, the flanking regions can be subjected to the reaction, mutagenesis. Therefore, transitions, transversions, deletions and insertions can be carried out in the sequence present in nature. In addition, all or part of the sequence can be synthesized. In the structural gene, one or more codons can be modified to provide for a modified amino acid sequence, or one or more codon mutations can be introduced to provide a convenient restriction site. The structural gene can also be modified using synthetic adapters, interleavers to introduce one or more convenient restriction sites, or the like. The nucleic acid or amino acid sequences encoding a DAGAT of plants of this invention can be combined with other non-native or "heterologous" sequences in a variety of ways. By "heterologous" sequences is meant any sequence that is not naturally bound to plant DAGAT, including, for example, combinations of nucleic acid sequences from the same plant that is not naturally bound thereto. The DNA sequence encoding a DAGAT plant of this invention can be used in conjunction with all or part of the gene sequences normally associated with DAGAT. In its component parts, a DNA sequence encoding DAGAT is combined into 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 sequences encoding the plant DAGAT and a transcription and translation termination region.
The potential host cell includes prokaryotic and eukaryotic cells. The host cell may be unicellular or be in a differentiated or undifferentiated molecular organism depending on the intended use. The cells of this invention can be distinguished by having a wild-plant DAGAT to the wild-type cell present therein. For example, having a recombinant nucleic acid construct encoding a DAGAT of plants therein. Depending on the host, the regulatory regions could vary, including the regions of viral, plasmid or chromosomal genes or the like. For expression in prokaryotic or eukaryotic microorganisms, particularly unicellular hosts, a wide variety of constitutive or regulatable promoters can be employed. Expression in a microorganism can provide a ready source of the plant enzyme. Among the initiation regions transcripts that have been described, are the bacterial and yeast host regions, such as E. coli, b. subtilis, Sachromyces cerevisiae, including genes such as beta-galactosidase, T7 polymerase, tritophane E, and the like. For the most part, the constructions will involve functional regulatory regions in plants that provide modified production of DAGAT from plants, and possibly, the modification of the fatty acid composition. The open reading frame, which encodes the DAGAT of the plant, or a functional fragment thereof, can be joined at its end to a transcriptional initiation regulatory region. Modification through manipulation via mutagenesis, restriction enzymes and the like. In embodiments wherein the expression of the DAGAT protein is convenient in a plant host, the use of another part of the DAGAT gene of the whole plant is desired, namely all or part of the regions without coding upstream of 5 '. (promoter) together with the "sequence of structural genes and regions without current coding under 3 'may be employed, if a different promoter is desired, such as a native promoter for the host of the plant of interest or a modified promoter., that is, having the transcription initiation regions derived from a gene source and translational initiation regions derived from a different gene source, numerous transcription initiation regions are available that provide a wide variety of structured gene functions that can be induced, and of transcription. The transcription / translation initiation regions correspond to the structural genes that are immediately 5 'upstream at the respective start codons. Among the transcriptional initiation regions used for plants are in the regions associated with the T-DNA structural genes such as noaplin and aminopin synthases, the 19S and 35S promoters of CaMV and the 5 'downstream regions of other plant genes such as napin, ACP, SSU, PG, zein, phaseolin, and the like. Enhanced promoters, such as double 35S, are also available for the expression of DAGAT sequence for such applications when regions without upstream 5 'coding are desired are derived from other genes regulated during seed maturation, which are preferentially expressed in plant embryo tissues, such as ACP and transcriptional initiation control regions derived from napin. Said "seed specific promoters" may be obtained and used in accordance with the techniques of the U.S.A. Series. No. 07 / 147,781, filed on 01/25/88 (now the Series of E.U.A. No. 07 / 550,804, filed on 09/07/90) and the Series of E.U.A. DO NOT. 07 / 494,722 both filed on March 16, 1990 having a title "Novel Sequences Preferentially Expressed In Early Seed Deveiopmetn and Méthods Related Thereto" whose references are incorporated herein by references. The transcription initiation regions that are preferentially expressed in seed tissue, ie, not detected in other parts of the plant, are considered suitable for TAG modifications in order to minimize any destructive or adverse effects of the TAG. gene product. Regulatory transcription termination regions can be provided in the DNA constructs of the invention. The termination regions transcripts can 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 transcription termination region that is naturally associated with the transcription initiation region. Where the transcription termination region is from the different source of genes, which could contain at least 0.5 kb, preferably around 1-3 kb of 3 'sequence for the structural gene from which the termination region is derived. The expression of plants or transcription constructs having plant DAGAT as the DNA sequence of interest to increase or decrease the expression thereof can be employed with a wide variety of plant life, particularly, plant life involved in the production of vegetable oils for edible and industrial uses. More especially preferred are those of oily seeds of temperament. Plants of interest include, but are not limited to, rapeseed (Cañola and varieties with high Erucic Acid content). Sunflower, safflower, cotton, soybean, peanut, coconut and palm and corn oils. Depending on the method for introducing the recombinant constructs into the host cell, other DNA sequences may be required. Importantly, this invention is applicable to similar dicotyledonous and monocotyledonous species and which may be readily applicable to new and / or improved transformation and regulation techniques. Of particular interest, is the use of constructions of DAGAT in plants that have been genetically treated to produce a particular fatty acid in the oil seed of plants, where TAG in the seeds of plants or treated of the treated species, does not naturally contain the particular fatty acid. Therefore, the expression of novel DAGAT in plants can be convenient for the incorporation of the unique fatty acyl groups in the sn-3 position. In addition, the plant genetic engineering applications for the DAGAT proteins of this invention include their use in the preparation of structured plant lipids containing TAG molecules having the desired fatty acyl groups incorporated in particular at the positions in the TAG molecule. The transformation method obtained in the transgenic plants is not critical to present the invention and various plant transformation methods are currently available. In addition, new methods are available to transform crops, which can also be applied directly under it. For example, many species of plants naturally susceptible to infection by Agrobacterium can be transformed successively via tripartite or binary Agrobacterium-mediated transformation binary methods. In many cases, it will be convenient to have the construction of the sides on one of both sides by the T-DNA, particularly the left and right sides, more particularly the right side. This is particularly useful when the construction uses A. tumemafiens or A. rhizogenes as a mode for transformation, through the sides of T-DNA can contract use with other modes of transformation. In addition, microinjection techniques, bombardment of the DNA particle and electroporation have been developed to allow the transformation of several species of monocotyledonous or dicotyledonous plants. Normally, including the construction of DNA that could be a structural gene that has the regulatory regions necessary for expression in the host and providing the selection of transforming cells. The gene can provide resistance to a cyto-toxic agent, e.g., antibiotic, heavy metal, toxin, etc., complementation, providing prototrophy to an auxotrophic host, viral immunity or the like. Depending on the number of different host species, the expression construct or components thereof may be introduced, one or more markers may be employed, where different conditions for selection are used for the different hosts. When Agrobacterium is used for the transformation of plant cells, a vector can be used which can 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, can be armed (capable of causing scale formation) or disarmed (unable to cause embedding), the latter being permissible, while the vir genes are present in the Agrobacterium host. The armed plasmid can give a mixture of normal plant cells and embedding. In some cases where Agrobacterium is used as the vehicle for the transformation of host plant cells, the expression or transcription construct surrounded by the T-DNA side regions could be inserted into a wide range of host vectors capable of replication in the host. coli and Agrobacterium having a wide scale of host vectors described in the literature. PRK2 or derivatives thereof are commonly used. See, for example, Ditta, et al. (Proc. Nat. Acad. Sci., U.S.A. (1980) 77: 7347-7351) and EPA 0 120 515, which are incorporated herein by reference. Alternatively, one can be inserted into the sequences to be expressed in plant cells in a vector that contains replication sequences separately, one of which strains the vector in E. coli, and the other in Agrobacterium. See, for example, McBride and Summerfelt (Plant Mol. Biol. (1990) 14: 269-276), where the origin of pRiHRI (Jouanin, et al., Mol. Gen. Genet. (1985) 201-370-374). ) of replication is used and is provided to add stability of the expression vectors in plants of Agrobacterium host cells. Including the expression construct and the T-DNA there could be one or more markers, which allow the selection of transformed Agrobacterium and cells of transformed plants. A number of markers have been developed for use with plant cells, such as resistance to chloramphenicol, kanamycin, aminoglycoside G418, hygromycin or the like. The particular marker employed is not essential for this invention, one or the other marker I feel preferred depending on the particular host and the form of construction.
