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Timestamp: 2018-02-20 04:03:30
Document Index: 763449082

Matched Legal Cases: ['§ 1', 'Application No. 60', 'Application No. 60', 'Application No. 60', '§1', '§ 1']

Aspartate kinase - E. I. du Pont de Nemours and Company
United States Patent 7345220
Falco, Saverio Carl (Wilmington, DE, US)
11/034569
435/6.11, 435/6.15, 435/6.16, 435/69.1, 435/183, 435/252.3, 435/320.1, 435/419, 435/468, 530/370, 536/23.6, 800/278
A01H1/00; C12N9/12; C12N15/82; C07H21/04; C07K14/415; C12N5/14; C12N9/00
536/23.6, 435/468, 435/6, 530/370, 435/419, 435/69.1, 435/320.1, 435/252.3, 800/295, 800/278
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6459019 Chimeric genes and methods for increasing the lysine and threonine content of the seeds of plants 2002-10-01 Falco et al.
5850016 Alteration of amino acid compositions in seeds 1998-12-15 Jung et al. 800/287
5773691 Chimeric genes and methods for increasing the lysine and threonine content of the seeds of plants 1998-06-30 Falco et al.
5451516 Bifunctional protein from carrots (Daucus carota) with aspartokinase and homoserine dehydrogenase activities 1995-09-19 Matthews et al.
5258300 Method of inducing lysine overproduction in plants 1993-11-02 Glassman et al.
EP0485970 1992-05-20 TRANSGENIC PLANTS OVERPR
R. L. Phillips et. al., Cereal Chem., vol. 62:213-218, 1985, Elevated Protein-Bound Methionine in Seeds of a Maize Line Resistant to Lysine Plus Threonine.
James T. Madison et. al., Plant Cell, vol. 7:473-476, 1988, Characterization of Soybean Tissue Culture Cell Lines Resistant to Methionine Analogs.
National Center for Biotechnology Information General Identifier No. 7798569, May 13, 2000, Kiyota, S., Lysine Sensitive Aspartate Kinase From Rice.
National Center for Biotechnology Information General Identifier No. 4376158, Jul. 2, 1997, Frankard, V. et al., Molecular characterization of an Arabidopsis thaliana cDNA coding for a monofunctional aspartate kinases.
National Center for Biotechnology Information General Identifier No. 7529283, Apr. 7, 2000, Bevan, M. et al.
National Center for Biotechnology Information General Identifier No. 5305740, Jun. 24, 1999, Esau, B.D., et al., Isolation and Characterization of a cDNA Clone Encoding a Monofunctional Aspartokinase.
J. Theze et. al., Journal of Bacteriology, vol. 117:133-143, 1974, Mapping of the Structural Genes of the Three Aspartokinases and of the Two Homoserine Dehygrogenases of Escherichia coli K-12.
Valerie Frankard et. al., Plant Molecular Biology, vol 34:233-242, 1997, Molecular Characterization of an Arabidopsis thaliana cDNA Coding for a Monofunctional Aspartate Kinase.
EMBL Sequence Library Database Accession No. X98873, Jul. 2, 1997, Frankard V. et. al., Molecular Characterisation of an Arabidopsis thaliana cDNA Coding for a Monofunctional Aspartate Kinase.
EMBL Sequecne Library Database Accession No. 023152, Jan. 1, 1998, Frankard V. et. al., Molecular Characterization of an Arabidopsis thaliana cDNA Coding for a Monofunctional Aspartate Kinase.
EMBL Sequence Library Database Accession No. U62020, Jul. 16, 1997, Tang G. et. al., Cloning and Expression of an Arabidopsis thaliana cDNA Encoding a Monofunctional Aspartate Kinase Homologous to the Lysine-Sensitive Enzyme of Escherichia coli.
EMBL Sequence Library Database Accession No. 023653, Jan. 1, 1998, Tang G. et. al., Cloning and Expression of an Arabidopsis thaliana cDNA Encoding a Monofunctional Aspartate Kinase Homologous to the Lysine-Sensitive Enzyme of Escherichia coli.
