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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
  • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
• • 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
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8285—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for nematode resistance
• • 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
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• PLANT-OPTIMIZED POLYNUCLEOTIDES ENCODING APPROXIMATELY 15 kDa AND APPROXIMATELY 45 kDa PESTICIDAL PROTEINS
• Chemical pesticides have provided an effective method of pest control; however, the public has become concerned about the amount of residual chemicals which might be found in food, ground water, and the environment. Therefore, synthetic chemical pesticides are being increasingly scrutinized, and correctly so, for their potential toxic environmental consequences. Synthetic chemical pesticides can poison the soil and underlying aquifers, pollute surface waters as a result of runoff, and destroy non-target life forms. Synthetic chemical control agents have the further disadvantage of presenting public safety hazards when they are applied in areas where pets, farm animals, or children may come into contact with them. They may also provide health hazards to applicants, especially if the proper application techniques are not followed.
• synthetic chemical pesticides include the organochlorines, e.g., DDT, mirex, kepone, lindane, aldrin, chlordane, aldicarb, and dieldrin; the organophosphates, e.g., chlorpyrifos, parathion, malathion, and diazinon; and carbamates.
• a biological pesticidal agent that is being used with increasing popularity is the soil microbe Bacillus thuringiensis (B.t).
• the soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, spore-forming bacterium. Most strains of B.t. do not exhibit pesticidal activity. Some B.t. strains produce, and can be characterized by, parasporal crystalline protein inclusions. These " ⁇ -endotoxins," which typically have specific pesticidal activity, are different from exotoxins, which have a non-specific host range. These inclusions often appear microscopically as distinctively shaped crystals.
• the proteins can be highly toxic to pests and are specific in their toxic activity.
• B. thuringiensis subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests.
• B. thuringiensis var. kurstaki HD-1 produces a crystalline ⁇ -endotoxin which is toxic to the larvae of a number of lepidopteran insects.
• the cloning and expression of a B.t. crystal protein gene in Escherichia coli was described in the published literature more than 15 years ago (Schnepf, H.E., H.R. Whiteley [1981] Proc. Natl. Acad. Sci. USA 78:2893-2897.).
• B.t. pesticides with specificities for a much broader range of pests.
• B.t. namely israelensis and morrisoni (a.k.a. tenebrionis, a.k.a. B.t. M-7), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, F.H. [1989] "Cellular Delivery Systems for Insecticidal Proteins:
• B.t. toxins Obstacles to the successful agricultural use of B.t. toxins include the development of resistance to B.t. toxins by insects.
• certain insects can be refractory to the effects of B.t.
• the latter includes insects such as boll weevil and black cutworm as well as adult insects of most species which heretofore have demonstrated no apparent significant sensitivity to B.t. ⁇ -endotoxins.
• resistance management strategies in B.t. plant technology have become of great interest, and there remains a great need for new toxin genes.
• WO 97/40162 discloses 15 kDa and 45 kDa coleopteran-active proteins obtainable from B.t. isolates PS80JJ1 and PS149B1.
• the subject invention concerns materials and methods useful in the control of pests and, particularly, plant pests. More specifically, the subject invention provides new, plant-optimized polynucleotide sequences that encode pesticidal proteins.
• the polynucleotide sequences of the subject invention have certain modifications, compared to wild-type sequences, that make them particularly well-suited for optimized expression in plants.
• the transformation of plants can be accomplished, using techniques known to those skilled in the art, in order to confer pest resistance upon said plants.
• the subject invention provides plant-optimized polynucleotide sequences which encode approximately 15 kDa and approximately 45 kDa pesticidal proteins.
• SEQ ID NO. 1 is a polynucleotide sequence for a gene designated 80JJ1-15-PO5, which is optimized for expression in maize. This gene encodes an approximately 15 kDa protein. This gene and protein were disclosed in WO 97/40162.
• SEQ ID NO. 2 is a novel polynucleotide sequence for a gene designated 80JJ1-
• SEQ ID NO. 3 is an amino acid sequence for a novel pesticidally active protein encoded by the gene designated 80JJ1-15-PO7.
• SEQ ID NO. 4 is a polynucleotide sequence for a gene designated 80JJ1-45-PO, which is optimized for expression in maize. This gene encodes an approximately 45 kDa protein. This gene was disclosed in WO 97/40162.
• SEQ ID NO. 5 is a novel polynucleotide sequence for a gene designated 149B1-15-PO, which is optimized for expression in Zea mays. This gene encodes an approximately 15 kDa protein obtainable from PS149B1 that is disclosed in WO 97/40162.
• SEQ ID NO. 6 is a novel polynucleotide sequence for a gene designated 149B1-
• 45-PO which is optimized for expression in Zea mays.
• This gene encodes an approximately 45 kDa protein obtainable from PS149B1 that is disclosed in WO 97/40162.
• the subject invention concerns materials and methods useful in the control of pests and, particularly, plant pests. More specifically, the subject invention provides new, plant-optimized polynucleotide sequences that encode pesticidal proteins.
