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  • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
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  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
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  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• This invention relates to chimeric genes and methods for increasing the lysine content of the seeds of plants and, in particular, to two chimeric genes, a first encoding plant lysine ketoglutarate reductase (LKR) and a second encoding lysine-insensitive dihydrodipicolinic acid synthase (DHDPS) which is operably linked to a plant chloroplast transit sequence, all operably linked to plant seed- specific regulatory sequences.
• LLR plant lysine ketoglutarate reductase
• DHDPS lysine-insensitive dihydrodipicolinic acid synthase
• Quality Protein Maize (QPM) bred at CIMMYT using the opaque-2 and sugary-2 genes and associated modifiers has a hard endosperm and enriched levels of lysine and tryptophan in the kernels [Vasal, S. K., et al. Proceedings of the 3rd seed protein symposium, Gatersleben, August 31 - September 2, 1983].
• the gene pools represented in the QPM lines are tropical and subtropical.
• Quality Protein Maize is a genetically complex trait and the existing lines are not easily adapted to the dent germplasm in use in the United States, preventing the adoption of QPM by corn breeders.
• the amino acid content of seeds is determined primarily (90-99%) by the amino acid composition of the proteins in the seed and to a lesser extent (1-10%) by the free amino acid pools.
• the quantity of total protein in seeds varies from about 10% of the dry weight in cereals to 20-40% of the dry weight of legumes.
• Much of the protein-bound amino acids is contained in the seed storage proteins which are synthesized during seed development and which serve as a major nutrient reserve following germination. In many seeds the storage proteins account for 50% or more of the total protein.
• Lysine along with threonine, methionine and isoleucine, are amino acids derived from aspartate, and regulation of the biosynthesis of each member of this family is interconnected. Regulation of the metabolic flow in the pathway appears to be primarily via end products.
• the first step in the pathway is the phosphorylation of aspartate by the enzyme aspartokinase (AK), and this enzyme has been found to be an important target for regulation in many organisms.
• AK aspartokinase
• the aspartate family pathway is also believed to be regulated at the branchpoint reactions.
• lysine this is the condensation of aspartyl ⁇ -semialdehyde with pyruvate catalyzed by dihydrodipicolinic acid synthase (DHDPS), while for threonine and methionine the reduction of aspartyl ⁇ -semialdehyde by homoserine dehydrogenase (HDH) followed by the phosphorylation of homoserine by homoserine kinase (HK) are important points of control.
• DHDPS dihydrodipicolinic acid synthase
• HDH homoserine dehydrogenase
• HK homoserine kinase
• the E. coli dapA gene encodes a DHDPS enzyme that is about 20-fold less sensitive to inhibition by lysine than a typical plant DHDPS enzyme, e.g., wheat germ DHDPS.
• the E. coli dapA gene has been linked to the 35S promoter of Cauliflower Mosaic Virus and a plant chloroplast transit sequence.
• the chimeric gene was introduced into tobacco cells via transformation and shown to cause a substantial increase in free lysine levels in leaves [Glassman et al. (1989) PCT Patent Appl. PCT/US89/01309, Shaul et al. (1992) Plant Jour. 2:203-209, Galili et al. (1992) ⁇ PO Patent Appl.
• LLR lysine-ketoglutarate reductase
• GenBank accession ATU9579 presents the sequence of a full-length cDNA clone for the bifunctional enzyme from Arabidopsis thaliana.
• the protein encoded by this clone is a homologue of both LKR and SDH proteins from fungal organisms.
• the DNA sequence for the genomic clone from Arabidopsis is also available as GenBank accession U95758 (Tang, et al. (1997) Plant Cell 9:1305-1316 and Epelbaum, et al. (1997) Plant Mol. Biol. 35: 735-748).
• GenBank accession AF003551 discloses a cDNA from corn which would direct the synthesis of a polypeptide from within the SDH domain of LKR/SDH proteins.
• GenBank accession AF042184 discloses the sequence of a cDNA from Brassica napus that is homologous to a relatively short portion of the full length clone from Arabidodpsis. However, whether such catabolic pathways are widespread in plants and whether they affect the level of accumulation of free amino acids is unknown. Finally, the effects of over- accumulation of a free amino acid such as lysine or threonine on seed development and viability is not known.
• This invention concerns an isolated nucleic acid fragment comprising a nucleic acid sequence encoding all or part of lysine ketoglutarate reductase.
• this invention concerns a chimeric gene comprising the aforesaid nucleic acid fragment encoding all or part of lysine ketoglutarate reductase, or a subfragment thereof, operably linked to suitable seed-specific regulatory sequences wherein said chimeric gene reduces lysine ketoglutarate reductase activity in seeds of transformed plants, as well as a plant cell or plant seed transformed with the aforesaid chimeric gene.
• this invention concerns a plant cell wherein lysine ketoglutarate reductase activity is reduced due to a mutation in a gene encoding lysine ketoglutarate reductase.
• this invention concerns a plant seed wherein lysine ketoglutarate reductase activity is reduced due to a mutation in a gene encoding lysine ketoglutarate reductase.
• step (b) regenerating fertile mature plants from the transformed plant cells obtained from step (a) under conditions suitable to obtain seeds;
• step (c) screening progeny seed of step (b) for reduced lysine ketoglutarate reductase activity
• a first chimeric gene comprising the aforesaid nucleic acid fragment encoding all or part of lysine ketoglutarate or a subfragment thereof, operably linked to suitable seed-specific regulatory sequences wherein said chimeric gene reduces lysine ketoglutarate reductase activity in seeds of transformed plants and
• a second chimeric gene wherein a nucleic acid fragment encoding dihydrodipicolinic acid synthase which is insensitive to inhibition by lysine is operably linked to a plant chloroplast transit sequence and to a plant seed-specific regulatory sequence.
• a seventh embodiment of this invention concerns a plant and a seed comprising in its genome the aforesaid nucleic acid fragments or the first and second aforesaid chimeric genes.
• Figure 1 shows an alpha helix from the side and top views.
• Figure 2 shows end ( Figure 2a) and side ( Figure 2b) views of an alpha helical coiled-coil structure.
• Figure 3 shows the chemical structure of leucine and methionine emphasizing their similar shapes.
• Figure 4a shows a schematic representation of a leaf gene expression cassette
• Figure 4b shows a schematic representation of a seed-specific gene expression cassette.
• Figure 5 shows a map of the binary plasmid vector pZS97K.
• Figure 6 shows a map of the binary plasmid vector pZS97.
• Figure 7A shows a map of the binary plasmid vector pZS199;
• Figure 7B shows a map of the binary plasmid vector pFS926;
• Figure 7C shows a map of the binary plasmid vector pBT593;
• Figure 7D shows a map of the binary plasmid vector pBT597.
• Figure 8 A shows a map of the plasmid vector pBT603
• Figure 8B shows a map of the plasmid vector pBT614.
• Figure 9 shows the amino acid sequence similarity between the polypeptides encoded by two plant cDNAs and fungal SDH (glutamate-forming).
• Figure 10 depicts the strategy for creating a vector (pSK5) for use in construction and expression of the SSP gene sequences.
• Figure 11 shows the strategy for inserting oligonucleotide sequences into the unique Ear I site of the base gene sequence.
• Figure 12 shows the insertion of the base gene oligonucleotides into the Nco I/EcoR I sites of pSK5 to create the plasmid pSK6.
• This base gene sequence was used as in Figure 8 to insert the various SSP coding regions at the unique Ear I site to create the cloned segments listed.
• Figure 13 shows the insertion of the 63 bp "segment" oligonucleotides used to create non-repetitive gene sequences for use in the duplication scheme in Figure 12.
• Figure 14 shows the strategy for multiplying non-repetitive gene "segments" utilizing in-frame fusions.
• Figure 15 shows the vectors containing seed specific promoter and 3' sequence cassettes. SSP sequences were inserted into these vectors using the Nco I and Asp718 sites.
• Figure 16 shows a map of the plasmid vector pML63.
• Figure 17 shows a map of the plasmid vector pBT680.
• Figure 18 shows a map of the plasmid vector pBT681.
• Figure 19 shows a map of the plasmid vector pLH104.
• Figure 20 shows a map of the plasmid vector pLH105.
• Figure 21 shows a map of the plasmid vector pBT739.
• Figure 22 shows a map of the plasmid vector pBT756.
• SEQ ID NO:l shows the nucleotide and amino acid sequence of the coding region of the wild type E. coli lysC gene, which encodes AKIII, described in Example 1.
• SEQ ID NOS:2 and 3 were used in Example 2 to create an Nco I site at the translation start codon of the E. coli lysC gene.
• SEQ ID NOS:4 and 5 were used in Example 3 as PCR primers for the isolation of the Corynebacterium dapA gene.
• SEQ ID NO:6 shows the nucleotide and amino acid sequence of the coding region of the wild type Corynebacterium dapA gene, which encodes lysine- insensitive DHDPS, described in Example 3.
• SEQ ID NO: 7 was used in Example 4 to create an Nco I site at the translation start codon of the E. coli dapA gene.
• SEQ ID NOS: 8, 9, 10 and 11 were used in Example 6 to create a chloroplast transit sequence and link the sequence to the E. coli lysC, E. coli lysC-M4.
• E. coli dapA and Corynebacteria dapA genes were used in Example 6 to create a chloroplast transit sequence and link the sequence to the E. coli lysC, E. coli lysC-M4.
• E. coli dapA and Corynebacteria dapA genes were used in Example 6 to create a chloroplast transit sequence and link the sequence to the E. coli lysC, E. coli lysC-M4.
• E. coli dapA and Corynebacteria dapA genes were used in Example 6 to create a chloroplast transit sequence and link the sequence to the E. coli lysC, E. coli lysC-M4.
• SEQ ID NOS: 12 and 13 were used in Example 6 to create a Kpn I site immediately following the translation stop codon of the E. coli dapA gene.
• SEQ ID NOS: 14 and 15 were used in Example 6 as PCR primers to create a chloroplast transit sequence and link the sequence to the Corynebacterium dapA gene.
• SEQ ID NOS: 16-92 represent nucleic acid fragments and the polypeptides they encode that are used to create chimeric genes for lysine-rich synthetic seed storage proteins suitable for expression in the seeds of plants.
• SEQ ID NO:93 was used in Example 6 as a constitutive expression cassette for corn.
• SEQ ID NOS:94-99 were used in Example 6 to create a corn chloroplast transit sequence and link the sequence to the E. coli lysC-M4 gene.
• SEQ ID NOS: 100 and 101 were used in Example 6 as PCR primers to create a corn chloroplast transit sequence and link the sequence to the E. coli dapA gene.
• SEQ ID NOS: 102 and 103 are cDNAs for plant lysine ketoglutarate reductase/saccharopine dehydrogenase from Arabidopsis thaliana.
• SEQ ID NOS:104 and 105 are polypeptides homologous to fungal saccharopine dehydrogenase (glutamate-forming) encoded by SEQ ID NOS: 102 and 103, respectively.
• SEQ ID NOS: 106 and 107 were used in Example 25 as PCR primers to add Nco I and Kpn I sites at the 5' and 3' ends of the corn DHDPS gene.
• SEQ ID NOS: 108 and 109 were used for PCR amplification of a 2.24 kb DNA fragment from genomic Arabidopsis DNA.
• SEQ ID NO:l 10 shows the sequence of the Arabidopsis LKR/SDH genomic DNA fragment.
• SEQ ID NO: 111 shows the sequence of the Arabidopsis LKR SDH cDNA.
• SEQ ID NO:l 12 shows the deduced amino acid sequence of Arabidopsis LKR/SDH protein.
• SEQ ID NOS: 113 and 114 were used for PCR amplification of soybean and corn LKR/SDH cDNA fragment.
• SEQ ID NO:l 15 shows the sequence of a soybean LKR/SDH cDNA fragment.
• SEQ ID NO: 116 shows the sequence of a corn LKR/SDH cDNA fragment.
• SEQ ID NO:l 17 shows the deduced partial amino acid sequence of soybean LKR/SDH protein.
• SEQ ID NO:l 18 shows the deduced partial amino acid sequence of corn LRK/SDH protein.
• SEQ ID NO:l 19 shows the sequence of a 2582 nucleotide cDNA from soybean.
• SEQ ID NO: 120 shows the sequence of a 3265 nucleotide cDNA from corn.
• SEQ ID NO: 121 shows the deduced partial amino acid sequence of soybean LKR/SDH protein encoded by nucleotides 3 through 2357 of SEQ ID NO:l 19.
• SEQ ID NO: 122 shows the deduced partial amino acid sequence of soybean LKR SDH protein encoded by nucleotides 3 through 3071 of SEQ ID NO: 120.
• SEQ ID NO: 123 is a nucleotide sequence corresponding to nucleotides 1 through 1908 of SED ID NO: 120.
• SEQ ID NO:124 is the deduced amino acid sequence from SEQ ID NO:123.
• SEQ ID NO: 125 shows the sequence of a 720 nucleotide LKR/SDH cDNA from rice.
• SEQ ID NO: 126 shows the deduced partial amino acid sequence of rice LKR/SDH protein encoded by nucleotides 2 through 720 of SEQ ID NO:125.
• SEQ ID NO:127 shows the sequence of a 308 nucleotide LKR/SDH cDNA from rice.
• SEQ ID NO: 128 shows the deduced partial amino acid sequence of rice LKR/SDH protein encoded by nucleotides 1 through 129 of SEQ ID NO: 127.
• SEQ ID NO: 129 shows the sequence of a 429 nucleotide cDNA from wheat.
• SEQ ID NO: 130 shows the deduced partial amino acid sequence of wheat LKR/SDH protein encoded by nucleotides 1 through 252 of SEQ ID NO: 129.
• SEQ ID NO: 131 shows the SDH coding region of the Arabidopsis cDNA clone.
• SEQ ID NO: 132 shows the amino acid sequence of the SDH domain of the Arabidopsis LKR/SDH protein.
• Nucleic acid fragments and procedures are described which are useful for increasing the accumulation of lysine in the seeds of transformed plants, as compared to levels of lysine in untransformed plants.
• genes encoding enzymes in the pathway were isolated from bacteria. In some cases, mutations in the genes were obtained so that the enzyme encoded was made insensitive to end-product inhibition.
• Intracellular localization sequences and suitable regulatory sequences for expression in the seeds of plants were linked to create chimeric genes. The chimeric genes were then introduced into plants via transformation and assessed for their ability to elicit accumulation of the lysine in seeds.
• a unique first nucleic acid fragment which comprises two nucleic acid subfragments (subsequences), one encoding LKR and the other encoding DHDPS which is substantially insensitive to feedback inhibition by lysine.
• substantially insensitive will mean at least 20-fold less sensitive to feedback inhibition by lysine than a typical plant enzyme catalyzing the same reaction. It has been found that a combination of subfragments successfully increases the lysine accumulated in seeds of transformed plants as compared to untransformed host plants.
• lysine catabolism results in the accumulation of lysine breakdown products such as saccharopine and ⁇ -amino adipic acid.
• lysine catabolism is prevented through reduction in the activity of the enzyme lysine ketoglutarate reductase (LKR), which catalyzes the first step in lysine breakdown.
• LLR lysine ketoglutarate reductase
• Such mutations can be identified in lysine over-producer lines by screening mutants for a failure to accumulate the lysine breakdown products, saccharopine and ⁇ -amino adipic acid.
• several procedures to isolate plant LKR genes are provided; nucleic acid fragments containing plant LKR cDNAs are also provided.
• Chimeric genes for expression of antisense LKR RNA or for cosuppression of LKR in the seeds of plants can then be created.
• the chimeric LKR gene is linked to the chimeric genes encoding lysine insensitive DHDPS and both are introduced into plants via transformation simultaneously, or the chimeric genes are brought together by crossing plants transformed independently with each of the chimeric genes.
• excess free lysine is incorporated into a form that is insensitive to breakdown, e.g., by incorporating it into a di-, tri- or oligopeptide, or preferably a lysine-rich storage protein.
• the lysine-rich storage protein chosen should contain higher levels of lysine than average proteins. Ideally, these storage proteins should contain at least 15% lysine by weight.
• the design of a preferred class of polypeptides which can be expressed in vivo to serve as lysine-rich seed storage proteins is provided.
• Genes encoding the lysine-rich synthetic storage proteins (SSP) are synthesized and chimeric genes wherein the SSP genes are linked to suitable regulatory sequences for expression in the seeds of plants are created.
• the SSP chimeric gene is then linked to the chimeric DHDPS gene and both are introduced into plants via transformation simultaneously, or the genes are brought together by crossing plants transformed independently with each of the chimeric genes.
• a method for transforming plants is taught herein wherein the resulting seeds of the plants have at least ten percent, preferably ten percent to four-fold greater, lysine than do the seeds of untransformed plants.
• a number of terms shall be utilized.
• nucleic acid refers to a large molecule which can be single-stranded or double-stranded, composed of monomers (nucleotides) containing a sugar, phosphate and either a purine or pyrimidine.
• a "nucleic acid fragment” is a fraction of a given nucleic acid molecule.
• deoxyribonucleic acid DNA
• RNA ribonucleic acid
• a “genome” is the entire body of genetic material contained in each cell of an organism.
• nucleotide sequence refers to a polymer of DNA or RNA which can be single- or double-stranded, optionally containing synthetic, non- natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
• homologous to refers to the complementarity between the nucleotide sequence of two nucleic acid molecules or between the amino acid sequences of two protein molecules. Quantitative estimates of 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 [as described in Hames and Higgins (eds.) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.]; or by the comparison of sequence similarity between two nucleic acids or proteins.
• DNA sequences that may involve base changes that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. It is therefore understood that the invention encompasses more than the specific exemplary sequences. Modifications to the sequence, such as deletions, insertions, or substitutions in the sequence which produce silent changes that do not substantially affect the functional properties of the resulting protein molecule are also contemplated.
• 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.
• 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 biologically equivalent product.
• Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein. In some cases, it may in fact be desirable to make mutants of the sequence in order to study the effect of alteration on the biological activity of the protein.
• 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.
• "essentially similar" sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1X SSC, 0.1% SDS, 65°C), with the sequences exemplified herein.
• Gene refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding) and following (3' non- coding) the coding region.
• “Native” gene refers to the gene as found in nature with its own regulatory sequences.
• “Chimeric” gene refers to a gene comprising heterogeneous regulatory and coding sequences.
• “Endogenous” gene refers to the native gene normally found in its natural location in the genome.
• a “foreign” gene refers to a gene not normally found in the host organism but that is introduced by gene transfer.
• Coding sequence refers to a DNA sequence that codes for a specific protein and excludes the non-coding sequences.
• “Initiation codon” and “termination codon” refer to a unit of three adjacent nucleotides in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation). "Open reading frame” refers to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence.
• RNA transcript refers to the product resulting from RNA polymerase- catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript.
• Messenger RNA (mRNA) refers to RNA that can be translated into protein by the cell.