For the transformation of plant cells using Agrobacterium, the explants can be combined and incubated with the transformed Agrobacterium for a sufficient time of transformation, the bacteria destroyed and the cells of plants grown in an appropriate selective medium. Once the callus is formed, shoot formation can be promoted by employing the appropriate plant hormones according to known methods and the shoots are transferred to the root formation medium for plant regeneration. Plants can then develop into seeds and the seeds are used to establish repetitive generations and to isolate vegetable oils. The invention now being described generally, will be more readily understood with reference to the following examples which are included for purposes of illustration only and are not intended to limit the present invention. EXAMPLES Example 1 - Wax Synthase Analysis Methods for analyzing wax synthase activity in microsomal membrane preparations or solubilized protein preparations are described. A. Radiolabelling Material The substrate generally used in wax synthase analysis. Palmitoyl-CoA from [1-1 C], is understood Amersham (Arlington Heigts, IL). Other chain length substrates are synthesized in order to carry out the chain length specification studies. The long chain [1-14 C] fatty acids (specific activity 51-56 Ci / moles), namely 11-cis-eicosanoic acid, 13-cis-docosanoic acid and 15-cis-tetracosanoic acid are prepared by the reaction of potassium cyanide [14C] with the corresponding alcohol mesylate, followed by 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), the methyl esters of fatty acid are subsequently hydrolyzed to the free fatty acids. The radiochemical purity is evaluated by three methods of CCF: the normal phase silica CCF, silver nitrate CCF and C18 reverse phase CCF. The radiochemical purity evaluated by these methods was 92-98%. Long-chain [1-14C] acyl-CoA was prepared from the free fatty acids of [1-14C] by the method of Young and Lynen (J. Bio, Chem. (1969) 244: 377) , to a specific activity of 10 Ci / moles. Hexadecanol of [1-14C] was prepared by oxidation of hexadecan-1-ol dichroate of [1-14C], according to a micro-scale modification of the Pletcher and Tate method (Tet. Lett. (1978) 1601-1692). The product was purified by preparative silica TLC, and stored as a hexane solution at -70 ° C until use. B. Analysis of Wax Synthase Activity in a Microsomal Membrane Preparation The wax synthase activity in a microsomal membrane preparation was measured by incubating 40 μM acyl-CoA of [1-4C] (usually palmitoyl-CoA, 5.1-5.6 mCi / mmoles) and 200 mM oleyl alcohol with the sample can be analyzed in a total volume of 0.25 ml. The incubation mixture also contains either 25 mM HEPES (4- [2-hydroxyethyl] -1-piperazineethane sulfonic acid), pH 7.5, as the regulating agent with 20% w / v glycerol, 1 mM DTT , 0.5M NaCl or 25 mM Tricine-NaOH, pH 7.8, as the regulating agent with 0.28M NaCl, 10% glycerol and 2mM β-mercaptoethanol. Initial studies were carried out with the first regulatory system, when the pH was chosen to accommodate the preference of the acyl-CoA reductase enzyme. The membrane preparations were changed last to the second regulatory system to adapt the optimum upper pH of the wax synthase. The substrate mixture was prepared in a glass jar, with oleic alcohol being added immediately before use, and added to the samples. The incubation was carried out at 30 ° C for one hour. The analysis was terminated by placing the test tube on ice and immediately 0.25 ml of isopropane acetic acid (4: 1 v / v) was added. Unlabeled wax esters (0.1 mg) and oleic alcohol (0.1 mg) were added as vehicles. The lipids of [14C] were extracted by the small scale protocol of Hará and Radin (Anal. Biochem. (1978) 90: 420). Two ml of hexane / isopropanol (3: 2 v / v) were added to the finished analysis. The sample stirred, 1 ml of aqueous sodium sulfate solution (6.6% w / v) were added and the mixture was stirred again. C. Analysis for Solubilized Wax Synthase Activity The solubilized wax synthase was analyzed using up to 50 μl of sample in a 250 μl analysis containing CoA of 1-14C-16: 0 of 40 μM (5 Ci / moles) , 200 μM of 18.1-OH, 0.07% of phospholipid probe / Sigma P-3644), 0.2% of CHAPS, 280 mM of NaCl, 25 mM of tricine-NaOH, pH 7.8, 2 mM of ß-ME and 5.6% of glycerol. The phospholipid (50 mg / ml in 0.5% CHAPS) was added directly to the sample, which is 1% CHAPS, then diluted with a cocktail containing the remaining analysis components. The reconstitution of the activity is assumed to be based on the incorporation of wax synthase into the phospholipid vesicles. The wax synthase is sensitive to detergent and requires the amount of phospholipid (PL) and detergent (CHAPS) to equilibrate to 2.8 / 1 (CHAPS / PL, w / w) in the analysis for maximum activity. The analysis for the synthase activity in the samples concentrated by the ultrafiltration requires a readjustment of the sample volume analyzed due to the concentration of CHAPS. The introduction of the CHAPS in the analysis resulted in the inhibition of activity. The samples were concentrated by ultrafiltration, the optimal volume of the sample was analyzed and can be re-established to carry out a percentage CHAPS concentration curve in the analysis using a small amount of the sample and analyzing at a fixed concentration of phospholipid and NaCl. Wax synthase is less sensitive to changes in PL concentration that is for changes in CHAPS concentration. D. Analysis of Analyzed Products Analyzing the wax synthase analysis products of the microsomal membrane preparation or solubilized wax synthase analysis, two protocols have been developed. A protocol, described immediately as "extensive analysis" is slower, but gives more highly quantitative results. The other protocol, described immediately as "rapid analysis" also provides a measurement of wax synthase activity, but is faster, more convenient and less quantitative. 1. Extensive Analysis: after the addition of sodium sulfate and stirring the sample, the upper organic phase is removed and the lower aqueous phase is washed with 4 ml of hexane / isopropanol (7: 2 v / v). The organic phases were poor and evaporated to dryness under nitrogen. The lipid residue was resuspended in a small volume of hexane and an aliquot was analyzed for radioactivity by liquid scintillation counting. The rest of the sample was used for TLC analysis of the labeled classes and therefore gives a measurement of the wax Total produced. For analysis of lipid classes, the sample was applied to a silica TLC plate, and the plate was developed in hexane / diethyl ether / acetic acid (80.20.1 or 70: 30: 2 v / v / v). The distribution of radioactivity between the lipid classes, the largest wax esters, the free fatty acids, the fatty alcohols and the polar lipids at the origin, were measured using a radioanalytical imaging system of AMBIS (AMBIS Systems Inc. , San Diego, CA). If necessary, the individual lipid classes can be recovered from the TLC plate during further analysis. Reverse phase TLC systems using C18 plates developed in methanol have also been swept for analysis. 2. Rapid Analysis: after the addition of sodium sulfate and stirring the sample, a known percentage of the organic phase was removed and counted via the liquid scintillation counting. The calculation is used to estimate the total quantities in the organic phase. Another portion of the organic phase is then removed, washed under nitrogen, redissolved in hexane and stained in TLC plates and developed and scanned as described in the detailed analysis. In this way the percentage of the total quantities that are incorporated in the wax is determined.
Example 2 - Additional Studies for Synthase Activity of Characterized Wax A. Seed Development and Wax Synthase Activity Profiles Embryo development is tracked in two summers at five plants in Davis, CA. It was found that the fresh embryo and dry weights increase at a fairly stable rate from about day 80 to around day 130. Lipid extractions reveal that when the fresh embryo weight reaches approximately 300 mg (approximately at day 80) ), the ratio of lipid weight to dry weight reaches the maximum level of 50%. Synthase activity was measured in developing embryos as described in Example 1B. Since it was determined that the jojoba seed coatings can be the source of the inhibition factors, the seed coatings were removed before freezing the embryos in liquid nitrogen to be stored at -70 ° C. Development profiles for wax synthase activities were measured in a homogenate-free cell or a membrane fraction, indicating a long induction in activity with peaks of approximately 110-115 days after the anthesis. Embryos during enzymology studies where the harvest is between approximately 90 to 110 days post-synthesis, a period when wax synthase activity is high, lipid deposition does not reach maximum levels and seed coatings are easily summarized. The higher rate of increase in synthase activity is observed between days 80 and 90 post-synthesis. The embryos for the cDNA library construction were therefore recovered between 80 and 90 days post-synthesis when presumably the synthase regime of the wax synthase protein could be maximized. Correspondingly, it could be assumed that the level of the mRNA encoding the wax synthase may be maximum at this stage. B. Preparation of Microsomal Membrane Jojoba embryos were harvested at approximately 90-110 days after flowering, as calculated by measuring the water content of the embryos (45-70%). The outer husks and seed coatings were removed and the cotyledons were rapidly frozen in liquid nitrogen and stored at -70 ° C for future use. For the initial protein preparation, the frozen embryos were pulverized by grinding a steel mortar at liquid nitrogen temperature. In a normal experiment, 70 g of embryos were processed. Dust was added, in a ratio of 280 ml of solution per 70 g of embryos, to the following solution with high salt content: 3M NaCl, 0.3M sucrose, 100 mM HEPES, 2mm DTT and the protease inhibitors , 1 mM EDTA, 0.7 mg / ml leupeptin, 0.5 mg / ml pestatin and 17 mg / ml PMSF. A cell-free homogenate (CFH) was formed by dispersing the embryos in powder in the buffer solution with a homogenizing tissue (Cinemática, Switzerland, model PT10 / 35) for approximately 30 seconds and then filtering through of three layers of Miracloth (CalBioChem, LaJolla, CA). The filtrate was 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 was removed and the supernatant fraction was recovered and dialysed overnight (with three changes of pH buffer) against a solution containing 1M NaCl, 100 mM HEPES, 2 mM DTT and 0.5 M of EDTA. The dialysate was centrifuged at 200,000 x g for 1 1/2 hours to give a pellet, DP2. The pellet was suspended in 25 mM HEPES and 10% glycerol at 1/20 of the original CFH volume, to give the microsomal membrane preparation. The activity was analyzed as described in Example 1. The recovery of the wax synthase activity was calculated at 35% of the original activity in the cell-free homogenate. The wax synthase activity of this preparation is stable when stored at -70 ° C. C. Substrate specificity. Acyl-CoA and alcohol substrates having varying carbon chain lengths and degrees of unsaturation were added to the microsomal membrane fractions prepared as described above to determine the scale of substrates recognized by jojoba wax synthase. The synthase activity was measured as described in Example 1B, with specified acyl-mediated using 80 mM acyl-CoA substrate and 100 mM radiolabelled oleyl alcohol. The specified alcohol was measured using 100 mM of alcohol substrate and 40 mM of radiolabeled eicosenoyl-CoA. The results of these experiments are presented in Table 1 below.