Guilang Tang et. al., Plant Molecular Biology, vol. 34:287-294, 1997, Cloning and Expression of an Arabidopsis thaliana cDNA Encoding a Monofunctional Aspartate Kinase Homologous to the Lysine-Sensitive Enzyme of Escherichia coli.
EMBL Sequence Library Database Accession No. AF135862, Jul. 1, 1999, Esau B. D. et . al., Isolation and Characterization of a cDNA Clone Encoding a Monofunctional Aspartokinase.
EMBL Sequence Library Database Accession No. Q9XHC5, Nov. 1, 1999, Esau B. D. et. al., Isolation and Characterization of a cDNA Clone Encoding a Monofunctional Aspartokinase.
EMBL Sequence Library Database Accession No. AB042521, May 12, 2000, Kiyota S., Lysine Sensitive Aspartate Kinase From Rice.
EMBL Sequence Library Database Accession No. Q9MAX0, Oct. 1, 2000, Kiyota S., Lysine Sensitive Aspartate Kinase From Rice.
Hagai Karchi et. al., The Plant Journal, vol. 3:721-727, 1993, Seed-Specific Expression of a Bacterial Desensitized Aspartate Kinase Increases the Production of Seed Theronine and Methionine in Transgenic Tobacco.
John Giovanelli et al., Plant Phys., vol. 77:450-455, 1985, In Vivo Regulation of De Novo Methionine Biosynthesis in a Higher Plant (Lemna).
Faith C. Belanger and Alan L. Kriz, Plant Phys., vol. 91:636-643, 1989, Molecular Characterization of the Major Maize Embryo Globulin Encoded by the GLB1 Gene.
S. C. Falco et al., Transgenic Canola and Soybean Seeds with Increased Lysine, BioTechnology, Jun. 1995, vol. 13.
This case is a divisional application under 37 CFR § 1.53(b). Specifically, this application is a divsional of Application Ser. No. 09/890,813 filed Aug. 2, 2001, issued on Mar. 1, 2005 as U.S. Pat. No. 6,861,575, which claims the benefit of U.S. Provisional Application No. 60/172,944, filed Dec. 21, 1999, the entire contents of which are hereby incorporated by reference.
1. A transgenic plant having an increased level of at least one free amino acid in seed when compared to a nontransgenic plant of the same species, said transgenic plant comprising a nucleic acid fragment encoding a polypeptide having aspartate kinase activity, wherein the polypeptide has an amino acid sequence of at least 90% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:8, said nucleic acid fragment increases an endogenous level of said free amino acid when transformed and expressed in the plant; wherein said free amino acid is threonine, aspartate, lysine or methionine.
2. A transgenic corn plant having an increased level of free threonine in seed when compared to a nontransgenic corn plant, said transgenic plant comprising a nucleic acid fragment encoding a polypeptide having aspartate kinase activity, wherein the polypeptide has an amino acid sequence of at least 90% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:8, and wherein said nucleic acid fragment increases an endogenous level of said free threonine when transformed and expressed in the plant.
3. The plant of claim 1 wherein said plant is a monocot or a dicot.
4. The plant of claim 1 wherein said plant is corn or soybean.
5. The plant of claim 1 wherein said free amino acid is threonine.
6. The plant of claim 1 wherein the polypeptide has an amino acid sequence of at least 95% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 8.
