KR0145513B1 - Insecticidally effective peptides - Google Patents

Abstract

The present invention provides a peptide having an insecticidal effect, which can be isolated from the Diguetia spider poison, a method for preparing and using an insecticide, and a DNA encoding the peptide having the insecticidal effect.

Description

Peptides with Insecticidal Effects

The present invention relates to a peptide having an insecticidal effect. In particular, the present invention relates to a peptide having a pesticidal effect that is separable from Diguetia spider venom, DNA encoding the same, a method for preparing the same, and a method for inhibiting invertebrate pests thereby.

In recent years, there is a keen awareness of the environmental toxicity and mammalian toxicity associated with the use of synthetic pesticides. As a result, the use of such pesticides is rapidly decreasing. However, the need for effective insect control remains the same. Because of this, researchers are rushing to develop new insect control methods.

The most widely used microbial pesticides are derived from the bacterium Bacillus thuringiensis (hereinafter referred to as B. t.). This bacterial agent is used to control various leaf-eating caterpillars, Japanese beetles and mosquitoes. Karamata et al., U.S. Patent No. 4,797,279, filed Jan. 10, 1989 to B. t. Gene encoding the Kurstaki delta-endotoxin and B. t. Hybrid bacterial cells comprising genes encoding tenebrionis delta-endotoxins and methods for their preparation are described. B. t. Hybrid is B. t. B. t. As well as for pests susceptible to Teneblionis strains. It is also active against pests susceptible to Kurstarki strains. In general, the hybrids have far superior pesticides than the results observed by the physical mixture of the parent strains in terms of pesticidal activity level, in terms of activity range, or both. The pesticidal composition comprising the microorganism may be used to exterminate the insect by applying the hybrid to the insect or its environment in an insecticidal effective amount.

Another process of induction from the bacterium B. t. Is disclosed in European Patent Application Publication No. 0 325 400 A1 to Gilroy and Wilcox. The present invention relates to a hybrid toxin gene that is toxic to Repidoftera insects. Specifically, this invention is directed to B. t. Part of the variant Kurstaki HD-73 toxin gene and B. t. Hybrid delta-endogenous toxin genes comprising a portion of the toxin gene of variant Kurstaki strain HD-1. This application discloses a hybrid toxin gene (DNA) that encodes a protein that is active against Repidoptera insects.

The bacterium B. t. Also used its insecticidality in European Patent Publication No. 0 340 948 to Wilcox et al. The invention is described in B. t. The hybrid intestinal epithelial cell recognition region of the gene is fused to the diphtheria toxin B chain, thereby enabling hybrid B. t. A hybrid insecticidal toxin produced by preparing a toxin. In that publication, the hybrid B. t. It has been suggested that genes can be inserted into plants or cloned into baculovirus to produce recoverable toxins. In addition, hybrid B. t. It has been suggested that hosts containing genes can be applied directly to the surrounding environment of target insects and used as insecticides.

In the study of pesticidal compounds, delivery poisons have been identified as possible sources of compounds that provide pesticides. Zlotkin et al., An Excitatory and a Depressant Insect Toxin from Scorpion Venom both Affect Sodium conductance and Possess a Common Binding Site, Arch Brochem and Biophysics, 240: 877-87 (1985). In studies of their chemical and pharmacological properties, it was found that one toxin induces rapid excitatory contraction paralysis of fly larvae and the other toxin induces slow inhibitory relaxation paralysis. These all affected the sodium conductance.

Canadian Patent No. 2,005,658, issued June 19, 1990 to Zlotkin et al., Has a protein with insecticidal effect derived from Scorpion leurus quinquestriatus hebraus butidae, Leiurus quinquestriatus hebraeus Buthidae, Buthinae. It is starting. In the invention, the poison was lyophilized to separate into fractions. Fractions with the highest toxicity to larvae and the lowest toxicity to mice were further purified to name the final product LqhP35.

Grishin's Toxic components from Buthus eupeus and Lucosa singoriensis venoms, Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Moscow 117988, GSP-1, USSR, are scorpions from scorpions that are poisonous to insects. Four toxins isolated from the toxin of eupeus) are disclosed. The isolation and characterization of the toxin component of Taranto spider Lucosa singoriensis poison is also disclosed. Crude venom was nontoxic to insects.

