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
  • C12N15/63—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
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/50—Isolated enzymes; Isolated proteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
  • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/08—Fusion polypeptide containing a localisation/targetting motif containing a chloroplast localisation signal
• • 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
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/14011—Baculoviridae
  • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
  • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • 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

• This invention relates to insecticidally effective peptides. More particularly, the invention relates, inter alia , to insecticidally effective peptides isolatable from Diguetia spider venom, DNA encoding such insecticidally effective peptides, methods for producing said peptides, and methods for controlling invertebrate pests.
• the most widely used microbial pesticides are derived from the bacterium Bacillus thuringiensis (hereinafter B . t . ) .
• This bacterial agent is used to control a variety of leaf-eating caterpillars, Japanese beetles and mosquitos.
• U.S. Patent No. 4,797,279 issued January 10, 1989 to Karamata, et al. discloses hybrid bacterial cells comprising the gene coding for B . t . kurstaki delta-endotoxin and the gene coding for B . t . tenebrionis delta-endotoxin and their preparation.
• the B . t . hybrids are active against pests susceptible to B . t .
• hybrids have useful insecticidal properties which are superior to those observed by physical mixtures of the parent strains in terms of level of insecticidal activity, or in terms of spectrum of activity, or both.
• the insecticidal compositions comprising such microorganisms may be used to combat insects by applying the hybrids in an insecticidally effective amount to the insects or to their environment.
• Another derivation from the bacterium B . t . was disclosed in European Patent Application, Publication No. 0 325 400 A1, issued to Gilroy and Wilcox.
• This invention relates to a hybrid toxin gene which is toxic to lepidopteran insects.
• the invention comprises a hybrid delta-endotoxin gene comprising part of the B . t . var. kurstaki HD-73 toxin gene and part of the toxin gene from B . t . var. kurstaki strain HD-1.
• the hybrid toxin gene (DNA) encoding a protein having activity against lepidopteran insects was disclosed.
• the bacterium B . t . was also utilized for its insecticidal properties in European Patent Application, Publication No. 0 340 948, issued to Wilcox, et al.
• This invention concerns hybrid pesticidal toxins which are produced by the fusion of an insect gut epithelial cell recognition region of a B . t. gene to diphtheria toxin B chain to prepare a hybrid B . t. toxin which is active against lepidopteran insects. It was suggested that the hybrid B . t . gene may be inserted into a plant or cloned into a baculovirus to produce a toxin which can be recovered. Alternatively, the host containing the hybrid B . t . gene can be used as an insecticide by direct application to the environment of the targeted insect.
• scorpion venom was identified as a possible source of compounds providing insecticidal properties.
• Two insect selective toxins isolated from the venom of the scorpion Leiurus quinquestriatus quinquestriatus were revealed in Zlotkin, et al., "An Excitatory and a Depressant Insect Toxin from Scorpion Venom both Affect Sodium Conductance and Possess a Common Binding Site," Arch Biochem and Biophysics , 240:877-87 (1985).
• Zlotkin, et al. "An Excitatory and a Depressant Insect Toxin from Scorpion Venom both Affect Sodium Conductance and Possess a Common Binding Site," Arch Biochem and Biophysics , 240:877-87 (1985).
• Zlotkin, et al. "An Excitatory and a Depressant Insect Toxin from Scorpion Venom both Affect Sodium Conductance and Possess a Common Binding Site," Arch Bio
• Grishin "Toxic components from Buthus eupeus and Lucosa singoriensis venoms," Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Moscow 117988, GSP-1, USSR, discloses four toxins isolated from the venom of the scorpion Buthus eupeus which are toxic to insects. Also disclosed was the isolation and characterization of the toxic component of the venom of the tarantula Lucosa singoriensis. The crude venom was nontoxic to insects.
• U.S. Patent No. 4,879,236, issued November 7, 1989 to Smith and Summers relates to a method for incorporating a selected gene coupled with a baculovirus promoter into a baculovirus genome to produce a recombinant baculovirus expression vector capable of expression of the selected gene in an insect cell.
• the method involves cleaving baculovirus DNA to produce a DNA fragment comprising a polyhedrin gene or portion thereof, including a polyhedrin promoter.
• a recombinant transfer vector To prepare a recombinant transfer vector, the DNA fragment is inserted into a cloning vehicle and then a selected gene is inserted into this modified cloning vehicle such that it is under the control of the polyhedrin promoter. The recombinant transfer vector is then contacted with wild type baculovirus DNA so as to effect homologous recombination and incorporation of the selected gene into the baculovirus genome.
• the baculovirus Autographa californica (AcMNPV) and its associated polyhedrin promotor were found to be useful in producing a viral expression vector capable of extremely high levels of expression of a selected gene in a eukaryotic host cell.
• the inventors suggest that the expression vector might be used in a system for controlling insects by selecting a gene which produces a protein which is toxic to a specific insect or to a spectrum of insects and cloning that gene into the AcMNPV expression vector. They suggest that the vector could be applied to the plant or animal to be protected. The recombinant virus could invade the cells of the intestinal wall following ingestion by the insect and begin replication. An alternative suggestion is to insert the gene into the baculovirus genome so that it would be fused to the polyhedrin structural sequence in such a way that the polyhedron coating would be dissociated by the alkaline conditions of the insect gut and the toxic product would be released.
• the process consists of collecting pollen, germinating it in a germinating medium for 30-60 minutes after which the pollen tube will start to come out of the pollen grain, adding the desired DNA to the liquid suspension containing the pollen, administering an electric shock to open pores in the pollen tube, washing the excess DNA away, and putting the altered pollen on the stigma of a plant and waiting until seeds are formed.
• This may be an easy method to move any gene into crop plants.
• the protein-producing microbial cell itself is used as the delivery system so no purification of the produced compound is necessary.
• Any protein, polypeptide, amino acid, or compound, including insecticides, that may be produced by microbial means may be the starting material of the invention.
• European Patent Application publication number 0 431 829, discloses transgenic plants which effectively express in their cells an insect-specific toxin of an insect predator in an amount sufficient so as to cause toxicity to selective insects ingesting the plant tissues.
• T h e particular toxin described was isolated from the venom of the scorpion Androctonus australis.
• U.S. Patent No. 4,925,664 issued to Jackson and Parks on May 15, 1990, discloses methods of treating heart and neurological diseases by applying toxins derived from the spiders Agelenopsis aperta and Hololena curta .
• the toxins are also effective as specific calcium channel or excitatory amino acid receptor blockers that may be used against insects and related pests.
• European Patent Application discloses polyamines and polypeptides isolated from the venom of the spider Agelenopsis aperta.
• the polyamines antagonize excitatory amino acid neurotransmitters.
• the polypeptides and one of the polyamines block calcium channels in living cells of various organisms. The use of said calcium channel blockers in the control of invertebrate pests is suggested.
• European Patent Application publication number 0 374 940, discloses toxins isolated from the venom of the spider Hololena curta .
• the polypeptides are useful as insecticides and in pharmaceuticals, for example, as calcium channel and glutamate antagonists.
• spider toxins were discussed in Jackson and Parks, "Spider Toxins: Recent Applications in Neurobiology, " Ann Rev Neurosci 12:405-14 (1989). This article teaches that there is great heterogeneity in the toxins of different taxa. It recognizes that experiments have suggested species-specific properties of calcium channels and the spider venoms might provide calcium channel antagonists. The spider venoms discussed are found to affect vertebrates. The article also identifies spider venoms as possible sources of insect-specific toxins for agricultural applications.
• Yoshioka et al., discloses a receptor inhibitor obtained from Joro spider venom glands, and its manufacturing method.
• the compound has an insecticidal effect when insects contact the compound carried in a liquid or solid.
• U.S. Patent No. 4,918,107 issued April 17, 1990 to Nakajima et al. relates to a compound which has glutamate receptor inhibitor activity, a process for preparing the same, and an insecticidal composition containing the same.
• the compound is carried in a liquid or solid carrier with a dispersing agent added and applied directly to the plant or animal to be protected.