• cDNA refers to a double-stranded DNA that is complementary to and derived from mRNA.
• Sense RNA transcript that includes the mRNA.
• Antisense RNA refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene by interfering with the processing, transport and or translation of its primary transcript or mRNA.
• the complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence.
• antisense RNA may contain regions of ribozyme sequences that increase the efficacy of antisense RNA to block gene expression.
• “Ribozyme” refers to a catalytic RNA and includes sequence-specific endoribonucleases.
• regulatory sequences refer to nucleotide sequences located upstream (5 1 ), within, and/or downstream (3') to a coding sequence, which control the transcription and/or expression of the coding sequences, potentially in conjunction with the protein biosynthetic apparatus of the cell.
• regulatory sequences include promoters, translation leader sequences, transcription termination sequences, and polyadenylation sequences.
• Promoter refers to a DNA sequence in a gene, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription.
• a promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions. It may also contain enhancer elements.
• an “enhancer” is a DNA sequence which can stimulate promoter activity. It may be an innate element of the promoter or a heterologous element inserted to enhance the level and/or tissue-specificity of a promoter. "Constitutive promoters” refers to those that direct gene expression in all tissues and at all times. "Organ-specific” or “development-specific” promoters as referred to herein are those that direct gene expression almost exclusively in specific organs, such as leaves or seeds, or at specific development stages in an organ, such as in early or late embryogenesis, respectively.
• operably linked refers to nucleic acid sequences on a single nucleic acid molecule which are associated so that the function of one is affected by the other.
• a promoter is operably linked with a structure gene (i.e., a gene encoding aspartokinase that is lysine-insensitive as given herein) when it is capable of affecting the expression of that structural gene (i.e., that the structural gene is under the transcriptional control of the promoter).
• expression is intended to mean the production of the protein product encoded by a gene. More particularly, “expression” refers to the transcription and stable accumulation of the sense (mRNA) or antisense RNA derived from the nucleic acid fragment(s) of the invention that, in conjunction with the protein apparatus of the cell, results in altered levels of protein product. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of preventing the expression of the target protein. “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms.
• Coding refers to the expression of a foreign gene which has substantial homology to an endogenous gene resulting in the suppression of expression of both the foreign and the endogenous gene.
• altered levels refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.
• the "3' non-coding sequences” refers to the DNA sequence portion of a gene that contains a polyadenylation signal and any other regulatory signal capable of affecting mRNA processing or gene expression.
• the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
• translation leader sequence refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream (5') of the translation start codon.
• the translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency.
• “Mature” protein refers to a post-translationally processed polypeptide without its targeting signal.
• Precursor protein refers to the primary product of translation of mRNA.
• a “chloroplast targeting signal” is an amino acid sequence which is translated in conjunction with a protein and directs it to the chloroplast.
• Chloroplast transit sequence refers to a nucleotide sequence that encodes a chloroplast targeting signal.
• Transformation herein refers to the transfer of a foreign gene into the genome of a host organism and its genetically stable inheritance.
• methods of plant transformation include Agrobacterium-mediated transformation and particle-accelerated or “gene gun” transformation technology.
• amino acids herein refer to the naturally occurring L amino acids (Alanine, Arginine, Aspartic acid, Asparagine, Cystine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Proline, Phenylalanine, Serine, Threonine, Tryptophan, Tyrosine, and Valine).
• Essential amino acids are those amino acids which cannot be synthesized by animals.
• a “polypeptide” or “protein” as used herein refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
• Synthetic protein herein refers to a protein consisting of amino acid sequences that are not known to occur in nature.
• the amino acid sequence may be derived from a consensus of naturally occurring proteins or may be entirely novel.
• Primary sequence refers to the connectivity order of amino acids in a polypeptide chain without regard to the conformation of the molecule. Primary sequences are written from the amino terminus to the carboxy terminus of the polypeptide chain by convention.
• “Secondary structure” herein refers to physico-chemically favored regular backbone arrangements of a polypeptide chain without regard to variations in side chain identities or conformations.
• “Alpha helices” as used herein refer to right- handed helices with approximately 3.6 residues per turn of the helix.
• An “amphipathic helix” refers herein to a polypeptide in a helical conformation where one side of the helix is predominantly hydrophobic and the other side is predominantly hydrophilic.
• oiled-coil herein refers to an aggregate of two parallel right-handed alpha helices which are wound around each other to form a left-handed superhelix.
• Salt bridges as discussed here refer to acid-base pairs of charged amino acid side chains so arranged in space that an attractive electrostatic interaction is maintained between two parts of a polypeptide chain or between one chain and another.
• “Host cell” means the cell that is transformed with the introduced genetic material.
• the E. coli lysC gene has been cloned, restriction endonuclease mapped and sequenced previously [Cassan et al. (1986) J. Biol. Chem. 251:1052-1057].
• the lysC gene was obtained on a bacteriophage lambda clone from an ordered library of 3400 overlapping segments of cloned E. coli DNA constructed by Kohara, Akiyama and Isono [Kohara et al. (1987) Cell 50:595-508].
• the E. coli lysC gene encodes the enzyme AKIII, which is sensitive to lysine inhibition. Mutations were obtained in the lysC gene that cause the AKIII enzyme to be resistant to lysine.
• sequence of the wild type lysC gene and three mutant genes were determined.
• sequences of the three mutant IvsC genes that encoded lysine- insensitive aspartokinase each differed from the wild type sequence by a single nucleotide, resulting in a single amino acid substitution in the protein.
• One mutant (M2) had an A substituted for a G at nucleotide 954 of S ⁇ Q ID NO:l : resulting in an isoleucine for methionine substitution in the amino acid sequence of AKIII and two mutants (M3 and M4) had identical T for C substitutions at nucleotide 1055 of SEQ ID NO:l resulting in an isoleucine for threonine substitution.
• Example 1 Other mutations could be generated, either in vivo as described in Example 1 or in vitro by site-directed mutagenesis by methods known to those skilled in the art, that result in amino acid substitutions for the methionine or threonine residue present in the wild type AKIII at these positions. Such mutations would be expected to result in a lysine-insensitive enzyme. Furthermore, the method described in Example 1 could be used to easily isolate and characterize as many additional mutant lysC genes encoding lysine insensitive AKIII as desired.
• AK genes have been isolated and sequenced. These include the thrA gene of E. coli (Katinka et al. (1980) Proc. Natl. Acad. Sci. USA 77:5730-5733], the metL gene of E. coli (Zakin et al. (1983) J Biol. Chem. 255:3028-3031], the HOM3 gene of S. cerevisiae [Rafalski et al. (1988) J. Biol. Chem. 255:2146-2151].
• the thrA gene of E. coli encodes a bifunctional protein, AKI-HDHI. The AK activity of this enzyme is insensitive to lysine, but sensitive to threonine.
• the metL gene of E. coli also encodes a bifunctional protein, AKII-HDHII, and the AK activity of this enzyme is also insensitive to lysine.
• the HOM3 gene of yeast encodes an AK which is insensitive to lysine, but sensitive to threonine.
• lysine-insensitive AK In addition to these genes, several plant genes encoding lysine-insensitive AK are known. In barley lysine plus threonine-resistant mutants bearing mutations in two unlinked genes that result in two different lysine-insensitive AK isoenzymes have been described [Bright et al. (1982) Nature 299:278-279, Rognes et al. (1983) Planta 757:32-38, Arruda et al. (1984) Plant Physiol 76:442-446]. In corn, a lysine plus threonine-resistant cell line had AK activity that was less sensitive to lysine inhibition than its parent line [Hibberd et al. (1980) Planta 745:183-187].
• a subsequently isolated lysine plus threonine- resistant corn mutant is altered at a different genetic locus and also produces lysine-insensitive AK [Diedrick et al. (1990) Theor. Appl. Genet. 79:209-215, Dotson et al. (1990) Planta 752:546-552].
• tobacco there are two AK enzymes in leaves, one lysine-sensitive and one threonine-sensitive.
• a lysine plus threonine-resistant tobacco mutant that expressed completely lysine-insensitive AK has been described [Frankard et al. (1991) Theor. Appl. Genet. 52:273-282].
• These plant mutants could serve as sources of genes encoding lysine-insensitive AK and used, based on the teachings herein, to increase the accumulation of lysine and threonine in the seeds of transformed plants.
• IvsC genes in E. coli, a bacterial expression vector which employs the bacteriophage T7 RNA polymerase/T7 promoter system [Rosenberg et al. (1987) Gene 56:125-135] was used.
• the expression vector and IvsC gene were modified as described in Example 2 to construct a IvsC expression vector.
• the mutant lysC genes M2, M3 and M4
• the wild type lysC gene was replaced with the mutant genes as described in Example 2.
• each of the expression vectors was transformed into E. coli strain B121(DE3) [Studier et al. (1986) J. Mol. Biol. 759:113-130]. Cultures were grown, expression was induced, cells were collected, and extracts were prepared as described in Example 2. Supernatant and pellet fractions of extracts from uninduced and induced cultures were analyzed by SDS polyacrylamide gel electrophoresis and by AK enzyme assays as described in Example 2. The major protein visible by Coomassie blue staining in the supernatant and pellet fractions of induced cultures was AKIII. About 80% of the AKIII protein was in the supernatant and AKIII represented 10-20% of the total E. coli protein in the extract.
• AKIII enzyme activity was in the supernatant fraction.
• the specific activity of wild type and mutant crude extracts was 5-7 ⁇ moles product per minute per milligram total protein.
• Wild type AKIII was sensitive to the presence of L-lysine in the assay. Fifty percent inhibition was found at a concentration of about 0.4 mM and 90 percent inhibition at about 0.1 mM. In contrast, mutants AKIII-M2, M3 and M4 were not inhibited at all by 15 mM L-lysine.
• Wild type AKIII protein was purified from the supernatant of an induced culture as described in Example 2. Rabbit antibodies were raised against the purified AKIII protein.
• lysC expression vectors Many other microbial expression vectors have been described in the literature. One skilled in the art could make use of any of these to construct lysC expression vectors. These lysC expression vectors could then be introduced into appropriate microorganisms via transformation to provide a system for high level expression of AKIII.
• the E. coli dapA gene (ecodapAI has been cloned, restriction endonuclease mapped and sequenced previously [Richaud et al. (1986) J. Bacteriol. 766:297-300].
• the dapA gene was obtained on a bacteriophage lambda clone from an ordered library of 3400 overlapping segments of cloned E. coli. DNA constructed by Kohara, Akiyama and Isono [Kohara et al. (1987) Cell 50:595-508].
• the ecodapA gene encodes a DHDPS enzyme that is sensitive to lysine inhibition. However, it is about 20-fold less sensitive to inhibition by lysine than a typical plant DHDPS, e.g., wheat germ DHDPS.
• the Corynebacterium dapA gene (cordapA) was isolated from genomic DNA from ATCC strain 13032 using polymerase chain reaction (PCR).
• PCR polymerase chain reaction
• the nucleotide sequence of the Corynebacterium dapA gene has been published [Bonnassie et al. (1990) Nucleic Acids Res. 75:6421]. From the sequence it was possible to design oligonucleotide primers for polymerase chain reaction (PCR) that would allow amplification of a DNA fragment containing the gene, and at the same time add unique restriction endonuclease sites at the start codon and just past the stop codon of the gene to facilitate further constructions involving the gene.
• the details of the isolation of the cordapA gene are presented in Example 3.
• the cordapA gene encodes a DHDPS enzyme that is insensitive to lysine inhibition.
• the PCR primers In addition to introducing a restriction endonuclease site at the translation start codon, the PCR primers also changed the second codon of the cordapA gene from AGC coding for serine to GCT coding for alanine. Several cloned DNA fragments that expressed active, lysine-insensitive DHDPS were isolated, indicating that the second codon amino acid substitution did not affect enzyme activity.
• the PCR-generated Corynebacterium dapA gene was subcloned into the phagemid vector pGEM-9zf(-) from Promega, and single-stranded DNA was generated and sequenced (SEQ ID NO: 6). Aside from the differences in the second codon already mentioned, the sequence matched the published sequence except at two positions, nucleotides 798 and 799. In the published sequence these are TC, while in the gene shown in SEQ ID NO:6 they are CT. This change results in an amino acid substitution of leucine for serine. The reason for this difference is not known. The difference has no apparent effect on DHDPS enzyme activity. The isolation of other genes encoding DHDPS has been described in the literature.
• a cDNA encoding DHDPS from wheat [Kaneko et al. (1990) J. Biol. Chem. 265:17451-17455], and a cDNA encoding DHDPS from corn [Frisch et al. (1991) Mol. Gen. Genet. 225:287-293] are two examples. These genes encode wild type lysine-sensitive DHDPS enzymes. However, Negrutui et al. [(1984) Theor. Appl. Genet. 65:11-20], obtained two AEC-resistant tobacco mutants in which DHDPS activity was less sensitive to lysine inhibition than the wild type enzyme. These genes could be isolated using the methods already described for isolating the wheat or corn genes or, alternatively, by using the wheat or corn genes as heterologous hybridization probes.
• genes encoding DHDPS could be isolated by one skilled in the art by using either the ecodapA gene, the cordapA gene, or either of the plant DHDPS genes as DNA hybridization probes.
• other genes encoding DHDPS could be isolated by functional complementation of an E. coli dapA mutant, as was done to isolate the cordapA gene [Yeh et al. (1988) Mol. Gen. Genet. 272:105-111] and the corn DHDPS gene.
• each of the expression vectors was transformed into E. coli strain BL21(DE3) [Studier et al. (1986) J. Mol. Biol. 759:113-130]. Cultures were grown, expression was induced, cells were collected, and extracts were prepared as described in Example 4. Supernatant and pellet fractions of extracts from uninduced and induced cultures were analyzed by SDS polyacrylamide gel electrophoresis and by DHDPS enzyme assays as described in Example 4. The major protein visible by Coomassie blue staining in the supernatant and pellet fractions of both induced cultures had a molecular weight of 32-34 kd, the expected size for DHDPS. Even in the uninduced cultures this protein was the most prominent protein produced.
• E. coli DHDPS The specific activity of E. coli DHDPS in the supernatant fraction of induced extracts was about 50 OD540 units per milligram protein.
• E. coli DHDPS was sensitive to the presence of L-lysine in the assay. Fifty percent inhibition was found at a concentration of about 0.5 mM.
• enzyme activity was measured in the supernatant fraction of uninduced extracts, rather than induced extracts. Enzyme activity was about 4 OD530 units per minute per milligram protein.
• Corynebacterium DHDPS was not inhibited at all by L-lysine, even at a concentration of 70 mM.
• E. coli expression cassettes were inserted into expression vectors and then transformed into E. coli strain BL21(DE3) [Studier et al. (1986) J. Mol. Biol. 759:113-130] to induce E. coli to produce and excrete amino acids. Details of the procedures used and results are presented in Example 5.
• microbial expression vectors known to those skilled in the art could be used to make and combine expression cassettes for the IvsC and dapA genes. These expression vectors could then be introduced into appropriate microorganisms via transformation to provide alternative systems for production and excretion of lysine, threonine and methionine.
• a preferred class of heterologous hosts for the expression of the chimeric genes of this invention are eukaryotic hosts, particularly the cells of higher plants.
• Preferred among the higher plants and the seeds derived from them are soybean, rapeseed (Brassica napus, B. campestris), sunflower (Helianthus annus), cotton (Gossypium hirsutum), corn, tobacco (Nicotiana tabacum), alfalfa (Medicago sativd), wheat (Triticum sp), barley (Hordeum vulgare), oats (Avena sativa, L), sorghum ⁇ Sorghum bicolor), rice (Oryza sativa), and forage grasses.
• promoter chosen to drive the expression of the coding sequence is not critical as long as it has sufficient transcriptional activity to accomplish the invention by expressing translatable mRNA or antisense RNA in the desired host tissue.
• Preferred promoters for expression in all plant organs, and especially for expression in leaves include those directing the 19S and 35S transcripts in Cauliflower mosaic virus [Odell et al.(1985) Nature 575:810-812; Hull et al. (1987) Virology 56:482-493], small subunit of ribulose 1,5-bisphosphate carboxylase [Morelli et al.(1985) Nature 575:200; Broglie et al.
• promoters that are specific for expression in one or more organs of the plant.
• examples include the light-inducible promoters of the small subunit of ribulose 1,5-bisphosphate carboxylase, if the expression is desired in photosynthetic organs, or promoters active specifically in seeds.
• Preferred promoters are those that allow expression specifically in seeds. This may be especially useful, since seeds are the primary source of vegetable amino acids and also since seed-specific expression will avoid any potential deleterious effect in non-seed organs.
• seed-specific promoters include, but are not limited to, the promoters of seed storage proteins. The seed storage proteins are strictly regulated, being expressed almost exclusively in seeds in a highly organ-specific and stage-specific manner [Higgins et al.(1984) Ann. Rev. Plant Physiol. 55:191-221; Goldberg et al.(1989) Cell 56:149-160; Thompson et al. (1989) BioEssays 70:108-113]. Moreover, different seed storage proteins may be expressed at different stages of seed development.
• seed-specific expression of seed storage protein genes in transgenic dicotyledonous plants include genes from dicotyledonous plants for bean ⁇ -phaseolin [Sengupta-Goplalan et al. (1985) Proc. Natl. Acad. Sci. USA 52:3320-3324; Hoffman et al. (1988) Plant Mol. Biol. 77:717-729], bean lectin [Voelker et al. (1987) EMBOJ. 6: 3571-3577], soybean lectin [Okamuro et al. (1986) Proc. Natl. Acad. Sci.
• soybean kunitz trypsin inhibitor [Perez-Grau et al. (1989) Plant Cell 7:095-1109] soybean ⁇ -conglycinin [Beachy et al. (1985) EMBO J. 4:3047-3053; Barker et al. (1988) Proc. Natl. Acad. Sci. USA 55:458-462; Chen et al. (1988) EMBOJ. 7:297-302; Chen et al. (1989) E>ev. Genet. 70:112-122; Naito et al. (1988) Plant Mol. Biol. 77:109-123], pea vicilin [Higgins et al. (1988) Plant Mol.
• promoters of seed-specific genes also maintain their temporal and spatial expression pattern in transgenic plants.
• Such examples include Arabidopsis thaliana 2S seed storage protein gene promoter to express enkephalin peptides in Arabidopsis and B. napus seeds [Vandekerckhove et al.
• nucleic acid fragment of the invention will be the heterologous promoters from several extensively- characterized soybean seed storage protein genes such as those for the Kunitz trypsin inhibitor [Jofuku et al. (1989) Plant Cell 7:1079-1093; Perez-Grau et al. (1989) Plant Cell 7:1095-1109], glycinin [Nielson et al. (1989) Plant Cell 7:313-328], ⁇ -conglycinin [Harada et al. (1989) Plant Cell 7:415-425].