• the polynucleotide sequences of the subject invention have certain modifications, compared to wild-type sequences, that make them particularly well-suited for optimized expression in plants.
• the transformation of plants can be accomplished, using techniques known to those skilled in the art, in order to confer pest resistance upon said plants.
• the subject invention provides plant-optimized polynucleotide sequences which encode approximately 15 kDa and approximately 45 kDa pesticidal proteins.
• the genes of the subject invention can be obtained through several means.
• the subject genes may be constructed synthetically by using a gene synthesizer, for example.
• the specific genes exemplified herein can also be obtained by modifying, according to the teachings of the subject invention, certain wild-type genes (for example, by point-mutation techniques) from certain isolates deposited at a culture depository as discussed below.
• the subject invention includes, in preferred embodiments, polynucleotide sequences optimized for expression in plants, wherein said sequences are selected from the group consisting of S ⁇ Q ID NO. 2, S ⁇ Q ID NO. 5, and S ⁇ Q ID NO. 6.
• S ⁇ Q ID NO. 2 encodes a preferred protein that is shown in S ⁇ Q ID NO. 3.
• polynucleotides of the subject invention can be used to form complete "genes" to encode proteins or peptides in a desired host cell.
• S ⁇ Q ID NO. 2, S ⁇ Q ID NO. 5, and S ⁇ Q ID NO. 6 are shown without stop codons.
• S ⁇ Q ID NO. 2, S ⁇ Q ID NO. 5, and/or S ⁇ Q ID NO. 6 can be appropriately placed under the control of a promoter in a host of interest, as is readily known in the art.
• DNA can exist in a double- stranded form. In this arrangement, one strand is complementary to the other strand and vice versa.
• the "coding strand” is often used in the art to refer to the strand having a series of codons (a codon is three nucleotides that can be read three-at-a-time to yield a particular amino acid) that can be read as an open reading frame (ORF) to form a protein or peptide of interest.
• ORF open reading frame
• a strand of DNA is typically translated into a complementary strand of RNA which is used as the template for the protein.
• DNA is replicated in a plant (for example) additional, complementary strands of DNA are produced.
• the subject invention includes the use of either the exemplified polynucleotides shown in the attached sequence listing or the complementary strands.
• RNA and PNA peptide nucleic acids
• novel DNA molecules are included in the subject invention.
• DNA sequences of the subject invention have been specifically exemplified herein. These sequences are exemplary of the subject invention. It should be readily apparent that the subject invention includes not only the genes and sequences specifically exemplified herein but also equivalents and variants thereof (such as mutants, fusions, chimerics, truncations, fragments, and smaller genes) that exhibit the same or similar characteristics relating to expressing toxins in plants, as compared to those specifically disclosed herein.
• variants and “equivalents” refer to sequences which have nucleotide (or amino acid) substitutions, deletions (internal and/or terminal), additions, or insertions which do not materially affect the expression of the subject genes, and the resultant pesticidal activity, in plants. Fragments of polynucleotide proteins retaining pesticidal activity, and "pesticidal portions" of full- length proteins, are also included in this definition.
• Genes can be modified, and variations of genes may be readily constructed, using standard techniques. For example, techniques for making point mutations are well known in the art. In addition, commercially available exonucleases or endonucleases can be used according to standard procedures, and enzymes such as B ⁇ 1 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Useful genes can also be obtained using a variety of restriction enzymes.
• equivalent genes will encode toxins that have high amino acid identity or homology with the toxins encoded by the subject genes.
• the amino acid homology will be highest in critical regions of the toxin which account for biological activity or are involved in the determination of three-dimensional configuration which ultimately is responsible for the biological activity.
• certain substitutions are acceptable and can be expected if these substitutions are in regions which are not critical to activity or are conservative amino acid substitutions which do not affect the three-dimensional configuration of the molecule.
• amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound.
• Table 1 provides a listing of examples of amino acids belonging to each class.
• Nonpolar Ala Val, Leu, He, Pro, Met, Phe, Tip Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, Gin
• non-conservative substitutions can also be made.
• the critical factor is that these substitutions must not significantly detract from the ability of plants to express the subject DNA sequences or from the biological activity of the toxin.
• isolated polynucleotides and/or “purified” toxins refers to these molecules when they are not associated with the other molecules with which they would be found in nature and would include their use in plants. Thus, reference to “isolated” and/or “purified” signifies the involvement of the "hand of man” as described herein.
• Recombinant hosts The toxin-encoding genes of the subject invention can be introduced into a wide variety of microbial or plant hosts. In some embodiments of the subject invention, transformed microbial hosts can be used in preliminary steps for preparing precursors, for example, that will eventually be used to transform, in preferred embodiments, plant cells and plants so that they express the toxins encoded by the genes of the subject invention.
• Microbes transformed and used in this manner are within the scope of the subject invention.
• Recombinant microbes may be, for example, a B.t., E. coli, or Pseudomonas. Transformations can be made by those skilled in the art using standard techniques. Materials necessary for these transformations are disclosed herein or are otherwise readily available to the skilled artisan.