• a low dosage is effective as an insecticide and has very low mammalian and fish toxicity and small adverse influence to the environment.
• baculoviruses as bioinsecticides has also been explored.
• the major deficit of wild type baculoviruses as bioinsecticides is that they are slow-acting.
• Larvae that ingest wild type baculoviruses generally die within 5 to 7 days. The infected larvae continue to feed during a significant portion of this time and substantial crop damage can occur.
• insecticidally effective peptides of this invention are believed to have a high degree of selectivity for invertebrates and, in particular, insects. Furthermore, the insecticidally effective peptides of this invention have been demonstrated to be highly effective insecticides against agriculturally important pests and thus are believed to represent an important contribution to the field of invertebrate pest control.
• a novel recombinant expression vector comprising a DNA sequence encoding an insecticidally effective peptide substantially isolatable from Diguetia spider venom, wherein the vector is capable of effecting the expression of said coding sequence in transformed cells.
• a novel recombinant host cell transformed or transfected with a DNA sequence encoding an insecticidally effective peptide substantially isolatable from Diguetia spider venom in a manner allowing the host cell to express said peptide.
• baculovirus expression vector capable of expressing a DNA sequence encoding an insecticidally effective peptide substantially isolatable from Diguetia spider venom in a host or in a host insect cell.
• the method comprises the steps of culturing recombinant host cells wherein a recombinant expression vector transformed or transfected in said host cells has a DNA sequence encoding an insecticidally effective peptide substantially isolatable from Diguetia spider venom, said vector being capable of effecting the expression of said coding sequence in transformed cells; and recovering said insecticidally effective peptide from the recombinant host cell culture.
• a novel transgenic plant comprising a DNA sequence encoding an insecticidally effective peptide substantially isolatable from Diguetia spider venom introduced into the germ line of said plant, or an ancestor of said plant, such that the trait of expression of said DNA sequence is inherited by subsequent generations of said plant through sexual propagation or asexual propagation.
• this invention is a novel method of controlling invertebrate pests comprising contacting said pests with an effective amount of an insecticidally effective peptide substantially isolatable from Diguetia spider venom and agriculturally or horticulturally acceptable salts thereof.
• this invention is a novel method of controlling invertebrate pests comprising contacting said pests with a recombinant baculovirus capable of expressing an effective amount of an insecticidally effective peptide substantially isolatable from Diguetia spider venom and agriculturally or horticulturally acceptable salts thereof.
• novel insecticidal composition comprising an insecticidally effective amount of an insecticidally effective peptide substantially isolatable from Diguetia spider venom and agriculturally or horticulturally acceptable salts thereof in an agriculturally or horticulturally acceptable carrier therefor.
• this invention is a novel method of using the antibodies of this invention to detect the presence of an insecticidally effective peptide substantially isolatable from Diguetia spider venom comprising the steps of obtaining spider venom; contacting the spider venom with said antibodies coupled to a detectable label; and detecting the labeled antibody bound to said peptide.
• this invention is a novel method of using the antibodies of this invention to purify an insecticidally effective peptide substantially isolatable from Diguetia spider venom comprising the steps of conjugating the antibody to a solid support; contacting a solution containing said peptide with said antibody conjugated to said solid support whereby said peptide present in the solution attaches to said antibody; removing said peptide attached to said antibody conjugated to said solid support; and collecting said purified peptide.
• this invention is a novel method of detecting the presence of nucleic acid encoding an insecticidally effective peptide substantially isolatable from Diguetia spider venom comprising the steps of obtaining spider nucleic acids; contacting said nucleic acids with the DNA probe of this invention and detecting said probe bound to said nucleic acid.
• Figure 1 depicts the RP HPLC of whole Diguetia canities venom using a gradient of 0.1% TFA to acetonitrile showing the three groups of components tested in mace.
• Fraction 2 represents the TBW active components.
• FIG. 2 shows DK 9.2 tested at 1 ⁇ M on synaptic transmission (evoked population spike) at the Schaffer collateral-CA 1 pyramidal cell synapse in rat hippocampal slices.
• the data depicted represent the time-averaged population spike recordings (a) for 5 minutes prior to DK
• Figure 3 is a map of p WR9, a baculovirus transfer vector carrying the gene encoding preDK9.2.
• Figure 4 is a continuous viral feeding assay in neonate tobacco budworm (1000,000 PIB/gm diet).
• Figure 5 depicts the ability of TTX to prevent or reverse DK 9.2-induced bursting.
• an insecticidally effective peptide substantially isolatable from Diguetia spider venom includes insecticidally effective peptides, as well as insecticidally effective fragments of said peptides, from any source so long as the peptide could have been substantially isolated from Diguetia by any of the techniques known to those in the art.
• sources include, for example, recombinantly produced insecticidally effective peptides.
• expression vector includes vectors which are capable of expressing DNA sequences contained therein, where such sequences are operably linked to other sequences capable of effecting their expression. It is implied, although not always explicitly stated, that these expression vectors must be replicable in the host organisms either as episomes or as an integral part of the chromosomal DNA. Clearly a lack of replicability would render them effectively inoperable. In sum, “expression vector” is given a functional definition, and any DNA sequence which is capable of effecting expression of a specified DNA code disposed therein is included in this term as it is applied to the specified sequence.
• expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer to circular double stranded DNA which, in their vector form are not bound to the chromosome.
• Plasmid and “vector” are used interchangeably as the plasmid is the most commonly used form of vector.
• the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
• Recombinant host cells refers to cells which have been transformed with vectors constructed using recombinant DNA techniques.
• the spider family Diguetidae currently consists of a single genus, Diguetia .
• Diguetia species are generally found in the southeastern United States (including California) and Mexico. These small spiders spin expansive, irregular webs on many kinds of plants and shrubs, including cacti.
• the various species of Diguetia are, like most spiders, general predators, readily consuming most types of prey insects they encounter.
• the unusual symptomatology seen upon injection of the insecticidally effective peptides of this invention into insects is very similar to the symptoms seen when veratridine, an alkaloid Na + channel activator, or scorpion toxins from the family Buthidae and genus Buthus, known presynaptic Na + channel activators, are injected into insects. It is believed that said insecticidally effective peptides may be causing a partial depolarization of muscle and/or nerve membranes, possibly affecting Na + or K + channels. More specifically, it is believed that said insecticidally effective peptides induce repetitive burst discharges in nerves that are sensitive to block by tetrodotoxin. Thus, it is probably the voltage- sensitive sodium channel of nerve membranes that is the site of action of these peptides.
• Spider venom can be removed from Diguetia by any method known such as spider gland extraction from cephalothorax.
• the spider venom preferably is obtained by electrical stimulation of the spiders to cause release of the venom and subsequent suction to collect the released venom and prevent contamination of the venom by regurgitate or hemolymph as described in U.S. 4,925,664.
• the spider venom Once the spider venom is obtained by electrical milking techniques, it can be fractionated into its peptide (toxin) components using high performance liquid chromatography ("HPLC") or gel filtration chromatography or any other useful fractionation technique. In addition, it is frequently desirable for final fractionation of the spider venom to be performed by HPLC.
• HPLC high performance liquid chromatography
• HPLC gel filtration chromatography
• This invention in one of its aspects, provides an insecticidally effective peptide, and insecticidally effective fragments thereof, substantially purified and isolatable from Diguetia spider venom and agriculturally or horticulturally acceptable salts thereof.
• amino acid sequence determination can be performed in any way known to those in the art such as N-terminal amino acid sequencing and use of an automated amino acid sequencer.
• insecticidally effective proteins are expected to be within the scope of the invention. That is, it is believed other insecticidally effective peptides are isolatable from Diguetia in addition to the three detailed herein.
• the following relates to a family of insecticidally effective proteins isolatable from Diguetia .
• Members of this family of insecticidally effective peptides isolated from Diguetia appear to share the following characteristics:
• SEQ ID NOS:1, 3 and 5 have greater than 40% sequence homology
• SEQ ID NOS:1, 3 and 5 have about 7 or 8 cysteine residues
• DK 9.2 has been isolated and its amino acid sequence, as translated from the isolated cDNA, appears as defined in SEQ ID NO:l.