• Promoters of genes for ⁇ '- and ⁇ -subunits of soybean ⁇ -conglycinin storage protein will be particularly useful in expressing mRNAs or antisense RNAs in the cotyledons at mid- to late-stages of soybean seed development [Beachy et al. (1985) EMBOJ. 4:3047-3053; Barker et al. (1988) Proc. Natl. Acad. Sci. USA 55:458-462; Chen et al. (1988) EMBOJ. 7:297-302; Chen et al. (1989) Dev. Genet. 70:112-122; Naito et al. (1988) Plant Mol. Biol.
• heterologous promoters from several extensively characterized com seed storage protein genes such as endosperm-specific promoters from the 10 kD zein [Kirihara et al. (1988) Gene 77:359-370], the 27 kD zein [Prat et al. (1987) Gene 52:51-49; Gallardo et al. (1988) Plant Sci. 54:211-281], and the 19 kD zein [Marks et al. (1985) J Biol. Chem. 260: 16451-16459] .
• enhancers or enhancer-like elements into other promoter constructs will also provide increased levels of primary transcription to accomplish the invention.
• enhancers or enhancer-like elements would include viral enhancers such as that found in the 35S promoter [Odell et al. (1988) Plant Mol. Biol. 70:263-272], enhancers from the opine genes [Fromm et al. (1989) Plant Cell 7:977-984], or enhancers from any other source that result in increased transcription when placed into a promoter operably linked to the nucleic acid fragment of the invention.
• DNA sequence element isolated from the gene for the ⁇ '-subunit of ⁇ -conglycinin that can confer 40-fold seed-specific enhancement to a constitutive promoter [Chen et al. (1988) EMBOJ. 7:297-302; Chen et al. (1989) Dev. Genet. 70:112-122].
• One skilled in the art can readily isolate this element and insert it within the promoter region of any gene in order to obtain seed-specific enhanced expression with the promoter in transgenic plants. Insertion of such an element in any seed-specific gene that is expressed at different times than the ⁇ -conglycinin gene will result in expression in transgenic plants for a longer period during seed development.
• Any 3' non-coding region capable of providing a polyadenylation signal and other regulatory sequences that may be required for the proper expression can be used to accomplish the invention.
• DNA sequences coding for intracellular localization sequences may be added to the lysC and dapA coding sequence if required for the proper expression of the proteins to accomplish the invention.
• Plant amino acid biosynthetic enzymes are known to be localized in the chloroplasts and therefore are synthesized with a chloroplast targeting signal.
• Bacterial proteins such as DHDPS and AKIII have no such signal.
• a chloroplast transit sequence could, therefore, be fused to the dapA and lysC coding sequences.
• Preferred chloroplast transit sequences are those of the small subunit of ribulose 1,5-bisphosphate carboxylase, e.g. from soybean [Berry-Lowe et al. (1982) J. Mol. Appl. Genet. 7:483-498] for use in dicotyledonous plants and from com [Lebrun et al. (1987) Nucleic Acids Res. 75:4360] for use in monocotyledonous plants.
• a DNA sequence i.e., of transforming
• Such methods include those based on transformation vectors based on the Ti and Ri plasmids of Agrobacterium spp. It is particularly preferred to use the binary type of these vectors.
• Ti-derived vectors transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants, such as soybean, cotton and rape [Pacciotti et al. (1985) Bio/Technology 5:241; Byrne et al.
• the chimeric genes of the invention can be inserted into binary vectors as described in Examples 7-12 and 14-16.
• the vectors are part of a binary Ti plasmid vector system [Bevan, (1984) Nucl. Acids. Res. 72:8711-8720] of Agrobacterium tumefaciens.
• transformation methods are available to those skilled in the art, such as direct uptake of foreign DNA constructs [see EPO publication 0 295 959 A2], techniques of electroporation [see Fromm et al. (1986) Nature (London) 579:791] or high- velocity ballistic bombardment with metal particles coated with the nucleic acid constructs [see Kline et al. (1987) Nature (London) 527:70, and see U.S. Pat. No. 4,945,050]. Once transformed, the cells can be regenerated by those skilled in the art.
• the chimeric genes of the invention can be inserted into suitable vectors as described in Example 6.
• Transformed plants can be obtained as described in Examples 17-19.
• the AKIII or DHDPS proteins can be detected and quantitated enzymatically and/or immunologically by methods known to those skilled in the art. In this way lines producing high levels of expressed protein can be easily identified.
• free amino acids can be extracted by various methods including those as described in Example 7.
• extracts can be prepared by various methods including those as described in Example 8.
• the relatively small increases of free threonine or lysine achieved with the AKIII protein were not sufficient to yield detectable increases compared to untransformed plants, in the levels of total threonine or lysine in the seeds.
• the larger increases of free threonine achieved via expression of the AKIII-M4 protein were sufficient to yield detectable increases, compared to seeds from untransformed plants, in the levels of total threonine in the seeds.
• Sixteen to twenty-five percent increases in total threonine content of the seeds were observed.
• the lines that showed increased total threonine were the same ones that showed the highest levels of increase in free threonine and high expression of the AKIII-M4 protein.
• amino acid biosynthesis takes place in seeds and can be modulated by the expression of foreign genes encoding amino acid biosynthetic enzymes. Furthermore, they show that control of an amino acid biosynthetic pathway can differ markedly from one plant organ to another, e.g. seeds and leaves. The importance of this observation is emphasized upon considering the different effects of expressing a foreign DHDPS in leaves and seeds described below. It can be concluded that threonine biosynthesis in seeds is controlled primarily via end-product inhibition of AK. Therefore, threonine accumulation in the seeds of plants can be increased by expression of a gene, introduced via transformation, that encodes AK which is insensitive to lysine inhibition and which is localized in the chloroplast.
• the above teachings demonstrate that transformed plants which express higher levels of the introduced enzyme in seeds accumulate higher levels of free threonine in seeds. Furthermore, the teachings demonstrate that transformed plants which express a lysine-insensitive AK in seeds accumulate higher levels of free threonine in seeds than do transformed plants which express similar levels of a lysine-sensitive AK. To achieve commercially valuable increases in free threonine, a lysine-insensitive AK is preferred.
• Tobacco plants transformed with a vector carrying both E. coli DHDPS and AKIII-M4 genes linked to the 35S promoter are described in Example 11.
• the level of expression of E. coli DHDPS determines the level of lysine accumulation in leaves (Example 11, Table 6).
• the level of expression of each protein plays a role in controlling the level of lysine accumulation.
• Transformed lines that express DHDPS at comparable levels accumulate more lysine when AKIII-M4 is also expressed (Table 6, compare lines 564-18A, 564-56A, 564-36E, 564-55B, and 564-47 A).
• expression of a lysine-insensitive AK increases lysine accumulation in leaves when expressed in concert with a DHDPS enzyme that is 20-fold less sensitive to lysine than the endogenous plant enzyme.
• the E. coli DHDPS enzyme is less sensitive to lysine inhibition than plant DHDPS, but is still inhibited by lysine.
• the above teachings on the AK proteins indicate that expression of a completely lysine-insensitive enzyme can lead to a much greater accumulation of the aspartate pathway end-product threonine than expression of an enzyme which, while less sensitive than the plant enzyme, is still inhibited by lysine. Therefore vectors carrying both Corynebacterium DHDPS and AKIII-M4 genes linked to the seed-specific promoters were constructed as described in Examples 15 and 19. Tobacco plants transformed with vectors carrying both Corynebacterium DHDPS and AKIII-M4 genes linked to seed- specific promoters are described in Example 15.
• a seed meal can be prepared as described in Examples 16 or 19 or by any other suitable method.
• the seed meal can be partially or completely defatted, via hexane extraction for example, if desired.
• Protein extracts can be prepared from the meal and analyzed for AK and/or DHDPS enzyme activity. Alternatively the presence of the AK and/or DHDPS protein can be tested for immunologically by methods well-known to those skilled in the art.
• free amino acids can be extracted from the meal and analyzed by methods known to those skilled in the art (see Examples 8, 16 and 19 for suitable procedures).
• prevention of lysine catabolism by inactivation of lysine ketoglutarate reductase should further increase the accumulation of free lysine in the seeds.
• incorporation of lysine into a peptide or lysine-rich protein would prevent catabolism and lead to an increase in the accumulation of lysine in the seeds.
• prevention of lysine catabolism by inactivation of lysine ketoglutarate reductase should further increase the accumulation of free lysine in the soybean seeds.
• incorporation of lysine into a peptide or lysine-rich protein would prevent catabolism and lead to an increase in the accumulation of lysine in the soybean seeds.
• the soybean seeds expressing Corynebacteria DHDPS showed substantial increases in accumulation of total seed lysine. Seeds with a 5-35% increase in total lysine content, compared to the untransformed control, were observed. In these seeds lysine makes up 7.5-7.7% of the total seed amino acids.
• Soybean seeds expressing Corynebacteria DHDPS in concert with E. coli AKIII-M4 showed much greater accumulation of total seed lysine than those expressing Corynebacteria DHDPS alone. Seeds with a more than four-fold increase in total lysine content were observed. In these seeds lysine makes up 20-25% of the total seed amino acids, considerably higher than any previously known soybean seed.
• Com plants regenerated from transformed callus can be analyzed for the presence of the intact lysC and dapA transgenes via Southern blot or PCR. Plants carrying the genes are either selfed or outcrossed to an elite line to generate FI seeds. Six to eight seeds are pooled and assayed for expression of the Corynebacterium DHDPS protein and the E. coli AKIII-M4 protein by western blot analysis. The free amino acid composition and total amino acid composition of the seeds are determined as described above.
• Corynebacterium DHDPS protein, and/or the E. coli AKIII-M4 protein can be obtained in the embryo of the seed using regulatory sequences active in the embryo, preferably derived from the globulin 1 gene, or in the endosperm using regulatory sequences active in the endosperm, preferably derived from the glutelin 2 gene or the 10 kD zein gene (see Example 26 for details).
• Free lysine levels in the seeds is increased from about 1.4% of free amino acids in control seeds to 15-27% in seeds of transformants expressing Corynebacterium DHDPS alone from the globulin 1 promoter. The increased free lysine was localized to the embryo in seeds expressing Corynebacterium DHDPS from the globulin 1 promoter.
• Total lysine levels can be increased at least 130% in seeds expressing Corynebacterium DHDPS from the globulin 1 promoter. Greater increases in free lysine levels can be achieved by expressing E. coli AKIII-M4 protein from the globulin 1 promoter in concert with Corynebacterium DHDPS.
• Lysine catabolism is expected to be much greater in the com endosperm than the embryo.
• LLR ketoglutarate reductase
• LKR catalyzes the first step in lysine catabolism, the condensation of L-lysine with ⁇ -ketoglutarate into saccharopine using NADPH as a cofactor.
• LKR activity increases sharply from the onset of endosperm development in com, reaches a peak level at about 20 days after pollination, and then declines [Arruda et al. (1983) Phytochemistry 22:2687-2689].
• LKR gene This could be accomplished by cloning the LKR gene, preparing a chimeric gene for cosuppression of LKR or preparing a chimeric gene to express antisense RNA for LKR, and introducing the chimeric gene into plants via transformation.
• plant mutants could be obtained wherein LKR enzyme activity is absent.
• the protein can be purified from com endosperm, as described in Brochetto-Braga et al. [(1992) Plant Physiol 95:1139-1147] and used to raise antibodies. The antibodies can then be used to screen an cDNA expression library for LKR clones. Alternatively the purified protein can be used to determine amino acid sequence at the amino-terminal of the protein or from protease derived internal peptide fragments. Degenerate oligonucleotide probes can be prepared based upon the amino acid sequence and used to screen a plant cDNA or genomic DNA library via hybridization.
• Another method makes use of an E. coli strain that is unable to grow in a synthetic medium containing 20 ⁇ g/mL of L-lysine. Expression of LKR full- length cDNA in this strain will reverse the growth inhibition by reducing the lysine concentration. Construction of a suitable E. coli strain and its use to select clones from a plant cDNA library that lead to lysine-resistant growth is described in Example 20.
• Yet another method relies upon homology between plant LKR and saccharopine dehydrogenase.
• Fungal saccharopine dehydrogenase (glutamate- forming) and saccharopine dehydrogenase (lysine-forming) catalyze the final two steps in the fungal lysine biosynthetic pathway.
• Plant LKR and fungal saccharopine dehydrogenase (lysine-forming) catalyze both forward and reverse reactions, use identical substrates and use similar co-factors.
• plant saccharopine dehydrogenase which catalyzes the second step in the lysine catabolic pathway, works in both forward and reverse reactions, uses identical substrates and uses similar co-factors as fungal saccharopine dehydrogenase (glutamate-forming).
• fungal saccharopine dehydrogenases Several genes for fungal saccharopine dehydrogenases have been isolated and sequenced and are readily available to those skilled in the art [Xuan et al. (1990) Mol. Cell. Biol. 70:4795-4806, Feller et al. (1994) Mol. Cell. Biol 74:6411-6418].
• genes could be used as heterologous hybridization probes to identify plant LKR and plant saccharopine dehydrogenase (glutamate-forming) nucleic acid fragments, or alternatively to identify homologous protein coding regions in plant cDNAs.
• Plant LKR has been reported to have a molecular weight of about 140,000 indicating that it is like the animal catabolic protein wherein both LKR and saccharopine dehydrogenase (glutamate-forming) enzyme activities are present on a single protein.
• Two plant saccharopine dehydrogenase (glutamate-forming) nucleic acid fragments (SEQ ID NOS:102 and 103) containing cDNA derived from Arabidopsis thaliana are provided. These were identified as cDNAs that encode proteins homologous to fungal saccharopine dehydrogenase (glutamate-forming). These nucleic acid fragments were used to design and synthesize oligonucleotide primers (SEQ ID NO: 108 and SEQ ID NO: 109). The primers were synthesized and used for PCR amplification of a 2.24 kb DNA fragment from genomic Arabidopsis DNA.
• This DNA fragment was used to isolate a larger genomic DNA fragment, which included the entire coding region, as well as 5' and 3' flanking regions, via hybridization to a genomic DNA library.
• the sequence of this genomic DNA fragment is provided (SEQ ID NO:l 10); oligonucleotides were synthesized based on this sequence and used to isolate a full length cDNA via RT-PCR.
• the sequence of the full length cDNA (SEQ ID NO:l 11) is provided.
• the deduced amino acid sequence of Arabidopsis LKR/SDH protein is shown in SEQ ID NO: 112.
• the amino acid sequence shows that in plants LKR and SDH enzyme activities are carried on a single bi-functional protein, and that the protein lacks an N-terminal targeting sequence indicating that the lysine degradative pathway is located in the plant cell cytosol.
• the amino acid sequence of Arabidopsis LKR/SDH protein was compared to that of other LKR and SDH proteins thus revealing regions of conserved amino acid sequence.
• Degenerate oligonucleotides can be designed based upon this information and used to amplify genomic or cDNA fragments via PCR from other organisms, preferably plants.
• SEQ ID NO:l 13 and SEQ ID NO: 114 were designed and used to amplify soybean and com LKR/SDH cDNA fragments.
• the sequence of a partial soybean LKR/SDH cDNA is shown in SEQ ID NO:l 15, and the sequence of a partial com cDNA is shown in SEQ ID NO:l 16.
• SEQ ID NO:l 15 The sequence of a partial soybean LKR/SDH cDNA is shown in SEQ ID NO:l 15
• SEQ ID NO:l 16 The sequence of a partial com cDNA is shown in SEQ ID NO:l 16.
• These DNA fragments can be used to isolate larger genomic DNA fragments, which include the entire coding region, as well as 5' and 3' flanking regions, via hybridization to com or soybean genomic DNA or cDNA libraries, as was done for Arabidopsis.
• More complete sequence information from the coding regions for soybean and com LKR/SDH was obtained using the sequences in SEQ ID NOS: 115 and 116 as starting materials in protocols such as 5' RACE and hybridization to cDNA libraries.
• a near full-length cDNA for soybean LKR/SDH is shown in SEQ ID NO:l 19
• a near full-length cDNA for com LKR SDH is shown in SEQ ID NO: 120.
• a truncated version of the LKR/SDH cDNA from com is set forth in SEQ ID NO: 123.
• the deduced partial amino acid sequences of soybean LRK/SDH protein is shown in SEQ ID NOS: 117 and 121 and the deduced partial amino acid sequences of com LKR/SDH protein is shown in SEQ ID NO:l 18, 122 and 124.
• These amino acid sequences can be compared to other LKR/SDH protein sequences, e.g., the Arabidopsis LKR/SDH protein sequence, thus revealing regions of conserved amino acid sequence.
• oligonucleotide primers can be designed and synthesized to permit isolation of LKR/SDH genomic or cDNA fragments from any plant source.
• SEQ ID NOS: 125 and 127 set forth sequences for partial cDNA clones encoding LKR/SDH from rice
• SEQ ID NO: 129 set forth the sequence of a partial cDNA encoding a ffragment of LKR/SDH from wheat.
• the prdicted protein fragments encoded by the sequences presented in SEQ ID NOS: 125, 127 and 129 are set forth in SEQ ID NOS: 126, 128 and 130, respectively,
• LKR/SDH genes make it possible to block expression of the LKR/SDH gene in transformed plants.
• a chimeric gene designed for cosuppression of LKR can be constructed by linking the LKR gene or gene fragment to any of the plant promoter sequences described above.
• a chimeric gene designed to express antisense RNA for all or part of the LKR gene can be constructed by linking the LKR gene or gene fragment in reverse orientation to any of the plant promoter sequences described above.
• Patent 5,107,065 for methodology to block plant gene expression via antisense RNA.
• Either the cosuppression or antisense chimeric gene can be introduced into plants via transformation. Transformants wherein expression of the endogenous LKR gene is reduced or eliminated are then selected.
• Preferred promoters for the chimeric genes would be seed-specific promoters.
• strong seed- specific promoters from a bean phaseolin gene, a soybean ⁇ -conglycinin gene, glycinin gene, Kunitz trypsin inhibitor gene, or rapeseed napin gene would be preferred.
• a strong endosperm- specific promoter e.g., the 10 kD or 27 kD zein promoter, or a strong embryo- specific promoter, e.g., the FLB1 promoter, would be preferred.
• Transformed plants containing any of the chimeric LKR genes can be obtained by the methods described above.
• the cosuppression or antisense LKR gene could be linked to the chimeric gene encoding substantially lysine-insensitve DHDPS and the two genes could be introduced into plants via transformation.
• the chimeric gene for cosuppression of LKR or antisense LKR could be introduced into previously transformed plants that express substantially lysine-insensitive DHDPS, or the cosuppression or antisense LKR gene could be introduced into normal plants and the transformants obtained could be crossed with plants that express substantially lysine-insensitive DHDPS.
• LKR/SDH genes make it possible to express the proteins in heterologous systems.
• a DNA fragment which includes the Arabidopsis SDH coding region (SEQ ID NO:l 19) was generated using PCR primers and ligated into a prokaryotic expression vector.