Corresponding to research and development work involving various compositions with pesticidal properties, the researchers have developed a method for constructing insecticidal genes and introducing them into target pests. Smith and Summers, U.S. Patent No. 4,879,236, issued November 7, 1989, inserts a selected gene linked to a baculovirus promoter into the baculovirus genome to express this selected gene in insect cells. A method for producing a recombinant baculovirus expression vector. The method includes cutting baculovirus DNA to produce a DNA fragment comprising a polyhedrin gene or portion thereof comprising a polyhedrin promoter. To prepare a recombinant transport vector, a DNA fragment is inserted into a cloning vehicle and then the selected gene is inserted into the modified cloning, vehicle, so that the gene is under the control of the polyhedrin promoter. The recombinant transport vector is then contacted with wild type baculovirus DNA to perform homologous recombination and integration of the selected gene into the baculovirus genome. Baculovirus Autographa and California (AcMNPV) and related polyhedrin promoters have been found to be useful for preparing viral expression vectors capable of expressing very high levels of selected genes in eukaryotic host cells. The present inventors suggest that the expression vectors may be used in insect control systems by selecting genes that produce proteins toxic to a particular insect or a wide range of insects and cloning the gene into an AcMNPV expression vector. It is also suggested that the vector can be applied to a plant or animal to be protected. Recombinant viruses can be ingested by insects and invade cells of the intestinal wall to initiate replication. It is also shown that the gene is inserted into the baculovirus genome so that the gene is fused to the polyhedrin structural sequence such that the polyhedron coat is separated by alkaline conditions in the insect gut to release toxic products.

Another method of constructing insecticidal genes and introducing them to the target to be protected is described in Cutler's Electroporation: Being Developed to Transform Crops: Success with Model Crop Confirmed, AG Biotech. News Vol. 7 (5): 3 17 (1990). This document teaches that when DNA is electroporated directly to germination pots, and then returned to flowers to produce seeds, the seeds grow into transformed plants. The method has been used successfully in tobacco plants and can also be used successfully in corn and alfalfa. This method may be easier than electroporating the protoplasts, rather than regenerating the plant and pollinating the flowers so that the flowers do their work. This process collects the pots, germinates for 30 to 60 minutes in the germination medium, allowing the potted tube to emerge from the potted particles, adds the target DNA to the liquid suspension containing the potted plants, and applies an electric shock to open the potted tube's holes. , Washing and removing excess DNA, placing the modified pollen on the stigma of the plant and waiting for the seed to form. This may be an easy way to deliver certain genes to crops.

Another delivery system is disclosed in US Pat. No. 4,861,595, filed August 29, 1989 to Barnes and Edwards. The invention relates to the use of treated, almost intact microbial cells as a delivery system for delivering protein compounds to animals and humans. Microbial cells initially produce proteins intracellularly by homologous genes. Protein-producing microorganisms are treated by chemical or physical means, but the cells are rarely damaged. The manipulation of the treatment produces non-proliferative treated microbial cells without a significant loss of the activity of intracellular compounds. Since the cells will not replicate and will have a stable cell wall that can be destroyed in the target region of the animal or human digestive system, the release of the encapsulated product according to the invention can be timed or targeted. After proper treatment, the protein-producing microbial cells themselves are used as the delivery system, so purification of the resulting compounds is not necessary. Any protein, polypeptide, amino acid or compound that may be produced by microbial means, including pesticides, may be a starting material of the invention.

Synthesis of a genecoding for an insect-specific scorpion neurotoxin and attempts to express it using baculovirus vectors, Gene for the possibility of using DNA techniques to insert synthetic genes encoding neurotoxins found in scorpion venom 73: 409-18 (1988). This document describes the possibility of improving baculovirus pesticides by using DNA techniques to insert a synthetic gene encoding the neurotoxin found in the poison of the Scorpion Buthus eupeus into the baculovirus genome. Three expression methods were studied using a toxin producing polyhedron promoter-based AcMNPV expression system. Expression of the 36 codon gene alone produced very small amounts of toxins. Successful attachment of signal peptides to toxins. Fusion of the toxin gene to the N-terminus of the polyhedron gene produced significant levels of protein. However, it was produced 10-20 times less than that observed with the polyhedron itself. Restrictions on expression were thought not to be at the level of transcription but to post-transcriptional levels including protein translation and protein stability. No paralytic activity of the toxin product was detected.

European Patent Application Publication No. 0 431 829 is a transgenic that effectively expresses intracellularly insect-specific toxins of animals that eat insects in sufficient amounts to induce toxicity to selective insects that consume plant tissue. The plant is disclosed. Certain toxins described in this European patent application have been isolated from the poison of the scorpion Androctonus australis.