• Mass spectroscopy data suggest two isozymes containing a conservative substitution at position 26.
• One isozyme contains a threonine at position 26 and the other contains a glutamine at position 26.
• the glutamine isozyme is about 27 atomic mass units greater than the threonine isozyme.
• any reference to DK 9.2 should be interpreted as a reference to the either and/or both isozymes.
• DK 9.2 is characterized by a molecular weight of about 6371-6397 daltons as determined by mass spectrometry and movement as a single peak on reverse phase HPLC.
• DK 11 has been isolated and is characterized by a molecular weight of about 6700 daltons or about 6740 daltons as determined by mass spectrometry and movement as a single peak on reverse phase HPLC. More particularly, this active HPLC fraction has been identified to have an amino acid sequence, as translated from the isolated cDNA, as shown in SEQ ID NO: 3.
• DK 12 a third member of this insecticidally effective protein family, DK 12, has been characterized by a molecular weight of about 7100 daltons or about 7080 daltons as determined by mass spectrometry and moves as a single peak on reverse phase HPLC. More particularly this active fraction has been tentatively shown to have an amino acid sequence as shown in SEQ ID NO: 5.
• Such peptides may exist in nature or may be produced by methods of recombinant DNA technology known to those of ordinary skill in the art. Such peptides, whether naturally occurring or recombinantly produced, are contemplated as within the scope of this invention
• a substantially isolated DNA sequence encoding an insecticidally effective peptide substantially isolatable from Diguetia spider venom is provided.
• the genes responsible for the production of proteins isolatable from the spider can be isolated and identified. Numerous methods are available to obtain the gene responsible for the production of a peptide.
• Patent No. 4,703,008 "DNA Sequences Encoding Erythropoietin" which patent is incorporated by reference.
• a DNA molecule is synthesized which encodes the determined amino acid sequence or which represents the complementary DNA strand to such a DNA molecule which encodes the determined amino acid sequence.
• DNA molecule may then be used to probe for DNA sequence homology in cell clones containing recombinant DNA molecules comprising, in part, DNA sequences derived from the genomic DNA of an organism such as a spider or derived from cDNA copies of mRNA molecules isolated from cells or tissues of an organism such as a spider.
• DNA molecules of fifteen (15) nucleotides or more are required for unique identification of an homologous DNA, said number requiring unique determination of at least five (5) amino acids in sequence. It will be appreciated that the number of different DNA molecules which can encode the determined amino acid sequence may be very large since each amino acid may be encoded for by up to six (6) unique trinucleotide DNA sequences or codons.
• PCR Polymerase Chain Reaction
• RNA is isolated from the spider and purified.
• a deoxythymidylate-tailed oligonucleotide is then used as a primer in order to reverse transcribe the spider RNA into cDNA.
• a synthetic DNA molecule or mixture of synthetic DNA molecules as in the degenerate probe described above is then prepared which can encode the amino-terminal amino acid sequence of the venom protein as previously determined. This DNA mixture is used together with the deoxythymidylate-tailed oligonucleotide to prime a PCR reaction. Because the synthetic DNA mixture used to prime the PCR reaction is specific to the desired mRNA sequence, only the desired cDNA will be effectively amplified.
• the resultant product represents an amplified cDNA which can be ligated to any of a number of known cloning vectors.
• "families" of peptides may exist in spider venoms which will have similar amino acid sequences and that in such cases, the use of mixed oligonucleotide primer sequences may result in the amplification of one or more of the related cDNAs encoding these related peptides.
• Genes encoding related peptides are also within the scope of the invention as the related peptides also have useful insecticidal activities.
• the produced cDNA sequence can be cloned into an appropriate vector using conventional techniques, analyzed and the nucleotide base sequence determined.
• DNA sequences, encoding insecticidally effective proteins, are presented e.g. in SEQ ID NO: 2 and 4. A direct amino acid translation of these PCR products will reveal that they corresponded to the complete coding sequence for the mature protein.
• DK 9.2 is synthesized as a precursor protein comprising a signal peptide, propeptide and the mature toxin.
• the function of the signal peptide is thought to be important for targeting the synthesized polypeptide for secretion.
• a signal sequence plays an important role in ensuring the proper localization of a newly synthesized protein. Generally they provide "topogenic signals" (Blobel, G. Proc.Nat .Acad.Sci . , U.S.A.
• the signal peptide encoded by the cDNA coding for the precursor protein of DK 9.2 is believed to be comprised of 17 amino acids of the following sequence:
• propeptide is a 21 amino acid peptide of the following sequence:
• precursor peptides may contribute greatly to the stability, expression and folding of DK 9.2 or other recombinant proteins when expressed in vivo and/or in vitro. That these sequences or portions thereof could prove useful in the expression of other molecules, for example in a gene construct, is also anticipated. As part of this invention we will also supply data to confirm that other signal and/or propeptide sequences could be used for the expression of recombinant DK 9.2. E. Recombinant expression
• a recombinant expression vector comprising a DNA sequence which encodes an insecticidally effective peptide substantially isolatable from Diguetia spider venom.
• the vector is capable of effecting the expression of the coding sequence in transformed cells.
• recombinant host cells transformed or transfected with a DNA sequence encoding an insecticidally effective peptide substantially isolatable from Diguetia spider venom in a manner allowing the host cell to express the peptide.
• Provision of a suitable DNA sequence encoding the desired protein permits the production of the protein using recombinant techniques now known in the art.
• the coding sequence can be obtained by retrieving a cDNA or genomic sequence from a native source of the protein or can be prepared synthetically using the accurate amino acid sequence determined from the nucleotide sequence of the gene.
• advantage can be taken of known codon preferences of the intended host.
• control sequences such as promoters, and preferably enhancers and termination controls, are readily available and known in the art for a variety of hosts. See e . g. , Sambrook et al., Molecular Cloning a Laboratory Manual , Second Ed. Cold Spring Harbor Press (1989).
• the desired proteins can be prepared in both procaryotic and eucaryotic systems, resulting, in the case of many proteins, in a spectrum of processed forms.
• procaryotic system The most commonly used procaryotic system remains E. coli , although other systems such as B . subtilis and Pseudomonas are also expected to be useful.
• Suitable control sequences for procaryotic systems include both constitutive and inducible promoters including the lac promoter, the trp promoter, hybrid promoters such as tac promoter, the lambda phage PI promoter.
• foreign proteins may be produced in these hosts either as fusion or mature proteins.
• the sequence produced may be preceded by a methionine which is not necessarily efficiently removed.
• the peptides and proteins claimed herein may be preceded by an N-terminal Met when produced in bacteria.
• constructs may be made wherein the coding sequence for the peptide is preceded by an operable signal peptide which results in the secretion of the protein. When produced in procaryotic hosts in this matter, the signal sequence is removed upon secretion.
• eucaryotic hosts are also now available for production of recombinant foreign proteins.
• eucaryotic hosts may be transformed with expression systems which produce the desired protein directly, but more commonly signal sequences are provided to effect the secretion of the protein.
• Eucaryotic systems have the additional advantage that they are able to process introns which may occur in the genomic sequences encoding proteins of higher organisms.
• Eucaryotic systems also provide a variety of processing mechanisms which result in, for example, glycosylation, oxidation or derivatization of certain amino acid residues, conformational control, and so forth.
• eucaryotic systems include yeast, insect cells, mammalian cells, avian cells, and cells of higher plants.
• Suitable promoters are available which are compatible and operable for use in each of these host types as well as are termination sequences and enhancers, as e.g. the baculovirus polyhedrin promoter.
• promoters can be either constitutive or inducible.
• the MTII promoter can be induced by the addition of heavy metal ions.
• the DNA encoding it is suitably ligated into the expression system of choice, and the system is then transformed into the compatible host which is then cultured and maintained under conditions wherein expression of the foreign gene takes place.