7. The plant of claim 1 wherein the polypeptide comprises SEQ ID NO:8.
8. The plant of claim 1 wherein the nucleic acid fragment comprises SEQ ID NO:7.
The organization of the pathway leading to biosynthesis of lysine, threonine, methionine, cysteine and isoleucine indicates that over-expression or reduction of expression of several genes encoding key regulatory enzymes of the pathway in corn, soybean, wheat and other crop plants could be used to alter levels of these amino acids in human food and animal feed. For example, methionine, along with threonine, lysine and isoleucine, are amino acids derived from aspartate. The first step in the pathway is the phosphorylation of aspartate by the enzyme aspartate kinase (Tang et al. (1997) Plant Mol Biol 34:287-293; Frankard et al. (1997) Plant Mol. Biol 34:233-242), and this enzyme has been found to be an important target for regulation of the pathway in many organisms. The aspartate family pathway is also believed to be regulated at the branch-point reactions. For methionine the reduction of aspartyl β-semialdehyde by homoserine dehydrogenase (HDH) may be an important point of control. Some aspartate kinases only carry aspartate kinase activity, in which case they are referred to as monofunctional, whereas there are bifunctional proteins found in bacteria and plants that carry both aspartate kinase and homoserine dehydrogenase enzymatic activities in two separate domains on one polypeptide. The first committed step to methionine, the production of cystathionine from O-phosphohomoserine and cysteine by cystathionine γ-synthase (CS), appears to be an important point of control of flux through the methionine pathway [Giovanelli et al., Plant Physiol., (1984), 77, 450-455]. The final step in methionine biosynthesis is catalyzed by the enzyme 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, also known as methionine synthase. Accordingly, availability of nucleic acid sequences encoding all or a portion of aspartate kinase would facilitate development of nutritionally improved crop plants.
The present invention concerns an isolated polynucleotide comprising: (a) a first nucleotide sequence encoding a first polypeptide comprising at least 50 or 100 amino acids, wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO:10 have at least 95% identity based on the Clustal alignment method, (b) a second nucleotide sequence encoding a second polypeptide comprising at least 95 or 100 amino acids, wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:2 have at least 90% or 95% identity based on the Clustal alignment method, (c) a third nucleotide sequence encoding a third polypeptide comprising at least 100 amino acids, wherein the amino acid sequence of the third polypeptide and the amino acid sequence of SEQ ID NO:4 have at least 70%, 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, (d) a fourth nucleotide sequence encoding a fourth polypeptide comprising at least 100 amino acids, wherein the amino acid sequence of the fourth polypeptide and the amino acid sequence of SEQ ID NO:14 have at least 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, (e) a fifth nucleotide sequence encoding a fifth polypeptide comprising at least 250 amino acids, wherein the amino acid sequence of the fifth polypeptide and the amino acid sequence of SEQ ID NO: 12 have at least 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, (f) a sixth nucleotide sequence encoding a sixth polypeptide comprising at least 400 amino acids, wherein the amino acid sequence of the sixth polypeptide and the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:8 have at least 85%, 90%, or 95% identity based on the Clustal alignment method, (g) a seventh nucleotide sequence encoding a seventh polypeptide comprising at least 400 amino acids, wherein the amino acid sequence of the seventh polypeptide and the amino acid sequence of SEQ ID NO:16 have at least 90% or 95% identity based on the Clustal alignment method, or (h) the complement of the first, second, third, fourth, fifth, sixth, or seventh nucleotide sequence, wherein the complement and the first, second, third, fourth, fifth, sixth, or seventh nucleotide sequence contain the same number of nucleotides and are 100% complementary. The first polypeptide preferably comprises the amino acid sequence of SEQ ID NO:10, the second polypeptide preferably comprises the amino acid sequence of SEQ ID NO:2, the third polypeptide preferably comprises the amino acid sequence of SEQ ID NO:4, the fourth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:14, the fifth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:12, the sixth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:8, and the seventh polypeptide preferably comprises the amino acid sequence of SEQ ID NO:16. The first nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:9, the second nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO: 1, the third nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:3, the fourth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:13, the fifth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:11, the sixth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:7, and the seventh nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:15. The first, second, third, fourth, fifth, sixth, and seventh polypeptides preferably are aspartate kinases.
In a second embodiment, the present invention relates to a chimeric gene comprising any of the isolated polynucleotides of the present invention operably lined to a regulatory sequence, and a cell, a plant, and a seed comprising the chimeric gene.