The researchers were also able to isolate the toxins extracted from the spider's poison. Geren et al. (Neurotoxins and Necrotoxins of Spider Venoms, J. Toxicol.-Toxin Reviews 5 (2): 161-170 (1986)) review a study related to neurotoxins and necrosis, with spider poison molecules specific It is also proposed to provide a model for pesticides.

U.S. Patent No. 4,925,664, filed May 15, 1990 to Jackson and Parks, applies a toxin derived from spider agelenopsis aperta and Holona curta. And methods of treating neurological diseases. 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 Publication No. 0 395 357 discloses polyamines and polypeptides isolated from the venom of spider agelenopsis aperta. Polyamines antagonize excitatory amino acid neurotransmitters. One of these polypeptides and polyamines blocks calcium channels in living cells of various organisms. The use of said calcium channel blockers has been suggested for the inhibition of invertebrate pests.

European Patent Application Publication No. 0 374 940 discloses toxins isolated from the venom of the spider Hololena Kurta. This polypeptide is useful as an insecticide, for example as a calcium channel and glutamate antagonist in pharmaceuticals.

Bowers et al., Identification and purification of an irreversible presynaptic neurotoxin from the venom of the spider Hololena curta, Proc. Natl. Acad. Sci. 84: 3506-3510 (1987) discloses the inhibition of neuromuscular transmission in Drosophila larvae and protein neurotoxins isolated from the venom of spider hololona kurta. Bowers et al suggest that the toxin blocks presynaptic calcium channels in the drozophyla motor neuron.

Quistad et al., Paralytic and Insecticidal Toxins from the Funnel Web Spider, Hololena Curta, toxicon 29 (3): 329-336 (1991), describe one peptide purified from the venom of Hollolena Kurta and 10. It describes the effects of when injected into the dog's curtatoxin and Repidofterra larvae.

Stapleton et al. Curtatoxins: Neurotoxic insecticidal polypeptides isolated from the funnel-web spider Hololena curta, J. Biol. Chem. 265 (4): 2054-2059 (1990) discloses the effect on three polypeptide neurotoxins and crickets Acheta domestica isolated from the venom of spider hololona kurta.

Quicke and Usherwood, Extended Summaries Pesticides Group and Physicochemical and Biophysical Panel Symposium Novel Approaches in Agrochemical Research, Pestic. Sci. 20: 315-317 (1987) disclose that toxins present in the venom of parasitic wasps and the venom of some orb-web spiders cause rapid paralysis upon injection into insects. Kickke et al suggest that spider toxin interacts with open channels acted by glutamate receptors in grasshopper skeletal muscle and is a blocker of cation-selective ion channels acted by glutamate receptors. This publication relates to toxins isolated from the low molecular weight toxins of Argiope and Araneus spider venom as well as the venom of Nephila clavata, the Joro spider.

Another study related to the properties of isolated spider toxins revealed the ability to reversibly bind the calcium channels of low molecular weight elements isolated from funnel-web spider poisons. International Publication No. WO 89/07608, published on August 24, 1989 to Cherksey et al., Said active low molecular weight elements reversibly bind to calcium channels with sufficient specificity and affinity to eliminate calcium conductance in neurons and to prevent calcium channels. It is disclosed to enable the isolation and purification of the constructs. The poison has been found to be toxic to mammals. Another use of spider toxins has been described in Jackson and Parks, Spider Toxins: Recent Applications in Neurobiology, Ann Rev Neurosci 12: 405-14 (1989). The document teaches that large heterogeneity exists in toxins of different taxa. Experiments suggest that the species suggest species-specific properties of calcium channels and that spider venom can provide calcium channel antagonists. The spider venom considered has been found to affect vertebrates. The document also holds spider poisons as a possible source of agricultural insect-specific toxins.

Adams et al., Isolation and Biological Activity of Synaptic Toxins from the Venom of the Funnel Web Spider, Agelenopsis Aperta, in Insect Neurochemistry and Neurophysiology 1986, Borkovec and Gelman eds., Humana Press, New Jersey, 1986 It has been taught that multiple peptide toxins that antagonize synaptic transmission in E. coli were isolated from spider agelenopsis aperta.

U.S. Patent No. 4,855,405 to August 8, 1989 to Yoshioka et al. Discloses a receptor inhibitor obtained from Joro spider venom and a method of making the same. When an insect comes into contact with the compound in a liquid or solid carrier, the compound has an insecticidal effect.

