Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/93—Ligases (6)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/8255—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving lignin biosynthesis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
  • C12N9/1007—Methyltransferases (general) (2.1.1.)

Definitions

• This invention relates materials and methods for modifying the content, composition and metabolism of isoprenoids in plants and other organisms. More particularly, this invention relates to polypeptides involved in the synthesis of isoprenoid compounds, such as terpenoid and steroid compounds, polynucleotides encoding such polypeptides, expression of such polypeptides, and methods for modulating the composition and/or expression levels of such polypeptides, thereby modulating isoprenoid content, composition, and metabolism.
• Isoprenoids form a large family of naturally occurring compounds, with over 20,000 distinct compounds having been described.
• the isoprenoids include vitamins A, D, E, and K, first recognized as fatty materials essential to the normal growth of animals, and numerous biological pigments.
• isoprenoid compounds including terpenoid and steroid compounds, include hormones such as gibberellic acid and abscisic acid, pigments, electron carriers, membrane components (phytosterols), phytotoxins, antibiotics, flavors such as menthol, vitamins, macromolecular compounds such as rubber and guttapercha, and others.
• Isoprene compounds or prenyl lipids, are composed of one or more basic isoprene skeleton(s) (C 5 ) formed by the decarboxylation of mevalonate-5-pyrophosphate. From the isopentenyl pyrophosphate ("active isoprene” or "IPP") and the isomeric dimethylallyl pyrophosphate, the geranyl pyrophosphate (Cio) may be formed by "head-tail” condensation. By linkage of a further C 5 unit, farnesyl pyrophosphate (Cu) is formed.
• FIG. 1 A schematic diagram of the basic biosynthetic pathways of isoprene compounds is shown in Fig. 1.
• IPP is the branching point for a large variety of biologically significant molecules, including isoprenoids, carotenoids, and various sterols in different eukaryotic organisms (mycosterols, phytosterols and zoosterols).
• mycosterols phytosterols and zoosterols
• cholesterols are precursors for several hormones and bile acids.
• Fungal ergosterol and mammalian cholesterol arise from IPP via squalene oxide and lanosterol, while higher plant sterols, like campesterol and sitosterol, are produced by cyclization of squalene oxide to cycloartol and by further plant-specific enzymes.
• Terpenoids include isoprene (C 5 Hg) compounds, including isopen-tenylpyroposphate and active isoprene; monoterpene (CioHi ⁇ ) compounds, including geraniol, and from which menthol, camphor, pinene and citronellal are derived; sesquiterpene (C] 5 H 2 4) compounds, including farnesol, from which zingiberene, ubiquinone, plastoquinone, abscisic acid and rishitine are derived; diterpene (C2 0 H 3 ?) compounds, such as geranylgeraniol, from which phytol, kaurene, giberrerellic acid and fusicoccin are derived; triterpene (C 3 oH 48 ) compounds, including squalene, from which steriods and saponins are derived; tetraterpene (C 4 oH 64 ) compounds, including phytoene and ca
• Synthase enzymes producing terpenes are thought to be of common evolutionary origin, lacking close similarity to other enzymes (except prenyltransferases). Most synthase enzymes have the ability to produce a variety of end-products from a single substrate. This may explain, in part, the enormous diversity of terpenoid compounds found in plants (Mitchell-Olds et al, Trends in Plant Science 3(9):362-365, 1998). Complex te ⁇ ene mixtures are thought to be important plant defensive compounds, their diversity and synergistic action delaying development of resistance in herbivores and pathogens (Langenheim J, J. Chem. Ecol. 20:1223-1280, 1994).
• Plant te ⁇ enoids also have many known medicinal effects, and some plant isoprenoid compounds are administered as drugs.
• Taxol which has proven to be efficacious in treating cancer, for example, is derived from te ⁇ enoid compounds. Dietary isoprenoids have been suggested to suppress mevalonate pathway, thereby affecting cancer and cardiovascular disease (Elson CE, J. Nutr. 125(6 Suppl): 1666S-1672S, 1995).
• Farnesol the last precursor common to all branches of the mevalonate pathway, has been demonstrated to inhibit calcium channels in muscle cells (Roullette J-B, J Biol. Chem. 51 :32240-32246, 1997).
• Ubiquinone and plastoquinone which are also isoprenoid derivatives, function as electron carriers in the production of ATP in mitocondria and chloroplasts.
• ubiquinone also called coenzyme Q
• Plastoquinone is the plant equivalent of ubiquinone. In their role as electron carriers, both ubiquinone and plastoquinone can accept either one or two electrons and either one or two protons to be reduced.
• isoprenyl intermediates A remarkable role for isoprenyl intermediates has recently been discovered in studies of a protein that is implicated in human cancers and is known to associate with membranes through a covalently bound isoprenyl lipid.
• This protein the RAS PROTEIN, is the product of the gene, a mutant version of a normal protein and a number of related GTP-binding proteins.
• the normal protein and the number of related GTP -binding proteins are known to art in signal transductions triggered by neurotransmitters, hormones, growth factors and other extracellular signals.
• the present invention is therefore directed to providing novel polynucleotides encoding polypeptides involved in the biosynthesis of isoprenoids, and providing methods for modifying the expression and composition of such polypeptides, thereby modulating isoprenoid content, composition, and metabolism.
• Sequencing of the genomes, or portions of the genomes, of numerous biological materials, including humans, animals, microorganisms and various plant varieties, has been and is being carried out on a large scale.
• Polynucleotides identified using sequencing techniques may be partial or full-length genes, and may contain open reading frames, or portions of open reading frames, that encode polypeptides. Putative polypeptides may be determined based on polynucleotide sequences. The sequencing data relating to polynucleotides thus represents valuable and useful information.
• Polynucleotides may be analyzed for novelty by comparing identified sequences to sequences published in various public domain databases, such as EMBL. Newly identified polynucleotides and putative polypeptides may also be compared to polynucleotides and polypeptides contained in databases to ascertain homology to known polynucleotides and polypeptides. In this way, the degree of similarity or identity or homology of polynucleotides and polypeptides having an unknown function may be determined relative to polynucleotides and polypeptides having known functions.
• Patent 5,589,619 discloses materials and methods for increasing squalene and sterol accumulation in higher plants by modifying the copy number of a gene encoding a polypeptide having HMG-CoA reductase activity. Genetic materials, including polynucleo-tides, polypeptides, DNA molecules, and the like, relating to HMG-CoA reductase activity are disclosed, as well as methods for transforming plant cells and producing transgenic plants.
• U.S. Patent 5,689,047 discloses stilbene synthase genes derived from grapevines, as well as the use of those genes in vectors and transformed microorganisms, as well as transformed plant cells and plants.
• U.S. Patent 5,753,507 discloses plant polynucleotides encoding geraniol/nerol 10 - hydroxylase (GioH), as well as methods for using complete and partial polynucleotides as probes, and methods for expressing GioH and enhancing levels of te ⁇ enoid indole alkaloid and ividoid insect pheromone produced by a plant.
• GioH geraniol/nerol 10 - hydroxylase
• U.S. Patents disclose isoprenoid compounds or related compounds, or methods for using such compounds: U.S. Patent 5,429,939; U.S. Patent 5,444, 166; U.S. Patent 5,460,949; U.S. Patent 5,470,832; U.S. Patent 5,474,925; U.S. Patent 5,495,070; U.S. Patent5,521 ,078; U.S. Patent 5,545,816; U.S. Patent 5,547,856; U.S. Patent 5,569,832; U.S. Patent 5,580,963; U.S. Patent 5,597,718; U.S. Patent 5,670,349; U.S. Patent 5,674,485; U.S.
• Patent 5,684,238 U.S. Patent 5,689,056; U.S. Patent 5,691 ,147; U.S. Patent 5,693,476; and U.S. Patent 5,443,978.
• the U.S. Patents cited above are inco ⁇ orated by reference herein in their entireties. Summary of the Invention
• the present invention provides isolated polynucleotides encoding polypeptides involved in the production and modification of isoprenoids. Genetic constructs comprising such sequences and methods for the use of such genetic constructs are also provided, together with transgenic cells and plants inco ⁇ orating such genetic constructs and exhibiting modified isoprenoid content, composition, and metabolism.
• the present invention provides isolated polynucleotide sequences identified in the attached Sequence Listing as SEQ ID NOS: 1-53 and 78-164, variants of those sequences, extended sequences comprising the sequences set out in SEQ ID NOS: 1-53, 78-164 and their variants, probes and primers corresponding to the sequences set out in SEQ ID NOS: 1-53, 78-164 and their variants, polynucleotides comprising at least a specified number of contiguous residues of any of the polynucleotides identified as SEQ ID NOS: 1-53 and 78-164 (x-mers), and extended sequences comprising portions of the sequences set out in SEQ ID NOS: 1-53 and 78-164, all of which are referred to herein, collectively, as "polynucleotides ofthe present invention.”