• High level expression of Arabidopsis SDH was achieved in E. coli and the SDH protein has been purified from the bacterial extracts, and used to raise rabbit antibodies to the protein.
• These antibodies can be used to screen for plant mutants in order to find variants which do not produce LKR/SDH protein, or produce reduced amounts of the protein compared to the parent plant.
• the plant mutants that express reduced LKR/SDH protein, or no protein at all could be crossed with plants that express substantially lysine-insensitive DHDPS.
• lysine produced may be desirable to convert the high levels of lysine produced into a form that is insensitive to breakdown, e.g., by incorporating it into a di-, tri- or oligopeptide, or a lysine-rich storage protein. No natural lysine-rich proteins are known.
• polypeptides which can be expressed in vivo to serve as lysine-rich seed storage proteins.
• Polypeptides are linear polymers of amino acids where the ⁇ -carboxyl group of one amino acid is covalently bound to the ⁇ -amino group of the next amino acid in the chain. Non- covalent interactions among the residues in the chain and with the surrounding solvent determine the final conformation of the molecule.
• electrostatic forces, hydrogen bonds, Van der Waals forces, hydrophobic interactions, and conformational preferences of individual amino acid residues in the design of a stable folded polypeptide chain [see for example: Creighton, (1984) Proteins, Structures and Molecular Properties, W. H.
• the synthetic storage proteins (SSPs) embodied in this invention are chosen to be polypeptides with the potential to be enriched in lysine relative to average levels of proteins in plant seeds. Lysine is a charged amino acid at physiological pH and is therefore found most often on the surface of protein molecules [Chothia, (1976) Journal of Molecular Biology 705:1-14].
• Applicants chose a molecular shape with a high surface-to-volume ratio for the synthetic storage proteins embodied in this invention.
• the altematives were either to stretch the common globular shape of most proteins to form a rod-like extended structure or to flatten the globular shape to a disk-like structure.
• Coiled-coils constitute a well-studied subset of the class of fibrous proteins [see Cohen et al., (1986) Trends Biochem. Sci. 77:245-248]. Natural examples are found in ⁇ -keratins, paramyosin, light meromyosin and tropomyosin. These protein molecules consist of two parallel alpha helices twisted about each other in a left-handed supercoil. The repeat distance of this supercoil is 140 A (compared to a repeat distance of 5.4 A for one turn of the individual helices). The supercoil causes a slight skew (10°) between the axes of the two individual alpha helices.
• the a and d amino acids of the heptad follow a 4,3 repeat pattern in the primary sequence and fall on one side of an individual alpha helix (See Figure 1). If the amino acids on one side of an alpha helix are all non-polar, that face of the helix is hydrophobic and will associate with other hydrophobic surfaces as, for example, the non-polar face of another similar helix.
• a coiled-coil structure results when two helices dimerize such that their hydrophobic faces are aligned with each other (See Figure 2a).
• the amino acids on the external faces of the component alpha helices (b, c, e, f, g) are usually polar in natural coiled-coils in accordance with the expected pattern of exposed and buried residue types in globular proteins [Schulz, et al., (1979) Principles of Protein Structure. Springer Verlag, New York, p 12; Talbot, et al , (1982) Ace. Chem. Res. 75:224-230; Hodges et al., (1981) Journal of Biological Chemistry 256:1214-1224].
• Charged amino acids are sometimes found forming salt bridges between positions e and g' or positions g and e' on the opposing chain (see Figure 2a).
• polypeptides of this invention are designed to dimerize with a coiled- coil motif in aqueous environments.
• Applicants have used a combination of hydrophobic interactions and electrostatic interactions to stabilize the coiled-coil conformation. Most nonpolar residues are restricted to the a and d positions which creates a hydrophobic stripe parallel to the axis of the helix. This is the dimerization face.
• Applicants avoided large, bulky amino acids along this face to minimize steric interference with dimerization and to facilitate formation of the stable coiled-coil structure.
• any destabilization of the coiled-coil that may be caused by methionine in the hydrophobic core appears to be compensated in sequences where the formation of salt bridges (e-g' and g-e') occurs at all possible positions in the helix (i.e., twice per heptad).
• Applicants minimized the unbalanced charges in the polypeptide. This may help to prevent undesirable interactions between the synthetic storage proteins and other plant proteins when the polypeptides are expressed in vivo.
• polypeptides of this invention are designed to spontaneously fold into a defined, conformationally stable structure, the alpha helical coiled-coil, with minimal restrictions on the primary sequence. This allows synthetic storage proteins to be custom-tailored for specific end-user requirements. Any amino acid can be incorporated at a frequency of up to one in every seven residues using the b, c, and f positions in the heptad repeat unit.
• novel synthetic storage proteins described in this invention represent a particular subset of possible coiled-coil polypeptides. Not all polypeptides which adopt an amphipathic alpha helical conformation in aqueous solution are suitable for the applications described here.
• the synthetic polypeptide comprises n heptad units (d e f g a b c), each heptad being either the same or different, wherein: n is at least 4; a and d are independently selected from the group consisting of
• e and g are independently selected from the group consisting of the acid/base pairs Glu Lys, Lys/Glu, Arg/Glu, Arg/Asp, Lys/Asp, Glu/Arg, Asp/Arg and Asp/Lys; and b, c and fare independently any amino acids except Gly or Pro and at least two amino acids of b, c and f in each heptad are selected from the group consisting of Glu, Lys, Asp, Arg, His, Thr, Ser, Asn, Gin, Cys and Ala.
• DNA sequences which encode the polypeptides described above can be designed based upon the genetic code. Where multiple codons exist for particular amino acids, codons should be chosen from those preferable for translation in plants. Oligonucleotides corresponding to these DNA sequences can be synthesized using an ABI DNA synthesizer, annealed with oligonucleotides corresponding to the complementary strand and inserted into a plasmid vector by methods known to those skilled in the art. The encoded polypeptide sequences can be lengthened by inserting additional annealed oligonucleotides at restriction endonuclease sites engineered into the synthetic gene.
• a chimeric gene designed to express RNA for a synthetic storage protein gene encoding a lysine-rich polypeptide can be constructed by linking the gene to any of the plant promoter sequences described above.
• Preferred promoters would be seed-specific promoters.
• rapeseed and other dicotyledonous plants strong seed-specific promoters from a bean phaseolin gene, a soybean ⁇ -conglycinin gene, glycinin gene, Kunitz trypsin inhibitor gene, or rapeseed napin gene would be preferred.
• a strong endosperm-specific promoter e.g., the 10 kD or 27 kD zein promoter, or a strong embyro-specific promoter, e.g., the com globulin 1 promoter, would be preferred.
• plants can be transformed by any of the methods described above.
• the SSP gene could be linked to the chimeric genes encoding substantially lysine-insensitive DHDPS and AK and the three genes could be introduced into plants via transformation.
• the chimeric SSP gene could be introduced into previously transformed plants that express substantially lysine-insensitive DHDPS and AK, or the SSP gene could be introduced into normal plants and the transformants obtained could be crossed with plants that express substantially lysine-insensitive DHDPS and AK.
• Lysine plus threonine inhibition is thought to result from feedback inhibition of endogenous AK, which reduces flux through the pathway leading to starvation for methionine.
• endogenous AK endogenous AK
• AKIII-M4 Expression of active lysine and threonine insensitive AKIII-M4 also reverses lysine plus threonine growth inhibition (Table 2, Example 7). There is a good correlation between the level of AKIII-M4 protein expressed and the resistance to lysine plus threonine. Expression of lysine-sensitive wild type AKIII does not have a similar effect. Since expression of the AKIII-M4 protein permits growth under normally inhibitory conditions, a chimeric gene that causes expression of AKIII-M4 in plants can be used as a selectable genetic marker for transformation as illustrated in Examples 13 and 17.
• EXAMPLE 1 Isolation of the E. coli IvsC Gene and mutations in lysC resulting in lysine-insensitive AKIII
• the E. coli lysC gene has been cloned, restriction endonuclease mapped and sequenced previously [Cassan et al. (1986) J Biol. Chem. 261:1052-1057].
• the lysC gene was obtained on a bacteriophage lambda clone from an ordered library of 3400 overlapping segments of cloned E. coli DNA constructed by Kohara, Akiyama and Isono [Kohara et al. (1987) Cell 50:595-508].
• This library provides a physical map of the whole E. coli chromosome and ties the physical map to the genetic map. From the knowledge of the map position of IvsC at 90 min on the E. coli genetic map [Theze et al. (1974) J. Bacteriol 7 7:133-143], the restriction endonuclease map of the cloned gene [Cassan et al. (1986) J. Biol. Chem. 267:1052-1057], and the restriction endonuclease map of the cloned DNA fragments in the E. coli library [Kohara et al.
• E. coli strain GiflO ⁇ Ml E. coli Genetic Stock Center strain CGSC-5074
• This strain lacks all AK activity and therefore requires diaminopimelate (a precursor to lysine which is also essential for cell wall biosynthesis), threonine and methionine.
• diaminopimelate a precursor to lysine which is also essential for cell wall biosynthesis
• threonine a precursor to lysine which is also essential for cell wall biosynthesis
• methionine methionine
• lysine or diaminopimelate which is readily converted to lysine in vivo
• M9 media [see Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press] supplemented with the arginine and isoleucine, required for GiflO ⁇ Ml growth, and ampicillin, to maintain selection for the pBT436 plasmid, was used. This inhibition is reversed by addition of threonine plus methionine to the growth media.
• Plasmid DNA was prepared from eight of these and re- transformed into GiflO ⁇ Ml to determine whether the lysine resistance determinant was plasmid-bome. Six of the eight plasmid DNAs yielded lysine- resistant colonies. Three of these six carried lysC genes encoding AKIII that was uninhibited by 15 mM lysine, whereas wild type AKIII is 50% inhibited by 0.3-0.4 mM lysine and >90% inhibited by 1 mM lysine (see Example 2 for details).
• sequences of the wild type lysC gene and three mutant genes were determined.
• a method for "Using mini-prep plasmid DNA for sequencing double stranded templates with SequenaseTM” [Kraft et al. (1988) BioTechniques 6:544-545] was used. Oligonucleotide primers, based on the published lysC sequence and spaced approximately every 200 bp, were synthesized to facilitate the sequencing.
• the sequence of the wild type IvsC gene cloned in pBT436 (SEQ ID NO:l) differed from the published IvsC sequence in the coding region at 5 positions.
• sequences of the three mutant lysC genes that encoded lysine- insensitive AK each differed from the wild type sequence by a single nucleotide, resulting in a single amino acid substitution in the protein.
• Mutant M2 had an A substituted for a G at nucleotide 954 of SEQ ID NO:l resulting in an isoleucine for methionine substitution at amino acid 318 and mutants M3 and M4 had identical T for C substitutions at nucleotide 1055 of SEQ ID NO:l resulting in an isoleucine for threonine substitution at amino acid 352.
• mutations M3 and M4 had identical T for C substitutions at nucleotide 1055 of SEQ ID NO:l resulting in an isoleucine for threonine substitution at amino acid 352.
• Nco I (CCATGG) site was inserted at the translation initiation codon of the IvsC gene using the following oligonucleotides:
• oligonucleotides When annealed these oligonucleotides have BamH I and Asp718 "sticky" ends.
• the plasmid pBT436 was digested with BamH I, which cuts upstream of the lysC coding sequence and Asp718 which cuts 31 nucleotides downstream of the initiation codon.
• the annealled oligonucleotides were ligated to the plasmid vector and E. coli transformants were obtained.
• Plasmid DNA was prepared and screened for insertion of the oligonucleotides based on the presence of an Nco I site. A plasmid containing the site was sequenced to assure that the insertion was correct, and was designated pBT457.
• this oligonucleotide insertion changed the second codon from TCT, coding for serine, to GCT, coding for alanine. This amino acid substitution has no apparent effect on the AKIII enzyme activity.
• the bacterial expression vector pBT430 was used. This vector is a derivative of p ⁇ T-3a [Rosenberg et al. (1987) Gene 56:125-135] which employs the bacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 was constmcted by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector.
• Nde I site at the position of translation initiation was converted to an Nco I site using oligonucleotide-directed mutagenesis.
• the DNA sequence of pET-3aM in this region, 5'-CATATGG. was converted to 5'-CCCATGG in pBT430.
• the lysC gene was cut out of plasmid pBT457 as a 1560 bp Nco I-EcoR I fragment and inserted into the expression vector pBT430 digested with the same enzymes, yielding plasmid pBT461.
• pBT461 was digested with Kpn I-EcoR I, which removes the wild type lysC gene from about 30 nucleotides downstream from the translation start codon, and inserting the homologous Kpn I-EcoR I fragments from the mutant genes yielding plasmids pBT490, pBT491 and pBT492, respectively.
• each of the plasmids was transformed into E. coli strain BL21(DE3) [Studier et al. (1986) J. Mol. Biol. 759:113-130]. Cultures were grown in LB medium containing ampicillin (100 mg/L) at 25°C. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio- ⁇ -galactoside, the inducer) was added to a final concentration of 0.4 mM and incubation was continued for 3 h at 25°.
• ampicillin 100 mg/L
• IPTG isopropylthio- ⁇ -galactoside, the inducer
• the cells were collected by centrifugation and resuspended in l/20th (or 1/lOOth) the original culture volume in 50 mM NaCl; 50 mM Tris-Cl, pH 7.5; 1 mM EDTA, and frozen at -20°. Frozen aliquots of 1 mL were thawed at 37° and sonicated, in an ice-water bath, to lyse the cells. The lysate was centrifuged at 4° for 5 min at 15,000 rpm. The supernatant was removed and the pellet was resuspended in 1 mL of the above buffer.
• the supernatant and pellet fractions of uninduced and IPTG-induced cultures of BL21(DE3)/pBT461 were analyzed by SDS polyacrylamide gel electrophoresis.
• the major protein visible by Coomassie blue staining in the supernatant of the induced culture had a molecular weight of about 48 kd, the expected size for AKIII.
• AK activity was assayed as shown below: Assay mix (for 12 assay tubes): 4.5 mL H 2 0 1.0 mL 8M KOH 1.0 mL 8M NH 2 OH-HCl 0.5 mL 0.2M ATP (121 mg/mL in 0.2M NaOH) 50 ⁇ L 1M MgSO 4
• Each 1.5 mL eppendorf assay tube contained:
• Assay tubes were incubated at 30° for desired time (10-60 min). Then 0.4 mL FeCl 3 reagent (10% w/v FeCl 3 , 3.3% trichloroacetic acid, 0.7 M HCl) was added and the material centrifuged for 2 min in an eppendorf centrifuge. The supernatant was decanted. The OD was read at 540 nm and compared to the aspartyl-hydroxamate standard.
• AKIII activity was in the supernatant fraction.
• the specific activity of wild type and mutant crude extracts was 5-7 ⁇ M product per min per milligram total protein. Wild type AKIII was sensitive to the presence of L-lysine in the assay. Fifty percent inhibition was found at a concentration of about 0.4 mM and 90% inhibition at about 1.0 mM. In contrast, mutants AKIII-M2, M3 and M4 (see Example 1) were not inhibited at all by 15 mM L-lysine.
• Wild type AKIII protein was purified from the supernatant of the IPTG- induced culture as follows. To 1 mL of extract, 0.25 mL of 10% streptomycin sulfate was added and kept at 4° overnight. The mixture was centrifuged at 4° for 15 min at 15,000 rpm. The supernatant was collected and desalted using a Sephadex G-25 M column (Column PD-10, Pharmacia). It was then run on a Mono-Q HPLC column and eluted with a 0-1M NaCl gradient. The two 1 mL fractions containing most of the AKIII activity were pooled, concentrated, desalted and run on an HPLC sizing column (TSK G3000SW).
• TSK G3000SW HPLC sizing column
• EXAMPLE 3 Isolation of the E. coli and Corynebacterium glutamicum dap A genes
• the E. coli dapA gene (ecodapA) has been cloned, restriction endonuclease mapped and sequenced previously [Richaud et al. (1986) J Bacteriol 766:297-300].
• the dapA gene was obtained on a bacteriophage lambda clone from an ordered library of 3400 overlapping segments of cloned E. coli DNA constmcted by Kohara, Akiyama and Isono [Kohara et al. (1987) Cell 50:595-508, see Example 1].
• the phages were grown in liquid culture from single plaques as described [see Current Protocols in Molecular Biology (1987) Ausubel et al. eds., John Wiley & Sons New York] using LE392 as host [see Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press]. Phage DNA was prepared by phenol extraction as described [see Current Protocols in Molecular Biology (1987) Ausubel et al. eds., John Wiley & Sons New York]. Both phages contained an approximately 2.8 kb Pst I DNA fragment expected for the dapA gene [Richaud et al. (1986) J. Bacteriol. 766:297-300]. The fragment was isolated from the digest of phage 5A8 and inserted into Pst I digested vector pBR322 yielding plasmid pBT427.
• Corynebacterium dapA gene was isolated from genomic DNA from ATCC strain 13032 using polymerase chain reaction (PCR).
• PCR polymerase chain reaction
• the nucleotide sequence of the Corynebacterium dapA gene has been published [Bonnassie et al. (1990) Nucleic Acids Res. 75:6421]. From the sequence it was possible to design oligonucleotide primers for PCR that would allow amplification of a DNA fragment containing the gene, and at the same time add unique restriction endonuclease sites at the start codon (Nco I) and just past the stop codon (EcoR I) of the gene.
• the oligonucleotide primers used were:
• PCR was performed using a Perkin-Elmer Cetus kit according to the instructions of the vendor on a thermocycler manufactured by the same company.
• the reaction product when run on an agarose gel and stained with ethidium bromide, showed a strong DNA band of the size expected for the Corynebacterium dapA gene, about 900 bp.
• the PCR-generated fragment was digested with restriction endonucleases Nco I and EcoR I and inserted into expression vector pBT430 (see Example 2) digested with the same enzymes.
• the PCR primers also resulted in a change of the second codon from AGC coding for serine to GCT coding for alanine.
• Several clones that expressed active, lysine- insensitive DHDPS were isolated, indicating that the second codon amino acid substitution did not affect activity; one clone was designated FS766.
• the Nco I to EcoR I fragment carrying the PCR-generated Corynebacterium dapA gene was subcloned into the phagemid vector pGEM-9Zf(-) from Promega, single-stranded DNA was prepared and sequenced. This sequence is shown in SEQ ID NO:6.
• Putative mutants were screened for the presence of an Nco I site and a plasmid, designated pBT437, was shown to have the proper sequence in the vicinity of the mutation by DNA sequencing.
• the addition of an Nco I site at the translation start codon also resulted in a change of the second codon from TTC coding for phenylalanine to GTC coding for valine.
• the bacterial expression vector pBT430 (see Example 2) was used.