• the insecticidally effective protein of this invention thus produced is recovered from the culture, either by lysing the cells or from the culture medium as appropriate and known to those in the art.
• a “deletion” is defined as a polypeptide in which one or more internal amino acid residues are absent.
• An “addition” is defined as a polypeptide which has one or more additional internal amino acid residues as compared to the wild type.
• a “substitution” results from the replacement of one or more amino acid residues by other residues.
• a protein "fragment” is a polypeptide consisting of a primary amino acid sequence which is identical to a portion of the primary sequence of the protein to which the polypeptide is related.
• substitutions are those which are conservative, i.e., wherein a residue is replaced by another of the same general type.
• naturally-occurring amino acids can be subclassified as acidic, basic, neutral and polar, or neutral and nonpolar and/or aromatic. It is generally preferred that encoded peptides differing from the native form contain substituted codons for amino acids which are from the same group as that of the amino acid replaced.
• the basic amino acids Lys, Arg, and His are interchangeable; the acidic amino acids Asp and Glu are interchangeable; the neutral polar amino acids Ser, Thr, Cys, Gln, and Asn are interchangeable; the nonpolar aliphatic acids Gly, Ala, Val, lie, and Leu are conservative with respect to each other (but because of size, Gly and Ala are more closely related and Val, lie and Leu are more closely related), and the aromatic amino acids Phe, Trp, and Tyr are interchangeable.
• Polar amino acids which represent conservative changes include Ser, Thr, Gin, Asn; and to a lesser extent, Met.
• Ala, Gly, and Ser seem to be interchangeable, and Cys additionally fits into this group, or may be classified with the polar neutral amino acids.
• proteins of the invention can be made by recombinant techniques as well as by automated amino acid synthesizers. Because of the variety of post-translational characteristics conferred by various host cells, various modifications for the naturally-occurring proteins will also be obtained.
• a "modified" protein differs from the unmodified protein as a result of post-translational events which change the glycosylation, amidation or lipidation pattern, or the primary, secondary, or tertiary structure of the protein and are of course included within the scope of the invention as claimed. It should be further noted that if the proteins herein, such as SEQ ID NO:1 are made synthetically, substitution by amino acids which are not encoded by the gene may also be made.
• Alternative residues include, for example, the ⁇ amino acids of the formula H 2 N(CH 2 ) n COOH wherein n is 2-6. These are neutral, nonpolar amino acids, as are sarcosine (Sar), t-butylalanine (t-BuAla), t- butylglycine (t-BuGly), N-methyl isoleucine (N-MeIle), and norleucine (Nleu).
• Phenylglycine for example, can be substituted for Trp, Tyr or Phe an aromatic neutral amino acid; citrulline (Cit) and methionine sulfoxide (MSO) are polar but neutral, cyclohexyl alanine (Cha) is neutral and nonpolar, cysteic acid (Cya) is acidic, and ornithine (Orn) is basic.
• the conformation conferring properties of the proline residues may be obtained if one or more of these is substituted by hydroxyproline (Hyp).
• transgenic plants comprising a DNA sequence encoding an insecticidally effective peptide substantially isolatable from Diguetia spider venom introduced into the germ line of the plant, such that the trait of expression of the DNA sequence is inherited by subsequent generations of the plant through sexual propagation or asexual propagation.
• Genes encoding the insecticidally effective peptides according to the present invention can be introduced into a plant by genetic engineering techniques, which upon production of the peptide in the plant cell is expected to be useful as a means for controlling insect pests. Therefore, it is possible to produce a plant that is more insect-tolerant than the naturally occurring variety.
• the coding region for an insecticidally effective peptide gene that may be used to transform a plant may be the full-length or partial active length of the gene. It is necessary, however, that the genetic sequence coding for the peptide be expressed, and produced, as a functional peptide in the resulting plant cell. It is believed that DNA from both genomic DNA and cDNA and synthetic DNA encoding an insecticidally effective peptide may be used to transform. Further, a gene may be constructed partially of a cDNA clone, partially of a genomic clone, and partially of a synthetic gene and various combinations thereof. In addition, the DNA coding for a peptide gene may comprise portions from various species other than from the source of the isolated peptide.
• insecticidally effective peptide may be combined with another compound or compounds to produce unexpected insecticidal properties in the transformed plant, containing chimeric genes, expressing the compounds.
• these other compounds can include protease inhibitors, for example, which have oral toxicity to insects or polypeptides from Bacillus thuringiensis .
• the B . thuringiensis protein causes changes in potassium permeability of the insect gut cell membrane and is postulated to generate small pores in the membrane.
• Other pore-forming proteins could also be used in combination with the insecticidally effective peptides.
• pore-forming proteins examples include the magainins, the cecropins, the attacins, meiittin, gramicidin S, sodium channel proteins and synthetic fragments, the a-toxin of Staphylococcus aureus, apolipoproteins and their fragments, alamethicin and a variety of synthetic amphipathic peptides.
• Lectins which bind to cell membranes and enhance endocytosis are another class of proteins which could be used in combination with the insecticidally effective peptides of this invention to genetically modify plants for insect resistance.
• the promoter of the peptide gene is expected to be useful in expressing the chimeric genetic sequence, however, other promoters are also expected to be useful.
• An efficient plant promoter that may be useful is an overproducing promoter. This promoter in operable linkage with the genetic sequence for the peptide should be capable of promoting expression of the peptide such that the transformed plant has increased tolerance to insect pests. Overproducing plant promoters that are expected to be useful in this invention are known.
• the chimeric genetic sequence comprising an insecticidally effective peptide gene operably linked to a promoter can be ligated into a suitable cloning vector to transform the desired plant.
• a suitable cloning vector to transform the desired plant.
• plasmid or viral (bacteriophage) vectors containing replication and control sequences derived from species compatible with the host cell are used.
• the cloning vector will typically carry a replication origin, as well as specific genes that are capable of providing phenotypic selection markers in transformed host cells, typically resistance to antibiotics.
• the transforming vectors can be selected by these phenotypic markers after transformation in a host cell.
• Host cells that are expected to be useful include procaryotes, including bacterial hosts such as E. coli , Salmonella ryphimurium, and Serratia marcescens ; and eucaryotic hosts such as yeast or filamentous fungi.
• the cloning vector and host cell transformed with the vector are generally used to increase the copy number of the vector.
• the vectors containing the peptide gene can be isolated and, for example, used to introduce the genetic sequences described herein into the plant or other host cells.
• plant tissue can be transformed by direct infection of or co-cultivation of plants, plant tissue or cells with A. tumefaciens ; direct gene transfer of exogenous DNA to protoplasts; incubation with PEG; microinjection and microprojectile bombardment.
• the exogenous DNA may be added to the protoplasts in any form such as, for example, naked linear, circular or supercoiled DNA, DNA encapsulated in liposomes, DNA in spheroplasts, DNA in other plant protoplasts, DNA complexed with salts, and the like.
• All plant cells which can be transformed by Agrobacterium and whole plants regenerated from the transformed cells can also be transformed according to the invention so to produce transformed whole plants which contain the transferred insecticidally effective peptide gene. Transformation in rice has been confirmed by D.M. Raineri et al., "Agrobacterium-mediated transformation of rice (Oryza sativa 1. ) " , Biotechnology, Vol.8, pp 33-38 (January 1990).
• Another method of introducing the insecticidally effective peptide gene into plant cells is to infect a plant cell with A. tumefaciens transformed with the insecticidally effective peptide gene. Under appropriate conditions known in the art, the transformed plant cells are grown to form shoots, roots, and develop further into transformed plants.
• the insecticidally effective peptide genetic sequences can be introduced into appropriate plant cells, for example, by means of the Ti plasmid of A. tumefaciens .
• the Ti plasmid is transmitted to plant cells on infection by A. tumefaciens and is stably integrated into the plant genome.
• Ti plasmids contain two regions believed essential for the production of transformed cells. One of these, named transfer DNA (T DNA), induces tumor formation. The other, termed virulent region, is essential for the formation but not maintenance of tumors.