Table 1 Specificity of Acyl Substrate and Alcohol Structure Acyl Group Alcohol Group - 12: 0 12 100 14: 0 95 145 16: 0 81 107 18: 0 51 56 20: 0 49 21 22: 0 46 17 18: 1 22 110 18: 2 7 123 20: 1 122 72 22: 1 39 41 24: 1 35 24 The above results demonstrate that jojoba wax synthase utilizes a broad scale of fatty acyl-CoA and fatty alcohol substrates. In addition the forward synthase activity varies the acyl-thioester substrates were similarly tested using palmitoyl-CoA, palmitoyl-ACP and N-acetyl-S-palmitoyl cysteamide as acyl substrates. The greatest activity is observed with the acyl-CoA substrate. Significant activity (-10% acyl-CoA) was observed with acyl-ACP, but activity with the cysteamine substrate of N-acetyl-S-palmitoyl was not detectable. D. Effects of Activity Various sulfhydryl agents were screened for their effect on wax synthase activity. It was shown that the organomecurium compounds have strongly inhibited activity. Iodoacetamide and N-ethylamide were much less effective. The inhibition by para-hydroxymercuribenzoate was observed, but this inhibition could be reversed by the subsequent addition of DTT. These results demonstrate that inhibition by para-hydroxymercuribenzoate involves blocking an essential sulfhydryl group. Example 3 - Purification of Jojoba Wax Synthase The methods were described that can be used for the isolation of a jojoba membrane preparation having synthase activity, solubilization of synthase activity and further purification of the wax synthase protein. A. Preparation of Microsomal Membrane The following modification of the method described in Example 2 is employed and provides an improved membrane fraction useful for the purification of wax synthase from the solubilized membranes. Normally, 100 g of jojoba embryos were added to 400 ml of extraction pH buffer (40 mm of Tricine-NaOH, pH 7.8, 200 mM of KCI, 10 mM of EDTA, 5 mM of β-mercaptoethanol), crushed in a mixer, and homogenized with a Politron tissue switch. All subsequent steps were carried out at 4 ° C. The mixed material was filtered through Miracloth (CalBioChem). The centrifugation (20,000 x g, 20 min.) Of the yield filtrate of a floating wax layer, a turbid supernatant fraction and a dark green pellet. The supernatant fraction was recovered and centrifuged (100,000 xg, 2 hours) to obtain the membrane pellets which were then resuspended in 40 ml of buffer A (25 mM Tricine-NaOH, pH 7.8, 200 mM KCL, 5 mM of EDTA, 5 mM of β-mercaptoethanol) containing 50% (w / v) of sucrose. This homogenate was distributed in four centrifuge tubes SW28 (Beckman) and each was overlapped with 10 ml of buffer A containing 20% sucrose and then with 12 ml of buffer A. After centrifugation (28,000 rpm; Hours), a membrane fraction was recovered from the 20% / 50% sucrose interface, diluted with four volumes of buffer solution and recovered by centrifugation (200,000 xg, 1 hour). The membranes were then homogenized in 10 ml of stored buffer [25 mM Tricine-NaOH, pH 7.8, 1 mM NaCl, 10% (w / v) glycerol, 5 mM β-mercaptoethanol)]. The protein concentration of the membranes prepared via this protocol is usually between 7 and 9 mg / ml. The concentrations were calculated as described in (Bradford, 1976) using BSA as the normal protein. B. Solubilization of Wax Synthase Protein The membrane suspension was adjusted to approximately 0.83 mg of the protein by me by dilution with stored buffer (25 mM Tricine-NaOH, pH 7.8, 1 M NaC, 10% glycerol, 5 mM of β-mercaptoethanol). 3 - ([3-colamidopropyl] dimethyl-ammonium) -1-propanesulfate solid (CHAPS) was added to achieve a final concentration of 2% (w / v) and a detergent for the protein ratio of 24: 1. After incubation on ice for 1 hour, the sample was centrifuged (200,000 g for 1 hour) and the supernatant fraction recovered. C. Purification of the Wax Synthase Activity The 200,000 g supernatant was diluted (with 0.57% CHAPS, 25 mM Tricine-NaOH, pH 7.8, 20% glycerol) to give the final concentrations of NaCl and CHAPS of 0.3M and 1%, respectively, the sample was loaded onto a Blue A agarose column (Amicon, Inc., Beverly, MA) that was equilibrated with the B reagent solution (25 mM Tricine-NaOH, pH 7.8, 1 % of CHAPS, 20% glycerol), which contain 0.3 M NaCl. After washing with the buffer solution in equilibrium, the wax synthase activity was eluted with buffer B containing 2M NaCl. Active fractions eluted from the Blue A column were poor (Poor Blue) and used for additional chromatography.
Two purification protocols were used for band identification and additional purification of the wax synthase protein. In Protocol 1 (Figure 1), Poor Blue was concentrated 5.4 times by ultrafiltration in a pressure cell placed with a YM 30 membrane (Am? Con, Inc., Beverly, MA). One half of the concentrate was applied to a Ceramic Hydroxyapatite (CHT) column (Bio-Scale CHT-2; Bio-Rad, Hercules, CA) equilibrated in buffer B containing 2M NaCl. The column was washed with 6 column volumes of the equilibrium buffer and the binding proteins were eluted with buffer B containing 0.1M dipotassium phosphate and 2M NaCl. After rebalancing, the concentrate was chromatographed in the same manner. In order to detect the activity, the wax synthase was analyzed according to the protocol for the concentrated samples by ultrafiltration. The wax synthase activity, measured in CTH 1 operation, was found in the flow through the wash. The protein profiles of the two CTH operations were identical so that the operation of CTH 2 was not analyzed. The active fractions of the two CTH operations were poured and concentrated 10 times and applied to an HR S100 column of Sephacril. (2.5 x 90 cm) balanced in buffer solution b with 1.0 M NaCl. Protein and activity determinations were made and the active fractions were selected from the retentate portion of the maximal activity and protein operation minimized. Poor S100 (fractions 64-70) was applied to a column of crystalline hydroxylapatite (HA) (Bio-Gel HAT, Bio-Rad, Hercules, CA, 1 x 19.3 cm) equilibrated in buffer solution B with 1M NaCl. Again, most of the wax synthase activity was present in the flow through the wash. The binding proteins were eluted in buffer B with 0.1M dipotassium phosphate and 1M NaCl. The fractions of the final HA operation were examined by SDS-PAGE. A single 33 kD migratory protein on SDS-PAGE was correlated in the presence of wax synthase activity.
In a second preparation (Protocol 2, Figure 2) the Poor Blue was applied directly to a crystalline HA column (1 x 11.7 cm), equilibrated in buffer solution to B with 1M NaCl. Without concentration. The two fractions were selected during the additional purification by size exclusion chromatography on a Superdex 75 HR 10/30 column (Bio-Rad, Hercules, CA, scale size: 5000-75,000 daltons) equilibrated with 25 mM Tricine-NaOH , pH 7.8, 1% of CHAPS, 20% of glycerol, 1M of NaCl. The wax synthase activity was measured according to the protocol described for the samples solubilized in Example 1C. A fraction easily eluted in the flow through the HA column (fraction 31) and the other fraction eluted with the wash (fraction 67). The protein profiles of the two fractions were different based on the SDS-PAGE analysis. Both the Superdex 75 operations were examined by the SDS-PAGE gradient and a protein of approximately 33 kD was identified that chromatographed with an activity. A calibration curve was generated using normal molecular mass chromatography under the same buffer and column conditions. The comparison of the elution volume of the peak of the Wax Synthase activity to this normal curve yield and a value of 48 kDa for the molecular mass of the solubilized enzyme. A plotted purification of the wax synthase of Protocol 1 (Table 2) shows a 150-fold purification of the enzyme from the solubilized protein fraction.