• the present invention also provides isolated polypeptide sequences identified in the attached Sequence Listing as SEQ ID NOS: 165-304, polypeptide variants of those sequences, polypeptides comprising the isolated polypeptide sequences and variants of those sequences, polypeptides comprising at least a specified number of contiguous residues of any of the polypeptides identified as SEQ ID NOS: 165-304; and polypeptides comprising portions of the sequences set out in SEQ ID NOS: 165-304.
• polynucleotide sequences identified as SEQ ID NOS: 1-53 and 78-164 were derived from plant sources, namely from Eucalyptus grandis and Pinus radiata.
• the polynucleotides of the present invention are primarily "partial" sequences, in that they do not represent a full length gene encoding a full length polypeptide. Such partial sequences may be extended by analyzing and sequencing various DNA libraries using primers and/or probes and well known hybridization and/or PCR techniques.
• the partial sequences identified as SEQ ID NOS: 1-53 and 78-164 may thus be extended until an open reading frame encoding a polypeptide, a full length polynucleotide and/or gene capable of expressing a polypeptide, or another useful portion of the genome is identified.
• Such extended sequences including full length polynucleotides and genes, are described as "corresponding to" a sequence identified as one of the sequences of SEQ ID NOS: 1-53 and 78-164 or a variant thereof, or a portion of one of the sequences of SEQ ID NOS: 1-53 and 78-164 or a variant thereof, when the extended polynucleotide comprises an identified sequence or its variant, or an identified contiguous portion (x-mer) of one ofthe sequences of SEQ ID NOS: 1-53 and 78-164 or a variant thereof.
• RNA sequences, reverse sequences, complementary sequences, anti-sense sequences, and the like, corresponding to the polynucleotides of the present invention may be routinely ascertained and obtained using the cDNA sequences identified as SEQ ID NOS: 1-53 and 78-164.
• the polynucleotides identified as SEQ ID NOS: 1-53 and 78-164 may contain open reading frames ("ORFs") or partial open reading frames encoding polypeptides. Additionally, open reading frames encoding polypeptides may be identified in extended or full length sequences corresponding to the sequences set out as SEQ ID NOS: 1-53 and 78-164. Open reading frames may be identified using techniques that are well known in the art. These techniques include, for example, analysis for the location of known start and stop codons, most likely reading frame identification based on codon frequencies, etc. Suitable tools and software for ORF analysis are available, for example, on the Internet at http://www.ncbi.nlm.nih.gov/eorf/gorf.html.
• Open reading frames and portions of open reading frames may be identified in the polynucleotides of the present invention. Once a partial open reading frame is identified, the polynucleotide may be extended in the area of the partial open reading frame using techniques that are well known in the art until the polynucleotide for the full open reading frame is identified. Thus, polynucleotides and open reading frames encoding polypeptides may be identified using the polynucleotides of the present invention.
• the open reading frames may be isolated and/or synthesized.
• Expressible DNA constructs comprising the open reading frames and suitable promoters, initiators, terminators, etc., which are well known in the art, may then be constructed.
• DNA constructs may be introduced into a host cell to express the polypeptide encoded by the open reading frame.
• Suitable host cells may include various prokaryotic and eukaryotic cells, including plant cells.
• Polypeptides encoded by the polynucleotides of the present invention may be expressed and used in various assays to determine their biological activity. Such polypeptides may be used to raise antibodies, to isolate corresponding interacting proteins or other compounds, and to quantitatively determine levels of interacting proteins or other compounds.
• the present invention also contemplates methods for modulating the polynucleotide and/or polypeptide content and composition of an organism, such methods involving, according to one embodiment, stably inco ⁇ orating into the genome of the organism a genetic construct comprising one or more polynucleotides of the present invention.
• the target organism is a plant, preferably a woody plant, more preferably a woody plant of the Pinus or Eucalyptus species, and most preferably Eucalyptus grandis or Pinus radiata.
• a method for producing an organism having an altered genotype or phenotype comprising transforming a host cell with a genetic construct of the present invention to provide a transgenic cell, and cultivating the transgenic cell under conditions conducive to growth and regeneration.
• Organisms having an altered genotype or phenotype as a result of modulation of the level or content of a polynucleotide or polypeptide of the present invention compared to a wild-type organism, as well as components (seeds, etc.) of such organisms and progeny of such organisms, are contemplated by and encompassed within the present invention.
• the isolated polynucleotides of the present invention have utility in genome mapping, in physical mapping, and in positional cloning of genes. Additionally, the polynucleotide sequences identified as SEQ ID NOS: 1-53, 78-164, and their variants, may be used to design oligonucleotide probes and primers. Oligonucleotide probes and primers have sequences that are substantially complementary to the polynucleotide of interest over a certain portion of the polynucleotide.
• Oligonucleotide probes designed using the polynucleotides of the present invention may be used to detect the presence and examine the expression patterns of genes in any organism having sufficiently similar DNA and RNA sequences in their cells using techniques that are well known in the art, such as slot blot DNA hybridization tecliniques.
• Oligonucleotide primers designed using the polynucleotides of the present invention may be used for PCR amplifications.
• Oligonucleotide probes and primers designed using the polynucleotides of the present invention may also be used in connection with various microarray technologies, including the microarray technology used by Synteni (Palo Alto, CA).
• the polynucleotides of the present invention may also be used to tag or identify an organism or reproductive material therefrom. Such tagging may be accomplished, for example, by stably introducing a non-disruptive non-functional heterologous polynucleotide identifier into an organism, the polynucleotide comprising one of the polynucleotides ofthe present invention.
• the polynucleotides of the present invention encode polypeptides that have activity in an isoprenoid biosynthetic pathway.
• the isoprenoid metabolism-related polynucleotides were isolated from pine and eucalyptus, and putatively identified by DNA and protein similarity searches.
• Various isoprenoid compounds are well characterized and have useful properties.
• Methods of the present invention relating to modulating the polynucleotide and/or polypeptide content and composition of an organism and, thereby, modulating the isoprenoid content, composition and metabolism of an organism, are applicable to a wide range of activities.
• manipulating isoprenoid pathways or isoprenoid composition may, for example, affect plant development, pest resistance, and the value of extractives (pinene, myrcene, etc.).
• isoprenoids affect the nutritional quality and pharmacological properties of the ingested material, e.g, cholesterol or phytosterol composition of animal- derived and plant-derived foods for human or animal consumption. Additionally, isoprenoid pathways control the production of vitamins A, E, and K; plant pigments such as carotene and the phytol chain of chlorophyll; natural rubber; many essential oils, such as the fragrant principles of lemon oil, eucalyptus, and musk; insect juvenile hormone, which controls metamo ⁇ hosis; dolichols, which serve as lipid-soluble carriers in complex polysaccharide synthesis; and ubiquinone and plastoquinone, electron carriers in mitochondria and chloroplasts.
• the ubiquitous and varied roles of isoprenoids thus make these compounds and the polynucleotides encoding them attractive targets for biotechnical applications in a variety of fields.
• the present invention provides isolated polynucleotides encoding polypeptides involved in the synthesis of isoprenoids.
• the polynucleotides and polypeptides of the present invention have demonstrated similarity to polypeptides that are known to be involved in the synthesis of isoprenoids as shown below in Table 1. TABLE 1
• the isolated polynucleotides comprise a sequence selected from the group consisting of: (a) sequences recited in SEQ ID NOS: 1-53 and 78-164; (b) complements of the sequences recited in SEQ ID NOS: 1-53 and 78-164; (c) reverse complements of the sequences recited in SEQ ID NOS: 1-53 and 78-164; (d) reverse sequences of the sequences recited in SEQ ID NOS: 1-53 and 78-164; and (e) sequences having either 40%, 60%, 75% or 90% identity, as defined herein, to a sequence of (a) - (d) or a specified region of a sequence of (a) - (d).
• polypeptides encoded by the polynucleotides of the present invention are provided.
• such polypeptides comprise an amino acid sequence encoded by polynucleotides of the present invention, including polynucleotides comprising a sequence set out in the group consisting of SEQ ID NOS: 1- 53 and 78-164, as well as polypeptides comprising an amino acid sequence recited in SEQ ID NOS: 165- 304.
• the invention provides genetic constructs comprising a polynucleotide of the present invention, either alone, in combination with one or more additional polynucleotides of the present invention, or in combination with one or more known polynucleotides, together with transgenic cells comprising such constructs.
• the present invention provides genetic constructs comprising, in the 5 '-3' direction, a gene promoter sequence; an open reading frame coding for at least a functional portion of an enzyme encoded by an inventive polynucleotide or a variant thereof; and a gene termination sequence.
• the open reading frame may be oriented in either a sense or antisense direction.
• Genetic constructs comprising a non-coding region of a gene coding for an enzyme encoded by the above polynucleotide or a nucleotide sequence complementary to a non-coding region, together with a gene promoter sequence and a gene termination sequence, are also provided.
• Genetic constructs comprising, in the 5' - 3' direction, a promoter sequence; a polynucleotide sequence comprising at least one of the following: (1) a polynucleotide comprising a polynucleotide of the present invention; or (2) a polynucleotide comprising a polynucleotide of the present invention and including a non-coding region of a gene coding for a polypeptide having activity in an isoprenoid biosynthetic pathway, are also contemplated.