• T DNA region which transfers to the plant genome, can be increased in size by the insertion of an enzyme's genetic sequence without its transferring ability being affected. By removing the tumor-causing genes so that they no longer interfere, the modified Ti plasmid can then be used as a vector for the transfer of the gene constructs of the invention into an appropriate plant cell.
• the genetic material may also be transferred into the plant cell by using polyethylene glycol (PEG) which forms a precipitation complex with the genetic material that is taken up by the cell.
• PEG polyethylene glycol
• Transfer of DNA into plant cells can also be achieved by injection into isolated protoplasts, cultured cells and tissues and injection into meristematic tissues of seedlings and plants.
• Transgenic plants and progeny therefrom are obtained by conventional methods known in the art.
• Another method to introduce foreign DNA sequences into plant cells comprises the attachment of the DNA to particles which are then forced into plant cells by means of a shooting device, "gene guns".
• Any plant tissue or plant organ may be used as the target for this procedure, including but not limited to embryos, apical and other meristems, buds, somatic and sexual tissues in vivo and in vitro .
• Transgenic cells and callus are selected following established procedures. Targeted tissues are induced to form somatic embryos or regenerate shoots to give transgenic plants according to established procedures known in the art. The appropriate procedure may be chosen in accordance with the plant species used.
• Transgenic maize plants have been prepared by using high-velocity microprojectiles to transfer genes into embryogenic cells. "Inheritance and expression of chimeric genes in the progeny of transgenic maize plants", Biotechnology, Vol. 8, pp 833-838 (September 1990).
• the regenerated plant may be chimeric with respect to the incorporated foreign DNA. If the cells containing the foreign DNA develop into either micro- or macrospores, the integrated foreign DNA will be transmitted to sexual progeny. If the cells containing the foreign DNA are somatic cells of the plant, non-chimeric transgenic plants are produced by conventional methods of vegetative (asexual) propagation either in vivo, from buds or stem cuttings, or in vitro following established procedures known in the art. Such procedures may be chosen in accordance with the plant species used.
• phenotypic markers include, but are not limited to, antibiotic resistance. Other phenotypic markers are known in the art and may be used in this invention.
• Additional plant genera that may be transformed by Agrobacterium include Ipomoea , Passiflora, Cyclamen , Malus , Prunus, Rosa, Rubus , Populus , Santalion , Allium, Lilium, Nacissus, Ananas, Arachis , Phaseolus, and Pisum.
• Regeneration varies from species to species of plants, but generally a suspension of transformed protoplasts containing multiple copies of the insecticidally effective peptide gene is first provided. Embryo formation can then be induced from the protoplast suspensions, to the stage of ripening and germination as natural embryos.
• the culture media will generally contain various amino acids and hormones. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable.
• the mature plants, grown from the transformed plant cells, can be selfed to produce an inbred plant.
• the inbred plant produces seed containing the gene for the insecticidally effective peptide. These seeds can be grown to produce plants that express the insecticidally effective peptide.
• the inbreds can, e.g., be used to develop insect tolerant hybrids. In this method, an insect tolerant inbred line is crossed with another inbred line to produce the hybrid.
• diploid plants typically one parent may be transformed by the insecticidally effective peptide (toxin) genetic sequence and the other parent is the wild type. After crossing the parents, the first generation hybrids
• F 1 will show a distribution of 1/2 toxin/wild type: 1/2 toxin/wild type.
• first generation hybrids (F 1 ) are selfed to produce second generation hybrids (F 2 ).
• the genetic distribution of the F 2 hybrids is 1/4 toxin/toxin: 1/2 toxinwild type: 1/4 wild type/wild type.
• the F 2 hybrids with the genetic makeup of toxin/toxin are chosen as the insect tolerant plants.
• variant describes phenotypic changes that are stable and heritable, including heritable variation that is sexually transmitted to progeny of plants, provided that the variant still expresses an insecticidally effective peptide of the invention.
• mutant describes variation as a result of environmental conditions, such as radiation, or as a result of genetic variation in which a trait is transmitted meiotically according to well- established laws of inheritance. The mutant plant, however, must still express the peptide of the invention.
• the ideal insecticidally effective protein chosen to be expressed in a transgenic plant will be one that is characterized by its safety to non-target insects and vertebrates. Expression systems will be chosen such that the level of expression affords insecticidal efficacy. Thus, this technical feasibility of obtaining such transgenic agriculturally important plants is expected to offer farmers an additional weapon to use in an integrated pest management system to reduce insect damage to crops in an environmentally responsible manner.
• insecticidally effective peptides of this invention are believed to be useful in controlling invertebrate pests such as the order of Lepidoptera, by contacting the pests with an effective amount of a peptide of this invention.
• invertebrate pests such as the order of Lepidoptera
• insects are the preferred pest.
• Methods of contacting an invertebrate pest with a peptide to control said pests are known. Examples include synthetically encapsulating the protein for oral ingestion by the pest. Recombinant hosts expressing the proteins of this invention, such as Pseudomonas fluorescens , can be heat killed and applied to pests for subsequent oral ingestion and control.
• invertebrate pests using the proteins of this invention can be used in combination with other methods of controlling pests.
• the transgenic plants and E. coli mentioned above can be engineered to express other invertebrate toxins depending on the type of pests to be controlled and other important variables present.
• An insecticidal composition comprising an insecticidally effective amount of a peptide according to this invention and agriculturally or horticulturally acceptable salts thereof in an agriculturally or horticulturally acceptable carrier therefor is also provided.
• Another aspect of this invention are antibodies to the insecticidally effective peptides of this invention.
• An antibody is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.
• epitope is meant to refer to that portion of a peptide antigen which can be recognized and bound by an antibody.
• An antigen may have one or more than one epitope.
• An "antigen” is capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen.
• the specific reaction referred to above is meant to indicate that the antigen will immunoreact, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
• antibody or “monoclonal antibody” (Mab) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab and F(ab') 2 fragments) which are capable of binding an antigen.
• Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an insect antibody.
• the antibodies of the present invention may be prepared by any of a variety of methods. Methods for the production of such antibodies are well known and described fully in the literature. See e.g., Sambrook et al., "Molecular Cloning a laboratory manual", second ed. Cold Spring Harbor Press, Vol. 3, Ch. 18 (1989).
• cells expressing the insecticidally effective peptide or a fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies that are capable of binding the insecticidally effective peptide.
• an insecticidally effective peptide fragment is prepared and purified to render it substantially free of natural contaminants or an insecticidally effective peptide fragment is synthesized, according to means known in the art. Either the purified fragment or the synthesized fragment or a combination of purified natural fragments and/or synthesized fragment may be introduced into an animal in order to produce polyclonal antisera of greater specific activity.
• Monoclonal antibodies can be prepared using known hybridoma technology. In general, such procedures involve immunizing an animal with an insecticidally effective peptide antigen. The splenocytes of such animals are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention. After fusion, the resulting hybridoma cells are selectively maintained in a suitable medium and then cloned by limiting dilution. The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the insecticidally effective peptide antigen.
• hybridoma cells will produce antibodies capable of binding to the peptide (other hybridoma cells will produce antibody capable of binding to the peptide contaminants).
• Screen among the hybridoma cells for those which are capable of secreting an antibody which is capable of binding to the peptide. Such screening is preferably accomplished by incubating a sample of the peptide (or venom) in the presence of monoclonal antibody secreted from each of a group of particular hybridoma cells and identifying any hybridoma cell capable of secreting an antibody which is able to neutralize or attenuate the ability of the venom to paralyze an insect. Once such a hybridoma cell has been identified, it may be clonally propagated by means known in the art in order to produce the peptide-specific monoclonal antibody.
• an antibody capable of binding to the insecticidally effective peptide it is necessary to employ an antibody capable of binding to the insecticidally effective peptide.
• an antibody will be a monoclonal antibody.
• a peptidespecific monoclonal antibody Once a peptidespecific monoclonal antibody has been obtained, it may be immobilized by binding to a solid support and used to purify the peptide from natural venom or other sources using immunoaffinity chromatography in accordance to methods which are well known in the art. Such methods are capable of mediating a high degree of purification and of thereby producing a peptide which is substantially free of natural contaminants.