Nakajima et al., US Pat. No. 4,918,107, filed April 17, 1990, relates to compounds having glutamate receptor inhibitor activity, methods of making the same, and pesticidal compositions containing the same. The compound is applied directly to the plant or animal to be contained and protected in a liquid or solid carrier to which the dispersant is added. Low doses as insecticides are effective, have very low mammalian and fish toxicity, and less adversely affect the environment.

The use of baculovirus as a biopesticide has also been studied. The main drawback of wild type baculovirus as a biopesticide is that it works slowly. Larvae that consume wild-type baculovirus generally die within 5 to 7 days. Infected larvae, however, continue to be ingested for much of the period, resulting in significant crop damage.

Due to the combination of problems associated with some synthetic insecticides, including loss of efficiency due to toxicity, environmental risks and resistance, genetically engineered to express protein toxins that can render the host incapacitated faster than the baculovirus infection itself. There is a continuing need for the development of novel invertebrate control means, including the development of recombinant baculovirus.

The present invention provides novel peptides and agricultural or horticulturally acceptable salts thereof that are substantially purified from the Diguetia spider venom and have an isolated pesticidal effect.

Peptides having an insecticidal effect of the present invention are believed to have a high degree of selectivity for invertebrates, especially insects. Moreover, since the peptide having the pesticidal effect of the present invention has been proved to be a very effective insecticide against agriculturally important pests, it is considered to be an important contribution to the field of invertebrate pest control.

Also provided by the present invention are novel, substantially isolated DNA sequences encoding peptides having an insecticidal effect that is substantially isolable from Diguetia spider venom.

Also provided by the present invention is a novel recombinant expression vector comprising a DNA sequence encoding a peptide having a pesticidal effect substantially isolated from the Diguetia spider venom, wherein the vector is the coding sequence in a transformed cell. Can be expressed.

The present invention also provides a novel recombinant host cell transformed or transfected with a DNA sequence encoding the peptide in such a way that the host cell expresses a peptide having a substantially isolated insecticidal effect from the Diuetia spider venom. Is provided.

The present invention also provides a novel recombinant baculovirus expression vector capable of expressing a DNA sequence encoding a peptide having a substantially isolateable insecticidal effect from the Diguetia arachnid in a host or in a host insect cell.

The present invention also provides a novel method for producing a peptide having a substantially isolateable pesticidal effect from a Diguetia spider venom and a peptide produced thereby. The method has a DNA sequence encoding a peptide having a pesticidal effect such that a recombinant host cell, wherein the recombinant expression vector transformed or transfected within the host cell can be substantially isolated from the Diguetia spider poison, Cultivating the peptide having the pesticidal effect from the recombinant host cell culture medium.

The invention also relates to a germ cell of a plant, such that a DNA sequence encoding a peptide having a substantially insulated insecticidal effect from the Diguetia spider venom is such that its expression characteristics are inherited by the later generations of the plant through sexual or asexual growth. Provided is a novel transgenic plant comprising the DNA sequence introduced into the plant's aid.

The invention also provides a method for controlling invertebrate pests, the method comprising contacting invertebrate pests with a peptide having substantially insecticidal effect from Diguetia spider poison and an agriculturally or horticulturally acceptable salt effect. New methods are provided.

According to the present invention, in contact with an invertebrate pest with a recombinant baculovirus capable of expressing a peptide having substantially insecticidal effect from Diguetia spider poison and its agriculturally or horticulturally acceptable salt effective amount Also provided are novel methods of controlling invertebrate pests.

The present invention also includes a peptide having a pesticidal effect substantially isolated from the Diguetia spider poison and an agriculturally or horticulturally acceptable salt thereof in an agriculturally or horticulturally acceptable carrier, in an insecticidal effective amount. Provided are novel pesticidal compositions.

In addition, the present invention provides novel antibodies that substantially immunoreact with peptides and their agriculturally or horticulturally acceptable salts that have a substantially insulated insecticidal effect from the Diguetia spider venom.

According to the present invention, there is also provided a method of obtaining a spider venom, contacting the spider venom with an antibody bound to a detectable label, and detecting the labeled antibody bound to the spider venom peptide. Novel methods are provided for detecting the presence of a peptide having an insecticidal effect that is substantially isolable from a Diegtia spider venom using antibodies.