• the genetic construct may further include a marker for the identification of transformed cells.
• transgenic host cells such as transgenic plant cells, comprising the genetic constructs of the present invention are provided, together with plants comprising such transgenic cells, and fruits, seeds, and progeny of such plants.
• Other useful host cells include bacterial cells, insect cells, yeast cells and mammalian cells.
• the target organism is a plant and the plant is a woody plant, preferably selected from the group consisting of eucalyptus, pine, acacia, poplar, sweetgum, teak and mahogany species, more preferably from the group consisting of pine and eucalyptus species, and most preferably from the group consisting of Eucalyptus grandis and Pinus radiata.
• a method for producing an organism having modified isoprenoid content comprising transforming a host cell with a genetic construct of the present invention to provide a transgenic cell and cultivating the transgenic cell under conditions conducive to growth and regeneration.
• the present invention provides methods for modifying the activity of a polypeptide in a target organism such as a plant, comprising stably inco ⁇ orating into the genome of the organism a genetic construct ofthe present invention.
• the target organism is a plant
• the plant is a woody plant, preferably selected from the group consisting of eucalyptus, pine, acacia, poplar, sweetgum, teak and mahogany species, more preferably from the group consisting of pine and eucalyptus species, and most preferably from the group consisting of Eucalyptus grandis and Pinus radiata.
• the present invention provides methods for modulating one or more of the content, the composition and the metabolism of an isoprenoid compound in an organism by administering an isolated polypeptide of the present invention to the organism.
• administration of the polypeptide may be topical, such as by spraying or similar topical application.
• administration of the polypeptide may be systemic, such as by injection, intradermal delivery, oral delivery, delivery via nasal passageways or airways, or the like.
• Fig. 1 shows a schematic diagram illustrating basic biosynthetic pathways of isoprene compounds.
• Fig. 2 illustrates genomic DNA samples from tobacco plants created in a tagging experiment using a unique sequence identifier from Pinus (left panel) and a unique sequence identifier from Eucalyptus (right panel). In both panels, Lanes A and B contain
• Lanes C-E contain DNA samples from plants transformed with a unique sequence identifier.
• Fig. 3 illustrates detection of a Pinus unique sequence identifier in transformed tobacco plants.
• Lanes A and B show the hybridization of a probe from SEQ ID NO: 76 to the genomic DNA of tobacco plants which lack the Pinus unique sequence identifier (empty-vector transformed control plants).
• Lanes C-E show the hybridization of the probe to the genomic DNA of tobacco plants containing one to three copies of the Pinus unique sequence identifier.
• Fig. 4 illustrates detection of a Eucalyptus unique sequence identifier in transformed tobacco plants.
• Lanes A and B show the hybridization of a probe from SEQ ID NO: 77 to the genomic DNA of tobacco plants which lack the Eucalyptus unique sequence identifier (empty-vector transformed control plants).
• Lanes C-E show the hybridization of the probe to the genomic DNA of tobacco plants containing one to two copies of the Eucalyptus unique sequence identifier.
• isoprenoids are important components in a variety of eukaryotic functions. Modification of isoprenoid content, composition, and metabolism in Jhe earlier parts of the pathway, especially the steps up to the formation of isopentenyl- diphosphate (IPP), geranyl-diphosphate (GPP), famesyl-diphosphate (FPP) and squalene, may have a profound influence on the synthesis of the isoprenoid compounds deriving from these two precursors.
• IPP isopentenyl- diphosphate
• GPP geranyl-diphosphate
• FPP famesyl-diphosphate
• squalene squalene
• Blocking one or more of the downstream steps branching from isopentenyl-diphosphate and squalene may also have a substantial effect on the pool of isopentenyl-diphosphate and squalene available for synthesis of te ⁇ enes or steroids.
• the isoprenoid content of a plant may be modified by inco ⁇ orating sense or antisense copies of polynucleotides encoding polypeptides involved in the synthesis of isoprenoids into the genome of a target organism.
• the number of copies and combination of polynucleotides encoding for different enzymes in the biosynthetic pathway of isoprenoids may be manipulated to modify the relative amounts of isoprenoids synthesized, thereby producing biological materials having an altered composition and/or altered isoprenoid metabolism.
• isoprenoid composition for direct application in a target organism, or for production of polypeptides for separate use, is advantageous for a variety of applications, as evidenced by the references cited above and inco ⁇ orated herein by reference.
• the present invention provides isolated polynucleotides encoding, or partially encoding, polypeptides having similarity to polypeptides known to be involved in isoprenoid synthesis and modification.
• the polynucleotides of the present invention were isolated from eucalyptus and pine species, but may alternatively be isolated from other plant sources and may be synthesized using conventional synthesis techniques.
• isolated polynucleotides of the present invention comprise: the polynucleotides identified as SEQ ID NOS: 1-53 and 78-164; complements of the sequences identified as SEQ ID NOS: 1-53 and 78-164; reverse sequences of the sequences identified as SEQ ID NOS: 1-53 and 78-164; reverse complements of the sequences identified as SEQ ID NOS: 1 -53 and 78-164; at least a specified number of contiguous residues (x-mers) of any of the above-mentioned polynucleotides; polynucleotides complementary to any of the above polynucleotides; anti-sense sequences corresponding to any of the above polynucleotides; and variants of any of the above polynucleotides, as that term is described in this specification.
• the isolated polynucleotides recited in SEQ ID NOS: 1-53 and 78-164 encode, or partially encode, polypeptides demonstrating sequence similarity to polypeptides known to be involved in an isoprenoid biosynthetic pathway, as indicated in Table 1 above. More specifically, the isolated polynucleotides listed in the first column of Table 1 encode, or partially encode the polypeptides listed in alignment in the second column of Table 1, above. Predicted amino acid sequences corresponding to the polynucleotides set out in SEQ ID NOS: 1-53, 78-164, based on information available at the time of filing this application, are provided in SEQ ID NOS: 165-304, as indicated in Table 1.
• polynucleotide(s), means a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and corresponding RNA molecules, including HnRNA and mRNA molecules, both sense and anti-sense strands, and comprehends cDNA, genomic DNA and recombinant DNA, as well as wholly or partially synthesized polynucleotides.
• An HnRNA molecule contains introns and corresponds to a DNA molecule in a generally one-to-one manner.
• An mRNA molecule corresponds to an HnRNA and DNA molecule from which the introns have been excised.
• a polynucleotide may consist of an entire gene, or any portion thereof.
• a gene is a polypeptide that codes for a functional polypeptide or RNA molecule.
• Operable anti- sense polynucleotides may comprise a fragment of the corresponding polynucleotide, and the definition of "polynucleotide” therefore includes all such operable anti-sense fragments.
• Anti-sense polynucleotides and techniques involving anti-sense polynucleotides are well known in the art and are described, for example, in Robinson- Benion et al., Methods in Enzymol. 254(23):363-375, 1995; and Kawasaki et al., Artific.
• Polynucleotides of the present invention also encompass polynucleotide sequences that differ from the disclosed sequences but which, as a result of the degeneracy of genetic code, encode a polypeptide which is the same as that encoded by a polynucleotide of the present invention.
• DNAs can be accomplished by standard DNA/DNA hybridization techniques, under appropriately stringent conditions, using all or part of a cDNA sequence as a probe to screen an appropriate library.
• PCR techniques using oligonucleotide primers that are designed based on known genomic DNA, cDNA and protein sequences can be used to amplify and identify genomic and cDNA sequences.
• Synthetic DNAs corresponding to the identified sequences and variants may be produced by conventional synthesis methods. All of the polynucleotides described herein are isolated and purified, as those terms are commonly used in the art.
• the present invention provides isolated polypeptides encoded, or partially encoded, by the above polynucleotides.
• polypeptide encompasses amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
• polypeptide encoded by a polynucleotide includes polypeptides encoded by a polynucleotide which comprises an isolated polypeptide or variant provided herein.
• polypeptides of the present invention comprise an amino acid sequence selected from the group consisting of sequences provided in SEQ ID NOS: 165-304, as well as variants of such sequences.
• polypeptides of the present invention comprise at least a specified number of contiguous residues (x-mers) of any of the sequences provided in SEQ ID NOS: 165-304.
• Polypeptides of the present invention may be produced recombinantly by inserting a polynucleotide that encodes the polypeptide into an expression vector and expressing the polypeptide in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art may be employed. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a polypeptide encoding a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are Escherichia coli, insect, yeast or a mammalian cell line such as COS or CHO. The polynucleotide(s) expressed in this manner may encode naturally occurring polypeptides, portions of naturally occurring polypeptides, or other variants thereof.
• polypeptides are provided that comprise at least a functional portion of a polypeptide having an amino acid sequence selected from the group consisting of sequences provided in SEQ ID NOS: 165-304, and variants thereof.
• a "functional portion" of a polypeptide is that portion which contains the active site essential for affecting the function of the polypeptide, for example, the portion of the molecule that is capable of binding one or more reactants.