• the E. coli dapA gene was cut out of plasmid pBT437 as an 1150 bp Nco I-Hind III fragment and inserted into the expression vector pBT430 digested with the same enzymes, yielding plasmid pBT442.
• the 910 bp Nco I to EcoR I fragment of SEQ ID NO:6 inserted in pBT430 (pFS766, see Example 3) was used.
• each of the plasmids was transformed into E. coli strain BL21(DE3) [Studier et al. (1986) J. Mol. Biol. 759:113-130]. Cultures were grown in LB medium containing ampicillin (100 mg/L) at 25°. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio- ⁇ -galactoside, the inducer) was added to a final concentration of 0.4 mM and incubation was continued for 3 h at 25°.
• ampicillin 100 mg/L
• IPTG isopropylthio- ⁇ -galactoside, the inducer
• the cells were collected by centrifugation and resuspended in l/20th (or 1/100th) the original culture volume in 50 mM NaCl; 50 mM Tris-Cl, pH 7.5; 1 mM EDTA, and frozen at -20°. Frozen aliquots of 1 mL were thawed at 37° and sonicated, in an ice-water bath, to lyse the cells. The lysate was centrifuged at 4° for 5 min at 15,000 rpm. The supernatant was removed and the pellet was resuspended in 1 mL of the above buffer.
• the supernatant and pellet fractions of uninduced and IPTG-induced cultures of BL21(DE3)/pBT442 or BL21(DE3)/pFS766 were analyzed by SDS polyacrylamide gel electrophoresis.
• the major protein visible by Coomassie blue staining in the supernatant and pellet fractions of both induced cultures had a molecular weight of 32-34 kd, the expected size for DHDPS. Even in the uninduced cultures this protein was the most prominent protein produced.
• coli DHDPS or Corynebacterium DHDPS were solubilized in 50 mM NaCl; 50 mM Tris-Cl, pH 7.5; 1 mM EDTA, 0.2 mM dithiothreitol, 0.2% SDS and sent to Hazelton Research Facility (310 Swampridge Road, Denver, PA 17517) to have rabbit antibodies raised against the proteins.
• DHDPS enzyme activity was assayed as follows: Assay mix (for 10 X 1.0 mL assay tubes or 40 X 0.25 mL for microtiter dish); made fresh, just before use: 2.5 mL H 2 0
• ASA DL-Aspartic- ⁇ -semialdehyde
• Enzyme activity was about 4 OD530 units per min per milligram protein in a 0.25 mL assay.
• Corynebacterium DHDPS was not inhibited at all by L-lysine, even at a concentration of 70 mM.
• the E. coli expression cassette with the E. coli dapA gene linked to the T7 RNA polymerase promoter was isolated by digesting pBT442 (see Example 4) with Bgl II and BamH I separating the digestion products via agarose gel electrophoresis and eluting the approximately 1250 bp fragment from the gel. This fragment was inserted into the BamH I site of plasmids pBT461 (containing the T7 promoter/lvsC gene) and pBT492 (containing the T7 promoter/lysC-M4 gene).
• Inserts where transcription of both genes would be in the same direction were identified by restriction endonuclease analysis yielding plasmids pBT517 (T7/dapA + T7/lvsC-M4) and pBT519 (T7/daoA + T7/lvsC).
• these plasmids were transformed into E. coli strain BL21(DE3) [Studier et al. (1986) J Mol Biol. 759:113-130]. All of these plasmids, but especially pBT517 and pBT519, are somewhat unstable in this host strain, necessitating careful maintenance of selection for ampicillin resistance during growth.
• All of the plasmids lead to the excretion of lysine into the culture medium.
• Expression of the lysC or the lvsC-M4 gene lead to both lysine and threonine excretion.
• Expression of lvsC-M4 + dapA lead to excretion of lysine, methionine, aspartic acid and glutamic acid, but not threonine.
• alanine and valine were not detected in the culture supernatant. Similar results were obtained with lysC + dapA. except that no glutamic acid was excreted.
• a leaf expression cassette ( Figure 4a) is composed of the 35S promoter of cauliflower mosaic vims [Odell et al.(1985) Nature 575:810-812; Hull et al. (1987) Virology 56:482-493], the translation leader from the chlorophyll a/b binding protein (Cab) gene, [Dunsmuir (1985) Nucleic Acids Res. 75:2503-2518] and 3' transcription termination region from the nopaline synthase (Nos) gene [Depicker et al. (1982) J Mol.
• a seed-specific expression cassette ( Figure 4b) is composed of the promoter and transcription terminator from the gene encoding the ⁇ subunit of the seed storage protein phaseolin from the bean Phaseolus vulgaris [Doyle et al. (1986) J. Biol. Chem. 267:9228-9238].
• the phaseolin cassette includes about 500 nucleotides upstream (5') from the translation initiation codon and about 1650 nucleotides downstream (3') from the translation stop codon of phaseolin. Between the 5' and 3' regions are the unique restriction endonuclease sites Nco I (which includes the ATG translation initiation codon), Sma I, Kpn I and Xba I. The entire cassette is flanked by Hind III sites.
• a second seed expression cassette was used for the cordapA gene. This was composed of the promoter and transcription terminator from the soybean Kunitz tyrosine inhibitor 3 (KTI3) gene [Jofuku et al. (1989) Plant Cell 7:427-435].
• the KTI3 cassette includes about 2000 nucleotides upstream (5') from the translation initiation codon and about 240 nucleotides downstream (3') from the translation stop codon of phaseolin. Between the 5' and 3' regions are the unique restriction endonuclease sites Nco I (which includes the ATG translation initiation codon), Xba I, Kpn I and Sma I. The entire cassette is flanked by BamH I sites.
• a constitutive expression cassette for com was used for expression of the lysC-M4 gene and the ecodapA gene. It was composed of a chimeric promoter derived from pieces of two com promoters and modified by in vitro site-specific mutagenesis to yield a high level constitutive promoter and a 3' region from a com gene of unknown function. Between the 5' and 3' regions are the unique restriction endonuclease sites Nco I (which includes the ATG translation initiation codon), Sma I and Bgl II. The nucleotide sequence of the constitutive com expression cassette is shown in SEQ ID NO:93.
• Plant amino acid biosynthetic enzymes are known to be localized in the chloroplasts and therefore are synthesized with a chloroplast targeting signal. Bacterial proteins such as DHDPS and AKIII have no such signal.
• a chloroplast transit sequence (cts) was therefore fused to the ecodapA. cordapA. lysC. and lysC-M4 coding sequence in some chimeric genes. The cts used was based on the cts of the small subunit of ribulose 1,5-bisphosphate carboxylase from soybean [Berry-Lowe et al. (1982) J. Mol. Appl. Genet. 7:483-498].
• the oligonucleotides SEQ ID NOS: 8-11 were synthesized and used as described below.
• the cts used was based on the cts of the small subunit of ribulose 1,5-bisphosphate carboxylase from com [Lebrun et al. (1987) Nucleic Acids Res. 75:4360] and is designated mcts to distinguish it from the soybean cts.
• the oligonucleotides SEQ ID NOS: 17-22 were synthesized and used as described below. Fourteen chimeric genes were created:
• HH534 5* region/mcts/ecodaEA/HH2-l 3' region A 1440 bp Nco I-Hpa I fragment containing the entire lysC coding region plus about 90 bp of 3' non-coding sequence was isolated from an agarose gel following electrophoresis and inserted into the leaf expression cassette digested with Nco I and Sma I (chimeric gene No. 1), yielding plasmid pBT483.
• Oligonucleotides SEQ ID NO:8 and SEQ ID NO:9 which encode the carboxy terminal part of the chloroplast targeting signal, were annealed, resulting in Nco I compatible ends, purified via polyacrylamide gel electrophoresis, and inserted into Nco I digested pBT461. The insertion of the correct sequence in the correct orientation was verified by DNA sequencing yielding pBT496.
• Oligonucleotides SEQ ID NO: 10 and SEQ ID NO:l 1, which encode the amino terminal part of the chloroplast targeting signal were annealed, resulting in Nco I compatible ends, purified via polyacrylamide gel electrophoresis, and inserted into Nco I digested pBT496. The insertion of the correct sequence in the correct orientation was verified by DNA sequencing yielding pBT521. Thus the cts was fused to the lysC gene.
• pBT521 was digested with Sal I, and an approximately 900 bp DNA fragment that included the cts and the amino terminal coding region of IvsC was isolated. This fragment was inserted into Sal I digested pBT492, effectively replacing the amino terminal coding region of lysC-M4 with the fused cts and the amino terminal coding region oflysC. Since the mutation that resulted in lysine-insensitivity was not in the replaced fragment, the new plasmid, pBT523, carried the cts fused to lysC-M4.
• the 1600 bp Nco I-Hpa I fragment containing the cts fused to lysC plus about 90 bp of 3' non-coding sequence was isolated and inserted into the leaf expression cassette digested with Nco I and Sma I (chimeric gene No. 2), yielding plasmid pBT541 and the seed-specific expression cassette digested with Nco I and Sma I (chimeric gene No. 4), yielding plasmid pBT543.
• the 1600 bp Nco I-Hpa I fragment containing the cts fused to lysC-M4 plus about 90 bp of 3' non-coding sequence was isolated and inserted into the leaf expression cassette digested with Nco I and Sma I (chimeric gene No. 3), yielding plasmid pBT540 and the seed-specific expression cassette digested with Nco I and Sma I (chimeric gene No. 5), yielding plasmid pBT544.
• the ecodapA gene was modified to insert a restriction endonuclease site, Kpn I, just after the translation stop codon.
• the oligonucleotides SEQ ID NOS: 12- 13 were synthesized for this purpose:
• Oligonucleotides SEQ ID NO:12 and SEQ ID NO:13 were annealed, resulting in an Sph I compatible end on one end and a Hind III compatible end on the other and inserted into Sph I plus Hind III digested pBT437. The insertion of the correct sequence was verified by DNA sequencing yielding pBT443.
• Nco I-Kpn I fragment from pBT443 containing the entire ecodapA coding region was isolated from an agarose gel following electrophoresis and inserted into the leaf expression cassette digested with Nco I and Kpn I (chimeric gene No. 6), yielding plasmid pBT450 and into the seed-specific expression cassette digested with Nco I and Kpn I (chimeric gene No. 8), yielding plasmid pBT494.
• Oligonucleotides SEQ ID NO:8 and SEQ ID NO:9 which encode the carboxy terminal part of the chloroplast targeting signal, were annealed resulting in Nco I compatible ends, purified via polyacrylamide gel electrophoresis, and inserted into Nco I digested pBT450. The insertion of the correct sequence in the correct orientation was verified by DNA sequencing yielding pBT451.
• a 950 bp Nco I-Kpn I fragment from pBT451 encoding the carboxy terminal part of the chloroplast targeting signal fused to the entire ecodapA coding region was isolated from an agarose gel following electrophoresis and inserted into the seed-specific expression cassette digested with Nco I and Kpn I, yielding plasmid pBT495.
• Insertion of the correct sequence in the correct orientation was verified by DNA sequencing yielding pBT455 and pBT520, respectively.
• the cts was fused to the ecodapA gene in the leaf expression cassette (chimeric gene No. 7) and the seed-specific expression cassette (chimeric gene No. 9).
• An 870 bp Nco I-EcoR I fragment from pFS766 containing the entire cordapA coding region was isolated from an agarose gel following electrophoresis and inserted into the leaf expression cassette digested with Nco I and EcoR I, yielding plasmid pFS789.
• a DNA fragment containing the entire cts was prepared using PCR.
• the template DNA was pBT540 and the oligonucleotide primers used were:
• PCR was performed using a Perkin-Elmer Cetus kit according to the instructions of the vendor on a thermocycler manufactured by the same company.
• the PCR-generated 160 bp fragment was treated with T4 DNA polymerase in the presence of the 4 deoxyribonucleotide triphosphates to obtain a blunt-ended fragment.
• the cts fragment was inserted into pFS789 which had been digested with Nco I and treated with the Klenow fragment of DNA polymerase to fill in the 5' overhangs.
• the inserted fragment and the vector/insert junctions were determined to be correct by DNA sequencing, yielding pFS846 containing chimeric gene No. 10.
• a 1030 bp Nco I-Kpn I fragment from pFS846 containing the cts attached to the cordapA coding region was isolated from an agarose gel following electrophoresis and inserted into the phaseolin seed expression cassette digested with Nco I and Kpn I, yielding plasmid pFS889 containing chimeric gene No. 11.
• the 1030 bp Nco I-Kpn I fragment from pFS846 was inserted into the KTI3 seed expression cassette digested with Nco I and Kpn I, yielding plasmid pFS862 containing chimeric gene No. 12.
• Oligonucleotides SEQ ID NO:94 and SEQ ID NO:95 which encode the carboxy terminal part of the com chloroplast targeting signal, were annealed, resulting in Xba I and Nco I compatible ends, purified via polyacrylamide gel electrophoresis, and inserted into Xba I plus Nco I digested pBT492 (see Example 2). The insertion of the correct sequence was verified by DNA sequencing yielding pBT556.
• Oligonucleotides SEQ ID N ⁇ :96 and SEQ ID NO:97 which encode the middle part of the chloroplast targeting signal, were annealed, resulting in Bgl II and Xba I compatible ends, purified via polyacrylamide gel electrophoresis, and inserted into Bgl II and Xba I digested pBT556. The insertion of the correct sequence was verified by DNA sequencing yielding pBT557.
• Oligonucleotides SEQ ID NO:98 and SEQ ID NO:99 which encode the amino terminal part of the chloroplast targeting signal, were annealed, resulting in Nco I and Afl II compatible ends, purified via polyacrylamide gel electrophoresis, and inserted into Nco I and Afl II digested pBT557. The insertion of the correct sequence was verified by DNA sequencing yielding pBT558. Thus the mcts was fused to the lysC-M4 gene.
• a DNA fragment containing the entire mcts was prepared using PCR as described above.
• the template DNA was pBT558 and the oligonucleotide primers used were:
• SEQ ID NO:100 GCGCCCACCG TGATGA
• SEQIDNO:101 CACCGGATTC TTCCGC
• the mcts fragment was inserted into pBT450 (above) which had been digested with Nco I and treated with the Klenow fragment of DNA polymerase to fill in the 5' overhangs.
• the inserted fragment and the vector/insert junctions were determined to be correct by DNA sequencing, yielding pBT576.
• Plasmid pBT576 was digested with Asp718, treated with the Klenow fragment of DNA polymerase to yield a blunt-ended fragment, and then digested with Nco I.
• the resulting 1030 bp Nco I-blunt-ended fragment containing the ecodapA gene attached to the mcts was isolated from an agarose gel following electrophoresis.
• This fragment was inserted into the constitutive com expression cassette digested with Bgl II, treated with the Klenow fragment of DNA polymerase to yield a blunt-ended fragment, and then digested with Nco I, yielding plasmid pBT583 containing chimeric gene No. 14.
• Transformation of tobacco with the 35S promoter/lvsC chimeric genes was effected according to the following: The 35S promoter/Cab leader/lvsC/Nos 3', 35S promoter/Cab leader/cts/lvsC/Nos 3', and 35S promoter/Cab leader/cts/lvsC-M4/Nos 3' chimeric genes were isolated as 3.5-3.6 kb BamH I-EcoR I fragments and inserted into BamH I-EcoR I digested vector pZS97K ( Figure 5), yielding plasmids ⁇ BT497, pBT545 and pBT542, respectively.
• the vector is part of a binary Ti plasmid vector system [Bevan, (1984) Nucl. Acids. Res. 72:8711-8720] of Agrobacterium tumefaciens.
• the vector contains: (1) the chimeric gene nopaline synthase promoter/neomycin phosphotransferase coding region (nos:NPT II) as a selectable marker for transformed plant cells [Bevan et al. (1983) Nature 504:184-186]; (2) the left and right borders of the T-DNA of the Ti plasmid [Bevan (1984) Nucl Acids. Res. 72:8711-8720]; (3) the E.
• the 35S promoter/Cab leader/cts/lvsC/Nos 3', and 35S promoter/Cab leader/cts/lysC-M4/Nos 3' chimeric genes were also inserted into the binary vector pBT456, yielding pBT547 and pBT546, respectively.
• This vector is pZS97K, into which the chimeric gene 35S promoter/Cab leader/cts/dapA/Nos 3' had previously been inserted as a BamH I-Sal I fragment (see Example 9). In the cloning process large deletions of the dapA chimeric gene occurred.
• these plasmids are equivalent to pBT545 and pBT542, in that the only transgene expressed in plants (other than the selectable marker gene, NPT II) was 35 S promoter/Cab leader/cts/lvsC/Nos 3' or 35S promoter/Cab leader/cts/lysC-M4/Nos 3'.
• the binary vectors containing the chimeric IvsC genes were transferred by tri-parental matings [Ruvkin et al. (1981) Nature 289:85-88] to Agrobacterium strain LBA4404/pAL4404 [Hockema et al (1983), Nature 505:179-180].
• the Agrobacterium transformants were used to inoculate tobacco leaf disks [Horsch et al. (1985) Science 227:1229-1231]. Transgenic plants were regenerated in selective medium containing kanamycin.
• protein was extracted as follows. Approximately 2.5 g of young plant leaves, with the midrib removed, were placed in a dounce homogenizer with 0.2 g of polyvinyl polypyrrolidone and 11 mL of 50mM Tris-HCl pH8.0, 50mM NaCl, ImM EDTA (TNE) and ground thoroughly. The suspension was further homogenized by a 20 sec treatment with a Brinkman Polytron Homogenizer operated at setting 7. The resultant suspensions were centrifuged at 16,000 rpm for 20 min at 4° in a Dupont-Sorvall superspeed centrifuge using an SS34 rotor to remove particulates.
• the supernatant was decanted, the volume was adjusted to be 10 mL by addition of TNE if necessary, and 8 mL of cold, saturated ammonium sulfate was added. The mixture was set on ice for 30 min and centrifuged as described above. The supernatant was decanted and the pellet, which contained the AKIII protein, was resuspended in 1 mL of TNE and desalted by passage over a Sephadex G-25 M column (Column PD-10, Pharmacia).
• the membranes were exposed to the AKIII antibodies prepared as described in Example 2 at a 1 :5000 dilution of the rabbit serum using standard protocol provided by BioRad with their Immun-Blot Kit. Following rinsing to remove unbound primary antibody, the membranes were exposed to the secondary antibody, donkey anti-rabbit Ig conjugated to horseradish peroxidase (Amersham) at a 1:3000 dilution. Following rinsing to remove unbound secondary antibody, the membranes were exposed to Amersham chemiluminescence reagent and X-ray film.
• AKIII could be distinguished from endogenous AK activity, if it were present, by its increased resistance to lysine plus threonine. Unfortunately, however, this assay was not sensitive enough to reliably detect AKIII activity in these extracts.