In a fifth embodiment, the present invention concerns an isolated polypeptide comprising: (a) a first amino acid sequence comprising at least 50 or 100 amino acids, wherein the first amino acid sequence and the amino acid sequence of SEQ ID NO: 10 have at least 95% identity based on the Clustal alignment method, (b) a second amino acid sequence comprising at least 95 or 100 amino acids, wherein the second amino acid sequence and the amino acid sequence of SEQ ID NO:2 have at least 90% or 95% identity based on the Clustal alignment method, (c) a third amino acid sequence comprising at least 100 amino acids, wherein the third amino acid sequence and the amino acid sequence of SEQ ID NO:4 have at least 70%, 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, (d) a fourth amino acid sequence comprising at least 100 amino acids, wherein the fourth amino acid sequence and the amino acid sequence of SEQ ID NO: 14 have at least 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, (e) a fifth amino acid sequence comprising at least 250 amino acids, wherein the fifth amino acid sequence and the amino acid sequence of SEQ ID NO:12 have at least 80%, 850%, 90%, or 95% identity based on the Clustal alignment method, (f) a sixth amino acid sequence comprising at least 400 amino acids, wherein the sixth amino acid sequence and the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:8 have at least 85%, 90%, or 95% identity based on the Clustal alignment method, or (g) a seventh amino acid sequence comprising at least 400 amino acids, wherein the seventh amino acid sequence and the amino acid sequence of SEQ ID NO:16 have at least 90% or 95% identity based on the Clustal alignment method. The first amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:10, the second amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:2, the third amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:4, the fourth amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:14, the fifth amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:12, the sixth amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:8, and the seventh amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:16. The polypeptide preferably is an aspartate kinase.
In a twelfth embodiment, this invention concerns a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or an expression cassette of the present invention; and (b) growing the transformed host cell preferably a plant cell, such as a monocot or a dicot, under conditions which allow expression of the aspartate kinase polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.
FIGS. 1A and 1B, together, shows an alignment of the amino acid sequences of aspartate kinase encoded by nucleotide sequences derived from corn clone cho1c.pk002.k6 (SEQ ID NO:6), corn clone cpd1c.pk010.k1 (SEQ ID NO:8), and Glycine max (NCBI GenBank Identifier (GI) No. 5305740; SEQ ID NO:17). Amino acids which are conserved among all and at least two sequences with an amino acid at that position are indicated with an asterisk (*). Dashes are used by the program to maximize alignment of the sequences.
Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing. Table 1 also identifies the cDNA clones as individual ESTs (“EST”), the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”), contigs assembled from two or more ESTs (“Contig”), contigs assembled from an FIS and one or more ESTs or PCR fragment sequence (“Contig*”), or sequences encoding the entire protein derived from an FIS, a contig, or an FIS and PCR fragment sequence (“CGS”). Nucleotide SEQ ID NOs:3, 9, and 13 correspond to nucleotide SEQ ID NOs:1, 3, and 5, respectively, presented in U.S. Provisional Application No. 60/172,944, filed Dec. 21, 1999. Amino acid SEQ ID NOs:4, 10, and 14 correspond to amino acid SEQ ID NOs:2, 4, and 6, respectively, presented in U.S. Provisional Application No. 60/172,944, filed Dec. 21, 1999. The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.
Protein) (Amino
(Plant Source) Clone Designation Status (Nucleotide) Acid)
Aspartate bms1.pk0008.e5 FIS 1 2
Kinase (Corn)
Aspartate cho1c.pk002.k6 EST 3 4
Aspartate cho1c.pk002.k6 (FIS) CGS 5 6
Aspartate cpd1c.pk010.k1 (FIS) CGS 7 8
Aspartate rdr1f.pk005.f20 EST 9 10
Kinase (Rice)
Aspartate rdr1f.pk005.f20 FIS 11 12
Aspartate wr1.pk0046.b11 EST 13 14
Kinase (Wheat)
Aspartate wr1.pk0046.b11 FIS 15 16
SEQ ID NOS:18-21 are PCR primers used to amplify portions of the cDNA insert in clone cpd1c.pk010.k1 to create an aspartate-kinase-encoding construct for expression in E. coli.
SEQ ID NOS:22 and 23 are PCR primers used to introduce a site-specific mutation to change S (serine) to L (leucine) in the corn mono functional aspartate kinase as described in Example 8.
SEQ ID NO: 26 is the Region 1 of the E.coli monofunctional Aspartate kinase described in Example 8.
SEQ ID NO: 28 is the Recilon 2 in the E.coli monofunctional Aspartate kinase described in Example 8.
The Sequence Listing contains the one letter code for nucleotide sequence characters and three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB spandards described in Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbolspand format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. § 1.822.