Table 2 Purification of Jojoba Wax Synthase Purification Step Performance Activity Protein Activity Purification Enzyme% (mg) specific (fold) (nmol / min.) (Nmol / min / mg) Fraction 274.4 100 415 0.7 1 solubilized Agarose Blue A 214.7 78.2 15 14.3 22 Hydroxyapatite 176.6 64.3 6.4 27.6 42 Sephacrylic S-100 ceramic 41.3 15.1 1.2 33.1 50 (sizing) Hydroxyapatite 18.8 6.9 0-2 99.2 150 (crystalline) D. Analysis of SDS PAGE The sample of the column fractions were diluted in the sample buffer SDS-PAGE (1x buffer = 2% SDS, 250 mM β-mercaptoethanol, 0.0025% bromophenol blue) and analyzed by electrophoresis. The polyacrylamide gradient gel electrophoresis (10-13%) was carried out according to the method of Laemmii (Nature (1970) 227: 680-685) with a gap of the Delepelaie modifications (Proc. Nat. Acad. Sci. (1979) 76: 111-115). Sodium dodecyl sulfate was used in the bottom buffer solution higher than 0.1% but was omitted from the bottom bottom watering solution, piling and stirring the gels. The stacked gel containing 5% of a 30% existing acrylamide (29.2% acrylamide, 0.8% of N.N'-bis-methyleneacrylamide, p / b). 0.06% ammonium persulphate (w / v) and 0.1% TEMED (v / v). The stirred gel contains a linear gradient of 10-15% of existing acrylamide stabilized by a linear gradient of 0-10% sucrose. Electrophoresis was carried out at room temperature at 150V, constant voltage, for 9-10 hours. The proteins were visualized by staining them with silver according to the method of Blum et al., (Electrophoresis (1987) 8: 93-99 or with Coomassie Blue (0.1% Coomassie Blue R-250, 505 methane, 10% acetic acid The 33 kDa protein was identified as wax synthase that does not appear as a main component of the active fraction until purification through the hydroxyapatite column After purification protocol 1 (example 3C) of the single protein which correlates with the activity in the final column is a 33 kDa Example 4 - Preparation of Jojoba Wax Synthase Protein for Gel Digestion A. Preparation of SDS-PAGE Samples by Concentration Odd numbered fractions of the flow through the washing of the final HA column (Protocol 1) were poured and concentrated three times by ultrafiltration in a pressure wax placed with a YM 30 membrane (Amicon, Inc., Beverly, MA). concentrated using two Centricon-30 units (Amicon, Inc., Beverly, MA) at volumes of approximately 50 μl. Each sample was treated with 6 μl of SDS cocktail (4 μl of 20% SDS, 1 μl of 14.3M of β-mercaptoethanol and 1 μl of 0.1% Bromophenol Blue). After stirring at room temperature for 15 minutes. The samples were applied to a 10-13% acrylamide gradient gel (Example 3D) (16 x 16 cm x 1 mm thick) and the proteins were dissolved by electrophoresis at 150V, the constant voltage, for 9.5 hours. The gel was treated with 0.1% Coomassie Blue in 50% methanol, 10% acetic acid for 15 minutes after fade with 50% methane, 10% acetic acid for 2 x 20 minutes. The 33 kD Sintasa Wax band was excised from the gel and destained in 505 ethanol for 3 x 20 minutes. A line that contains a protein streak and was not used in the final digestion. B. Preparation of Samples for SDS-PAGE by Precipitation Aliquots (0.8 mL) of the enumerated fractions of the final HA column (Protocol 1) were combined in groups of three on the column profile. The protein was precipitated by the addition of 0.2 ml of 40% TCA. After 30 minutes on ice the samples were centrifuged (12,000 x g, 15 minutes at 4 ° C) to pellet the precipitated protein. The supernatants were removed and the pellets were washed twice with 0.6 ml of acetone cooled on ice. The final three pellets from each pool of the samples were resuspended with the same 50 μl of the SDS sample buffer solution by transferring the buffer from one vial the next. Vacuum flasks that were easily resuspended were washed with 10 μl of the buffer solution for a total resuspended volume of 60 μl of each poured sample. Samples were applied to 12% Tris / Glycine mini-gel acrylamide (Novex, San Diego, CA 1.5 mm. x 10 wells) and the proteins were dissolved by electrophoresis at 150 V, constant voltage during 20 minutes of the gel fat dye elution. The gel was stained with Coomassie Blue and destained using Gel-Clear (Novex, San Diego, CA). The Wax Synthase was excised from three non-equivalent forms in the gel representation of the peak and the back fractions of the column. Gel sections were placed in 1.5 ml flasks and destained with 1 ml of 50% methanol, 10% acetic acid for 2 hours. The destained solution was removed from the gel sections that were frozen in liquid nitrogen and sent on dry ice overnight to the WM Keck Foundation Biotechnology Resource Laboratory at Yale University for gel digestion. A gel cut of the sample was concentrated by ultrafiltration and three gel sections of the samples were concentrated by precipitation were poured during tryptic gel digestion. Example 5 - Determination of Amino Acid Sequence Protein sequencing was carried out in W.M. Keck Foundation Biotechnology Resource Laboratory, Yale University. The procedures include amino acid analysis of a portion (10-15%) of the gel cut for the quantification and composition of amino acids, the digestion of the protein with one of the proteolytic enzymes (trypsin or lysyl endopeptidase) and the fractionation of the products of reverse phase CLAR. The absorbance peaks were selected from the HPLC operation and subjected to laser desorption mass spectrometry to determine the presence, amount and mass of the peptide prior to protein sequencing. The lower peptides were selected for micro-sequencing. The amino acid sequences of jojoba wax synthase peptides obtained by trypsin digestion are presented in Table 3 below using the last code. Table 3 Amino Acid Sequence of Synthetic Triphosphate Synthetic Peptide Peptides Wspep29 FVPAVAPHGGALR Wspep33 TIDEYPVMNFNYTQK The isolation of the wax synthase nucleic acid sequences from the cDNA or genomic DNA libraries are described. A. Construction of Jojoba ADNc Banks RNA was isolated from jojoba embryos recovered at 80-90 days post-anthesis using a polyribosome isolation method, initially described by Jackson and Larkins (Planta Physiol. (1976) 57: 5- 10), as modified by Goldberg et al. (Developmental Biol. (1981) 83: 201-217). In this procedure all steps, less specifically state, were carried out at 4 ° C. 10 gm of tissue were seeded in liquid nitrogen in a Waring Mixer until the tissue became a fine powder. After the liquid nitrogen was evaporated, 170 ml of the extraction buffer (200 mM Tris pH 9.0, 16 mM KCI, 25 mM EGTA, 70 mM MgCl12, 1% Triton x-100, 0.5% disoxycholate sodium, 1 mM spermidine, 10 mM β-mercaptoethanol and 500 mM sucrose) were added and the tissue was homogenized for approximately 2 minutes. The homogenate was filtered through sterile Miracloth and centrifuged at 12,000 x g for 20 minutes. The supernatant was decanted into 500 ml of sterile flask and 1/19 volume of a 20% detergent solution (20% Brij 35, 20% Tween 40, 205 Noidet p-40 w / v) were added to room temperature. The solution was stirred at 4 ° C for 30 minutes at a moderate rate and the supernatant was then centrifuged at 12,000 x g for 30 minutes. Approximately 30 ml of the supernatant will be aliquoted into sterile Ti 60 centrifuge tubes and extended under 7 ml of a solution containing 40 mm. of Tris pH 9.0, 5 mM EGTA, 200 mM KCI, 30 mM MgCl12, 1.8 M sucrose, 5 mm. of β-mercaptoethanol. The tubes were filled with the extraction buffer, one rotation at 60,000 rpm for 4 hours at 4 ° C in a Ti60 rotor. After centrifugation, the supernatant was dispersed and 0.5 ml of the buffer solution was resuspended (40 mM Tris pH 9.0, 5 mM EGTA, 200 mM KCI, 30 mM MgCl 2, 5 mM β-mercaptoethanol) were added to each tube. The tubes were placed on ice for 10 minutes. After which the pellets were resuspended and poured completely. The supernatant was then centrifuged at 120 x g for 10 minutes to remove the insoluble material. A volume of 1 mg / ml of self-digested proteinase K in 20 mM tris pH 7.6, 200 mM EDTA, 2% N-lauryl-sarcosinate were added to the supernatant and the mixture was incubated at room temperature for 30 minutes. The RNA was precipitated by adding 1/10 volume of sodium acetate and 2 volume of ethanol. After several hours at 20 ° C the RNA was formed into pellets by centrifugation 12,000 x g at 4 ° C for 30 minutes. The pellets were resuspended in 10 ml of TE buffer (10 mM Tris, 1 mM EDTA) and extracted with an equal column of Tris pH 7.5 of saturated phenol. The phases were separated by centrifuging at 10,000 x g for 20 minutes at 4 ° C. The aqueous phase was removed and the organic phase was re-extracted with a volume of TE buffer. The aqueous phases were then poured out and extracted with a volume of chloroform. The phases were again separated by centrifugation and the aqueous phase of precipitated ethanol was previously described, to give the polyribosomal RNA. The polysaccharide contaminants in the polyribosomal RNA preparation were removed by the operation of RNA on a cellulose column (Sigma-cell 50) in high salt buffer (0.5 m NaCl, 20 mM Tris pH 7.5, 1 mM EDTA, 0.1% SDS). The contaminant junctions in the column and the RNA were recovered in the eluent. The fractions of the eluent were poured and the RNA is precipitated ethanol. The total RNA precipitated was then resuspended in a smaller volume and applied to an oligo d (T) cellulose column to isolate the polyadenylated RNA. Polyadenylated RNA was used to construct a library of ANDc in the cloning vector of plasmid pCGN1703, derived from the commercial cloning vector Bluescribe M13- (Stratagene Cloning Systems; San Diego, CA) and they were done in the following way. The Bluescribe M13 polylinker was altered by digestion with ßat? HI, bean endonuclease treatment and blunt end joining to create a BamYW suppression plasmid, pCGN1700. PCGN1700 was digested with EcoRI and Sst \ (adjacent restriction sites) and annealed with a synthetic linker having restriction sites for ßamHI, Pst \, Xba \, Apa \ and Sma \, 5 'overhang of AATT and 3' overhang of TCGA. The insertion of the linker in pCGN1700 removes the coRI site, recreates the site of Sst \ (also, some times not named as "Sacl" in the present) found in Bluescribe and aggregates in the new restriction sites contained in the linker. The resulting plasmid pCGN1702 was digested with Hind \\\ and turned blunt with Klenow enzyme; the linear DNA was partially digested with PvuW and a ligand with T4 DNA wax synthase in diluted solution. A transformant having the deleted lac promoter region is selected (pCGN1703) and used as the plasmid cloning vector. In summary, the cloning method for cDNA synthesis is as follows. The plasmid cloning vector was targeted with Sst and the T homopolymer tails were generated at the 3 'pendant retention ends using the deoxynucleotidyl transferase. The terminated plasmid was separated from undigested or unfinished plasmid by oligo (dA) -cellulose chromatography. The resulting vector serves as the primer for cDNA synthesis of cDNA major normals covalently linked either to the end of the vector plasmid. The cDNA-mRNA vector complexes were treated with terminal transferase in the presence of deoxyguanosidine triphosphate, generating G endings at the ends of the normal cDNAs. The extra cDNA-mRNA complex, adjacent to the Bam \? \ Site, was removed by the BamH \ digestion, allowing a cDNA-mRNA vector complex with a Bam retention end at one end and a G termination at the other. This complex was cyclized using a recognized synthetic cyclisation linker having a BamHl retention endpoint 57 recognition sequences for the restriction enzymes? / Ofl, EcoRI and Sst? And a C 3 'terminus terminus. After binding and repair of the circular complexes they were transformed into E. coli strain DH5a (BRL, Gaithersburg, MD) to generate the cDNA library. The cDNA library of jojoba embryos containing between approximately 1.5x106 clones with an average cDNA insert size of approximately 500 base pairs. Additionally, polyadenylated jojoba RNA was also used to construct a cDNA library in the IZAPU / EcoRI cloning vector (Stratagene, San Diego, CA). The bank was constructed using protocols, DNA and bacterial strains were supplied by the manufacturer. The clones were packed using Gigapack Gold packaging extracts (Stratagene), also according to the manufacturer's recommendations. The cDNA library was constructed in this form containing approximately 1 x 10 6 clones with an average cDNA insert size of approximately 400 base pairs. B. Synthetic oligonucleotides In general, to be used as PCR primers of the pattern of DNA a single strand transcribed in reverse form of mRNA, the oligonucleotides containing the sense orientation sequence corresponding to the wax synthase peptide encoding the sequences were prepared. These oligonucleotides were used as primers for the "forward" amplification reaction as described above. Where the wax synthase peptide sequences contain amino acids that can be encoded by a number of different codons, the forward or reverse primers can "degenerate" oligonucleotides, i.e. they contain a mixture of all or some of the possible coding sequences for a particular peptide region. To reduce the number of different oligonucleotides present in said mixture, it is preferable to select the peptide regions having at least a number of possible coding sequences when preparing the synthetic oligonucleotides for the PCR indicators. Similarly, where synthetic oligonucleotides can be used to directly screen a library for wax synthase sequences, oligonucleotides of lower degeneracy are preferred. The following is an example of the Wspep29 peptide sequence (center line) and the forward peptide (upper line) and the reverse peptide (lower line) the DNA sequences that can encode the WSpep29 peptide. 5 'TTY GTN CCN GCN GTN GCN CCN CAY GGN GGN GCN YTN MGN 3' F V P A V A P H G G A L R 3 'AAR CAN GGN CGN CAN CGN GGN GTR CCN CCN CGN RAN KNC 5' - An example of the WSpep33 peptide sequence (central line) and the forward (upper line) and reverse (lower line) DNA sequences that can encode the Wspep33 peptide are given below.