• the active site may be made up of separate portions present on one or more polypeptide chains and will generally exhibit high binding affinity.
• Functional portions of a polypeptide may be identified by first preparing fragments of the polypeptide by either chemical or enzymatic digestion of the polypeptide, or by mutation analysis of the polynucleotide that encodes the polypeptide and subsequent expression of the resulting mutant polypeptides. The polypeptide fragments or mutant polypeptides are then tested to determine which portions retain biological activity, using, for example, the representative assays provided below.
• a functional portion comprising an active site may be made up of separate portions present on one or more polypeptide chains and generally exhibits high substrate specificity.
• polypeptide encoded by a polynucleotide includes polypeptides encoded by a polynucleotide comprising a partial isolated polynucleotide of the present invention.
• Portions and other variants of the inventive polypeptides may also be generated by synthetic or recombinant means.
• Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids may be generated using techniques that are well known to those of ordinary skill in the art.
• such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2154, 1963.
• Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/ Applied Biosystems, Inc.
• Variants of a native polypeptide may be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagensis (Kunkel T, Proc. Natl. Acad. Sci. USA 82: 488-492, 1985). Sections of DNA sequences may also be removed using standard techniques to permit preparation of truncated polypeptides.
• the polypeptides disclosed herein are prepared in an isolated, substantially pure form.
• the polypeptides are at least about 80% pure; more preferably at least about 90% pure; and most preferably, at least about 99%) pure.
• the isolated polypeptides are inco ⁇ orated into pharmaceutical compositions or vaccines for use in the treatment of skin disorders.
• variant comprehends polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variant polynucleotide sequences preferably exhibit at least 40%; more preferably at least 60%; more preferably yet at least 75%; and most preferably at least 90% identity to a sequence of the present invention. Variant polypeptide sequences preferably exhibit at least 50%; more preferably at least 75%; more preferably yet at least 90%>; and most preferably at least 95% identity to a sequence of the present invention. The percentage identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portion, dividing that number by the total number of residues in the inventive (queried) sequence, and multiplying the result by 100.
• Polynucleotide and polypeptide sequences may be aligned, and percentage of identical residues in a specified region may be determined against another polynucleotide or polypeptide, using computer algorithms that are publicly available. Two exemplary algorithms for aligning and identifying the similarity of polynucleotide sequences are the BLASTN and FASTA algorithms. Polynucleotides may also be analyzed using the BLASTX algorithm, which compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database. ' The percentage identity of polypeptide sequences may be examined using the BLASTP algorithm.
• the BLASTN, BLASTX and BLASTP programs are available on the NCBI anonymous FTP server (ftp://ncbi.nlm.nih.gov) under /blast/executables/.
• the BLASTN algorithm Version 2.0.4 [Feb-24-1998] and Version 2.0.6 [Sept-16-1998], set to the parameters described below, is preferred for use in the determination of polynucleotide variants according to the present invention.
• the BLASTP algorithm set to the parameters described below, is preferred for use in the determination of polypeptide variants according to the present invention.
• BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX, is described at NCBI's website at URL http://www.ncbi.nlm.nih.gov BLAST/newblast.htmI and in the publication of Altschul, et al, Nucleic Acids Res. 25: 3389-3402, 1997.
• the computer algorithm FASTA is available on the Internet at the ftp site ftp://ftp.virginia.edu/pub/fasta/. Version 2.0u4 [February 1996], set to the default parameters described in the documentation and distributed with the algorithm, may be also used in the determination of variants according to the present invention.
• the use of the FASTA algorithm is described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988; and Pearson WR, Methods in Enzymol. 183: 63-98, 1990.
• the following running parameters are preferred for determination of alignments and similarities using BLASTN that contribute to the E values and percentage identity for polynucleotide sequences: Unix running command: blastall -p blastn -d embldb -e 10 -GO -E0 -r 1 -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -r Reward for a nucleotide match (BLASTN only) [Integer]; -v Number of one-line descriptions (V) [Integer]; -b Number of alignments to show (B) [Integer]; -i Query File [File In]; and
• the following running parameters are preferred for determination of alignments and similarities using BLASTP that contribute to the E values and percentage identity of polypeptide sequences: blastall -p blastp -d swissprotdb -e 10 -G 0 -E 0 -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; -d Database [String]; - e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -v Number of one-line descriptions (v) [Integer]; -b Number of alignments to show (b) [Integer]; -I Query File [File In]; -o BLAST report Output File [File Out] Optional.
• the "hits" to one or more database sequences by a queried sequence produced by BLASTN, FASTA, BLASTP or a similar algorithm align and identify similar portions of sequences.
• the hits are arranged in order of the degree of similarity and the length of sequence overlap.
• Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.
• the BLASTN, FASTA, and BLASTP algorithms also produce "Expect" values for alignments.
• the Expect value (E) indicates the number of hits one can "expect" to see over a certain number of contiguous sequences by chance when searching a database of a certain size.
• the Expect value is used as a significance threshold for determining whether the hit to a database, such as the preferred EMBL database, indicates true similarity.
• a database such as the preferred EMBL database
• an E value of 0.1 assigned to a polynucleotide hit is inte ⁇ reted as meaning that in a database of the size of the EMBL database, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance.
• the aligned and matched portions of the polynucleotide sequences then have a probability of 90%> of being the same.
• the probability of finding a match by chance in the EMBL database is 1% or less using the BLASTN or FASTA algorithm.
• variant polynucleotides and polypeptides preferably comprise sequences producing an E value of 0.01 or less when compared to the polynucleotide or polypeptide of the present invention. That is, a variant polynucleotide or polypeptide is any sequence that has at least a 99% probability of being the same as the polynucleotide or polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTN, FASTA, or BLASTP algorithms set at parameters described above.
• a variant polynucleotide is a sequence having the same number or fewer nucleic acids than a polynucleotide of the present invention that has at least a 99% probability of being the same as the polynucleotide of the present invention, measured as having an E value of 0.01 or less using the BLASTN or FASTA algorithms set at parameters described above.
• a variant polypeptide is a sequence having the same number or fewer amino acids than a polypeptide ofthe present invention that has at least a 99% probability of being the same as a polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTP algorithm set at the parameters described above.
• variant polynucleotides or polypeptides of the present invention comprise a sequence exhibiting at least 40%; more preferably at least 60%; more preferably yet at least 75%; and most preferably at least 90% 0 identity to a polynucleotide or polypeptide of the present invention, determined as described below.
• the percentage identity is determined by aligning sequences using one of the BLASTN, FASTA, or BLASTP algorithms, set at the running parameters described above, and identifying the number of identical nucleic or amino acids over the aligned portions; dividing the number of identical nucleic or amino acids by the total number of nucleic or amino acids of the polynucleotide or polypeptide of the present invention; and then multiplying by 100 to determine the percentage identity.
• a polynucleotide of the present invention having 220 nucleic acids has a hit to a polynucleotide sequence in the EMBL database having 520 nucleic acids over a stretch of 23 nucleotides in the alignment produced by the BLASTN algorithm using the parameters described above.
• the 23 nucleotide hit includes 21 identical nucleotides, one gap and one different nucleotide.
• the percentage identity of the polynucleotide of the present invention to the hit in the EMBL library is thus 21/220 times 100, or 9.5%.
• the polynucleotide sequence in the EMBL database is thus not a variant of a polynucleotide ofthe present invention.
• variant polynucleotides of the present invention hybridize to the polynucleotide sequences recited in SEQ ID NOS: 1-53 and 78-164, or complements, reverse sequences, or reverse complements of those sequences under stringent conditions.
• stringent conditions refers to prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1 % SDS at 65° C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65°C.
• the present invention also encompasses polynucleotides that differ from the disclosed sequences but that, as a consequence of the discrepancy of the genetic code, encode a polypeptide having similar enzymatic activity as a polypeptide encoded by a polynucleotide of the present invention.
• polynucleotides comprising sequences that differ from the polynucleotide sequences recited in SEQ ID NOS: 1-53 and 78-164, or complements, reverse sequences, or reverse complements of those sequences as a result of conservative substitutions are contemplated by and encompassed within the present invention.
• polynucleotides comprising sequences that differ from the polynucleotide sequences recited in SEQ ID NOS: 1-53 and 78-164, or complements, reverse complements, or reverse sequences as a result of deletions and/or insertions totaling less than 10% of the total sequence length are also contemplated by and encompassed within the present invention.
• polypeptides comprising sequences that differ from the polypeptide sequences recited in SEQ ID NOS: 165-304 as a result of amino acid substitutions, insertions, and/or deletions totaling less than 10%> of the total sequence length are contemplated by an encompassed within the present invention, provided the variant polypeptide has activity in an isoprenoid biosynthetic pathway.
• the polynucleotides of the present invention may be isolated from various libraries, or may be synthesized using techniques that are well known in the art.
• the polynucleotides may be synthesized, for example, using automated oligonucleotide synthesizers (e.g., Beckman Oligo 1000M DNA Synthesizer) to obtain polynucleotide segments of up to 50 or more nucleic acids.