• a peptide is said to be "substantially free of natural contaminants” if it is present in a form which lacks compounds with which it is naturally and normally associated (i.e. other proteins, lipids, carbohydrates, etc.).
• the peptide Once the peptide has been purified, it can be used to immunize an animal (such as a mouse or rabbit) in order to elicit the production of peptide-specific polyclonal antibody.
• an animal such as a mouse or rabbit
• DNA probes of suitable size can be derived from a DNA sequence encoding an insecticidally effective peptide substantially isolatable from Diguetia spider venom.
• Such probes can be used to detect the presence of DNA encoding an insecticidally effective peptide substantially isolatable from Diguetia spider venom by contacting a venom with the DNA probe and detecting the probe conjugated to said DNA by ways known to those in the art.
• insecticidally effective peptide alone or in combination with another insect toxin is expected to be useful in potentiating or enhancing the toxicity of microbes such as baculoviruses and hybrid bacteria.
• baculoviruses including those that infect Heliothis virescens (cotton bollworm), Orgyia pseudotsugata (Douglas fir tussock moth), Lymantia dispar (gypsy moth), Autographa californica (alfalfa looper), Neodiprion sertifer (European pine fly), and Lanspcyresia pomonella (codling moth) have been registered in some countries and used as pesticides. Introduction of at least one insect-selective toxin into the genome is expected to significantly enhance the potency of such pesticides.
• a recombinant expression vector expected to be particularly suitable for use in this invention is a baculovirus expression vector such as the type disclosed in U.S. Patent 4,879,236, which patent is incorporated by reference as if fully set forth herein. See also Carbonell et al. "Synthesis of a gene coding for an insect-specific scorpion neurotoxin and attempts to express it using baculovirus vectors," Gene , 73:409-418 (1988).
• the vector is expected to be useful in a system where a DNA sequence encoding an insecticidally effective peptide substantially isolatable from Diguetia spider venom can be cloned into baculovirus such as Autographa californica (AcMNPV) expression vector as described in U.S. 4,879,236 and Miller et al., Science , 219, 715-721 (1983).
• AcMNPV Autographa californica
• the recombinant expression vector virus could then be applied to the plant or animal upon which the insect is a pest, and when the virus is ingested by the pest insect, the recombinant virus will invade the cells of the intestinal wall and begin replication.
• the gene for the insecticidally effective protein will be expressed, resulting in the disablement or death of the insect in a shorter period than if the insect had ingested the wild type AcMNPV virus.
• a hybrid virus also expected to be useful is taught in European Patent Application 0 340948.
• the hybrid virus expressing the DNA of this invention is expected to yield a virus having an altered insect host range.
• fusion proteins could be expressed as a single polypeptide product of a hybrid gene consisting of DNA of this invention and a specific insect gut cell recognition protein to direct the expressed insecticidally effective peptide to the host insect target.
• prokaryotic and eukaryotic microbes can be transformed to express a hybrid toxin gene encoding an insecticidally effective protein by the method taught in European Patent Application 0 325 400.
• Hybrid bacterial cells comprising a plasmid with the gene coding for the protein of this invention are expected to be useful in the method of this invention. Insects would be controlled by applying the hybrids to insects. See e . g. , U.S. Patent 4,797,279 which patent is incorporated by reference as if fully set forth herein.
• DNA probes of suitable size can be derived from a
• DNA sequence of this invention can be used to detect the presence of nucleic acid encoding an insecticidally effective peptide of this invention by hybridization with nucleic acids from other sources. Screening with oligonucleotide probes encoding the signal sequence, fragments of the cDNA, or even the entire cDNA under conditions of reduced stringency will allow access to other active peptides with functional homology to the family of toxin molecules described herein.
• Sources of nucleic acids which would be good candidates for cross-hybridization with nucleotide probes generated from DNA sequences of this invention would include, but are not limited to spiders of the same genera but of different species, spiders of related genera, and spiders of the same genera but different locations.
• Another aspect of this invention provides for the promoter sequence which controls the synthesis of DK 9.2 in the spider.
• the organization of the gene for DK 9.2 was examined by screening a genomic library with the mature toxin cDNA as probe. Analysis of the genomic DNA upstream of the transcriptional start (assumed to be the 5' end of the cDNA), does indicate the presence of a putative promoter.
• a DNA sequence for 421 base pairs of the promoter region is presented in SEQ ID NO: 8. This putative promoter contains many of the essential control signals that are generally present in the region directly upstream of the translational start site. (For review of promoter control signals see McKnight et al., 1982. Transcriptional Control Signals of an Eucaryotic Protein-Coding Gene.
• a canonical TATA box appears at position -30 from the proposed transcriptional start site and is suggested to be important in controlling the start point for RNA synthesis. Further upstream there are two distal palindromic sequences, one of which contains the consensus CAAT box thought to be important for binding of the RNA polymerase II enzyme. It is anticipated that this promoter, or regions of this promoter will have utility in the transcription and expression of a variety of genes in plant, animal or bacterial cells, for example, in a recombinant construct.
• Spiders were obtained from, and species identification provided by, Spider Pharm, Inc. of Black Canyon City, AZ. Diguetia canities spiders were electrically milked for venom using a method that employs safeguards to prevent contamination of venom by regurgitate or hemolymph.
• Toxin Purification - Crude venom (stored at -80°C) was thawed, mixed thoroughly and dissolved in 0.1% trifluoroacetic acid (TFA) prior to chromatography. Crude venom was fractionated by reverse phase liquid chromatography (RPLC) incorporating Beckman System Gold 126 solvent and 168 photodiode-array detector modules. Acetonitrile or isopropanol was used in combinations with TFA as an ion pairing reagent.
• RPLC reverse phase liquid chromatography
• Dynamax 300 A RP C 18 column 25 cm ⁇ 4.6 mm i.d., 12 ⁇ m particle size
• Vydac 300 A C 18 analytical column 25 cm ⁇ 4.6 mm i.d., 5 ⁇ m particle
• Vydac 300 A C 18 semi-preprative column 25 cm ⁇ 10 mm i.d. 5 ⁇ m particle size. Peak detection was accomplished by monitoring at 220 nm and collecting fractions with a GILSON 208 microfraction collector. All fractions were lyophilized to dryness following fractionation and stored at -80°C.
• TFA trifluoroacetic acid
• a linear gradient of eluant was used beginning with an 85:15 mixture of aqueous 0.1% TFA: 50% acetonitrile, 0.1% TFA and ending with a 50:50 mixture after 180 minutes.
• each fraction was concentrated by lyophilization from the eluant followed by lyophilization from water. The residues were stored at -80° C. Each fraction was dissolved in 25 ⁇ L of a buffered physiological saline solution for insecticidal evaluation. The insecticidal activity of some fractions was confirmed by testing against the tobacco budworm (Heliothis virescens) "TBW" (Table I).
• TBW larvae five individuals for each fraction, were injected with 3 ⁇ L of the test solution using a 50 ⁇ L Hamilton syringe fitted with a 33 gauge needle and a PB 600 repeating microdispenser.
• the injections were made by insertion of the needle into the lateral midline of the abdomen (near one of the prolegs) at a shallow angle in order to avoid damaging internal organs.
• each insect was placed into a separate container containing artificial diet. Observations were made periodically; paralysis was measured at 24 hours following injection. Mean body mass of 0.3 gm for tobacco budworms was used in calculating doses.
• a set of control insects was injected with the buffered saline solution only.
• Paralysis is defined as the inability of the insect to right itself when turned on its side or back.
• the major components of the TBW active Fraction 9 were purified to homogeneity (one visual band each by SDS-PAGE electrophoresis, approximately in the molecular weight range of 6,500 daltons) by one additional chromatography through Vydac RP C 18 (25 cm ⁇ 10 mm i.d.) using a linear (1.0%/min) solvent gradient of iso-propanol/0.1% TFA at 3.5 mL/min, monitoring at 220 nm.