'ACN ATH GAY GAR TAY CCN GTN ATG TTY AAY TAY ACN CAR AAR 3' T I D E Y P V M F N Y T Q K 3 'TGN TAD CTR CTY ATR GGN CAN TAC AAT TTR ATR TGN GTY TTY 5' Synthetic oligonucleotide sequences that can be used to obtain the wax synthase sequences are given below. The oligonucleotide names reflect the numbers of particular wax synthase peptide fragments as listed in Example 6. The last "F" in the name of oligonucleotides designates a forward PCR reaction primer. The last "R" designates a reverse reaction initiator of PCR. WSpep29-F1 5 'TTYGTNCCNGCNGTNGC 3' WSpep29-F2 5 'GCNCCNCAYGGNGGNGC 3' WSpep29-R1 5'GCNCCNCCRTGNGGNGC 3 'WSpep29-R2 5' GCNACNGCNGGNACRAA 3 'WSpep33-F1 5' ACNATHGAYGARTAYCCNGT 3 'WSpep33-F2 5' CCNGTNATGTTYAAYTAYAC 3 'WSpep33- R1 5 'TTYTGNGTRTARTTRAACAT 3' WSpep33-R2 5 'AACATNACNGGRTAYTCRTC 3' An oligonucleotide, TSYN is used for reverse transcription of poly (A) + or total RNA to prepare single stranded DNA to be used as the PCR standard. In addition a poly (T) region for binding to the poly (A) tail of mRNA, the oligonucleotide containing the restriction digest sequences for HlndlW, Pst and Sst. The sequence of TSYN is as follows: TSYN 5 'CCAGCTTCTGCAGGAGCTCTTTTTTTTTTTTTTT 3' An oligonucleotide, TAMP, is useful in the reverse reaction of RCP for amplification of the contrasense thread of a wax synthase coding sequence. It was observed that where the PCR pattern is a single-stranded DNA-transcription mRNA, the reverse reaction did not occur until the first forward reaction was completed. The first yarn reaction results in the production of a normal sense pattern which can then be used in the amplification of the DNA counter-sense yarn from the reverse primer. The sequence of TAMP in the following way: TAMP 5 'CCAAGCTTCTGCAGGAGCTC 3' The base codes of nucleotides for the above oligonucleotides according to the normal of IUPAC are as follows. A = adenine T = thymine Y = cytosine or thymine C = cytosine U = uracil R = adenine or guanine G = guanine I = inosine O = inosine or cytosine H = adenine, cytosine or thymine D = adenine, guanine or thymine N = adenine , cytosine, guanine or thymine. W = adenine or thymine "S = guanine or cytosine B = guanine, cytosine or thymine K = a guanine or thymine M = adenine or cytosine C. Poly (A) + RNA PCR reactions were isolated from the total RNA prepared from jojoba tissue as described above. Single stranded cDNA was prepared from poly (A) + or total RNA by reverse transcription using Superscript reverse transcriptase (BRL) and TSYN as the oligonucleotide primer. The reaction was carried out according to the directions of the manufacturer, except that the reaction was operated at 45 ° C instead of 37 ° C. The single-stranded jojoba cDNA was used in the PCR 1-2 reactions as discussed below. RCP was carried out on a Perkin Elmer Cetus GeneAmp RCP System 9600 RCP machine using single-stranded reverse transcription cDNA as a standard. The commercially available PCR reaction and the optimization reagents were used according to the manufacturer's specifications. The portion of the cDNA located in 30 of the above primers can be amplified in the following reactions: Reaction Initiator Forward Reverse Initiator 1 WSpep29-F1 TAMP 2 WSpep29-F2 TAMP 3 WSpep33-F1 TAMP 4 WSpep33-F2 TAMP 5 WSpep29-F1 WSpep33- R1 6 WSpep29-F2 WSpep33-R1 7 WSpep33-F1 Wspep29-R1 8 WSpep33-F2 Wspep29-R2 9 WSpep29-F1 Wpep33-R2 10 WSpep29-F2 WSpep33-R2 11 WSpep33-F1 WSpep29-R2 12 WSpe ~ p33-F2 WSpep29 -R2 If further amplification is necessary, the PCR products of the reactions containing the primers were designated - F1 which can be used as the template for a second round of the PCR reactions using the designated primers -F2. Similarly, the PCR products of the reactions containing the primers were designated -R1 which can be used as a standard for a second cycle of the PCR reactions using the designated primers -R2. Alternatively, the total cDNA can be amplified using 5 'and 3' RACE (Frohman et al., 1988) using the Marathon CNA Amplification Kit (Clontech Laboratories Inc.) according to the manufacturer's instructions. The primers WSpep29-F1, WSpep29-F1, WSpep33-F1 and WSpep33-F2 were used for the 3 'RACE reactions. The primers WSpep29-R1, WSpep29-R1, WSpep33-R1 and WSpep33-R2 were used for the 5 'RACE reactions. The DNA fragments generated in the PCR reactions were cloned into pCR2.1 according to the manufacturer's protocol (Invitrogen Corp). The DNA sequence of the cloned fragments was determined to confirm that the cloned fragments encoding the wax synthase peptides. D. Sieving Banks for the Wax Synthase Sequences The wax synthase DNA fragments obtained by PCR were labeled and used as a probe to screen the clones from the cDNA libraries described above. Screening techniques of DNA libraries are known to those skilled in the art and are described, for example, in Maniatis et al. (Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press). In this form, the wax synthase nucleic acid sequences were obtained which can be analyzed for the nucleic acid sequence and used for the expression of wax synthase in various hosts, both prokaryotic and eukaryotic. Example 6 - Wax Synthase and Reductase Constructs for Plant Expression Constructs that provide for the expression of wax synthase and reductase sequences in plant cells can be prepared in the following manner. The expression ribbons containing the 5 'and 3' regulatory regions of the genes preferentially expressed in seed tissues can be prepared from genes of napin, Bce4 and ACP as described in the example in WO 92/03564. For example, a napkin expression tape, pCGN1808, which can be used for the expression of wax synthase constructs or reductase gene are described in Kridl et al. (Seed Science Research (1991) 1: 209-219). An additional napkin expression band, pCGN3223, containing a background of ampicillin resistance, and essentially identical 1,725 napin 5 'and 1,265 30 regulatory sequences as found in pCGN1808. The regulatory regions are flanked by restriction sites Hlnd \\\, Not \ and Kpn \ and cloning sites Sa / I, Sg / ll, Pst \ and Xho \ are located between the uncoded regions 5 'and 3'.
A cassette can also be used for the cloning of sequences for transcriptional regulation under the control of the 5 'and 3' regions of an oleosin gene. The sequence of an oleosin gene from Brassica napus was reported by Lee and Huang (Plant Phys. (1919) 96: 1395-1397). The sequence of an oleosin cassette, pCGN7636, is provided in Figure 4 of USPN 5,445,947. The oleosin tape is flanked by the restriction sites SssHIl, Kpn \ and Xal and contains the sites Sa / I, BamYW and Pst \ for the insertion of the wax synthase, reductase or other DNA sequences of interest between the regions of oleosin 5 'and 3'. Wax synthase and reductase gene sequences can be inserted into said cassettes to provide the expression constructs for plant transformation methods. For example, a construct for the expression of reductase plant cells using the 5 'and 3' regulatory regions of a napin gene was described in USPN 5,445,947. Constructs of the binary vector are transformed into Agrobacterium cells, such as strain EHA101 (Hood et al., J. Bacterio! (1986) 168: 1291-1301), by the method of Holsters et al. (Mol. Gen. Gente. (1978) 163: 181-187) and were used in plant transformation methods as described below. Example 7 - Diacylglycerol Acyltransferase (DAGAT) Analysis Methods for analyzing the activity of DAGAT in solubilized and solubilized protein preparations are described for Mortierella ramanniana.