• An alternative method to detect the expression of active AKIII enzyme was to evaluate the sensitivity or resistance of leaf tissue to high concentrations of lysine plus threonine. Growth of cell cultures and seedlings of many plants is inhibited by high concentrations of lysine plus threonine; this is reversed by addition of methionine (or homoserine which is converted to methionine in vivo). Lysine plus threonine inhibition is thought to result from feedback inhibition of endogenous AK, which reduces flux through the pathway leading to starvation for methionine. In tobacco there are two AK enzymes in leaves, one lysine-sensitive and one threonine sensitive [Negrutui et al. (1984) Theor. Appl.
• free amino acids were extracted as follows. Approximately 30-40 mg of young leaf tissue was chopped with a razor and dropped into 0.6 mL of methanol/ chloroform/water mixed in ratio of 12v/5v/3v (MCW) on dry ice. After 10-30 min the suspensions were brought to room temperature and homogenized with an Omni 1000 Handheld Rechargeable Homogenizer and then centrifuged in an eppendorf microcentrifuge for 3 min. Approximately 0.6 mL of supernatant was decanted and an additional 0.2 mL of MCW was added to the pellet which was then vortexed and centrifuged as above. The second supernatant, about 0.2 mL, was added to the first.
• BT542 transformants 35S promoter/Cab leader/cts/lvsC-M4/Nos 3'
• BT545 transformants 35S promoter/Cab leader/cts/lvsC/Nos 3'
• BT546 transformants 35S promoter/Cab leader/cts/lysC-M4/Nos 3'
• BT547 transformants 35S promoter/Cab leader/cts/lvsC/Nos 3'
• phaseolin promoter/lvsC chimeric gene cassettes, phaseolin 5' region/cts/lvsC/phaseolin 3' region, and phaseolin 5' region/cts/lysC-M4/phaseolin 3' region were isolated as approximately 3.3 kb Hind III fragments. These fragments were inserted into the unique Hind III site of the binary vector pZS97 ( Figure 6) yielding pBT548 and pBT549, respectively.
• This vector is similar to pZS97K described in Example 7 except for the presence of two additional unique cloning sites, Sma I and Hind III, and the bacterial ⁇ -lactamase gene (causing ampicillin resistance) as a selectable marker for transformed A. tumefaciens instead of the bacterial neomycin phosphotransferase gene.
• the binary vectors containing the chimeric lysC genes were transferred by tri-parental matings to Agrobacterium strain LBA4404/pAL4404, the Agrobacterium transformants were used to inoculate tobacco leaf disks and transgenic plants regenerated by the methods set out in Example 7.
• the membranes were exposed to the secondary antibody, donkey anti-rabbit Ig conjugated to horseradish peroxidase (Amersham) at a 1 :3000 dilution. Following rinsing to remove unbound secondary antibody, the membranes were exposed to Amersham chemiluminescence reagent and X-ray film.
• free amino acids were extracted from mature seeds as follows. Approximately 30-40 mg of seeds and an approximately equal amount of sterilized sand were put into a 1.5 mL disposable plastic microfuge tube along with 0.2 mL of methanol/chloroform/water mixed in ratio of 12v/5v/3v (MCW) at room temperature. The seeds were ground using a motorized grinder with disposable plastic shafts designed to fit into the microfuge tube. After grinding an additional 0.5 mL of MCW was added, the mixture was vortexed and then centrifuged in an eppendorf microcentrifuge for about 3 min.
• the samples were hydrolyzed in 6N hydrochloric acid, 0.4% (v/v) ⁇ -mercaptoethanol under nitrogen for 24 h at 110-120°; 1/4 of the sample was run on a Beckman Model 6300 amino acid analyzer using post-column ninhydrin detection. Relative free amino acid levels in the seeds were compared as ratios of lysine, methionine, threonine or isoleucine to leucine, thus using leucine as an internal standard.
• Normal was calculated as the average of 6 samples for free amino acid and 23 samples for total amino acids.
• 35S promoter/Cab leader/ecodapA/Nos 3' and 35S promoter/Cab leader/cts/ecodapA/Nos 3', chimeric genes were isolated as 3.1, and 3.3 kb BamH I-Sal I fragments, respectively and inserted into BamH I-Sal I digested binary vector pZS97K ( Figure 5), yielding plasmids pBT462 and pBT463, respectively.
• the binary vector is described in Example 7.
• the binary vectors containing the chimeric ecodapA genes were transferred by tri-parental matings to Agrobacterium strain LBA4404/pAL4404, the Agrobacterium transformants used to inoculate tobacco leaf disks and the resulting transgenic plants regenerated by the methods set out in Example 7.
• Example 7 To assay for expression of the chimeric genes in leaves of the transformed plants, protein was extracted as described in Example 7, with the following modifications. The supernatant from the first ammonium sulfate precipitation, approximately 18 mL, was mixed with an additional 12 mL of cold, saturated ammonium sulfate. The mixture was set on ice for 30 min and centrifuged as described in Example 7. The supernatant was decanted and the pellet, which contained the DHDPS protein, was resuspended in 1 mL of TNE and desalted by passage over a Sephadex G-25 M column (Column PD-10, Pharmacia).
• E. coli DHDPS could be distinguished from tobacco DHDPS activity by its increased resistance to lysine; E. coli DHDPS retained 80-90% of its activity at O.lmM lysine, while tobacco DHDPS was completely inhibited at that concentration of lysine.
• Free amino acids were extracted from leaves as described in Example 7. Expression of the chimeric gene, 35S promoter/Cab leader/cts/ecodapA/Nos 3', but not 35S promoter/Cab leader/ecodapA/Nos 3' resulted in substantial increases in the level of free lysine in the leaves. Free lysine levels from two to 90-fold higher than untransformed tobacco were observed.
• the transformed plants were allowed to flower, self-pollinate and go to seed. Seeds from several lines transformed with the 35S promoter/Cab leader/ cts/ecodapA/Nos 3' gene were surface sterilized and germinated on agar plates in the presence of kanamycin. Lines that showed 3 kanamycin resistant to 1 kanamycin sensitive seedlings, indicative of a single site of insertion of the transgenes, were identified. Progeny that were homozygous for the transgene insert were obtained from these lines using standard genetic analysis. The homozygous progeny were then characterized for expression of E. coli DHDPS in young and mature leaves and for the levels of free amino acids accumulated in young and mature leaves and in mature seeds.
• the binary vectors containing the chimeric ecodapA genes were transferred by tri-parental matings to Agrobacterium strain LBA4404/pAL4404, the Agrobacterium transformants used to inoculate tobacco leaf disks and the resulting transgenic plants were regenerated by the methods set out in Example 7.
• Example 8 To assay for expression of the chimeric genes, the transformed plants were allowed to flower, self-pollinate and go to seed. Total seed proteins were extracted as described in Example 8 and immunologically analyzed as described in Example 7, with the following modification. The Western blot membranes were exposed to the DHDPS antibodies prepared in Example 4 at a 1 :5000 dilution of the rabbit serum using standard protocol provided by BioRad with their Immun-Blot Kit.
• E. coli DHDPS could be distinguished from tobacco DHDPS activity by its increased resistance to lysine; E. coli DHDPS retained about 50% of its activity at 0.4 mM lysine, while tobacco DHDPS was completely inhibited at that concentration of lysine. High levels of E. coli DHDPS activity were seen in all four seed extracts tested eliminating this explanation.
• oligonucleotide adaptor was synthesized to convert the BamH I site at the 5' end of the 35S promoter/Cab leader/cts/lysC-M4/Nos 3' chimeric gene (see Figure 4a) to an EcoR I site.
• the 35S promoter/Cab leader/cts/lvsC-M4/Nos 3' chimeric gene was then isolated as a 3.6 kb EcoR I fragment from plasmid pBT540 (Example 6) and inserted into pBT463 (Example 9) digested with EcoR I, yielding plasmid pBT564.
• This vector has both the 35S promoter/Cab leader/cts/ecodapA/Nos 3', and 35S promoter/Cab leader/cts/lysC-M4/Nos 3' chimeric genes inserted in the same orientation.
• the binary vector containing the chimeric ecodapA and lysC-M4 genes was transferred by tri-parental matings to Agrobacterium strain LB A4404/pAL4404, the Agrobacterium transformants used to inoculate tobacco leaf disks and the resulting transgenic plants regenerated by the methods set out in Example 7.
• Example 7 for AKIII protein was extracted as described in Example 7 for AKIII, and as described in Example 9 for DHDPS.
• the leaf extracts were assayed for DHDPS activity as described in Examples 4 and 9.
• E. coli DHDPS could be distinguished from tobacco DHDPS activity by its increased resistance to lysine; E. coli DHDPS retained 80-90% of its activity at 0.1 mM lysine, while tobacco DHDPS was completely inhibited at that concentration of lysine. Extracts were characterized immunologically for expression of AKIII and DHDPS proteins via Western blots as described in Examples 7 and 10.
• Free amino acids were extracted from mature seeds derived from self- pollinated plants and quantitated as described in Example 8. There was no significant difference in the free amino acid content of seeds from untransformed plants compared to that from the plants showing the highest free lysine accumulation in leaves, i.e. plants 564-18A, 564-21A, 564-36E, 564-56A.
• phaseolin 5' region/cts/ecodapA/phaseolin 3' region and phaseolin 5' region/cts/lvsC-M4/phaseolin 3' were combined in the binary vector pZS97 ( Figure 6).
• the binary vector is described in Example 8.
• the phaseolin 5' region/cts/ecodapA/phaseolin 3' chimeric gene was isolated as a 2.7 kb Hind III fragment and inserted into the Hind III site of vector pUC 1318 [Kay et al (1987) Nucleic Acids Res. 6:2778], yielding pBT568.
• the binary vector pBT570 was transferred by tri-parental mating to Agrobacterium strain LBA4404/pAL4404, the Agrobacterium transformants used to inoculate tobacco leaf disks and the resulting transgenic plants regenerated by the methods set out in Example 7.
• ⁇ -aminoadipic acid In the highest expressing lines it was possible to detect a high level of ⁇ -aminoadipic acid.
• This compound is known to be an intermediate in the catabolism of lysine in cereal seeds, but is normally detected only via radioactive tracer experiments due to its low level of accumulation. The build-up of high levels of this intermediate indicates that a large amount of lysine is being produced in the seeds of these transformed lines and is passing through the catabolic pathway. The build-up of ⁇ -aminoadipic acid was not observed in transformants expressing only E. coli DHDPS or only AKIII-M4 in seeds. These results show that it is necessary to express both enzymes simultaneously to produce high levels of free lysine.
• phaseolin 5'region/cts/lysC-M4/phaseolin 3' region phaseolin 5'region/cts/ecodapA phaseolin 3' region
• EXAMPLE 13 Use of the cts/lvsC-M4 Chimeric Gene as a Selectable Marker for Tobacco Transformation
• the 35S promoter/Cab leader/cts/lysC-M4/Nos 3' chimeric gene in the binary vector pZS97K (pBT542, see Example 7) was used as a selectable genetic marker for transformation of tobacco.
• High concentrations of lysine plus threonine inhibit growth of shoots from tobacco leaf disks.
• Expression of active lysine and threonine insensitive AKIII-M4 reverses this growth inhibition (see Example 7).
• the binary vector pBT542 was transferred by tri-parental mating to Agrobacterium strain LB A4404/pAL4404, the Agrobacterium transformants used to inoculate tobacco leaf disks and the resulting transformed shoots were selected on shooting medium containing 3 mM lysine plus 3 mM threonine. Shoots were transferred to rooting media containing 3 mM lysine plus 3 mM threonine. Plants were grown from the rooted shoots. Leaf disks from the plants were placed on shooting medium containing 3 mM lysine plus 3 mM threonine. Transformed plants were identified by the shoot proliferation which occurred around the leaf disks on this medium.
• the 35S promoter/Cab leader/cts/cordapA/Nos 3' chimeric gene was isolated as a 3.0 kb BamH I-Sal I fragment and inserted into BamH I-Sal I digested binary vector pZS97K ( Figure 5), yielding plasmid pFS852.
• the binary vector is described in Example 7.
• the binary vector containing the chimeric cordapA gene was transferred by tri-parental mating to Agrobacterium strain LBA4404/pAL4404, the Agrobacterium transformant was used to inoculate tobacco leaf disks and the resulting transgenic plants were regenerated by the methods set out in Example 7.
• Example 7 To assay for expression of the chimeric gene in leaves of the transformed plants, protein was extracted as described in Example 7, with the following modifications. The supernatant from the first ammonium sulfate precipitation, approximately 18 mL, was mixed with an additional 12 mL of cold, saturated ammonium sulfate. The mixture was set on ice for 30 min and centrifuged as described in Example 7. The supernatant was decanted and the pellet, which contained the DHDPS protein, was resuspended in 1 mL of TNE and desalted by passage over a Sephadex G-25 M column (Column PD-10, Pharmacia).
• the leaf extracts were assayed for DHDPS protein and enzyme activity as described in Example 4. Corynebacteria DHDPS enzyme activity could be distinguished from tobacco DHDPS activity by its insensitivity to lysine inhibition. Eight of eleven transformants showed Corynebacteria DHDPS expression, both as protein detected via western blot and as active enzyme.
• Free amino acids were extracted from leaves as described in Example 7. Expression of Corynebacteria DHDPS resulted in large increases in the level of free lysine in the leaves (Table 8). However, there was not a good correlation between the level of expression of DHDPS and the amount of free lysine accumulated. Free lysine levels from 2 to 50-fold higher than untransformed tobacco were observed. There was also a 2 to 2.5-fold increase in the level of total leaf lysine in the lines that showed high levels of free lysine.
• the plants were allowed to flower, self-pollinate and go to seed. Mature seed was harvested and assayed for free amino acid composition as described in Example 8. There was no difference in the free lysine content of the transformants compared to untransformed tobacco seed.
• KTI3 5' region/cts/cordapA/ KTI3 3' region chimeric gene cassette was isolated as a 3.3 kb BamH I fragment and inserted into BamH I digested pBT549 (Example 8), yielding pFS883.
• This binary vector has the chimeric genes, KTI3 5' region/cts/cordapA/KTI3 3' region and phaseolin 5' region/cts/lvsC-M4/phaseolin 3' region inserted in opposite orientations.
• phaseolin 5' region/cts/cordapA/phaseolin 3'region chimeric gene cassette was modified using oligonucleotide adaptors to convert the Hind HI sites at each end to BamH I sites.
• the gene cassette was then isolated as a 2.7 kb BamH I fragment and inserted into BamH I digested pBT549 (Example 8), yielding pFS903.
• This binary vector has both chimeric genes, phaseolin 5' region/cts/cordapA/phaseolin 3' region and phaseolin 5' region/cts/lysC-M4/phaseolin 3' region inserted in the same orientation.
• the binary vectors pFS883 and pFS903 were transferred by tri-parental mating to Agrobacterium strain LBA4404/pAL4404, the Agrobacterium transformants were used to inoculate tobacco leaf disks and the resulting transgenic plants were regenerated by the methods set out in Example 7.
• Free amino acid composition and expression of bacterial DHDPS and AKIII proteins was also analyzed in developing seeds of two lines that segregated as single gene cassette insertions (see Table 10). Expression of the DHDPS protein under control of the KTI3 promoter was detected at earlier times than that of the AKIII protein under control of the Phaseolin promoter, as expected. At 14 days after flowering both proteins were expressed at a high level and there was about an 8-fold increase in the level of free lysine compared to normal seeds. These results confirm that simultaneous expression of lysine insensitive DHDPS and lysine-insensitive AK results in the production of high levels of free lysine in seeds. Free lysine does not continue to accumulate to even higher levels, however.
• phaseolin 5' region/cts/cordapA/ phaseolin 3' region was isolated as a 2.7 kb BamH I fragment (as described in Example 15) and inserted into BamH I digested pZS199, yielding plasmid pFS926 ( Figure 7B).
• This binary vector has the chimeric gene, phaseolin 5' region/cts/cordapA/phaseolin 3' region inserted in the same orientation as the 35S/NPT II/nos 3' marker gene.
• phaseolin 5' region/cts/lvsC-M4/phaseolin 3' region was isolated as a 3.3 kb EcoR I to Spe I fragment and inserted into EcoR I plus Xba I digested pZS199, yielding plasmid pBT593 ( Figure 7C).
• This binary vector has the chimeric gene, phaseolin 5' region/cts/lvsC-M4/phaseolin 3' region inserted in the same orientation as the 35S/NPT II/nos 3' marker gene.
• the EcoR I site of pBT593 was converted to a BamH I site using oligonucleotide adaptors, the resulting vector was cut with BamH I and the phaseolin 5' region/cts/cordapA/ phaseolin 3' region gene cassette was isolated as a 2.7 kb BamH I fragment and inserted, yielding pBT597 ( Figure 7D).
• This binary vector has both chimeric genes, phaseolin 5' region/cts/cordapA/phaseolin 3' region and phaseolin 5' region/cts/lysC- M4/phaseolin 3* region inserted in the same orientation as the 35S/NPT II/nos 3' marker gene.
• Brassica napus cultivar "Westar” was transformed by co-cultivation of seedling pieces with disarmed Agrobacterium tumefaciens strain LBA4404 carrying the appropriate binary vector.
• B. napus seeds were sterilized by stirring in 10% (v/v) Clorox, 0.1% SDS for thirty min, and then rinsed thoroughly with sterile distilled water. The seeds were germinated on sterile medium containing 30 mM CaCl 2 and 1.5% agar, and grown for 6 d in the dark at 24°.
• Liquid cultures of Agrobacterium for plant transformation were grown overnight at 28°C in Minimal A medium containing 100 mg/L kanamycin.
• the bacterial cells were pelleted by centrifugation and resuspended at a concentration of 10& cells/mL in liquid Murashige and Skoog Minimal Organic medium containing 100 uM acetosyringone.
• B. napus seedling hypocotyls were cut into 5 mm segments which were immediately placed into the bacterial suspension. After 30 min, the hypocotyl pieces were removed from the bacterial suspension and placed onto BC-35 callus medium containing 100 uM acetosyringone. The plant tissue and Agrobacteria were co-cultivated for 3 d at 24°C in dim light.
• the co-cultivation was terminated by transferring the hypocotyl pieces to BC-35 callus medium containing 200 mg/L carbenicillin to kill the Agrobacteria, and 25 mg/L kanamycin to select for transformed plant cell growth.
• the seedling pieces were incubated on this medium for three weeks at 24° under continuous light. After three weeks, the segments were transferred to BS-48 regeneration medium containing 200 mg/L carbenicillin and 25 mg/L kanamycin.
• Plant tissue was subcultured every two weeks onto fresh selective regeneration medium, under the same culture conditions described for the callus medium. Putatively transformed calli grew rapidly on regeneration medium; as calli reached a diameter of about 2 mm, they were removed from the hypocotyl pieces and placed on the same medium lacking kanamycin
• Plants were grown under a 16:8-h photoperiod, with a daytime temperature of 23° and a nighttime temperature of 17°.
• a daytime temperature 23°
• a nighttime temperature 17°.