• Peak detection and fraction collection were accomplished as described in Example 1. Two fractions were collected: the first eluting at 21.81 minutes (Fraction 9.1) and the second eluting at 22.39 minutes (Fraction 9.2). Fractions 9.1 and 9.2 were concentrated by lyophilization from the eluant followed by lyophilization from water, leaving residues 9.1 and 9.2, respectively. Purity of the lyophilized fractions was estimated to be at least 99%. Residue 9.2 obtained from 25 ⁇ l of whole venom was estimated to contain approximately 6 ⁇ g of pure protein.
• Residue 9.2 was tested for insecticidal activity against tobacco budworm as described in Example 1 by injecting each of five insects with 6.3 ⁇ g of residue in a buffered saline solution and five insects as controls with the buffered saline solution only. After 24 hours and 48 hours the insects were examined. At the 24 hr reading all those insects treated with the residue 9.2 solution were paralyzed and subsequently died while the control insects appeared normal. No changes were noticed at 48 hr.
• Example 3 The LD 50 of Diquetia toxin 9.2 in H. virescens (TBW) was approximately 1.0 nmol/gm. Symptoms were similar to those associated with administration of whole venom as described in Example 1.
• TW H. virescens
• Example 2 Fraction 11 isolated from Diguetia canities whole venom as in Example 1 was further purified by reverse phase liquid chromatography, obtaining one fraction. This fraction was concentrated by lyophilization from the eluant followed by lyophilization from water, leaving a residue, labeled residue 11. This residue was tested for insecticidal activity against tobacco budworm at 5.0 WVE/g as outlined in Example 2. After 24 hours 80% bf the insects treated with residue 11 exhibited paralysis, and after 48 hours 60% of the insects treated with this peptide exhibited paralysis. The control insects appeared normal.
• Fraction 12 In a manner similar to that of Example 2, Fraction 12 isolated from Diguetia canities whole venom as in Example 1 was further purified by reverse phase liquid chromatography, obtaining one fraction. This fraction was concentrated by lyophilization from the eluant followed by lyophilization from water, leaving a residue, labeled residue 12. This residue was tested for insecticidal activity against tobacco budworm as outlined in Example 2. After 24 hours 100% of the insects treated with residue 12 exhibited paralysis and after 48 hours 80% of the insects treated with residue 12 exhibited paralysis. The control insects appeared normal.
• RNA was reverse transcribed to cDNA with murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, MD) using the manufacturer's protocol.
• the 20 ⁇ l reaction mixture contained the enzyme buffer as supplied in a cDNA synthesis kit (Boehringer Mannheim, IN), 50 ng of mRNA, 2 units of RNase H, 30 ng of d(T) Not I primer (Promega, Madison, WI), 1 mM each deoxynucleoside triphosphates, and 100 ⁇ g of reverse transcriptase.
• the reaction mixture was incubated for 1 h at 37° C and continued for 10 minutes at 42° C.
• the reaction mixture was ethanol precipitated and resuspended in 20 ⁇ l water.
• a degenerate primer DNA sequence mixture which could code for amino acid residues 1 through 8 according to SEQ ID N0:1 was designed using some Drosophila codon preferences to reduce degeneracy. This primer was synthesized by the University of Utah, Howard Hughes Medical Institute contract facility. Step #4: Amplification
• thermostable DNA polymerase Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase was initially described by Saikki et al.. Science, 239:487 (1988). For our applications, 5 ⁇ l of the Diguetia cephalothorax cDNA was used as template in a polymerase chain reaction containing reagents contained in the GeneAmpTM DNA amplification kit (Perkin Elmer Cetus, CA). The amplification reaction contained the sense and antisense primers in a 2 ⁇ M concentration, 100 ⁇ M of each deoxynucleotide triphosphate, and 4 units of the thermostable recombinant Taq I polymerase. The reaction was run in a DNA Thermal Cycler manufactured by Perkin Elmer Cetus.
• Step #5 Cloning of PCR Products
• PCR products from both high and low stringency reactions were purified to remove unincorporated primers using a Centricon-100 (Amicon) molecular size separation unit.
• the retained products were then digested with the restriction enzyme Not I (MBR), Milwaukee, WI), which cleaves within the downstream (3' end) primer leaving a sticky end.
• the vector, pKS Stratagene, LaJolla, CA
• EcoR V US Biochemical
• Not I to generate sites specific for directional cloning.
• Vector and insert were ligated and transformed into compex DH5 ⁇ F'. Colony lifts were screened with the 32 P labeled internal probe and candidate colonies were further characterized by sequencing (US Biochemical's Sequenase Version 2.0) miniprep DNA using the internal probe as primer.
• SEQ ID NO: 2 and 4 The entire DNA sequences of the cDNA inserts of two clones are shown in SEQ ID NO: 2 and 4 . Only the former was obtained in clones derived from the stringent PCR reaction, while both types of cDNA inserts were found in clones obtained from the products of the low stringency PCR reaction.
• the amino acid sequence of the polypeptide encoded by SEQ ID NO: 2 is shown in SEQ ID NO:1. This polypeptide has an N-terminal sequence identical with that determined for residue 9.2. The calculated molecular weight of this polypeptide is 6377.9 Daltons.
• mice Whole venom, 5 ⁇ l, obtained from D. canities as described herein was fatal to mice by intraperitoneal injection (IP) in three separate tests. Treated mice were initially indistinguishable from saline controls. About 10 to 15 minutes after injection, treated mice became moderately hyperactive, displaying a characteristic hopping gait; this was followed by a short period of uncoordination, labored breathing and convulsions. Death followed within 2 minutes of the initial onset of symptoms.
• IP intraperitoneal injection
• Residues 9.2, 11 and 12 were injected intraperitoneally in mice in approximate doses of 4.2, 0.9 and 1.2 mg/kg respectively with no effects seen within 24 hours for all three peptides.
• Residue 9.2 was injected into the intracerebral ventricles in four mice (approx. 28 gm) at a dose of approximately 30 ⁇ g per animal (approximately 1 mg/kg). No effects were noted at any time up to 48 hours post injection. Another mouse was (IP) with approximately 125 ⁇ g (4.2 mg/kg) of residue 9.2 and no effects were seen.
• the assay is capable of detecting a variety of effects on various mammalian ion channels and neurotransmitter receptors (T.V. Dunwiddie, The Use of In Vitro Brain Slices in Neuropharmacology, in Electrophysiological Techniques in Pharmacology, edited by H.M. Geller, Alan R.
• upstream cDNA sequences encoding the precursor of DK 9.2
• an internal oligonucleotide corresponding to nucleic acid residues #159 to 178 on the antisense strand of the DNA sequences presented in SEQ ID NO: 2 was synthesized.
• An Eco RI restriction site is present at the 5' end of this primer.
• Ten microliters of single stranded venom gland cDNA was tailed at its 3' end with deoxyguanosine residues using the enzyme, terminal deoxynucleotide transferase (Bethesda Research Laboratories).
• a 20 ⁇ l reaction containing 14 ⁇ of enzyme and 500 ⁇ M of dGTP was incubated at 37°C for 15 minutes. The sample was ethanol precipitated and resuspended in 20 ⁇ l H 2 O.
• DNA sequences upstream of the gene specific primer were amplified using an anchored PCR technique similar to that used for the downstream/mature toxin cDNA sequnces.
• the amplification reaction contained the sense, (a d(C) tailed primer), and antisense primers in a 2 ⁇ M concentration, 100 ⁇ M of each deoxynucleotide triphosphate, and 4 units of the thermostable recombinant Tag polymerase.
• the temperature profile was as follows: 2 min at 94°C, 2 min at 37°C, 1 min at 37°C. This cycle was repeated twice and the program then switched to an identical profile incorporating an elevated annealing temperature of 54°C at the second step. This cycle was repeated 32 times.
• Anchored PCR yielded at 380bp fragment as evidenced on a 4% agarose gel in the presence of ethidium bromide.