A. Non-solubilized samples The activity of DAGAT was analyzed with 3.67 μm of the coenzyme of 1-14C-18: 1 A (53.5-54.5 Ci / moles, New England Nuclear, Boston, MA) and 1.5 mM of 1,2- 18: 1 diacylglycerol (DAG) (Sigma D-0128, prepared as 140 mM reserves in 2-methoxyethanol) in a buffer containing 10 mM potassium phosphate (pH 7.0), 100-150 mM KCI and 0.1 % TX-100 (w / v) in a total volume of 100 μl as similarly described by Kamisaka et al. (1993, 1994). The analyzes were carried out at 30 ° C for 5 minutes and were terminated with the addition of 1.5 ml of heptane: isopropanol: 0.5M H2SO (10: 40.1, v / v / v). If necessary, the samples can be diluted with the above buffer to be analyzed in order to maintain a linear regimen of product formation during the analysis. B. Solubilized samples. The analyzes were carried out as described for the samples not solubilized with the following changes: the amount of 1,2-18: 1 of DAG is reduced to 0.5 mM, the amount of Triton x-100 was increased to 0.2% and the concentration of KCI was maintained between 110-125 mM. Also if necessary to include La-phosphatidic acid (Sigma P-9511, prepared as 40 mM reserves in 1% Triton X-100 (w / v)) to recover the activity after solubilization with detergent as described by Kamisaka et al. (1996, 1997), with slight modification of the protocol. We found that using 300 μM of phosphatidic acid instead of 500 μM gives a superior stimulation of the DAGAT activity following the treatment by Triton x-100. We also found that the DAGAT activity is sensitive to the amount of KCI entering the analysis with the optimal level between 100-125 mM. The analyzes were carried out at 30 ° for 5-30 minutes and were finished as described for the non-solubilized samples. C. Sample Analysis Processing After the analyzes were stopped, they could be stored at 4 ° C during processing to a data process last or immediately by the addition of 0.1 ml of NaHCO3 followed by 1 ml of heptane containing 15 ml. mmoles / ml of triolein as a vehicle for extraction. The contents were stirred and, after separation of the aqueous and organic phases, the upper organic phase was stirred into a new glass jar and washed with 1 ml of 1M NaCl. Forty percent of the final organic phase was removed for liquid scintillation counting and the remaining organic phase was transferred to a clear flask and evaporated to dryness under nitrogen gas. The residue was resuspended in 45 μl of hexane and stained on a silica gel-G, glass, thin layer chromatography plate (TLC) with a pre-absorbent loading zone (Analtech # 31011, Newark, Delaware). The CCF plate was developed in hexane: dithyl ether: acetic acid (50.50: 1, v / v / v) to then stop the dryness and sweep it by a radioimaging analyzer (AMBIS 3000, San Diego, CA) to determine the portion of the radioactivity incorporated in triacylglycerol. The activity was reported in units as pmol / min. Example 8 - Growth and Crop of Mortierella ramanniana Cultures Mortierella ramanniana was cultivated by inoculation of 1 liter of Defined Glucose Medium (30 g of glucose, 1.5 g of (NH) 2SO4, 3 g of K2HPO4, 0.3 g of MgSO4 »7H2O, 0.1 g of NaCl, 5 g of CH3COONA.3H2O, 10 mg of FeSO4» 7H2O, 1.2 mg of CaCl2 «2H2O, 0.2 mg of CuSO4« 5H2O, 1.0 mg of ZnSO4 »7H2O, 1.0 mg of MnCI» 4H2O, 2 mg of thiamine-HCI and 0.02 mg of biotin in 1 liter of purified water by reverse osmosis (pH 5.7 )) with 1.5-3 x 106 spores and incubated at 30 ° C with shaking at 200 rpm for 9-11 days. The cultures were harvested by filtration through a Miracloth layer (CaLbiochem, La Jolla, CA). The excess liquid was removed by manual compression. The average yield of packed cells harvested per liter is 22.5 g. Example 9 - Gradient Gel Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis (SDS-PAGE) The column fraction samples are diluted in SDS-PAGE sample buffer (1x watering solution = 2% SDS, 250 mM of β-mercaptoethanol, 0.0025% of bromophenol blue) and were analyzed by electrophoresis. The polyacrylamide gradient gel electrophoresis (10-13%) was carried out according to the method of Laemmii (1970) with some modifications of Delepelaire (1979). Sodium dodecyl sulfate was used in the reserve solution in excess of 0.1% but was omitted from the lower stock solution, stacking them and stirring the gels. The stacked gel contains 5% of a stock of acylamide (acrylamide: N, N'-Methylene-acrylamide, 37.5: 1, Bio-Rad, Hercules, CA), 0.06% ammonium persulfate and 0.1% TEMED (v / v). Scrambled gels containing a linear gradient of 10-13% acrylamide stock stabilized by a linear gradient of 0-10% sucrose. Electrophoresis was carried out at room temperature at 150V, constant voltage, for 7-9 hours. The proteins were visualized by tifiándolas with plant according to the method of Blum and others (987) or with Coomassie Blue (0.15 Coomassie Blue R-250, 505 methanol (v / v), 105 acetic acid (v / v). Example 10 - Evaluation of the Chromatography Used by Kamisaka et al. (1997) in the purification of DAGAT of Mortierella ramanniana A. Preparation of the Body Fraction of Lipids The following steps were carried out at 4 ° C. Normally 70-75 g of humid packaging cells (stored at -70 ° C) were used for each lipid body preparation Just before use, the cells were melted on ice and resuspended in 150 ml of buffer solution A (10 mM potassium phosphate). (pH 7.0), 0.15 M KCI, 0.5 M sucrose, and 1 mM EDTA.) The following protease inhibitors were added to reduce proteolysis: 0.1 μM Aprotinin, 1 μM Leupeptin, and 100 μM Pefabloc (all from Boehringer Mannheim, Germany) .The cells were divided into zinc or 50 ml tubes and were used with a Polytron Tissue Homogenizer (Kinematic GmbH, Brinkman Instruments, Switzerland) at position # 7 with a 1 cm diameter probe for 7 x 1 minutes. The resulting slurry was transferred to centrifuge tubes (29 x 104 mm.) And the solids residues were made into pellets by rotating at 1500 xg (Beckman Instruments, rotor J2-21, JA-20, 3500 rpm) for 10 minutes. minutes at 4 ° C. The supernatant was removed and the pellets were washed with another 5 ml of buffer A. After centrifugation, the volumes of supernatants were combined. This fraction is called "S1". The S1 was divided into six ultracentrifuge tubes (25 x 89 mm, Beckman Instruments, Fullerton, CA) and each is coated with 5 ml of buffer B (10 mM potassium phosphate pH 7.0, KCI 0.15 M, sucrose 0.3 M and 1 mM EDTA). The samples were 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 coating, was recovered with a spatula and transferred to a glass homogenizer (Potter-Elvehjem). Small amounts of LBF remaining in the centrifuge tube were recovered by pipetting by removing 4 ml of buffer B and coating and combining with LBF in the homogenizer. The final LBF was homogenized in 40 ml of Regulatory Solution B. The remaining fractions were recovered as follows: the fraction interfaces (the interfaces between 0.3 and 0.5 M of sucrose buffer solutions), the soluble fraction (the interfaces of low volume of liquids) and the membrane fraction (a bronze / coffee pellet at the bottom of each tube). All were frozen and stored at -70 ° C awaiting further solubilization and purification. B. Solubilization of the DAGAT Activity of the Lipid Body Fraction LBF was frozen on ice and the solubility was improved by the addition of Triton x-100 (Boehringer Mannheim, Mannheim, Germany) from a reserve of 10% (w / v ) at a final concentration of 1.3% (w / v). Solid sucrose (Mallinckrodt, Paris, Kentucky) was added to achieve a final concentration of 0.5 M. The sample treated with detergent was balanced at 4 ° C for one hour then divided into six ultracentrifuge tubes (25 x 89 mm. , Beckman Instruments). Each tupe was coated with 5 ml of buffer B. The samples were centrifuged at 100,000 x g (Beckman Instruments, L8-M, SW-28 rotor, 21000 rpm) at 4 ° C for 3 hours. The solubilized material, termed as the "Triton x-100 extract" was recovered by inserting a thin tube through the coating within 1 cm of the bottom of each ultracentrifuge tube and removing the lower sucrose of 0.5M. the layer with gentle suction allowing the coating of sucrose of 0.3M higher (including a layer of floating grease) and the back pellet. In the protocol described by Kamisaka et al. (1997), the Body Fraction of lipids was solubilized with 0.1% Triton x-100 and then centrifuged at 100,000 x g or filtered through a 0.2 μm filter. We found that it is necessary to increase the concentration of Triton X-100 to 1.5% for the DAGAT activity to join the first column. C. Chromatography used in the DAGAT Purification of M. ramanniana Regulating solution C for chromatography was used, containing 10 mM potassium phosphate (pH 7.0), 0.1% Triton x-100 (w / v) (Boehringer Mannehim, Mannheim, Germany), 10% glycerol (w / v), 0.1 μM Aprotinin, 1 μM Leupeptin, 100 μM Pefabloc (all from Boehringer Mannheim, Mannheim, Germany) and varying amounts of potassium chloride (75-500) mM). This regulatory solution differs from the buffer solution of the corresponding column used by Kamisaka et al. (1997) in the glycerol substituent for ethylene glycol and EDTA, DTT and PMSF were omitted while including Aprotinin, Leupeptin and Pefabloc. Following the protocol of Kamisaka et al. (1997), a yellow Agarose column 86 (Sigma R-8504, St. Louis, MO) was prepared (1.5 cm x 5.8 cm) and equilibrated with 150 mM KCl in buffer solution C The DAGAT activity, present in the Triton X-100 extract, was combined to the Yellow Agarose 86 column under these conditions but we found that most DAGATs do not bind to the column. They were able to bind a