• a plurality of such polynucleotide segments may then be ligated using standard DNA manipulation techniques that are well known in the art of molecular biology.
• One conventional and exemplary polynucleotide synthesis technique involves synthesis of a single stranded polynucleotide segment having, for example, 80 nucleic acids, and hybridizing that segment to a synthesized complementary 85 nucleic acid segment to produce a 5 nucleotide overhang. The next segment may then be synthesized in a similar fashion, with a 5 nucleotide overhang on the opposite strand. The "sticky" ends ensure proper ligation when the two portions are hybridized. In this way, a complete polynucleotide of the present invention may be synthesized entirely in vitro.
• polynucleotides identified as SEQ ID NOS: 1-53 and 78-164 are referred to as "partial" sequences, in that they do not represent the full coding portion of a gene encoding a naturally occurring polypeptide.
• the partial polynucleotide sequences disclosed herein may be employed to obtain the corresponding full length genes for various species and organisms by, for example, screening DNA expression libraries using hybridization probes based on the polynucleotides of the present invention, or using PCR amplification with primers based upon the polynucleotides ofthe present invention.
• polynucleotide of the present invention upstream and downstream of the corresponding mRNA, as well as identify the corresponding genomic DNA, including the promoter and enhancer regions, of the complete gene.
• the present invention thus comprehends isolated polynucleotides comprising a sequence identified in SEQ ID NOS: 1-53 and 78-164, or a variant of one of the specified sequences, that encode a functional polypeptide, including full length genes.
• Such extended polynucleotides may have a length of from about 50 to about 4,000 nucleic acids or base pairs, and preferably have a length of less than about 4,000 nucleic acids or base pairs, more preferably yet a length of less than about 3,000 nucleic acids or base pairs, more preferably yet a length of less than about 2,000 nucleic acids or base pairs.
• extended polynucleotides of the present invention may have a length of less than about 1,800 nucleic acids or base pairs, preferably less than about 1,600 nucleic acids or base pairs, more preferably less than about 1,400 nucleic acids or base pairs, more preferably yet less than about 1,200 nucleic acids or base pairs, and most preferably less than about 1,000 nucleic acids or base pairs.
• Polynucleotides of the present invention also comprehend polynucleotides comprising at least a specified number of contiguous residues (x-mers) of any of the polynucleotides identified as SEQ ID NOS: 1-53 and 78-164, complements, reverse sequences, and reverse complements of such sequences, and their variants.
• polypeptides of the present invention comprehend polypeptides comprising at least a specified number of contiguous residues (x-mers) of any of the polypeptides identified as SEQ ID NOS: 165-304, and their variants.
• x-mer refers to a sequence comprising at least a specified number ("x") of contiguous residues of any of the polynucleotides identified as SEQ ID NOS: 1-53 and 78-164, or the polypeptides identified as SEQ ID NOS: 165-304.
• the value of x is preferably at least 20; more preferably, at least 40; more preferably yet, at least 60; and most preferably, at least 80.
• polynucleotides and polypeptides of the present invention comprise a 20-mer, a 40- mer, a 60-mer, an 80-mer, a 100-mer, a 120-mer, a 150-mer, a 180-mer, a 220-mer, a 250- mer, or a 300-mer, 400-mer, 500-mer or 600-mer of a polynucleotide or polypeptide identified as SEQ ID NOS: 1-53, and 78-304, and variants thereof.
• oligonucleotide probes and primers complementary to and or corresponding to SEQ ID NOS: 1-53 and 78-164, and variants of those sequences are also comprehended by the present invention.
• Such oligonucleotide probes and primers are substantially complementary to the polynucleotide of interest.
• the term "oligonucleotide” refers to a relatively short segment of a polynucleotide sequence, generally comprising between 6 and 60 nucleotides, and comprehends both probes for use in hybridization assays and primers for use in the amplification of DNA by polymerase chain reaction.
• oligonucleotide probe or primer is described as "corresponding to" a polynucleotide of the present invention, including one of the sequences set out as SEQ ID NOS: 1-53 and 78-164, or a variant, if the oligonucleotide probe or primer, or its
• ?? complement is contained within one of the sequences set out as SEQ ID NOS: 1-53 and 78-164, or a variant of one of the specified sequences.
• Two single stranded sequences are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared, with the appropriate nucleotide insertions and or deletions, pair with at least 80%>, preferably at least 90% to 95%, and more preferably at least 98% to 100%, of the nucleotides of the other strand.
• substantial complementarity exists when a first DNA strand selectively hybridizes to a second DNA strand under stringent hybridization conditions.
• Stringent hybridization conditions for determining complementarity include salt conditions of less than about 1 M, more usually less than about 500 mM and preferably less than about 200 mM.
• Hybridization temperatures may be as low as 5°C, but are generally greater than about 22°C, more preferably greater than about 30°C and most preferably greater than about 37°C. Longer DNA fragments may require higher hybridization temperatures for specific hybridization. Since the stringency of hybridization may be affected by other factors such as probe composition, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone.
• the DNA from plants or samples or products containing plant material can be either genomic DNA or DNA derived by preparing cDNA from the RNA present in the sample. In addition to DNA-DNA hybridization, DNA-RNA or RNA-RNA hybridization assays are also possible.
• the oligonucleotide probes and/or primers comprise at least about 6 contiguous residues, more preferably at least about 10 contiguous residues, and most preferably at least about 20 contiguous residues complementary to a polynucleotide sequence of the present invention. Probes and primers of the present invention may be from about 8 to 100 base pairs in length or, preferably, from about 10 to 50 base pairs in length or, more preferably, from about 15 to 40 base pairs in length.
• the probes can be easily selected using procedures well known in the art, taking into account DNA-DNA hybridization stringencies, annealing and melting temperatures, potential for formation of loops and other factors, which are well known in the art.
• Tools and software suitable for designing probes, and especially suitable for designing PCR primers are available on the Internet, for example, at URL http://www.horizonpress.com/pcr/.
• Preferred techniques for designing PCR primers are also disclosed in Dieffenbach CW and Dyksler GS, PCR primer: a laboratory manual. CSHL Press: Cold Spring Harbor, NY, 1995.
• kits generally comprise multiple DNA or oligonucleotide probes, each probe being specific for a polynucleotide sequence.
• Kits of the present invention may comprise one or more probes or primers corresponding to a polynucleotide of the present invention, including a polynucleotide sequence identified in SEQ ID NOS: 1-53 and 78-164.
• the oligonucleotide probe kits of the present invention comprise multiple probes in an array format, wherein each probe is immobilized in a predefined, spatially addressable location on the surface of a solid substrate.
• Array formats which may be usefully employed in the present invention are disclosed, for example, in U.S. Patent Nos. 5,412,087 and 5,545,531 ; and PCT Publication No. WO 95/00530, the disclosures of which are hereby inco ⁇ orated by reference.
• Probes preferably in the form of an array, may be employed to screen for differences in organisms or samples or products containing genetic material using high throughput screening techniques that are well known in the art.
• the significance of using probes in high-throughput screening systems is apparent for applications such as plant breeding and quality control operations in which there is a need to identify large numbers of seed lots and plant seedlings, to examine samples or products for unwanted plant materials, to identify plants or samples or products containing plant material for quarantine pu ⁇ oses, etc., or to ascertain the true origin of plants or samples or products containing plant material.
• oligonucleotide probe kits of the present invention may be employed to examine the presence/absence (or relative amounts in case of mixtures) of polynucleotides in different samples or products containing different materials rapidly and in a cost-effective manner.
• plant species that may be examined using the present invention, include forestry species, such as pine and eucalyptus species, other tree species, and agricultural and horticultural plants.
• a collection of a plurality ofthe polynucleotides of the present invention may be recorded and/or stored on a storage medium and subsequently accessed for p poses of analysis, comparison, etc.
• Suitable storage media include magnetic media such as magnetic diskettes, magnetic tapes, CD-ROM storage media, optical storage media, and the like.
• Suitable storage media and methods for recording and storing information, as well as accessing information such as polynucleotide sequences recorded on such media, are well known in the art.
• the polynucleotide information stored on the storage medium is preferably computer-readable and may be used for analysis and comparison of the polynucleotide information.
• the storage medium includes a collection of at least 4, preferably at least 10, more preferably at least 15, and most preferably at least 20 of the polynucleotides ofthe present invention, preferably the polynucleotides identified as SEQ ID NOS: 1-53 and 78-164, and variants of those polynucleotides.
• an open reading frame may be inserted into a genetic construct in a sense or antisense orientation, such that transformation of a target plant with the genetic construct produces a change in the expression level of the polypeptide compared to the expression in a wild-type organism. Transformation with a genetic construct comprising an open reading frame in a sense orientation will generally result in modulation of expression of the selected gene, while transformation with a genetic construct comprising an open reading frame in an antisense orientation generally produces reduced expression of the selected gene.
• a population of plants transformed with a genetic construct comprising an open reading frame of the present invention in either a sense or antisense orientation may be screened for increased or reduced expression of the gene in question using techniques well known to those of skill in the art, and plants having the desired phenotypes may thus be isolated.