• expression of a gene of this invention results, directly or indirectly, in the intracellular production and maintenance of the protein of interest.
• the pests When transformed plants are ingested by the pest, the pests will ingest the toxin. The result is a control of the pest.
• the B.t. toxin gene can be introduced via a suitable vector into a host, preferably a plant host.
• a host preferably a plant host.
• crops of interest such as com, wheat, rice, cotton, soybeans, and sunflowers.
• the genes of the subject invention are particularly well suited for providing stable maintenance and expression, in the transformed plant, of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.
• the subject invention includes recombinant hosts comprising a polynucleotide sequence optimized for expression in a plant, wherein said sequence is selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 5, and SEQ ID NO. 6.
• the recombinant host can be, for example, a plant cell. Entire plants comprising the subject polynucleotides are also within the scope of the subject invention.
• a plant can be made resistant to com rootworm damage by being transformed to express a polynucleotide, such as SEQ ID NO. 2, that encodes an approximately 15 kDa protein, together with a second polynucleotide, such as SEQ ID NO. 4, which encodes a 45 kDa protein.
• SEQ ID NO. 5 and SEQ ID NO. 6 can be used together, under one promoter or separate promoters, such as the ubiquitin promoter.
• the polynucleotide of SEQ ID NO. 2 and SEQ ID NO. 6 can be used together, or SEQ ID NO. 4 and SEQ ID NO. 5 can be used, for example.
• the subject invention provides specific embodiments of synthetic genes, other genes that are functionally equivalent to the genes exemplified herein can also be used to transform hosts, preferably plant hosts. Additional guidance for the production of synthetic genes can be found in, for example, U.S. Patent No. 5,380,831. All of the publications and patent references referred to or cited herein are hereby incorporated by reference in their entirety to the extent that they are not inconsistent with the explicit teachings of this specification.
• One aspect of the subject invention is the transformation of plants with the subject polynucleotide sequences encoding insecticidal toxins.
• the transformed plants are resistant to attack by the target pest.
• the genes of the subject invention are optimized for use in plants.
• a promoter region capable of expressing the gene in a plant is needed.
• the DNA of the subject invention is under the control of an appropriate promoter region.
• Techniques for obtaining inplanta expression by using such constructs is known in the art.
• a preferred promoter region used for expression of both 15 kDa and 45 kDa transgenes is the Zea mays ubiquitin promoter plus Z. mays exon 1 and Z. mays intron 1 (Christensen, A.H., et al. 1992 Plant Mol. Biol. 18:675-689).
• a preferred transcriptional terminator for both transgenes is the potato proteinase inhibitor II (Pinll) terminator (An, G. et al.
• Genes encoding pesticidal toxins, as disclosed herein, can be inserted into plant cells using a variety of techniques which are well known in the art. For example, in preferred embodiments, maize plants containing 14 kDa and 44 kDa transgenes were obtained by microprojectile bombardment using the Biolistics®O PDS-lOOHe particle gun manufactured by Bio-Rad, essentially as described by Klein et al. (1987). A large number of cloning vectors comprising a replication system in E. coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants.
• the vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly, the sequence encoding the B.t. toxin can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli. The E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids.
• the inserted D ⁇ A Once the inserted D ⁇ A has been integrated in the genome, it is relatively stable there and, as a rule, does not come out again. It normally contains a selection marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as kanamycin, G 418, bleomycin, hygromycin, or chloramphenicol, ter alia.
• the individually employed marker should accordingly permit the selection of transformed cells rather than cells that do not contain the inserted D ⁇ A.
• D ⁇ A D ⁇ A into a plant host cell.
• Those techniques include transformation with T-D ⁇ A using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, fusion, injection, biolistics (microparticle bombardment), or electroporation as well as other possible methods.
• Agrobacteria Agrobacteria are used for the transformation, the D ⁇ A to be inserted has to be cloned into special plasmids, namely either into an intermediate vector or into a binary vector.
• the intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination owing to sequences that are homologous to sequences in the
• the Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-D ⁇ A.
• Intermediate vectors cannot replicate themselves in Agrobacteria.
• the intermediate vector can be transferred into Agrobacterium tumefaciens by means of a helper plasmid (conjugation).
• Binary vectors can replicate themselves both in E. coli and in Agrobacteria. They comprise a selection marker gene and a linker or polylinker which are framed by the right and left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters et al. [1978] Mol. Gen. Genet. 163:181-187).
• the intermediate vector can be transferred into Agrobacterium tumefaciens by means of a helper plasmid (conjugation).
• Binary vectors can replicate themselves both in E. coli and in Agrobacteria. They comprise a selection marker gene and a linker or polylinker which are framed by the
• Agrobacterium used as host cell is to comprise a plasmid carrying a vtr region.
• the vtr region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained.
• the bacterium so transformed is used for the transformation of plant cells.
• Plant explants can advantageously be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant cell.
• Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension-cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection.
• the plants so obtained can then be tested for the presence of the inserted DNA. No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives.
• the transformed cells grow inside the plants in the usual manner. They can form germ cells and transmit the transformed trait(s) to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.

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