• Self-pollination was facilitated by shaking the plants several times each day. Mature seeds derived from self-pollinations were harvested about three months after planting.
• a partially defatted seed meal was prepared as follows: 40 mg of mature dry seed was ground with a mortar and pestle under liquid nitrogen to a fine powder. One milliliter of hexane was added and the mixture was shaken at room temperature for 15 min. The meal was pelleted in an eppendorf centrifuge, the hexane was removed and the hexane extraction was repeated. Then the meal was dried at 65° for 10 min until the hexane was completely evaporated leaving a dry powder. Total proteins were extracted from mature seeds as follows.
• the membranes were exposed to the secondary antibody, donkey anti-rabbit Ig conjugated to horseradish peroxidase (Amersham) at a 1 :3000 dilution. Following rinsing to remove unbound secondary antibody, the membranes were exposed to Amersham chemiluminescence reagent and X-ray film.
• free amino acids were extracted from 40 mg of the defatted meal in 0.6 mL of methanol/chloroform/water mixed in ratio of 12v/5v/3v (MCW) at room temperature. The mixture was vortexed and then centrifuged in an eppendorf microcentrifuge for about 3 min. Approximately 0.6 mL of supernatant was decanted and an additional 0.2 mL of MCW was added to the pellet which was then vortexed and centrifuged as above. The second supernatant, about 0.2 mL, was added to the first. To this, 0.2 mL of chloroform was added followed by 0.3 mL of water.
• MCW 12v/5v/3v
• the mixture was vortexed and then centrifuged in an eppendorf microcentrifuge for about 3 min, the upper aqueous phase, approximately 1.0 mL, was removed, and was dried down in a Savant Speed Vac Concentrator.
• the samples were hydrolyzed in 6 N hydrochloric acid, 0.4% (v/v) ⁇ -mercaptoethanol under nitrogen for 24 h at 110-120°; 1/4 of the sample was run on a Beckman Model 6300 amino acid analyzer using post-column ninhydrin detection. Relative free amino acid levels in the seeds were compared as ratios of lysine or threonine to leucine, thus using leucine as an internal standard.
• Corynebacterium DHDPS lead to large increases in accumulation of free lysine in rapeseed transformants.
• the highest expressing lines showed a greater than 100-fold increase in free lysine level in the seeds.
• the transformant that expressed AKIII-M4 in the absence of Corynebacteria DHDPS showed a 5-fold increase in the level of free threonine in the seeds. Concomitant expression of both enzymes resulted in accumulation of high levels of free lysine, but not threonine.
• AA is ⁇ -amino adipic acid
• EXAMPLE 17 Transformation of Maize Using a Chimeric lysC-M4 Gene as a Selectable Marker Embryogenic callus cultures were initiated from immature embryos (about 1.0 to 1.5 mm) dissected from kernels of a com line bred for giving a "type II callus" tissue culture response.
• the embryos were dissected 10 to 12 d after pollination and were placed with the axis-side down and in contact with agarose- solidified N6 medium [Chu et al. (1974) Sci Sin 75:659-668] supplemented with 0.5 mg/L 2,4-D (N6-0.5).
• the embryos were kept in the dark at 27°C.
• Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryos and somatic embryos borne on suspensor stmctures proliferated from the scutellum of the immature embryos.
• Clonal embryogenic calli isolated from individual embryos were identified and sub-cultured on N6-0.5 medium every 2 to 3 weeks.
• the particle bombardment method was used to transfer genes to the callus culture cells.
• a BiolisticTM PDS-1000/He BioRAD Laboratories, Hercules, CA was used for these experiments.
• the plasmid pBT573, containing the chimeric gene HH534 5' region/ mcts/lysC-M4/HH2- 1 3' region was precipitated onto the surface of gold particles.
• 2.5 ⁇ g of pBT573 in water at a concentration of about 1 mg/mL was added to 25 mL of gold particles (average diameter of 1.5 ⁇ m) suspended in water (60 mg of gold per mL).
• Calcium chloride (25 mL of a 2.5 M solution) and spermidine (10 mL of a 1.0 M solution) were then added to the gold-DNA suspension as the tube was vortexing.
• the gold particles were centrifuged in a microfuge for 10 s and the supernatant removed. The gold particles were then resuspended in 200 mL of absolute ethanol, were centrifuged again and the supernatant removed. Finally, the gold particles were resuspended in 25 mL of absolute ethanol and sonicated twice for one sec. Five ⁇ L of the DNA-coated gold particles were then loaded on each macro carrier disk and the ethanol was allowed to evaporate away leaving the DNA-covered gold particles dried onto the disk.
• Embryogenic callus (from the callus line designated #132.2.2) was arranged in a circular area of about 6 cm in diameter in the center of a 100 X 20 mm petri dish containing N6-0.5 medium supplemented with 0.25M sorbitol and 0.25M mannitol. The tissue was placed on this medium for 2 h prior to bombardment as a pretreatment and remained on the medium during the bombardment procedure. At the end of the 2 h pretreatment period, the petri dish containing the tissue was placed in the chamber of the PDS-1000/He. The air in the chamber was then evacuated to a vacuum of 28 inch of Hg.
• the macrocarrier was accelerated with a helium shock wave using a mpture membrane that bursts when the He pressure in the shock tube reaches 1100 psi.
• the tissue was placed approximately 8 cm from the stopping screen.
• Four plates of tissue were bombarded with the DNA-coated gold particles. Immediately following bombardment, the callus tissue was transferred to N6-0.5 medium without supplemental sorbitol or mannitol.
• the chimeric gene cassettes HH534 5' region/ mcts/ecodapA/HH2- 1 3' region plus HH534 5' region/ mcts/lvsC-M4/HH2- 1 3' region, (Example 6) were inserted into the vector pGem9z to generate a com transformation vector.
• Plasmid pBT583 (Example 6) was digested with Sal I and an 1850 bp fragment containing the HH534 5' region/mcts/ecodapA/HH2- 1 3' region gene cassette was isolated.
• pBT573 (Example 6), which carries the HH534 5' region/mcts/ lvsC-M4/HH2-l 3' region, digested with Xho I.
• the resulting vector with both chimeric genes in the same orientation was designated pBT586.
• Vector pBT586 was introduced into embryogenic com callus tissue using the particle bombardment method. The establishment of the embryogenic callus cultures and the parameters for particle bombardment were as described in Example 17.
• plasmids containing selectable markers were used in the transformations.
• One plasmid, pALSLUC [Frornm et al. (1990) Biotechnology 5:833-839] contained a cDNA of the maize acetolactate synthase (ALS) gene.
• the ALS cDNA had been mutated in vitro so that the enzyme coded by the gene would be resistant to chlorsulfuron.
• This plasmid also contains a gene that uses the 35S promoter from Cauliflower Mosaic Vims and the 3' region of the nopaline synthase gene to express a firefly luciferase coding region [de Wet et al. (1987) Molec.
• the other plasmid, pDETRIC contained the bar gene from Streptomvces hygroscopicus that confers resistance to the herbicide glufosinate [Thompson et al. (1987 The EMBO Journal 6:2519-2523].
• the bacterial gene had its translation codon changed from GTG to ATG for proper translation initiation in plants [De Block et al. (1987) The EMBO Journal 6:2513-2518].
• the bar gene was driven by the 35S promoter from Cauliflower Mosaic Vims and uses the termination and polyadenylation signal from the octopine synthase gene from Agrobacterium tumefaciens.
• each plasmid, pBT586 and one of the two selectable marker plasmids was co-precipitated onto the surface of gold particles as described in Example 17. Bombardment of the embryogenic tissue cultures was also as described in Example 17.
• the tissue bombarded with the selectable marker pALSLUC was transferred to N6-0.5 medium that contained chlorsulfuron (30 ng/L) and lacked casein or proline.
• the tissue bombarded with the selectable marker, pDETRIC was transferred to N6-0.5 medium that contained 2 mg/L glufosinate and lacked casein or proline. The tissue continued to grow slowly on these selective media. After an additional 2 weeks the tissue was transferred to fresh N6-0.5 medium containing the selective agents.
• Chlorsulfuron- and glufosinate-resistance callus clones could be identified after an additional 6-8 weeks. These clones continued to grow when transferred to the selective media.
• the chimeric gene cassettes, phaseolin 5' region/ cts/cordapA/phaseolin 3' region plus phaseolin 5' region/cts/lysC-M4/phaseolin 3', (Example 6) were inserted into the soybean transformation vector pBT603 ( Figure 8A).
• This vector has a soybean transformation marker gene consisting of the 35S promoter from Cauliflower Mosaic Vims driving expression of the E. coli ⁇ -glucuronidase gene [Jefferson et al. (1986) Proc. Natl. Acad. Sci. USA 55:8447-8451] with the Nos 3' region in a modified pGEM9Z plasmid.
• phaseolin 5' region/cts/lysC-M4/ phaseolin 3' region was isolated as a 3.3 kb Hind III fragment and inserted into Hind III digested pBT603, yielding plasmid pBT609.
• This binary vector has the chimeric gene, phaseolin 5' region/ cts/lysC-M4/phaseolin 3' region inserted in the opposite orientation from the 35S/GUS/Nos 3' marker gene.
• phaseolin 5' region/cts/cordapA/ phaseolin 3'region 3' the gene cassette was isolated as a 2.7 kb BamH I fragment (as described in Example 15) and inserted into BamH I digested pBT609, yielding plasmid pBT614 ( Figure 8B).
• This vector has both chimeric genes, phaseolin 5' region/cts/lvsC-M4/phaseolin 3' region and phaseolin 5' region/cts/cordapA/phaseolin 3' region inserted in the same orientation, and both are in the opposite orientation from the 35S/GUS/Nos 3' marker gene.
• Soybean was transformed with plasmid pBT614 according to the procedure described in United States Patent No. 5,015,580. Soybean transformation was performed by Agracetus Company (Middleton, WI). Seeds from five transformed lines were obtained and analyzed.
• a meal was prepared from a fragment of individual seeds by grinding into a fine powder.
• Total proteins were extracted from the meal by adding 1 mg to 0.1 mL of 43 mM Tris-HCl pH 6.8, 1.7% SDS, 4.2% (v/v) ⁇ -mercaptoethanol, 8% (v/v) glycerol, vortexing the suspension, boiling for 2-3 min and vortexing again.
• the resultant suspensions were centrifuged for 5 min at room temperature in a microfuge to remove particulates and 10 ⁇ L from each extract were run per lane on an SDS polyacrylamide gel, with bacterially produced DHDPS or AKIII serving as a size standard.
• the proteins were then elecfrophoretically blotted onto a nitrocellulose membrane.
• the membranes were exposed to the DHDPS or AKIII antibodies, at a 1:5000 or 1:1000 dilution, respectively, of the rabbit serum using standard protocol provided by BioRad with their Immun-Blot Kit. Following rinsing to remove unbound primary antibody the membranes were exposed to the secondary antibody, donkey anti-rabbit Ig conjugated to horseradish peroxidase (Amersham) at a 1 :3000 dilution. Following rinsing to remove unbound secondary antibody, the membranes were exposed to Amersham chemiluminescence reagent and X-ray film.
• free amino acids were extracted from 8-10 milligrams of the meal in 1.0 mL of methanol/chloro- form/water mixed in ratio of 12v/5v/3v (MCW) at room temperature. The mixture was vortexed and then centrifuged in an eppendorf microcentrifuge for about 3 min; approximately 0.8 mL of supernatant was decanted. To this supernatant, 0.2 mL of chloroform was added followed by 0.3 mL of water.
• the mixture was vortexed and then centrifuged in an eppendorf microcentrifuge for about 3 min, the upper aqueous phase, approximately 1.0 mL, was removed, and was dried down in a Savant Speed Vac Concentrator.
• the samples were hydrolyzed in 6 N hydrochloric acid, 0.4% (v/v) ⁇ -mercaptoethanol under nitrogen for 24 h at 110-120°; 1/10 of the sample was run on a Beckman Model 6300 amino acid analyzer using post-column ninhydrin detection. Relative free amino acid levels in the seeds were compared as ratios of lysine to leucine, thus using leucine as an internal standard.
• Soybean transformants expressing Corynebacteria DHDPS alone and in concert with E. coli AKIII-M4 accumulated high levels of free lysine in their seeds. From 20 fold to 120-fold increases in free lysine levels were observed (Table 13). A high level of saccharopine, indicative of lysine catabolism, was also observed in seeds that contained high levels of lysine. Thus, prevention of lysine catabolism by inactivation of lysine ketoglutarate reductase should further increase the accumulation of free lysine in the seeds. Altematively, incorporation of lysine into a peptide or lysine-rich protein would prevent catabolism and lead to an increase in the accumulation of lysine in the seeds.
• the soybean seeds expressing Corynebacteria DHDPS showed substantial increases in accumulation of total seed lysine. Seeds with a 5-35% increase in total lysine content, compared to the untransformed control, were observed. In these seeds lysine makes up 7.5-7.7% of the total seed amino acids.
• Soybean seeds expressing Corynebacteria DHDPS in concert with E. coli AKHI-M4 showed much greater accumulation of total seed lysine than those expressing Corynebacteria DHDPS alone. Seeds with a more than four-fold increase in total lysine content were observed. In these seeds lysine makes up 20-25% of the total seed amino acids, considerably higher than any previously known soybean seed.
• Lysine Ketoglutarate Reductase (LKR) enzyme activity has been observed in immature endosperm of developing maize seeds [Arruda et al. (1982) Plant Physiol. 69:988-989]. LKR activity increases sharply from the onset of endosperm development, reaches a peak level at about 20 d after pollination, and then declines [Arruda et al. (1983) Phytochemistry 22:2687-2689].
• Two phagemid libraries were generated using the mixtures of the Lambda Zap II phage and the filamentous helper phage of 100 ⁇ L to 1 ⁇ L. Two additional libraries were generated using mixtures of 100 ⁇ L Lambda Zap II to 10 ⁇ L helper phage and 20 ⁇ L Lambda Zap II to 10 ⁇ L helper phage.
• the titers of the phagemid preparations were similar regardless of the mixture used and were about 2 x 10 3 ampicillin-resistant-transfectants per mL with E. coli strain XL 1 -Blue as the host and about 1 x 10 3 with D ⁇ 126 (see below) as host.
• DEI 26 was constmcted. Constmction of DEI 26 occurred in several stages.
• a generalized transducing stock of coliphage Plvir was produced by infection of a culture of TST1 [F"> araD139, ⁇ (argF-lac)205, flb5301, ptsF25, reJAl, rpsL150. malE52::TnlO. deoCl, ⁇ "] (E. coli Genetic Stock Center #6137) using a standard method (for Methods see J. Miller, Experiments in Molecular Genetics).
• This phage stock was used as a donor in a transductional cross (for Method see J. Miller, Experiments in Molecular Genetics) with strain GIF106M1 [F-, arg-, ilvA296. l sClOOl, thrAHOl, metL 1000. ⁇ "> rpsL9, malTl. x l-7, mtl-2, thil(?). su ⁇ E44(?)l (E. coli Genetic Stock Center #5074) as the recipient. Recombinants were selected on rich medium [L supplemented with DAP] containing the antibiotic tetracycline.
• transposon TnlO conferring tetracycline resistance
• Tetracycline- resistant transductants derived from this cross are likely to contain up to 2 min of the E. coli chromosome in the vicinity ofmalE.
• the genes malE and lysC are separated by less than 0.5 minutes, well within cotransduction distance.
• DEI 25 has the phenotype of tetracycline resistance, growth requirements for arginine, isoleucine and valine, and sensitivity to lysine.
• the genotype of this strain is F" malE52::TnlO arg- ilvA296 thrAl 101 metL 1000 lambda- rpsL9 malTl x ⁇ i-7 mtl-2 Jhil(?) SUPE44(?).
• This step involves production of a male derivative of strain DEI 25.
• Strain DE125 was mated with the male strain AB1528 [F' 16/delta(gpt-proA)62. lacYl or lacZ4. glnV44.
• AB 1528 Growth of AB 1528 is prevented by the inclusion of the antibiotic tetracycline and the omission of proline and histidine from the synthetic medium. A patch of cells grew on this selective medium. These recombinant cells underwent single colony isolation on the same medium.
• the phenotype of one clone was determined to be Ilv + > Arg", TetR, Lysine-sensitive, male specific phage (MS2)-sensitive, consistent with the simple transfer of F'16 from AB1528 to DEI 25.
• This clone was designated DEI 26 and has the genotype F'16/malE52::TnlO, arg", ilvA296. thrAHOl, metL 100. lvsC+. ⁇ ", rpsL9. malTl. xyl-7. mtl-2. thi-1?. supE44?. It is inhibited by 20 ⁇ g/mL of L-lysine in a synthetic medium
• phagemid library 100 ⁇ L was mixed with 100 ⁇ L of an overnight culture of DEI 26 grown in L broth and the cells were plated on synthetic media containing vitamin Bl, L-arginine, glucose as a carbon and energy source, 100 ⁇ g/mL ampicillin and L-lysine at 20, 30 or 40 ⁇ g/mL.
• Four plates at each of the three different lysine concentrations were prepared.
• the amount of phagemid and DEI 26 cells was expected to yield about 1 x 10 5 ampicillin-resistant transfectants per plate. Ten to thirty lysine-resistant colonies grew per plate (about 1 lysine- resistant per 5000 ampicillin-resistant colonies).
• Plasmid DNA was isolated from 10 independent clones and retransformed into DE126. Seven of the ten DNAs yielded lysine-resistant clones demonstrating that the lysine-resistance trait was carried on the plasmid. Several of the cloned DNAs were sequenced and biochemically characterized. The inserted DNA fragments were found to be derived from the E. coli genome, rather than a com cDNA indicating that the cDNA library provided by Clontech was contaminated.
• plant saccharopine dehydrogenase which catalyzes the second step in the lysine catabolic pathway, works in both forward and reverse reactions, uses identical substrates and uses similar co-factors as fungal saccharopine dehydrogenase (glutamate-forming).
• Plant LKR has been reported to have a molecular weight of about 140,000 indicating that it is like the animal catabolic protein wherein both LKR and saccharopine dehydrogenase (glutamate-forming) enzyme activities are present on a single protein.
• DNA sequencing of the fragment confirmed that it encoded LKR/SDH.
• the fragment was labeled with digoxigenin (DIG) using Boehringer Mannheim's Dig- High Prime kit and protocol. This probe was used to screen a CD4-8 Landsberg erecta genomic library by plaque hybridization. Approximately 2.7 X 10 ⁇ recombinant phage were plated on the host E. coli LE392, grown overnight at 37°. The protocol was as described in the DIG Wash and Block Set (Boehringer Mannheim) with the hybridization temperature set at 55°. Five positive clones were isolated; one was subcloned into plasmid vector pBluescript ® SK +/- (Stratagene), transformed into DH5 ⁇ TM competent cells (GibcoBRL) and sequenced.