In addition, the present invention is in solution by contacting the antibody to a solid support, contacting the solution containing the peptide having a pesticidal effect substantially isolated from the Diguetia spider poison with the antibody bound to the solid support Using the antibody of the present invention, the method comprising attaching the peptide to the antibody, removing the peptide attached to the antibody bound to the solid support, and collecting the purified peptide. Provided are novel methods of purifying the peptides.

The present invention also provides a novel DNA probe derived from a DNA sequence encoding a peptide having a pesticidal effect that is substantially isolated from the Diueta spider venom.

According to the present invention substantially from a Diguetia spider poison, comprising obtaining a spider nucleic acid, contacting the nucleic acid with a DNA probe of the invention, and detecting the probe bound to the nucleic acid. Also provided are novel methods for detecting the presence of a nucleic acid encoding a peptide having an isolated pesticidal effect.

Figure 1 shows the reverse phase high performance liquid chromatography (RP HPLC) results of Diguetia canities whole venom using a gradient of 0.1% TFA to acetonitrile showing three groups of components tested in mice. Indicates. Fraction 2 represents an ingredient that is active against tobacco budworm (TBW).

2 shows DK 9.2 tested at 1 μM for synaptic transmission (induced population spikes) at Schaffer lateral-CA 1 vertebral cell synapses in the hippocampus slices of rats. Data shown represent time-averaged population spike records recorded for (a) 5 minutes before DK 9.2 addition and (b) 15 to 20 minute intervals after DK 9.2 addition. The two records are identical. This indicates that at this concentration DK 9.2 does not have activity in the rat CNS that can be detected by the assay.

3 is a map of the baculovirus transport vector pWKR9 with the gene encoding preDK 9.2.

FIG. 4 shows the results of continuous virus feeding (100,000 PIB / gm food) to newborn tobacco budworms.

5 shows the ability of tetrodotoxin (TTX) to prevent or reverse DK 9.2-induced excitability.

A. Definition

Peptides having an insecticidal effect that is substantially isolable from the Diguetia spider venom as used herein are obtained from any source so long as the peptide is substantially isolable from Diguetia by any technique known in the art. Peptides with pesticidal effects and fragments with pesticidal effects thereof. Such sources include, for example, peptides with pesticidal effects produced by recombinant techniques.

Expression vectors as used herein include vectors capable of expressing DNA sequences by operably linked DNA sequences contained therein to other sequences capable of expressing them. Although not always explicitly mentioned, it is implied that the expression vector must be replicated in the host organism either as an episome or as an integral part of chromosomal DNA. Clearly, lack of replication makes these vectors ineffective. In sum, expression vectors are given functional definitions and include any DNA sequence capable of expressing a particular DNA code arranged in the vector as applied to a particular sequence. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids that refer to circular double-stranded DNA that is not bound to the chromosome in the vector form. Plasmids and vectors are used interchangeably because plasmids are most commonly used in vector form. However, in the present invention, it refers to a cell which provides an equivalent action and transforms a recombinant host cell with a vector constructed using the recombinant DNA technique. It is also intended to include other forms of expression vectors that will be known in the art.

Diguetidae and spiders now consist of a single genus Diguetia. Diguetia species are commonly found in the southwestern United States (including California) and Mexico. These tiny spiders build a wide range of irregular cobwebs on many kinds of plants and shrubs, including cacti. Diguetia of various species, like most spiders, is a common predator that easily eats most of the types of trapping insects they encounter.

Although the present invention is not limited to any theoretical basis, an unexpected syndrome which can be seen when injecting a peptide having insecticidal effect of the present invention into an insect is a bootie known as an alkaloid Na + channel activator veratridine or a display synaptic Na + channel activator It is very similar to the symptoms of scorpion toxins from the genus Buthidae and Buthus. It is contemplated that peptides with this pesticidal effect may cause partial depolarization of muscle and / or neural membranes, possibly affecting Na + or K + channels. More specifically, the peptide having the insecticidal effect is thought to induce a repetitive excitatory discharge in nerves sensitive to blocking by tetrodotoxin. Thus, the site of action of the peptide would be the voltage-sensitive sodium channel of the neural membrane.

B. Isolation of Peptides from Diguetia Toxin

Spider poison can be removed from Diguetia by any known method, such as extraction of venom from the head. However, to avoid spider poisons and impurities in isolated toxins, as described in US Pat. No. 4,925,664, the spider is electrically stimulated to induce the release of venom, which is then inhaled to collect the released toxins and to reflux or hemolymph. It is desirable to obtain spider poison by preventing contamination of the poison by.