In the context of this disclosure, a number of terms spall be utilized. The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acid sequence”, and “nucleic acid fragment”/“isolated nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolated polynucleotide of the present invention may include at least 60 contiguous nucleotides, preferably at least 40 contiguous nucleotides, most preferably at least 30 contiguous nucleotides derived from SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, or 15, or the complement of such sequences.
Substantially similar nucleic acid fragments may be selected by screening nucleic acid fragments representing subfragments or modifications of the nucleic acid fragments of the instant invention, wherein one or more nucleotidespare substituted, deleted and/or inserted, for their ability to affect the level of the polypeptide encoded by the unmodified nucleic acid fragment in a plant or plant cell. For example, a substantially similar nucleic acid fragment representing at least 30 contiguous nucleotides derived from the instant nucleic acid fragment can be constructed and introduced into a plant or plant cell. The level of the polypeptide encoded by the unmodified nucleic acid fragment present in a plant or plant cell exposed to the substantially similar nucleic fragment can then be compared to the level of the polypeptide in a plant or plant cell that is not exposed to the substantially similar nucleic acid fragment.
For example, it is well known in the art that antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by using nucleic acid fragments that do not spare 100% sequence identity with the gene to be suppressed. Moreover, alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded polypeptide, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Consequently, an isolated polynucleotide comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, and 15, and the complement of such nucleotide sequences may be used in methods of selecting an isolated polynucleotide that affects the expression of an aspartate kinase polypeptide in a host cell. A method of selecting an isolated polynucleotide that affects the level of expression of a polypeptide in a virus or in a host cell (eukaryotic, such as plant or yeast, prokaryotic such as bacterial) may comprise the steps of: constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; measuring the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of a polypeptide or enzyme activity in a host cell that does not contain the isolated polynucleotide.
Moreover, substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min. then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C.
A “substantial portion” of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification of the protein or gene that the amino acid or nucleotide sequence comprises. Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer-based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410. In general, a sequence of ten or more contiguouspamino acids or thirty or more contiguous nucleotides is necespary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12 or more nucleotides may be used aspamplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprisespa nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The instant speciflcation teaches amino acid and nucleotide sequences encoding polypeptides that comprise one or more particular plant proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in thispart. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequencespas defined above.
The term “operably linked” refers to the association of two or more nucleic acid fragments on a single polynucleotide so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory, sequences in sense or antisense orientation.
“Null mutant” refers here to a host cell which either jacks the expression of a certain polypeptide or expresses a polypeptide which is inactive or does not have any detectable expected enzymatic function.
A “chloroplast transit peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. “Chloroplast transit sequence” refers to a nucleotide sequence that encodes a chloroplast transit peptide. A “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear location signal included (Raikhel (1992) Plant Phys. 100:1627-1632).
The present invention concerns an isolated polynucleotide comprising: (a) a first nucleotide sequence encoding a first polypeptide comprising at least 50 or 100 amino acids, wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO:10 have at least 95% identity based on the Clustal alignment method, (b) a second nucleotide sequence encoding a second polypeptide comprising at least 95 or 100 amino acids, wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:2 have at least 90% or 95% identity based on the Clustal alignment method, (c) a third nucleotide sequence encoding a third polypeptide comprising at least 100 amino acids, wherein the amino acid sequence of the third polypeptide and the amino acid sequence of SEQ ID NO:4 have at least 70%, 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, (d) a fourth nucleotide sequence encoding a fourth polypeptide comprising at least 100 amino acids, wherein the amino acid sequence of the fourth polypeptide and the amino acid sequence of SEQ ID NO: 14 have at least 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, (e) a fifth nucleotide sequence encoding a fifth polypeptide comprising at least 250 amino acids, wherein the amino acid sequence of the fifth polypeptide and the amino acid sequence of SEQ ID NO:12 have at least 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, (f) a sixth nucleotide sequence encoding a sixth polypeptide comprising at least 400 amino acids, wherein the amino acid sequence of the sixth polypeptide and the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:8 have at least 85%, 90%, or 95% identity based on the Clustal alignment method, (g) a seventh nucleotide sequence encoding a seventh polypeptide comprising at least 400 amino acids, wherein the amino acid sequence of the seventh polypeptide and the amino acid sequence of SEQ ID NO:16 have at least 90% or 95% identity based on the Clustal alignment method, or (h) the complement of the first, second, third, fourth, fifth, sixth, or seventh nucleotide sequence, wherein the complement and the first, second, third, fourth, fifth, sixth, or seventh nucleotide sequence contain the same number of nucleotides and are 100% complementary. The first polypeptide preferably comprises the amino acid sequence of SEQ ID NO:10, the second polypeptide preferably comprises the amino acid sequence of SEQ ID NO:2, the third polypeptide preferably comprises the amino acid sequence of SEQ ID NO:4, the fourth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:14, the fifth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:12, the sixth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:8, and the seventh polypeptide preferably comprises the amino acid sequence of SEQ ID NO:16. The first nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:9, the second nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:1, the third nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:3, the fourth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:13, the fifth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:11, the sixth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:7, and the seventh nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:15. The first, second, third, fourth, fifth, sixth, and seventh polypeptides preferably are aspartate kinases.