• expression of a gene involved in the biosynthesis of isoprenoids may be inhibited by inserting a portion of an open reading frame of the present invention, in either sense or antisense orientation, in the genetic construct.
• Such portions need not be full-length but preferably comprise at least 25, and more preferably, at least 50 residues of polynucleotide of the present invention.
• a much longer portion, or even the full length polynucleotide corresponding to the complete open reading frame, may be employed.
• the portion of the open reading frame does not need to be precisely the same as the endogenous sequence, provided that there is sufficient sequence similarity to achieve inhibition of the target gene.
• a sequence derived from one species may be used to inhibit expression of a gene in a different species.
• the genetic constructs of the present invention comprise a polynucleotide including a non-coding region of a gene coding for a polypeptide encoded by a polynucleotide of the present invention, or a polynucleotide complementary to such a non-coding region.
• non-coding regions which may be usefully employed in such constructs include introns and 5 '-non-coding leader sequences.
• Transformation of a target plant with such a genetic construct may lead to a reduction in the amount of an isoprenoid compound synthesized by the plant by the process of cosuppression, in a manner similar to that discussed, for example, by Napoli et al., Plant Cell 2:279-290, 1990 and de Carvalho Niebel et al, Plant Cell 7:347-358, 1995.
• regulation may be achieved by inserting appropriate sequences or subsequences (e.g. DNA or RNA) in ribozyme constructs (Mclntyre CL and Manners JM,
• Ribozymes are synthetic RNA molecules that comprise a hybridizing region complementary to two regions, each of which comprises at least 5 contiguous nucleotides in a mRNA molecule encoded by one of the inventive polynucleotides. Ribozymes possess highly specific endonuclease activity, which autocatalytically cleaves the mRNA.
• the genetic constructs of the present invention further comprise a gene promoter sequence and a gene termination sequence, operably linked to the polynucleotide to be transcribed, which control expression of the polypeptide.
• the gene promoter sequence is generally positioned at the 5' end of the polynucleotide to be transcribed, and is employed to initiate transcription of the polynucleotide.
• Gene promoter sequences are generally found in the 5' non-coding region of a gene but they may exist downstream of the open reading frame or in introns (Luehrsen KR, Mol. Gen. Genet. 225:81-93, 1991); or in the coding region, as for example in a plant defence gene (Douglas et al, EMBO J.
• the gene promoter sequence When the construct includes an open reading frame in a sense orientation, the gene promoter sequence also initiates translation of the open reading frame.
• the gene promoter sequence For genetic constructs comprising either an open reading frame in an antisense orientation or a non-coding region, the gene promoter sequence consists only of a transcription initiation site having a RNA polymerase binding site.
• gene promoter sequences that may be usefully employed in the genetic constructs of the present invention are well known in the art.
• the gene promoter sequence, and also the gene termination sequence may be endogenous to the target plant host or may be exogenous, provided the promoter is functional in the target host.
• the promoter and termination sequences may be from other plant species, plant viruses, bacterial plasmids and the like.
• gene promoter and termination sequences are common to those of the polynucleotide being introduced.
• Factors influencing the choice of promoter include the desired tissue specificity of the construct, and the timing of transcription and translation.
• constitutive promoters such as the 35S Cauliflower Mosaic Virus (CaMV 35S) promoter with or without enhancers, such as the Kozak sequence or the Omega enhancer, and Agrobacterium tumefaciens nopalin synthase terminator, may be usefully employed in the present invention.
• Use of a tissue specific promoter will result in production of the desired sense or antisense RNA only in the tissue of interest.
• the rate of RNA polymerase binding and initiation can be modulated by external stimuli, such as light, heat, anaerobic stress, alteration in nutrient conditions and the like.
• Temporally regulated promoters can be employed to effect modulation of the rate of RNA polymerase binding and initiation at a specific time during development of a transformed cell.
• the original promoters from the enzyme gene in question, or promoters from a specific tissue-targeted gene in the organism to be transformed, such as eucalyptus or pine are used.
• gene promoters which may be usefully employed in the present invention include mannopine synthase (mas), octopine synthase (ocs) and those reviewed by Chua et al, Science 244: 174-181, 1989.
• the gene termination sequence which is located 3' to the polynucleotide to be transcribed, may come from the same gene as the gene promoter sequence or may be from a different gene. Many gene termination sequences known in the art may be usefully employed in the present invention, such as the 3' end of the Agrobacterium tumefaciens nopaline synthase gene. However, preferred gene terminator sequences are those from the original enzyme gene or from the target species to be transformed.
• the genetic constructs of the present invention may also contain a selection marker that is effective in target cells, such as plant cells, to allow for the detection of transformed cells containing the inventive construct. Such markers, which are well known in the art, typically confer resistance to one or more toxins.
• NPTII neuropeptide kinase inhibitor
• NPTII neuropeptide kinase inhibitor
• NPTII kanamycin or hygromycin
• Transformed cells can thus be identified by their ability to grow in media containing the antibiotic in question.
• the presence of the desired construct in transformed cells can be determined by means of other techniques well known in the art, such as Southern and Western blots.
• a transcription initiation site may additionally included in the genetic construct when the sequence to be transcribed lacks such a site.
• the DNA construct of the present invention may be linked to a vector having at least one replication system, for example E. coli, whereby after each manipulation, the resulting construct can be cloned and sequenced and the correctness of the manipulation determined.
• the genetic constructs of the present invention may be used to transform a variety of target organisms such as plants, both monocotyledonous (e.g., grasses, corn, grains, oat, wheat and barley); dicotyledonous (e.g., Arabidopsis, tobacco, legumes, alfalfa, oaks, eucalyptus, maple); gymnosperms (e.g., Scots pine (Aronen, Finnish Forest Res. Papers, Vol. 595, 1996); white spruce (Ellis et al, Biotechnology 11 : 84-89, 1993); and larch (Huang et al, In Vitro Cell 27:201-207, 1991).
• target organisms such as plants, both monocotyledonous (e.g., grasses, corn, grains, oat, wheat and barley); dicotyledonous (e.g., Arabidopsis, tobacco, legumes, alfalfa, oaks,
• the inventive DNA constructs are employed to transform woody plants, herein defined as a tree or shrub whose stem lives for a number of years and increases in diameter each year by the addition of woody tissue.
• the target plant is selected from the group consisting of eucalyptus and pine species, most preferably from the group consisting of Eucalyptus grandis and Pinus radiata.
• Pines such as Pinus banksiana, Pinus brutia, Pinus car ⁇ aea, Pinus clausa, Pinus contorta, Pinus coulteri, Pinus echinata, Pinus eldarica, Pinus ellwti, Pinus jeffreyi, Pinus lambertiana, Pinus monticola, Pinus nigra, Pinus palustrus, Pinus pinaster, Pinus ponderosa, Pinus resinosa, Pinus rigida, Pinus serotina, Pinus strobus, Pinus sylvestris, Pinus taeda, Pinus virginiana, other gymnosperm, such as Abies amabihs, Abies balsamea, Abies concolor, Abies grandis, Abies lasiocarpa, Abies magnifica, Abies procera, Chamaecyparis lawsonion
• Techniques for stably mco ⁇ orating genetic constructs into the genome of target plants are well known m the art and include Agrobacterium tumefaciens mediated introduction, electroporation, protoplast fusion, injection into reproductive organs, injection into immature embryos, high velocity projectile introduction, and the like
• the choice of technique will depend upon the target plant to be transformed
• dicotyledonous plants and certain monocots and gymnosperms may be transformed by Ag> obactei mm Ti plasmid technology, as described, for example by Bevan, Nucleic Acid Res 12 871 1-8721 , 1984
• Targets for the introduction of the genetic constructs of the present invention include tissues, such as leaf tissue, disseminated cells, protoplasts, seeds, embryos, meristematic regions; cotyledons, hypocotyls, and the like.
• the preferred method for transforming eucalyptus and pine is a biolistic method using pollen (see, for example
• cells having the inventive genetic construct inco ⁇ orated in their genome may be selected by means of a marker, such as the kanamycin resistance marker discussed above.
• Transgenic cells may then be cultured in an appropriate medium to regenerate whole plants, using techniques well known in the art.
• the cell wall is allowed to reform under appropriate osmotic conditions.
• an appropriate germination or callus initiation medium is employed.
• an appropriate regeneration medium is used for explants. Regeneration of plants is well established for many species.
• RNA in target plant cells can be controlled by choice of the promoter sequence, or by selecting the number of functional copies or the site of integration of the polynucleotides inco ⁇ orated into the genome of the target plant host.
• a target plant may be transformed with more than one genetic constructs of the present invention, thereby modulating the activity of more than one isoprenoid metabolism enzyme, affecting enzyme activity in more than one tissue, or affecting enzyme activity at more than one expression time.
• a genetic construct may be assembled containing more than one open reading frame coding for an enzyme encoded by a polynucleotide of the present invention or more than one non-coding region of a gene coding for such an enzyme.