• DIG digoxigenin
• the complete genomic sequence of the Arabidopsis LKR/SDH gene is shown in SEQ ID NO:l 10.
• the sequence includes approximately 2 kb of 5' noncoding sequence and 500 bp of 3' noncoding sequence and 23 introns. Overlapping fragments of the corresponding cDNA were isolated from total Arabidopsis RNA by RT-PCR. Sequence analysis of the LKR-SDH cDNA revealed an ORF of 3.16 kb, which predicts a protein of 117 kd, and confirms that the LKR and SDH enzymes reside on one polypeptide.
• the complete protein coding sequence of Arabidopsis LKR/SDH gene, derived from the cDNA is shown in SEQ ID NO: 111.
• the deduced amino acid sequence of Arabidopsis LKR/SDH protein is shown in SEQ ID NO: 112.
• the protein lacks an N-terminal targeting sequence implying that the lysine degradative pathway is located in the plant cell cytosol.
• Degenerate oligonucleotides SEQ ID NO:l 13 and SEQ ID NO:l 14, were designed based upon comparison of the Arabidopsis LKR/SDH amino acid sequence with that of other LKR proteins. These were used to amplify soybean and com LKR/SDH cDNA fragments using PCR from mRNA, or cDNA synthesized from mRNA, isolated from developing soybean or com seeds. The soybean and com PCR-generated cDNA fragments were cloned and sequenced. The sequence of the soybean LKR/SDH cDNA fragment is shown in SEQ ID NO:l 15, and the sequence of the com cDNA fragment is shown in SEQ ID NO: 116.
• the deduced partial amino acid sequence of soybean LKR/SDH protein is shown in SEQ ID NO:l 17 and the deduced partial amino acid sequence of com LKR/SDH protein is shown in SEQ ID NO: 118.
• the partial cDNAs encoding com and soybean LKR/SDH obtained by PCR, above, were used in protocols that extended the sequence information for these functions. These protocols, which included RACE and direct DNA:DNA hybridization to cDNA libraries for the identification of overlapping clones, are well known to persons skilled in the art. From these efforts, more complete sequences for the com and soybean cDNAs for LKR/SDH were obtained.
• SEQ ID NOS: 119 and 120 list, respectively, near full- length sequences for the LKR/SDH coding regions from soybean and com.
• the deduced protein sequences encoded by these soybean and com cDNAs are shown in SEQ ID NOS: 121 and 122, respectively.
• Partial cDNA clones for LKR/SDH from rice and wheat were identifid in libraries prepared from rice roots and leafs and from wheat seedlings.
• cDNA libraries were prepared in Uni-ZAPTM XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA). Conversion of the Uni-ZAPTM XR libraries into plasmid libraries was accomplished according to the protocol provided by Stratagene. Upon conversion, cDNA inserts were contained in the plasmid vector pBluescript.
• cDNA inserts from randomly picked bacterial colonies containing recombinant pBluescript plasmids were amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences or plasmid DNA was prepared from cultured bacterial cells.
• Amplified insert DNAs or plasmid DNAs were sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or "ESTs"; see Adams, M. D. et al., (1991) Science 252: 1651). The resulting ESTs were analyzed using a Perkin Elmer Model 377 fluorescent sequencer.
• Possible protein products encoded by the ESTs were compared to the full-length sequence of Arabidopsis LKR/SDH (SEQ ID NO: 112).
• a contig for a partial cDNA from rice was constmcted and is presented in SEQ ID: 125.
• the predicted prtein fragment from the cDNA contig is shown in SEQ ID NO: 126.
• Another cDNA from rice was identified which corresponds to the 3' end of a LKR SDH coding region and this sequence is set forth in SEQ ID NO: 127.
• the predicted protein fragment is shown in SEQ ID NO: 128.
• a partial wheat clone was identified and possesses the sequence presented in SEQ ID NO: 129.
• the predicted protein fragment encoded by this cDNA is set forth isn SEQ ID NO: 130.
• the SDH coding region encompasses 1.4 kb on 3' end of the Arabidopsis cDNA clone (SEQ ID NO: 131), and encodes a protein of about 52 kD (SEQ ID NO: 132).
• a DNA fragment encoding SDH was generated using PCR primers, which added desired restriction enzyme sites, and ligated into prokaryotic expression vector pBT430 (see Example 2). Addition of the restriction enzyme cleavage site resulted in a change from thr to ala encoded by the second codon. High level expression of Arabidopsis SDH was achieved in E.coli BL21(DE3)LysS host which expressed T7 RNA polymerase.
• Extracts from IPTG-induced cells that were transformed with the vector carrying the 1.4 kb insert were analyzed by SDS-PAGE and a protein of the expected size was overproduced in these cells. Separation of the cell extracts into its supernatant (soluble) and pellet (insoluble) fractions showed that substantial amounts of protein were present in both. SDH activity was measured in the soluble fraction of the bacterial extracts. No SDH activity was observed in extracts from cells transformed with an unmodified vector. Extracts from cells containing the SDH cDNA insert converted substantial amounts of NAD+ to NADH. The reaction was specific for SDH because no significant activity was observed in the absence of the SDH substrate saccharopine. The SDH protein has been purified from these bacterial extracts and used to raise rabbit antibodies to the protein.
• a chimeric gene designed for cosuppression of LKR is constructed by linking the LKR gene or gene fragment to any of the plant promoter sequences described above. (See U.S. Patent No. 5,231,020 for methodology to block plant gene expression via cosuppression.)
• the com LKR gene, SEQ ID NO: 120 was modified by introducing an Nco I site at position 7 and a Kpn I site at position 1265 using PCR.
• This Nco I and Kpn I DNA fragment containing the com LKR gene fragment was inserted into a plasmid containing the glutelin 2 promoter and 10 kD zein 3' region (see Example 25) to create a chimeric gene for suppression of LKR expression in com endosperm.
• the soybean LKR gene, SEQ ID NO:l 19, was modified by introducing an Nco I site at position 2 and a Kpn I site at position 690 using PCR.
• This Nco I and Kpn I DNA fragment containing the soybean LKR gene fragment was inserted into a plasmid containing the KTI3 promoter and the KTI3 3' region (see Example 6) to create a chimeric gene for suppression of LKR expression in soybean seeds.
• a chimeric gene designed to express antisense RNA for all or part of the LKR is constructed by linking the LKR gene or gene fragment in reverse orientation to any of the plant promoter sequences described above. (See U.S. patent 5,107,065 for methodology to block plant gene expression via antisense RNA.) Either the cosuppression or antisense chimeric gene is introduced into plants via transformation as described in other Examples, e.g. Example 18 or Example 19. Transformants wherein expression of the endogenous LKR gene is reduced or eliminated are selected.
• Plasmid pSKl which was a spontaneous mutant of pBR322 where the ampicillin gene and the Ear I site near that gene had been deleted. Plasmid pSKl retained the tetracycline resistance gene, the unique EcoR I restriction sites at base 1 and a single Ear I site at base 2353. To remove the Ear I site at base 2353 of pSKl a polymerase chain reaction (PCR) was performed using pSKl as the template. Approximately 10 femtomoles of pSKl were mixed with 1 ⁇ g each of oligonucleotides SM70 and SM71 which had been synthesized on an ABI1306B DNA synthesizer using the manufacturer's procedures.
• PCR polymerase chain reaction
• the priming sites of these oligonucleotides on the pSKl template are depicted in Figure 10.
• the PCR was performed using a Perkin-Elmer Cetus kit (Emeryville, CA) according to the instructions of the vendor on a thermocycler manufactured by the same company. The 25 cycles were 1 min at 95°, 2 min at 42° and 12 min at 72°.
• the oligonucleotides were designed to prime replication of the entire pSKl plasmid excluding a 30 b fragment around the Ear I site (see Figure 10).
• Ten microliters of the 100 ⁇ L reaction product were run on a 1% agarose gel and stained with ethidium bromide to reveal a band of about 3.0 kb corresponding to the predicted size of the replicated plasmid.
• the remainder of the PCR reaction mix (90 ⁇ L) was mixed with 20 ⁇ L of 2.5 mM deoxynucleotide triphosphates (dATP, dTTP, dGTP, and dCTP), 30 units of Klenow enzyme added and the mixture incubated at 37° for 30 min followed by 65° for 10 min.
• the Klenow enzyme was used to fill in ragged ends generated by the PCR.
• the DNA was ethanol precipitated, washed with 70% ethanol, dried under vacuum and resuspended in water. The DNA was then treated with T4 DNA kinase in the presence of 1 mM ATP in kinase buffer. This mixture was incubated for 30 min at 37° followed by 10 min at 65°.
• restriction endonucleases were purchased from NEN Biolabs (Beverly, MA) or Boehringer Mannheim (Indianapolis, IN). Both the ligated DNA samples were transformed separately into competent JM103 [supE thi ⁇ (lac-proAB) F' [traD36 proAB, lacl q lacZ ⁇ M15] restriction minus] cells using the CaCl method as described in Sambrook et al., [Molecular Cloning, A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press] and plated onto media containing 12.5 ⁇ g/mL tetracycline. With or without Ear I digestion the same number of transformants were recovered suggesting that the Ear I site had been removed from these constructs.
• Clones were screened by preparing DNA by the alkaline lysis miniprep procedure as described in Sambrook et al., [Molecular Cloning, A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press] followed by restriction endonuclease digest analysis. A single clone was chosen which was tetracycline-resistant and did not contain any Ear I sites. This vector was designated pSK2. The remaining EcoR I site of pSK2 was destroyed by digesting the plasmid with EcoR I to completion, filling in the ends with Klenow and ligating. A clone which did not contain an EcoR I site was designated pSK3.
• the bacteriophage T7 RNA polymerase promoter/terminator segment from plasmid pBT430 was amplified by PCR.
• Oligonucleotide primers SM78 (SEQ ID NO: 18) and SM79 (SEQ ID NO: 19) were designed to prime a 300b fragment from pBT430 spanning the T7 promoter/terminator sequences (see Figure 10).
• the PCR reaction was carried out as described previously using pBT430 as the template and a 300 bp fragment was generated. The ends of the fragment were filled in using Klenow enzyme and phosphorylated as described above.
• DNA from plasmid pSK3 was digested to completion with PvuII enzyme and then treated with calf intestinal alkaline phosphatase (Boehringer Mannheim) to remove the 5' phosphate. The procedure was as described in Sambrook et al., [Molecular Cloning, A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press]. The cut and dephosphorylated pSK3 DNA was purified by ethanol precipitation and a portion used in a ligation reaction with the PCR generated fragment containing the T7 promoter sequence.
• Plasmid DNA was prepared via the alkaline lysis mini prep method and restriction endonuclease analysis was performed to detect insertion and orientation of the PCR product. Two clones were chosen for sequence analysis: Plasmid pSK5 had the fragment in the orientation shown in Figure 10.
• the strategy for the constmction of repeated synthetic gene sequences based on the Ear I site is depicted in Figure 11.
• the first step was the insertion of an oligonucleotide sequence encoding a base gene of 14 amino acids.
• This oligonucleotide insert contained a unique Ear I restriction site for subsequent insertion of oligonucleotides encoding one or more heptad repeats and added an unique Asp 718 restriction site for use in transfer of gene sequences to plant vectors.
• the overhanging ends of the oligonucleotide set allowed insertion into the unique Nco I and EcoR I sites of vector pSK5.
• DNA from plasmid pSK5 was digested to completion with Nco I and EcoR I restriction endonucleases and purified by agarose gel electrophoresis.
• Purified DNA 0.1 ⁇ g was mixed with 1 ⁇ g of each oligonucleotide SM80 (SEQ ID NO: 14) and SM81 (SEQ ID NO:13) and ligated.
• the ligation mixture was transformed into E. coli strain JM103 [supE thi ⁇ (lac-proAB) F' [traD36 proAB, lacl q lacZ ⁇ M15] restriction minus] and tetracycline resistant transformants screened by rapid plasmid DNA preps followed by restriction digest analysis.
• oligonucleotides SM84 (SEQ ID NO:23) and SM85 (SEQ ID NO:24) code for repeats of the SSP5 heptad.
• Oligonucleotides SM82 (SEQ ID NO:25) and SM83 (SEQ ID NO:26) code for repeats of the SSP7 heptad.
• Multimeric forms which separated on the gel as 168 bp (8n) or larger were purified by cutting a small piece of polyacrylamide containing the band into fine pieces, adding 1.0 mL of 0.5 M ammonium acetate, 1 mM EDTA (pH 7.5) and rotating the tube at 37° overnight. The polyacrylamide was spun down by centrifugation, 1 ⁇ g of tRNA was added to the supernatant, the DNA fragments were precipitated with 2 volumes of ethanol at -70°, washed with 70% (v/v) ethanol, dried, and resuspended in 10 ⁇ L of water.
• pSK6 DNA Ten micrograms of pSK6 DNA were digested to completion with Ear I enzyme and treated with calf intestinal alkaline phosphatase.
• the cut and dephosphorylated vector DNA was isolated following electrophoresis in a low melting point agarose gel by cutting out the banded DNA, liquefying the agarose at 55°, and purifying over NACS PREP AC columns (BRL) following manufacturer's suggested procedures.
• Approximately 0.1 ⁇ g of purified Ear I digested and phosphatase treated pSK6 DNA was mixed with 5 ⁇ L of the gel purified multimeric oligonucleotide sets and ligated. The ligated mixture was transformed into E.
• Clones were screened by restriction digests of rapid plasmid prep DNA to determine the length of the inserted DNA. Restriction endonuclease analyses were usually carried out by digesting the plasmid DNAs with Asp 718 and Bgl II, followed by separation of fragments on 18% non-denaturing polyacrylamide gels. Visualization of fragments with ethidium bromide, showed that a 150 bp fragment was generated when only the base gene segment was present. Inserts of the oligonucleotide fragments increased this size by multiples of 21 bases. From this screening several clones were chosen for DNA sequence analysis and expression of coded sequences in E. coli.
• the first and last SSP5 heptads flanking the sequence of each constmct are from the base gene described above. Inserts are designated by underlining.
• the oligonucleotides were separated on the basis of length using a gradient elution and a two buffer mobile phase [Buffer A: 25 mM Tris-Cl, pH 9.0, and Buffer B: Buffer A + 1 M NaCl]. Both Buffers A and B were passed through 0.2 micron filters before use. The following gradient program was used with a flow rate of 1 mL per min at 30°:
• Fractions (500 ⁇ L) were collected between 3 min and 9 min. Fractions corresponding to lengths between 120 bp and 2000 bp were pooled as determined from control separations of restriction digests of plasmid DNAs.
• coli strain DH5 ⁇ [su ⁇ E44 ⁇ lacU169 ( ⁇ 80 lacZ ⁇ M15) hsdR17 recAl endAl gyrl96 thil relAl] and tetracycline-resistant colonies selected.
• Applicants chose to use the DH5 ⁇ [supE44 ⁇ lacU169 ( ⁇ 80 lacZ ⁇ M15) hsdR17 recAl endAl gyrl96 thil relAl] strain for all subsequent work because this strain has a very high transformation rate and is recA-.
• the recA- phenotype eliminates concerns that these repetitive DNA stmctures may be substrates for homologous recombination leading to deletion of multimeric sequences.
• Clones were screened as described above. Several clones were chosen to represent insertions of each of the six oligonucleotide sets.
• the first and last SSP5 heptads flanking the sequence represent the base gene sequence. Insert sequences are underlined. Clone numbers including the letter "H” designate HPLC-purified oligonucleotides.
• the loss of the first base gene repeat in clone 82-4 may have resulted from homologous recombination between the base gene repeats 5.5 before the vector pSK6 was transferred to the recA- strain.
• the HPLC procedure did not enhance insertion of longer multimeric forms of the oligonucleotide sets into the base gene but did serve as an efficient purification of the ligated oligonucleotides.
• Oligonucleotides were designed which coded for mixtures of the SSP sequences and which varied codon usage as much as possible. This was done to reduce the possibility of deletion of repetitive inserts by recombination once the synthetic genes were transformed into plants and to extend the length of the constmcted gene segments. These oligonucleotides encode four repeats of heptad coding units (28 amino acid residues) and can be inserted at the unique Ear I site in any of the previously constructed clones. SM96 and SM97 code for SSP(5)4, SM98 and SM99 code for SSP(7) 4 and SM100 plus SM101 code for SSP8.9.8.9.
• DNA from clones 82-4 and 84-H3 were digested to completion with Ear I enzyme, treated with phosphatase and gel purified. About 0.2 ⁇ g of this DNA were mixed with 1.0 ⁇ g of each of the oligonucleotide sets SM96 and SM97, SM98 and SM99 or SM100 and SM101 which had been previously phosphorylated. The DNA and oligonucleotides were ligated overnight and then the ligation mixes transformed into E. coli strain DH5 ⁇ . Tetracycline-resistant colonies were screened as described above for the presence of the oligonucleotide inserts. Clones were chosen for sequence analysis based on their restriction endonuclease digestion patterns. Table 16
• Clone 2-9 was derived from oligonucleotides SM100 (SEQ ID NO:71) and SM101 (SEQ ID NO:72) ligated into the Ear I site of clone 82-4 (see above).
• Clone 3-5 (SEQ ID NO:78) was derived from the insertion of the first 22 bases of the oligonucleotide set SM96 (SEQ ID NO:65) and SM97 (SEQ ID NO:66) into the Ear I site of clone 82-4 (SEQ ID NO:53). This partial insertion may reflect improper annealing of these highly repetitive oligos.
• Clone 5-1 (SEQ ID NO:76) was derived from oligonucleotides SM98 (SEQ ID NO:68) and SM99 (SEQ ID NO:69) ligated into the Ear I site of clone 84-H3 (SEQ ID NO:55).
• a second strategy for constmction of synthetic gene sequences was implemented to allow more flexibility in both DNA and amino acid sequence.
• This strategy is depicted in Figure 13 and Figure 14.
• the first step was the insertion of an oligonucleotide sequence encoding a base gene of 16 amino acids into the original vector pSK5.
• This oligonucleotide insert contained an unique Ear I site as in the previous base gene constmct for use in subsequent insertion of oligonucleotides encoding one or more heptad repeats.
• the base gene also included a BspH I site at the 3' terminus.
• the overhanging ends of this cleavage site are designed to allow "in frame" protein fusions using Nco I overhanging ends. Therefore, gene segments can be multiplied using the duplication scheme described in Figure 14.
• the overhanging ends of the oligonucleotide set allowed insertion into the unique Nco I and EcoR I sites of vector pSK5.
• the oligonucleotide set was inserted into pSK5 vector as described in Strategy I above.
• the resultant plasmid was designated pSK34.
• Oligonucleotide sets encoding 35 amino acid "segments" were ligated into the unique Ear I site of the pSK34 base gene using procedures as described above. In this case, the oligonucleotides were not gel or HPLC purified but simply annealed and used in the ligation reactions. The following oligonucleotide sets were used:

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