Nucleic acid fragments encoding at least a portion of several aspartate kinases have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art. The nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).
Overexpression of the proteins of the instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development. The chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3′Non-coding sequences encoding transcription termination signals may also be provided. The instant chimeric gene may also comprise one or more introns in order to facilitate gene expression.
All or a substantial portion of the polynucleotides of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and used as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. For example, the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragments of the instant invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics I:174-181) in order to construct a genetic map. In addition, the nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
bms1 Corn (BMS) Cell Culture 1 Day After bms1.pk0008.e5
cho1c Corn Embryo (Alexho Synthetic High cho1c.pk002.k6
Oil) 20 Days After Pollination
cpd1c Corn Pooled BMS Treated with Chemicals cpd1c.pk010.k1
rdr1f Developing Root of 10 Day Old Rice rdr1f.pk005.f20
wr1 Root From 7 Day Old Light Grown wr1.pk0046.b11
cDNA clones encoding aspartate kinase were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410; searches for similarity to sequences contained in the BLAST “nr” database (comprising all non-redundant GenBank CDS transpations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences obtained in Example 1 were analyzed for similarity to all publicly available DNA sequences contained in the “nr” database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were transpated in all reading framespand compared for similarity to all publicly available protein sequences contained in the “nr” database using the BLASTX algorithm (Gish and States (1993) Nat. Genet. 3:266-272) provided by the NCBI. For convenience, the P-value (probability) of observing a match of a cDNA sequence to a sequence contained in the sparched databases merely by chance as calculated by BLAST are reported herein as “pLog” values, which represent the negative of the logarithm of the reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cDNA sequence and the BLAST “hit” represent homologous proteins.
ESTs submitted for analysis are compared to the genbank database as described above. ESTs that contain sequences more 5- or 3-prime can be found by using the BLASTn algorithm (Altschul et al (1997) Nucleic Acids Res. 25:3389-3402.) against the DuPont proprietary database comparing nucleotide sequences that share common or overlapping regions of sequence homology. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences can be assembled into a single contiguous nucleotide sequence, thus extending the original fragment in either the 5 or 3-prime direction. Once the most 5-prime EST is identified, its complete sequence can be determined by Full Insert Sequencing as described in Example 1. Homologous genes belonging to different species can be found by comparing the ammo acid sequence of a known gene (from either a proprietary source or a public database) against an EST database using the tBLASTn algorithm. The tBLASTn algorithm searches an amino acid query against a nucleotide database that is translated in all 6 reading frames. This search allows for differences in nucleotide codon usage between different species, and for codon degeneracy.