• polynucleotides of the present inventive may also be employed in combination with other known sequences encoding enzymes involved in the synthesis of isoprenoids. Additionally, the polynucleotides of the present invention have particular application for use as non-disruptive tags for marking organisms, particularly plants. Genetic constructs comprising polynucleotides of the present invention may be stably introduced into an organism as heterologous, non-functional, non-disruptive tags. It is then possible to identify the origin or source of the organism at a later date by determining the presence or absence of the tag(s) in a sample of material. Organisms other than plants may also be tagged with the polynucleotides of the present invention, including commercially valuable animals, fish, bacteria and yeasts.
• Detection of the tag(s) may be accomplished using a variety of conventional techniques, and will generally involve the use of nucleic acid probes. Sensitivity in assaying the presence of probe can be usefully increased by using branched oligonucleotides, as described by Horn et al, Nucleic Acids Res. 25(23):4842-4849, 1997), enabling detection of as few as 50 DNA molecules in the sample.
• Pinus radiata and Eucalyptus grandis cDNA expression libraries were constructed and screened as follows. mRNA was extracted from the plant tissue using the protocol of
• a cDNA expression library was constructed from the purified mRNA by reverse transcriptase synthesis followed by insertion of the resulting cDNA clones in Lambda ZAP using a ZAP Express cDNA Synthesis Kit (Stratagene), according to the manufacturer's protocol. The resulting cDNAs were packaged using a Gigapack II Packaging Extract (Stratagene) employing 1 ⁇ l of sample DNA from the 5 ⁇ l ligation mix. Mass excision of the library was done using XLl -Blue MRF' cells and
• XLOLR cells (Stratagene) with ExAssist helper phage (Stratagene).
• the excised phagemids were diluted with NZY broth (Gibco BRL, Gaithersburg, MD) and plated out onto LB-kanamycin agar plates containing X-gal and isopropylthio-beta-galactoside (IPTG).
• Polynucleotides for positive clones were obtained using a Perkin Elmer/ Applied Biosystems Division Prism 377 sequencer. cDNA clones were sequenced first from the 5' end and, in some cases, also from the 3' end. For some clones, internal sequences were obtained using subcloned fragments. Subcloning was performed using standard procedures of restriction mapping and subcloning to pBluescript II SK+ vector and other standard sequencing vectors.
• the determined cDNA sequences including the polynucleotides of the present invention, were compared to and aligned with known sequences in the. Specifically, the polynucleotides identified in SEQ ID NOS. 1-53 were compared to polynucleotides in the EMBL database EMBL as of the end of August, 1998 using the BLASTN algorithm Version 2.0.4 [Feb-24-1998] set to the following running: Unix running command: blastall -p blastn -d embldb -e 10 -G 0 -E 0 -r 1 -v 30 -b 30 -i queryseq -o results.
• the polynucleotides identified in SEQ ID NOS: 78-164 were compared to polynucleotides in the EMBL database EMBL as of the end of May, 1999 using BLASTN algorithm Version 2.0.6 [Sep-16-1998], set to the following running parameters: Unix running command: blastall -p blastn -d embldb -e 10 -G 0 -E 0 -r 1 -v 30 -b 30 -i queryseq -o results. Multiple alignments of redundant sequences were used to build up reliable consensus sequences. Based on similarity to known sequences from other plant species, the isolated polynucleotides of the present invention identified as SEQ ID NOS. 1-53 and 78-164 were putatively identified as encoding polypeptides having similarity to the polypeptides shown above in Table 1.
• the isolated cDNA sequences were compared to sequences in the EMBL DNA database using the computer algorithm BLASTN.
• the corresponding predicted polypeptide sequences were determined and were compared to sequences in the SwissProt database using the computer algorithm BLASTP.
• Comparisons of DNA sequences provided in SEQ ID NOS: 78-164, to sequences in the EMBL DNA database (using BLASTN) and amino acid sequences provided in SEQ ID NOS: 165-304 to sequences in the SwissProt database (using BLASTP) were made as of May, 1999.
• the cDNA sequences of SEQ ID NOS: 3, 7, 14, 18, 20, 25, 34, 36, 53, 84, 85, 87, 88, 101, 1 14, 116, 118, 119, 131, 137, 159, 161 and 162 were determined to have less than 60% identity, determined as described above, to sequences in the EMBL database using BLASTN, as described above.
• the cDNA sequences of SEQ ID NOS: 16, 17, 26, 43, 45, 93, 94 and 121 were determined to have less than 75% identity, determined as described above, to sequences in the EMBL database using BLASTN, as described above.
• the cDNA sequences of SEQ ID NOS: 13, 24, 95, 102 and 103 were determined to have less than 90%> identity, determined as described above, to sequences in the EMBL database using BLASTN, as described above.
• the predicted amino acid sequences of SEQ ID NOS: 194-200, 202, 216, 223, 230, 235, 239, 240, 243, 250, 255. 259, 260, 263, 270, 272, 274, 278, 291 , 292, 293, 296, 303 and 304 were determined to have less than 50% identity, determined as described above, to sequences in the SwissProt database using the BLASTP computer algorithm as described above.
• the predicted amino acid sequences of SEQ ID NOS: 165, 167, 178, 182, 189-191, 193, 201, 206, 208, 214, 215, 217, 220, 222, 226, 233, 238, 241, 246-250, 254, 256, 257, 258, 261, 264, 265, 266, 275, 280, 283, 285 and 288 were determined to have less than 90%> identity, determined as described above, to sequences in the SwissProt database using the computer algorithm BLASTP, as described above.
• the predicted amino acid sequences of SEQ ID NOS: 180, 181 and 271, were determined to have less than 95% identity, determined as described above, to sequences in the SwissProt database using the computer algorithm BLASTP, as described above.
• the six-frame translations of the polynucleotide sequences of SEQ ID NOS: 78- 164 were compared to and aligned with six-frame translations of polynucleotides in the EMBL database using the TBLASTX program version 2.0.6 [Sept- 16- 1998] set to the following running parameters: Unix running command: blastall -p blastn -d embldb -e 10 -G 0 -E 0 -v 30 -b 30 -i queryseq -o results.
• the translations of the polynucleotides of SEQ ID NOS: 82, 83, 90, 107-1 13, 115, 120, 122, 124-126, 129, 134-136, 142-144, 146- 149, 152, 153, 155-158 and 164, were determined to have less than 50% identity, determined as described above, to translations of polynucleotides in the EMBL database using the computer algorithm TBLASTX.
• the translations of the polynucleotide sequences of SEQ ID NOS: 78, 80, 93, 95, 102, 104, 106, 119, 121, 127, 128, 130, 133, 151, 159 and 163, were determined to have less than 90% identity, determined as described above, to translations of polynucleotides in the EMBL database using the computer algorithm TBLASTX.
• the translations of the polynucleotide sequence of SEQ ID NO: 94 was determined to have less than 95% identity, determined as described above, to translations of polynucleotides in the EMBL database using the computer algorithm TBLASTX.
• O-methyltransferase (OMT) Gene to Modify Lignin Biosynthesis
• the constructs of sense DNA were made by first cloning the PBK-CMV cDNA inserts into pART7 vectors. The pART7 vectors were then cut by restriction endonuclease Notl to remove the 35S-Insert- OCS 3'UTR construct for cloning into the plant expression vector pART27 (Gleave A, Plant Mol. Biol. 20: 1203-1207, 1992). The presence and integrity of the transgenic constructs were verified by restriction digestion and D ⁇ A sequencing.
• Tobacco (Nicotiana tabacum cv. Samsun) leaf sections were transformed with the sense and anti-sense OMT constructs using the method of Horsch et al, Science 227: 1229-1231, 1985. Five independent transformed plant lines were established for the sense construct and eight independent transformed plant lines were established for the anti-sense construct for OMT. Transformed plants containing the appropriate gene construct were verified using Southern blot experiments. A "+" in the column labeled "Southern" in Table 2 below indicates that the transformed plant lines were confirmed as independent transformed lines.
• Total R ⁇ A was isolated from each independent transformed plant line created with the OMT sense and anti-sense constructs.
• the R ⁇ A samples were analyzed in Northern blot experiments to determine the level of expression of the transgene in each transformed line.
• OMT enzyme activity was not estimated in sense plant line number 3.
• OMT is an enzyme involved in the biosynthesis of lignin.
• concentration of lignin in the transformed tobacco plants was determined using the well-established procedure of thioglycolic acid extraction (Freudenberg et al, Constitution and Biosynthesis of Lignin, Springer-Verlag: Berlin, 1968). Briefly, whole tobacco plants, of an average age of 38 days, were frozen in liquid nitrogen and ground to a fine powder in a mortar and pestle.