• This reaction product was filled in at the ends using the large (Klenow) fragment of E. coli DNA Polymerase I (Molecular Biology Resources, Madison, WI), and precipitated by the addition of ethanol.
• the product was resuspended and digested with the restriction enzyme, Eco RI
• the digested fragment was kinated in the presence of 1 mM ATP by the enzyme T4 Kinase and subsequently ligated to Eco RI and Eco RV digested pBluescriptsKS vector.
• Subclones were analyzed by double-stranded DNA sequencing using Sequenase 2.0 (USB).
• the encoded precursor protein is comprised of a signal sequence and propeptide region (SEQ ID NO: 7) which are removed to yield the mature peptide toxin which is isolatable from spide venom. It is speculated that the signal and propeptide sequences are necessary for the production and secretion of DK 9.2 in the spide.
• a lepidopteran signal sequence (Jones et al., Molecular Cloning Regulation and Complete Sequence of a Hemocyanin-Related Juvenile Hormone-Supressible Proteim From Insect Hemolymphs, J. Biol . Chem . 265:8596 (1990)), was constructed from two synthetic oligonucleotides using the method of Rossi, et al. (J. Biol . Chem . 257:9226 (1982)). Two 48mers were purified by ion exchange chromatography. These two oligonucleotides share eleven base pairs of complementary sequence at their 3' termini.
• DNA sequencing confirmed an in-frame fusion between the two cDNA sequences.
• the entire synthetic gene construct was excised and adapted for cloning into the NheI site of pBlueBac, a baculovirus transfer vector [Vialard, J., et al., J. Virology 64:3-50 (1990)].
• Subclones weer sequenced to confirm the correct insertion of the construct.
• DNA sequencing of plasmid WR9 confirmed the insertion of the synthesized "caterspider" gene (preDK 9.2) in the baculovirus transfer vector pBlueBac ( Figure 3).
• the use of the pBlueBac vector expedites the screening process as insertion of our recombinant gene into the baculovirus genome is accompanied by co-expression of ⁇ -galactodidase and detectable by a color change when grown on indicating media.
• Recombinant baculoviruses encoding preDK 9.2 were produced by transfection of Spodoptera frugiperda strain Sf9 (ATCC# CRL1711) cells with a mixture of 1 ⁇ g AcMNPV viral DNA and 2 ⁇ g plasmid DNA using the protocol of Summers and Smith (in "A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures", Texas Agricultural Experiment Bulletin No. 1555, 1988).
• Four days posttransfection dilutions of the cell supernatant were plagued on 100 mm plates seeded with 5 ⁇ 10 6 Sf9 cells and covered with agarose containing Bluo-gal (Gibso BRL, Gaithersburg, MD) as substrate.
• Viral stocks were titered by the plaque assay method (Luria et al., "General Virology", 1978, pp. 21-32; John Wiley and Sons, New York). Titers were expressed in terms of plaque forming units (PFU) per unit volume, whereas doses were expressed as PFU/larva.
• PFU plaque forming units
• One PFU is the functional equivalent of one mature virion (virus) in preparation wherein every virion is capable of successfully infecting one host cell (Luria et al., ibid). For example, 103 host cells could be infected by each microliter of viral preparation containing 106 PFU/ml (i.e., 103 PFU/ ⁇ l).
• treated larvae were held in individual containers with a supply of food and observed periodically.
• Table II The combined results of two such viral injection assays are illustrated in Table II.
• vAcDK9.2 and wt-AcMNPV were compared in a series of injection assays.
• Last instar tobacco budworm (Heliothis virescens) larvae, cabbage looper (Trichoplusia ni) larvae, and beet armyworm (Spodoptera exigua) larvae were injected with 5 ⁇ 10 5 PFU/larva of vAcDK9.2 or wt-AcMNPV; control larvae were injected with tissue culture medium.
• Table III demonstrate that vAcDK9.2 is substantially more effective than wt-AcMNPV in all three species.
• SF-9 cells were simultaneously infected with vAcDK9.2 and wt-AcMNPV at multiplicities of infection (MOI) of 10 and 2, respectively.
• MOI multiplicities of infection
• inclusion bodies were harvested by cell disruption and differential centrifugation (e.g., Wood, 1980, Virology 104:392-399). Inclusion bodies were counted on a hemacytometer. Cells which were co-infected with vAcDK9.2 and wt-AcMNPV produced fewer and smaller inclusion bodies than cells infected with wt-Ac MNPV alone.
• the yield of polyhedral inclusion (PIB) from the mixed infection was 4.6 PIB/cell, while the yield from the pure wt-AcMNPV infection was 33.3 PIB/cell.
• a diet incorporation assay was used.
• Mixed or wild-type PIB were incorporated into a non-agar based insect diet at a concentration of 10 5 PIB/gm diet.
• Control larvae were given identical amounts of untreated diet. The larvae were allowed to feed ad libitum, and were observed periodically for the development of symptoms. Results are shown in Table II.
• DNA was prepared from spiders using a protocol adapted from Herrmann and Frischholz (in "Methods in Enzymology", Vol. 152, Academic Press, Inc. 1987, pp. 180-183). The DNA was partially digested with Sau 3A and fractionated by centrifugation in a 10% - 38% sucrose density gradient at 25,000 rpm for 16 h with a TLS-55 rotor (Beckman Co., Ltd). Pooled DNA fractions of 35-45 kb were prepared for insertion into an Xho I digested cosmid vector using the partial fill-in method.
• origonucleotides encoding the amino- terminus, the carboxy terminus and an internal probe.
• One cosmid, cDK2 hybridized with all of the probes.
• a southern blot of Eco R1digested cosmid DNA indicated a 3.0 kb fragment to hybridize with both the internal and C-terminal probes.
• DNA sequencing upstream of the signal sequence on the genomic DNA suggested another intron at bp - 11 from the initiation methionine codon.
• An oligonucleotide primer corresponding to the 5' region of the precursor DK 9.2 cDNA was synthesized and used to prime a PCR reaction to determine how large of an intervening sequence there is between the transcriptional and translational start sites.
• a 1,000 based pair amplification product confirmed the presence of the intron and provided an estimate of its size.
• DNA sequencing of the genomic DNA upstream to the transcriptional start (assumed to be equivalent to the 5' end of the cDNA), does indicate the presence of a promoter.
• the DNA sequence of the promoter region is presented in Sequence ID Listing No. 8.
• This putative promoter contains many of the essential control signals that are generally present in other eucaryotic promoters in the region directly upstream of the translational start site. This promoter or sections of this promoter can be used for the transcription/translation of other eucaryotic genes in bacteria, viruses, plants or animals.
• DK 9.2 induces repetitive burst discharges in nerves that are sensitive to block by tetrodotoxin.
• the site of action of this toxin is probably the voltage-sensitive sodium channel of nerve membrane.
• DK 9.2 consistently excites peripheral nerves at a threshold concentration of 10 nM. In addition, it is at least 50 times as potent as mammal toxin 4 of the scorpion Leiurus quinquestriatus.
• a stimulating suction electrode was attached to any convenient nerve trunk to record neuromuscular transmission.
• the threshold for stimulation was slowly raised until a contracting fiber of muscle 6 or 7 was observed. Then, this fiber was impaled with a recording intracellular microelectrode connected to an intracellular preamplifier used to monitor Excitatory Post Synaptic Potentials (EPSP) in response to nerve stimulation.
• EBP Excitatory Post Synaptic Potentials
• the suction electrode was attached to an AC preamplifier. Signals were amplified 100-fold and the output filtered at 0.3 and 1 kHz. All recordings were digitized on a MacLab computerized instrumentation system for display and analysis.
• DK 9.2 was tested on the peripheral nerve preparation starting at 1 nM, and increasing the concentration every five minutes or so until an increase in activity was observed. In this preparation, 1 nM and 5 nM DK 9.2 were inactive, but 10 nM induced activity in the preparation after a short delay. Results from five nerve preparations used to determine the effective threshold concentration of DK 9.2 on insect nerve are summarized in Table IV.
• AAAAAGC 275 AAAAAGC 275

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