significant portion of the DAGAT activity to the column by diluting the KCI concentration of the sample applied at 75 mM with an equal volume of regulatory solution C (without KCI). According to the Yellow Agarose column 86 it was also equilibrated in 75 mM KCl in Regulatory Solution C. After application of the sample at 0.56 ml / min, the column was washed with 4 column volumes of the buffer of balance. The DAGAT activity and the proteins were bound to the column and eluted with 500 mM KCl in Regulatory Solution C (Figure 4). The DAGAT Activity eluted from the Yellow Agarose column 86 (Fractions 17-20) were diluted in 1: 3.33 with Regulatory Solution C to recover the concentration of KCI at 150 mM. The diluted combination (103 ml) was applied to a CL-6B column of Heparin-Sepahrose (Pharmacia, Uppsala, Swede, 0.5 cm x 4.8 cm) equilibrated with 150 mM KCl in buffer C at 0.2 ml / min. The column was washed with 5 volumes of equilibrium buffer and the activity of DAGAT and the protein eluted in a linear gradient of 15 ml of 150-500 mM KCl in buffer C. DAGAT activity elidated in two overlapping peaks . The first peak elutes during the gradient, as found by Kamisaka and others, (1997) and a second peak, not found by Kamisaka and others, eluted at the end of the gradient with much less protein (Figure 5A). A portion (250 μl) of the two peak fractions of the Heparin column were 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 Regulatory Solution C. For calibration only, the column was equilibrated with 150 mM KCl in a modified C-regulatory solution in which Triton X-100 was replaced with Triton X-100 R (Calbiochem, La Jolla, CA). The column was calibrated using Normal Bio-Rad Gel Filtration. The DAGAT activity of each of the two peaks of Heparin-Sepharose CL-6B elutes at a calculated molecular mass of 99 kDa. Additional chromatography was carried out on the last elution peak of the Heparin column, which contains DAGAT at a higher specific activity. In this case, the second peak of the Heparin column (fractions 36-41) was diluted 1: 6.6 with Regulatory Solution C for a volume of 46.7 ml. The sample was applied to a Yellow 86 agarose column (1.0 cm x 6.4 cm) equilibrated with 75 mM KCI in buffer at 0.5 ml / min. After washing with 5 column volumes of the equilibrium watering solution, the binding proteins and all the DAGAT activity eluted in a linear gradient of 40 ml of 75-500 mM KCl in buffer C. DAGAT activity eluted as a single peak (Figure 6A). The protein composition of the fractions containing the DAGAT activity from the Heparin and second columns of Yellow 86 were analyzed by the SDS-PAGE gradient according to the protocol in Example 3. The protein bands were detected by the stained plant. The pattern of the elution bands of these columns was compared by fraction by fraction, in the respective DAGAT activity profile. The main protein candidates are in the present that correlate with the presence of DAGAT activity. It is our opinion that the purification protocol is insufficient to identify a candidate protein particle associated with DAGAT activity (Figure 5B, 6B). Example 11 - New purification protocol for identifying the DAGAT protein candidates purified from Mortierella ramanniana A. Preparation of the Lipid Body Fraction. The following steps were carried out at 4 ° C. Normally 70-75 g of wet packaging cell (stored at -70 ° C) were used for each lipid body preparation. Just before use, the cells were melted in wire and responded in 150 ml of buffer A (10 mM potassium phosphate (pH 7.0), 0.15 M KCI, 0.5 M sucrose, and 1 mM EDTA). The following protease inhibitors were added to reduce proteolysis: 0.1 μM of Aprotinin, 1 μM of Leupeptin and 100 μM of Pefabloc (all from Boehringer Mannheim, Germany). The samples were used with a glass bead switch. The sample chamber was filled with 180 ml of glass beads. The moist packing cells were melted on ice and resuspended in 150 ml of Regulatory Solution A. The cell slurry was poured onto glass beads. In general, an additional buffer solution of 40-50 ml is necessary to fill the chamber for proper operation. This volume was used to raise the slurry of remaining cells from their container originating so that it can be combined with the rest of the sample. The cells were prepared ("homogenized" gradation) for 45-90 seconds depending on the viscosity of the sample. The cellular slurry containing glass beads was divided into tubes (29 x 104 mm.) And centrifuged at 500 x g (Beckman Instruments, GP centrifuge, GH 3.7 Horizontal rotor at 150 rpm) and 4 ° C. The supernatant was removed and the pellets were washed with another 5 ml of buffer A. After centrifugation the volumes of the supernatant were combined. The fraction refers to "S1". The S1 was divided into six ultracentrifuge tubes (25 x 80 mm., Beckman Instruments) and each was coated with 5 ml of modified buffer solution B (10 mM potassium phosphate pH 7.0, 0.15 M KCI and 0.3 M sucrose). EDTA was omitted from buffer B (See Example 4) because it interferes with hydroxyapatite chromatography. The samples were 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 coating, was recovered with a spatula and transferred to a glass homogenizer (Potter-Elvehjem). Small amounts of LBF remaining in the centrifuge tube recovered by pipetting by removing 4 ml of buffer solution B coated and combined with LBF in the homogenizer. The final LBF was homogenized in 40 ml of Regulatory Solution B. The remaining fractions were recovered as follows: fraction of the interface (the interface between 0.3 and 0.5 M of sucrose buffer solutions), the soluble fraction (the interface with low volume of liquids) and the membrane fraction (one bronze / brown pellet at the bottom of each tube). All were frozen and stored at -70 ° C waiting for further solubilization and purification. B. Solubilization of the DAGAT Activity of the Body Fraction of Lipids Before solubilization, a protein determination is made with an aliquot of the Body Fraction of Lipids by the Bradford method (Bio-Rad _Reagent, Hercules, CA) using bovine serum albumin as a normal. The LBF was melted in yarn, then diluted to a concentration of 1 mg protein / ml and treated with Triton X-100 to a detergent for the protein ratio of 15: 1 (w / w, equivalent to 1.3% Triton x-100). Solid sucrose (Mallinckrodt, Paris, Kentucky) was added to achieve a final concentration of 0.5M. the sample treated with detergent was treated at 4C for one hour. Then it was divided into six ultracentrifuge tubes (25 x 80 mm., Beckman Instruments). Each tube was coated with 5 ml of Modified Buffer B. The samples were centrifuged at 100,000 xg (Beckman Instruments, L- (M, rotor SW-28, 21000 rpm) at 4 ° C for 3 hours.The solubilized material, termed as the "Triton X-100 extract" was recovered by inserting a thin tube through the liner within 1 cm of the bottom of each ultracentrifuge tube and removing the lower 0.5M sucrose layer with gentle suction while reducing the top 0.3M sucrose coating (including a layer of floating grease) and the back pellet C. DAGAT Column chromatography Our experience provides purification of acyltransferase protein from plant species was applied in the case of the DAGAT purification of Mortierella ramanniana, successively in the use of hydroxylapatite chromatography in the purification of jojoba wax synthase (Simmondisia chinesis, WO 95/15387, all of which is incorporated herein by reference) and lysophosphatidyl acyltransferase co (LPAAT) of coconut (Cocos nucifera) (Patent Application of E.U.A. 08 / 231,196, the entirety of which is incorporated herein by reference). This purification step was introduced after the Yellow Agarose column 86. The Yellow Agarose 86 purification protocol followed by the hydroxylapatite was carried out in two forms. In Protocol A, the activity is joined in the first column and after the elution, the fractions were analyzed for activity. The active fractions were then poured and applied in the second column. We have referred to this as a sequential operation. In Protocol B, the activity joins the first column after eluting and directly flows into the second column without pouring and analyzing between them. We have named this as a joint operation.
In Protocol A, the extract of Triton X-100 was applied to a column of Yellow Agarose 86 (2.6 cm x 6.4 cm) equilibrated with 75 mM KCl in buffer C (Example 4.c) to 2 ml / min. The column was washed with 5 column volumes of the equilibrium buffer solution then eluted with 500 mM KCI in buffer C at 0.5 ml / min (Figure 7). The two most active fractions (64 and 65), containing 93% of the activity eluted, were poured and loaded onto a hydroxylapatite column (Bio ^ Gel HT, Bio-Rad, 1 cm x 25.5 cm) equilibrated with 500 mM of KCl in Regulatory Solution C at 0.5 ml / min. DAGAT activity flows through the column while most proteins bind to the column. The columns were washed with 3 volumes of the equilibrium buffer solution. Binding proteins were 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 peak DAGAT activity are operated on a gradient gel SDS-PAGE as described in Example 3. The proteins were stained with plant and the pattern of the bands was compared fraction by fraction to the profile of activity (Figure 8B). Several protein DAGAT candidates correlated with the activity. In particular, attention was paid to the migratory bands in the positions corresponding to 43 kDa, 36.5 kD, 33 kDa, 29 kD, 28 kD and 27 kD. This does not appear to be a protein lock in the 53 kDa region that correlates with activity.