• Transformation of tobacco plants with a Pinus radiata 4CL gene Sense and anti-sense constructs containing a DNA sequence including the coding region of 4CL (SEQ ID NO: 55) from Pinus radiata were inserted into Agrobacterium tumefaciens LBA4301 by direct transformation as described above in Example 2. The presence and integrity of the transgenic constructs were verified by restriction digestion and DNA sequencing. Tobacco (Nicotiana tabacum cv. Samsun) leaf sections were transformed as described above. Five independent transfo ⁇ ned plant lines were established for the sense construct and eight independent transformed plant lines were established for the anti-sense construct for 4CL. Transformed plants containing the appropriate lignin gene construct were verified using Southern blot experiments. A "+" in the column labeled "Southern" in Table 3 indicates that the transformed plant lines listed were confirmed as independent transformed lines.
• the RNA samples were analyzed in Northern blot experiments to determine the level of expression of the transgene in each transformed line.
• the data shown in the column labeled "Northern" in Table 3 below shows that the transformed plant lines containing the sense and anti-sense constructs for 4CL all exhibit high levels of expression, relative to the background on the Northern blots. 4CL expression in anti-sense plant line number 1 was not measured because the RNA was not available at the time of the experiment. There was no detectable hybridization to RNA samples from empty vector-transformed control plants.
• Tobacco (Nicotiana tabacum cv. Samsun) leaf sections were transformed as described in Example 2. Up to twelve independent transformed plant lines were established for each sense construct and each anti-sense construct listed in the preceding paragraph. Transformed plants containing the appropriate lignin gene construct were verified using Southern blot experiments. All ofthe transformed plant lines analyzed were confirmed as independent transformed lines. This demonstrates that transgenic plants with an expressed novel gene can be made, starting the whole process from an isolated cDNA obtained as in Example 1.
• the concentration of lignin in empty vector-transformed control plant lines and in up to twelve independent transformed lines for each sense construct and each anti-sense construct described in Example 5 was determined as described in Example 3.
• the column labeled "TGA" in Table 3 shows the thioglycolic acid extractable lignins for all plant lines assayed, expressed as the average percentage of TGA extractable lignins in transformed plants versus control plants. The range of variation is shown in parentheses. TABLE 4 transgene orientation no. of lines Northern TGA control na 3 blank 100 (92-104)
• Transfo ⁇ ned plant lines containing the sense and the anti-sense lignin biosynthetic gene constructs all exhibited significantly decreased levels of lignin, relative to the empty vector-transformed control plant lines.
• the most dramatic effects on lignin concentration were seen in the F5H anti-sense plants with as little as 35% of the amount of lignin in control plants, and in the PAL anti-sense plants with as little as 37% of the amount of lignin in control plants.
• PAL SEQ ID NO: 60
• PNL SEQ ID NO: 64
• LAC SEQ ID NO: 65
• CGT SEQ ID NO: 59
• Transgenic tobacco plants were created using unique identifier sequences which are not found in tobacco.
• the unique identifier sequences inserted were isolated from Pinus radiata, SEQ ID NO: 76, and Eucalyptus grandis, SEQ ID NO: 77.
• the unique identifier sequences were inserted into Agrobacterium tumefaciens LBA4301 (provided as a gift by Dr. C. Kado, University of California, Davis, CA) by direct transformation using published methods (An et al, "Binary vectors," in Gelvin SB and Schilperoort RA, eds., Plant Molecular Biology Manual, Kluwer Academic Publishers: Dordrecht, 1988).
• the presence and integrity of the unique identifier sequences in the Agrobacterium transgenic constructs were verified by restriction digestion and DNA sequencing.
• Tobacco (Nicotiana tabacum cv. Samsun) leaf sections were transformed using the method of Horsch et al, Science 227: 1229-1231, 1985. Three independent transformed plant lines were established for each unique sequence identifier used. Two empty-vector control plant lines were established using an empty gene transfer vector that lacked a unique sequence identifier.
• sequence identifiers The uniqueness of the sequence identifiers was assayed using Southern blot analyses to test for the presence of the sequence identifier in the genome of the plants. If the sequence identifier is unique and therefore useful as a tag, then the sequence identifier should be clearly absent in plants which have not been tagged and it should be clearly present in plants which have been tagged. In the present example, the unique identifiers would be expected to be absent in the empty-vector transformed control plants. The unique identifier would be expected to be present in the transgenic plants transformed with the unique sequence identifiers.
• Genomic DNA was prepared from empty-vector transformed control plants and plants transformed with unique sequence identifiers using the cetyltrimethyl-ammonium bromide (CTAB) extraction method of Murray MG and Thompson WF, Nucleic Acids Res. 8:4321-4325, 1980.
• CTAB cetyltrimethyl-ammonium bromide
• the DNA samples were digested with the restriction enzyme EcoRI in the case of the plants transfo ⁇ ned with the Pinus unique sequence identifier (SEQ ID NO: 76) and the restriction enzyme Xbal in the case of the plants transformed with the Eucalyptus unique sequence identifier (SEQ ID NO: 77).
• the DNA fragments produced in the restriction digests were resolved on a 1% agarose gel; the left panel of Fig. 2 and the right panel of Fig. 2 show the DNA fragment patterns of the DNA samples from the Pinus and Eucalyptus experiments, respectively.
• the DNA samples were transferred to Hybond-N+ nylon membranes (Amersham Life Science, Little Chalfont, Buckinghamshire, England) using methods established by Southern, J. Mol. Biol. 98:503- 517, 1975.
• the nylon membranes were probed with radioactively-labeled probes for the unique sequence identifiers identified above and washed at high stringency (final wash: 0.5 X salt sodium citrate buffer (SSC) plus 0.1% sodium dodecyl sulfate (SDS), 15 minutes at 65°C).
• SSC 0.5 X salt sodium citrate buffer
• SDS sodium dodecyl sulfate
• Fig. 3 shows the hybridization pattern detected in the Southern blot analysis using a probe derived from the Pinus sequence identifier (SEQ ID NO: 76).
• Lanes A-B contain DNA samples from empty-vector transformed control plants and Lanes C-E contain DNA from plants transformed with SEQ ID NO: 76.
• There is no hybridization in Lanes A-B indicating that SEQ ID NO: 76 is not present in empty-vector transformed tobacco plants; that is, SEQ ID NO: 76 is a unique tag suitable for unambiguous marking of tobacco plants.
• There is strong hybridization in Lanes C-E indicating that the plants which received SEQ ID NO: 76 via transformation have been clearly and unambiguously tagged with the unique sequence contained in SEQ ID NO: 76.
• Fig. 4 shows the hybridization pattern detected in the Southern blot analysis using a probe derived from the Eucalyptus sequence identifier (SEQ ID NO: 77).
• Lanes A-B contain DNA samples from empty-vector transformed control plants and Lanes C-E contain DNA from plants transformed with SEQ ID NO: 77. There is no hybridization in Lanes A-B indicating that SEQ ID NO: 77 is not present in empty-vector transformed tobacco plants; that is, SEQ ID NO: 77 is a unique tag suitable for unambiguous marking of tobacco plants.
• a user of the invention disclosed in this specification has both a high likelihood of finding a sequence identifier, among the list which has been disclosed, which will be useful for tagging any given organism and an unequivocal method for demonstrating that a tagged organism could only have acquired a given tag through the deliberate addition of the unique sequence to the genome of the organism to be tagged. If the user of this invention maintains the precise sequence of the tag used in a given organism as a secret, then any disputes as to the origin and history of the organism can be unambiguously resolved using the tag detection techniques demonstrated in the present example.
• SEQ ID NOS: 1-304 are set out in the attached Sequence Listing.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Genetics & Genomics (AREA)
• Engineering & Computer Science (AREA)
• Chemical & Material Sciences (AREA)
• Zoology (AREA)
• Biotechnology (AREA)
• Organic Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Wood Science & Technology (AREA)
• Molecular Biology (AREA)
• General Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Microbiology (AREA)
• Nutrition Science (AREA)
• Physics & Mathematics (AREA)
• Cell Biology (AREA)
• Biophysics (AREA)
• Plant Pathology (AREA)
• Medicinal Chemistry (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
• Enzymes And Modification Thereof (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Peptides Or Proteins (AREA)

Applications Claiming Priority (5)

Publications (1)

Family

ID=26843909

Family Applications (1)

Country Status (10)

Families Citing this family (20)

Family Cites Families (1)

• 1999
  • 1999-12-13 AR ARP990106323 patent/AR021636A1/es not_active Application Discontinuation
  • 1999-12-16 JP JP2000588332A patent/JP2002541764A/ja active Pending
  • 1999-12-16 MX MXPA01006009A patent/MXPA01006009A/es unknown
  • 1999-12-16 CN CN 99816247 patent/CN1334866A/zh active Pending
  • 1999-12-16 WO PCT/NZ1999/000219 patent/WO2000036081A2/en not_active Ceased
  • 1999-12-16 BR BR9917002-7A patent/BR9917002A/pt not_active IP Right Cessation
  • 1999-12-16 AU AU19001/00A patent/AU1900100A/en not_active Abandoned
  • 1999-12-16 ID IDW00200101559A patent/ID29580A/id unknown
  • 1999-12-16 EP EP99962594A patent/EP1151081A2/de not_active Withdrawn
  • 1999-12-16 CA CA002353910A patent/CA2353910A1/en not_active Abandoned

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events