Clone Status NCBI GI No. pLog Score
cho1c.pk002.k6 EST 4376158 19.30
rdr1f.pk005.f20 EST 5305740 54.70
wr1.pk0046.b11 EST 5305740 48.70
The sequence of the entire cDNA insert in the clones listed in Table 3 was determined. Further sequencing and searching of the DuPont proprietary database allowed the identification of other corn clones encoding aspartate kinase. The BLASTX search using the EST sequences from clones listed in Table 4 revealed similarity of the polypeptides encoded by the cDNAs to aspartate kinase from Oryza saliva (NCBI GI No. 7798569), Arabidopsis thaliana (NCBI GI Nos. 4376158 and 7529283), or Glycine max (NCBI GI No. 5305740). Shown in Table 4 are the BLAST results for individual ESTs (“EST”), the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”), sequences of contigs assembled from two or more ESTs (“Contig”), sequences of contigs assembled from an FIS and one or more ESTs (“Contig*”), or sequences encoding the entire protein derived from an FIS, a contig, or an FIS and PCR (“CGS”):
bms1.pk0008.e5 FIS 7798569 32.70
cho1c.pk002.k6 (FIS) CGS 5305740 >180.00
cpd1c.pk010.k1 (FIS) CGS 5305740 >180.00
rdr1f.pk005.f20 FIS 7529283 100.00
wr1.pk0046.b11 FIS 7529283 >180.00
SEQ ID NO. NCBI GI No. 5305740; SEQ ID NO: 17
6 66.4
Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of an aspartate kinase. These sequences represent the first corn, rice and wheat sequences encoding aspartate kinase known to Applicant.
The chimeric gene described above can then be introduced into corn cells by the following procedure. Immature corn embryos can be dissected from developing caryopses derived from crosses of the inbred corn lines H99 and LH132. The embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1:5 mm long. The embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept in the dark at 27° C. Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos. The embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.
Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment may then be purified 1% low melting agarose gel. Buffer and agarose contain 10 μg/ml ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase™ (Epicentre Technologies, Madison, Wis.) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 μL of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs (NEB); Beverly, Mass.). The fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above. The vector pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as described above. The prepared vector pBT430 and fragment can then be ligated at 16° C. for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL). Transformants can be selected on agar plates containing LB media and 100 μg/mL ampicillin. Transformants containing the gene encoding the instant polypeptide are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.
5′-CTCTCTGCCATGGGGAA-3′ (SEQ ID NO:18)
5′-GACTGGTACCTCAGCCCACGAGTAGGT-3′ (SEQ ID NO:19)
The resulting PCR fragment, designated PCR fragment 1, was digested with Nco I and Kpn I and ligated into pTrcHis cut with the same enzymes. Then a different portion of the corn mono-functional aspartate kinase cDNA in clone cpd1c.pk010.k1 was amplified via PCR using the following primers, to remove the chloroplast transit sequence and create a NcoI-NcoI fragment:
Oligo 9:
5′-GACTCCATGGAGGGATTGGGGGA-3′ (SEQ ID NO:20)
Oligo 8:
5′-GTTTTCCCCATGGCAGAGA-3′ (SEQ ID NO:21)
The resulting PCR fragment, designated PCR fragment 3, was digested with Nco I and ligated into the pTrcHis-based expression vector containing a portion of cpd1c.pk010.k1 cDNA described above that was also cut with Nco I. Insertion of the Nco I fragment in the proper orientation was determined by sequencing of the inserted DNA. The resulting plasmid with cDNA encoding full-length monofunctional corn aspartate kinase without chloroplast transit sequence in the pTrcHis vector was designated pBT994.
To establish that the cloned monofunctional corn aspartate kinase cDNA was functional, pBT994 was transformed into E. coli strain Gif106M1 (E. coli Genetic Stock Center strain CGSC-5074) which has mutations in each of the three E. coli aspartate kinase genes [Theze et al. (1974) J. Bacteriol. 117:133-143]. Because this stain lacks all aspartate kinase activity, it requires lysine, threonine and methionine for growth M9 media [see Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press], supplemented with the arginine, isoleucine and valine, also required for Gif106M1 growth, was used. In the pBT994 transformed strain the nutritional requirement for lysine, threonine and methionine was relieved demonstrating that the cloned monofunctional corn aspartate kinase cDNA encoded functional aspartate kinase.