In Protocol B, Triton X-100 extract was applied to a Yellow 86 agarose column (1.5 cm x 5.8 cm) equilibrated with 75 mM KCl in buffer solution C at 1 ml / min. The column was washed with 5 column volumes of the equilibrium buffer. Then, the exit of the Yellow Agarose column 86 was connected to the hydroxylapatite column inlet (1.0 cm x 26 cm, Bio-Rad, Hercules, CA) equilibrated with 500 mM KCl in buffer C. DAGAT activity bound to the Yellow 86 column was eluted with 110 ml of buffer C containing 500 mM KCI and passing directly through the hydroxylapatite column at 0.2 ml / min. Finally, the hydroxyapatite column was disconnected from the Yellow Agarose column 86 and the hydroxylapatite column units proteins were eluted with 100 mM dipotassium phosphate and 600 mM KCl in buffer C. The DAGAT activity was found in the fractions of the hydroxylapatite column recovered during the washing of 100 ml with buffer C containing 500 mM KCI. The majority of the protein in the Triton x-100 extract did not bind to the Yellow 86 agarose column and was discharged. A small subgroup of proteins, including DAGAT, bound to the Yellow 86 Agarose column and eluted with 500 mM KCI in the water-repellent solution C. With this eluate was applied to the hydroxylapatite column, the DAGAT activity flows through from the majority of the remaining protein units to the column and separated (Figure 9A). A portion of the fractions containing the peak DAGAT activity were operated on a gradient gel SDS-PAGE and stained with plant. The pattern of the bands eluted from these columns were compared, fraction by fraction, to the respective DAGAT activity profile examination of the stained protein bands indicated a protein at 33 kDa correlations improved with the activity of DAGAT (Figure 9B). Example 12 - Preparation of the Protein for Gel digestion. A. Preparation of the Samples for SDS-PAGE by Concentration The enumerated fractions of the flow through the washing of the final HA column (Protocol 1) were poured and concentrated three times by ultrafiltration at a pressure of cells fixed with a membrane. YM 30 (Amicon, Inc., Beverly, MA). The sample was further concentrated using two units of Centricon-30 (Amicon, Inc., Beverly, MA) at volumes of approximately 50 μl. Each sample was treated with 6 μl of SDS cocktail (4 μl of 20% SDS, 1 μl of 14.3M of β-mercaptoethanol and 1 μl of 0.1% Bromophenol Blue). After being set at room temperature for 15 minutes, the samples were applied to a 10-13% acrylamide gradient gel (Example 3D) (16 x 16 cm x 1 mm in thickness) and the proteins were stirred by electrophoresis. 150V, constant voltage, for 9.5 hours. The gel was stained with 0.1% Coomassie Blue in 50% ethanol, 10% acetic acid for 15 minutes then destained in 50% ethanol, 10% acetic acid for 2 x 20 minutes. The 33 kD Sintasa Wax band was excised from the gel and destained in 50% ethanol for 3 x 20 minutes. A line contended on a breakdown of the protein and was not used in the final digestion. B. Sample Preparation for SDS-PAGE by Precipitation The aliquots (0.8 ml) of the enumerated fractions of the final HA column (Protocol 1) were poured in groups of three on the column profile. The spills were divided equally in three 1.5 ml flasks. The protein was precipitated by the addition of 0.2 ml of 40% TC. After 30 minutes on ice the samples were centrifuged (12,000 x g, 15 minutes at 4 ° C) to pellet the started protein. The supernatants were removed and the pellets were washed twice with acetone cooled in ice 0.5 ml. The three final pellets of each poured equipment of the samples were resuspended with the same sample of 50 μl of regulatory solution transferring the regulatory solution from one vial to the next. Bottles packed with 10 μl of the sample buffer for a total resuspended volume of 60 μl of each poured sample. Samples were applied to a Tris / Glycine acrylamide mini-gel (Novex, San Diego, CA, 1.5 mm x 20 wells) and protein were stirred by electrophoresis at 150 V, constant voltage, for 20 minutes of dye elution of the gel fat. The gel was stained with Coomassi Blue and destained using Gel-Clear (Novex, San Diego, CA). The Wax Synthase was excised from three non-equivalent lines in the gel representing the peak and the rear fractions of the column. The gel sections were placed in a 1.5 ml flask and stained with 1 ml of 50% methane, 10% acetic acid for 2 hours. The destained solution was removed and the gel sections were frozen in liquid nitrogen and sent on dry ice, overnight, to the WM Keck Foundation Biotechnology Resource Laboratory at Yale University for gel digestion. A gel cut of the sample was concentrated by ultrafiltration and all three gel sections of the samples were concentrated by precipitation were poured for tryptic gel digestion. Example 13 - Determination of the Amino Acid Sequence The sequencing of the protein was carried out in W.M. Keck Foundation Biotechnology Resource Laboratory, Yale University. The procedures include analysis of amino acids of a portion (10-15% of the gel cut for the quantification and composition of amino acids, the digestion of the protein with one of the proteolytic enzymes (trypsin or lysyl endopeptidase) and the fractionation of the products by reverse phase HPLC The absorbance peaks were selected from the HPLC operation and subjected to laser desorption mass spectrometry to determine the presence, amount and more of the above peptide from the protein sequencing. Select for Microsequencing Example 14 Isolation of DAGAT Nucleic Acid Sequences from Mortierella ramanniana In general, for the use as PCR primers of the single-stranded DNA standard transcribed in reverse form of mRNA, the oligonucleotides containing the sequence of sense orientation corresponding to the DAGAT peptide encoding the sequences were prepared. These oligonucleotides were used as indicators for the "forward" amplification reaction to produce the DNA of a single sense strand. For the "reverse" reaction for amplification with the DNA strand without coding, an oligonucleotide can be designated to be identical to a portion of an initiator used to prepare the DNA standard for PCR. Alternatively, the oligonucleotides containing the sequence complementary to the DAGAT peptide encoding the sequences can be used in combination with an "frontal" oligonucleotide primer DÁGAT as described above. Where the DAGAT peptide sequences contain amino acids that can be encoded by a number of different conditions, the forward or reverse primers can be "degenerate" oligonucleotides, i.e. they contain a mixture of all or some of the possible coding sequences for a particular peptide region. In order to reduce the number of different oligonucleotides present in said mixture, it is preferable to select the regions of peptides having at least the number of possible coding sequences when preparing the synthetic oligonucleotide for the PCR primers.
Similarly, where synthetic oligonucleotides are used directly to screen a bank for DAGAT sequences, lower degeneracy of oligonucleotides is preferred. The DAGAT DNA fragments obtained by PCR were labeled and used as a probe to screen the clones of the cDNA libraries. Complementary DNA and DNA construction and bank screening techniques are known to those skilled in the art and are described, for example in Maniatis et al. (Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press). In this form, the DAGAT nucleic acid sequences are obtained which can be analyzed by the nucleic acid sequence and used for the expression of DAGAT in several hosts. Both prokaryotic and eukaryotic. Example 15 - Construction of DAGAT of Mortierella ramanniana for Plant Expression The constructs provided for the expression of DAGAT sequences in plant cells can be prepared as follows: The expression tapes containing the regulatory regions 'and 3' of the genes preferentially expressed in seed tissues can be prepared from genes of napin, Bce4 and ACP as described in the example in WO 92/03564. - For example, a napkin expression tape, pCGN1808, which can be used for the expression of wax synthase constructs or reductase gene are described in Kridl et al. (Seed Science Research (1991) 1: 209-219). An additional napkin expression band, pCGN3223, containing a background of ampicillin resistance, and essentially identical 1,725 napin 5 'and 1,265 30 regulatory sequences as found in pCGN1808. The regulatory regions are flanked with restriction sites HlndWl, Not \ and Kpn \ and cloning sites Sa / I, BglW, Pst \ and Xho \ are located between the regions without 5 'and 3' coding. A cassette for the cloning of sequences for transcriptional regulation under the control of the 5 'and 3' regions of an oleosin gene can also be used. The sequence of an oleosin gene from Brassica napus was reported by Lee and Huang (Plant Phys. (1919) 96: 1395-1397). The sequence of an oleosin cassette, pCGN7636, is provided in Figure 4 of USPN 5,445,947. The oleosin cassette is flanked by the restriction sites SssHIl, Kpn \ and Xba \ and contains the sites Salí, ßamHI and Pst \ for the insertion of the wax synthase, reductase or other DNA sequences of interest between the oleosin regions. 5 'and 3'. The DAGAT gene sequences can be inserted into said cassettes to provide the expression constructs for plant transformation methods. For example, a construct for the expression of reductase plant cells using the 5 'and 3' regulatory regions of a napin gene was described in USPN 5,445,947.
The binary vector constructs are transformed into Agrobacterium cells, such as strain EHA101 (Hood et al., J.
Bacteriol (1986) 168: 1291-1301), by the method of Holsters and others (MoL Gen. People. (1978) 163: 181-187) and were used in plant transformation methods as described below. Example 16 - Methods of Plant Transformation and Analysis A variety of methods have been developed to insert a DNA sequence of interest into the genome of a host plant to obtain transcription or transcription and translation of the sequence to effect the phenotypic changes. Varieties with high erucic acid content, such as the Rest culture, or Cañola varieties of Brassica napus can be transformed using Agrobacterium as measured by the transformation methods as described by Radke et al. (Theor, Appl. Genet. ) 75: 685-694; Plant cell Reports (1992) 11: 499-505). Transgenic Arabidopsis thaliana plants can be obtained by the transformation measured by Agrobacterium as described in Valverkens and others, (Proc. Nat. Acad. Sci. (1988) 85: 5536-5540). Other plant species can be similarly transformed using related techniques. Alternatively, micropoiectile bombardment methods, such as those described by Klien et al., (Bio / Technology 70: 286-291) can also be used to obtain the transformed plants comprising the reductase and the wax synthase expression construct described at the moment. The seed or other plant material of the transformed plants can be analyzed for DAGAT activity using the DAGAT analysis methods described in Example 1. The above results demonstrate the ability to obtain partially purified DAGAT proteins that are active in the formation of fatty acyl triacylglycerols and diacylglycerol substrates. The methods for obtaining the DAGAT proteins and the amino acid sequences thereof are provided. In addition, the DAGAT nucleic acid sequences can also be obtained from the amino acid sequences using PCR and the screening methods of banks provided in the patents. Said nucleic acid sequences can be manipulated to provide transcription of the sequences and / or expression of DAGAT proteins in host cells, whose proteins can be used for a variety of applications. Such applications include the modification of the levels of triacylglycerols and the compositions of the host cells. All publications and patent applications cited in this specification are incorporated herein by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the above invention has been described in some detail by way of illustration and example for the purposes of clarity and understanding, it should be readily apparent to those of ordinary skill in the art in view of the teaching of this invention that certain changes can be made and modifications in it without departing from the spirit and scope of the appended claims.

Claims (5)

  1. CLAIMS 1. A substantially purified acyltransferase wherein said acyltransferase is active in the formation of TAG of diacylglycerol and fatty acyl-CoA substrates.
  2. 2. The acyltransferase according to claim 1 having the activity in the 10/10-DAG direction.
  3. 3. An acyltransferase protein, wherein the protein has an apparent molecular mass of approximately 33kD in SDS-PAGE, said protein being substantially produced in the form of membranes and other proteins of the native cell and capable of catalyzing the production of triglycerides of , 3-diacylglycerol and an acyl-CoA.
  4. 4. The diacylglycerol acyltransferase of claim 3 having activity in the 18: 1 direction of the fatty acyl-CoA substrate.
  5. 5. The diacylglycerol acyltransferase of claim 4 obtainable from Mortierella rammanniana.
MXPA/A/1999/011201A 1997-06-05 1999-12-03 Diacylglycerol acyl transferase proteins MXPA99011201A (en)

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