A second method used PCR mutagenesis to create a site-specific mutation in the corn monofunctional aspartate kinase gene that reduces the encoded enzyme's sensitivity to inhibition by L-lysine. The particular amino acid substitutions to yield lysine-insensitive monofunctional corn aspartate kinase were based upon the homology that was discovered between monofunctional corn aspartate kinase and monofunctional E. coli aspartate kinase. Specifically, in two regions where particular amino acid substitutions were known to yield lysine-insensitive monofunctional E. coli aspartate kinase (see U.S. Pat. No. 5,773,691) the monofunctional corn aspartate kinase was found to have similar amino acid sequence. These regionspare shown below:
monofunctional corn aspartate kinase TSEVSVSVSLD (SEQ ID NO:25)
monofunctional E. coli aspartate kinase TSEVSVALTLD (SEQ ID NO:26)
The lysine-insensitive mutant monofunctional E. coli aspartate kinase has the underlined T
(threonine) residue changed to I (isoleucine).
monofunctional corn aspartate kinase SSRMLGQYGFLA (SEQ ID NO:27)
monofunctional E. coli aspartate kinase SLNMLHSRGFLA (SEQ ID NO:28)
First, a 370 bp portion of the corn monofunctional aspartate kinase cDNA in clone cpd1c.pk010.k1 was amplified via PCR using Oligo 2 (SEQ ID NO: 19) and Oligo 3 (SEQ ID NO:22) as primers:
Oligo 3: 5′-TTAGTGTTTCTGTGTTACTTGATCCATCAAAG-3′
Then a 980 bp portion of the corn monofunctional aspartate kinase cDNA in clone cpd1c.pk010.k1 was amplified via PCR using Oligo 1 (SEQ ID NO:18) and Oligo 6 (SEQ ID NO:23) as primers:
Oligo 6: 5′-CTTTGATGGATCAAGTAACACAGAAACACTAAC-3′
The 370 bp and 980 bp PCR fragments were then mixed together, denatured and allowed to hybridize heterologously. Staggered ends were filled-in with Taq polymerase, and PCR was performed on the DNA mixture using Oligos 1 (SEQ ID NO:18) and 2 (SEQ ID NO:19) as primers. This generated a 1320 bp Nco I-Kpn I fragment, designated PCR fragment 6, with the desired mutation that changes S (serine) to L (leucine) in the corn monofunctional aspartate kinase.
The 1320 bp NcoI-KpnI fragment containing the lysine-resistant (i.e., reduced sensitivity to inhibition by lysine) mutant corn monofunctional aspartate kinase was digested with Nco I and Kpn I and ligated into pTrcHis cut with the same enzymes. PCR fragment 3 described in Example 7 was ligated into the resulting plasmid in the same way. PCR fragments 1 and 3 were combined into a single plasmid described in Example 7. The creation of a mutant corn monofunctional aspartate kinase gene which contains a single nucleotide change compared to the native corn monofunctional aspartate kinase gene resulting in a change of amino acid 441 (in SEQ ID NO:8) from serine to leucine was confirmed by DNA sequencing. That the mutant corn monofunctional aspartate kinase gene encodes an enzyme with reduced sensitivity to inhibition by lysine was confirmed by in vivo testing as described below.
globulin 1 promoter/monofunctional corn aspartate kinase/globulin 13′region
the 1320 base pair Nco I and Kpn I PCR fragment 1 (described in Example 7) containing the major part of the monofunctional corn aspartate kinase coding region was inserted into plasmid pHD1 between the globulin 15′ and 3′ regions creating pBT954. A 380 bp fragment, designated PCR fragment 2, which has Nco I sites on both ends and contains the amino end of the coding sequence including the plant chloroplast targeting sequence, was generated via PCR using oligo 7 (SEQ ID NO:24) and oligo 8 (SEQ ID NO:21) as primers:
oligo 7:
5′-GACTCCATGGCAATCCCAGTGCG-3′ (SEQ ID NO:24)
PCR fragment 2 was digested with Nco I and ligated into pBT954. Insertion of 380 bp PCR fragment 2 in the proper orientation was determined by DNA sequencing, yielding the plant expression vector pBT960. Similarly, the 1320 base pair Nco I and Kpn I PCR fragment 6 (described in Example 8) containing the major part of the lysine-resistant mutant corn monofunctional aspartate kinase was inserted into plasmid pHD1 between the globulin 15′ and 3′ regions creating pBT955. Then 380 bp PCR fragment 2 (above), which contains the amino end of the coding sequence including the plant chloroplast targeting sequence, was digested with Nco I and ligated into pBT955. Insertion of 380 bp PCR fragment 2 in the proper orientation was determined by DNA sequencing, yielding the plant expression vector pBT961.
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