Once spider venom is obtained by an electrically poisoning technique, fractionate spider venom into its peptide (toxin) components using high performance liquid chromatography (HPLC) or gel filtration chromatography or any other useful fractionation technique. You can. In most cases it is also preferred that the final fractionation of spider venom is carried out by HPLC.

Thus, using a technique of electrically poisoning from spiders, combined with gel filtration chromatography and / or other related techniques such as high performance liquid chromatography and hydrophobic interaction chromatography, it is possible to obtain substantially purified spider toxins. Can be. However, other equivalent techniques may be used within the scope of the present invention to isolate spider toxins. Toxins isolated as described above can be assessed for amino acid sequence and pesticidal activity by methods known in the art.

C. Peptides with Insecticidal Effects

The present invention provides, in one aspect thereof, peptides having substantially pesticidal effects and purified from Diguetia spider venom, and fragments having pesticidal effects thereof, and agriculturally or horticulturally acceptable salts thereof.

Once the peptide-containing fraction having pesticidal effect is isolated and purified as described above, the amino acid sequence can be determined by any method known in the art, such as using N-terminal amino acid sequencing and automated amino acid sequencing.

It will be expected from the present disclosure that proteins with additional pesticidal effects fall within the scope of the present invention. That is, it is contemplated that peptides having other insecticidal effects in addition to the three described above can be isolated from Diguea. The following relates to a class of proteins having an insecticidal effect that can be isolated from Diguetia. Members of the class of peptides with pesticidal effects isolated from Diguetia seem to share the following properties:

1) size: all in the range of about 6200 to about 7200 daltons, the length of the range of about 55 to about 65 amino acids;

2) conserved amino acid termini: SEQ ID NOs: 1, 3 and 5 are identical to the first 5 amino acids;

3) SEQ ID NOs: 1, 3 and 5 have at least 40% sequence homology;

4) SEQ ID NOs: 1,3 and 5 have about 7 or 8 cysteine residues;

5) Clustering of Genes: Genes for one or more of these toxins appear to be located in a common region of genomic DNA, indicating that all of these genes may be from ancestral genes of single origin.

More specifically, peptides with three pesticidal effects were isolated and characterized. First, DK 9.2 was isolated and its amino acid sequence as translated from isolated cDNA was defined by SEQ ID NO: 1. Mass spectrometric data show two isozymes containing conservative substituents at position 26. One isozyme contains threonine at position 26 and the other contains glutamine at position 26. Glutamine isozyme is about 27 atomic weight greater than threonine isozyme. Any reference to DK 9.2 hereafter should be interpreted as referring to either and / or both isozymes. DK 9.2 is characterized by a molecular weight of about 6371 to 6397 Daltons and a shift as single peak in reverse phase HPLC as measured by mass spectrometry. Secondly, DK 11 was isolated, which is characterized by a molecular weight of about 6700 Daltons or about 6740 Daltons as measured by mass spectroscopy and shift as single peak in reverse phase HPLC. More particularly, the active HPLC fraction was found to have an amino acid sequence as shown in SEQ ID NO: 3 when translated from the isolated cDNA. Finally, to date, the third member of the class of proteins with this pesticidal effect, DK 12, is characterized by a molecular weight of about 7100 Daltons or about 7080 Daltons as measured by mass spectroscopy and migrates as a single peak on reverse phase HPLC. More particularly, the active fraction has been found to have an amino acid sequence as shown experimentally by SEQ ID NO: 5. The three isolated peptides corresponding to SEQ ID NOs: 1, 3 and 5 exhibit substantially the same pesticidal activity. It is expected that other peptides having about 55-65 amino acids, at least 40% sequence homology with SEQ ID NOs 1, 3 or 5 and about 7 or 8 cysteine residues also exhibit substantially the same pesticidal activity. The peptides may be naturally present or may be prepared by synthetic DNA techniques known to those of ordinary skill in the art. Such peptides are within the scope of the present invention, whether natural or prepared by recombinant methods.

D. Identification of Coding Sequences of Peptides Having Insecticidal Effects of the Invention

In another aspect of the present invention, a substantially isolated DNA sequence is provided that encodes a peptide having a substantially isolateable insecticidal effect from a Diguetia spider venom.

The partial amino acid sequence data obtained as described above can be used to isolate and identify users who are capable of producing isolateable proteins from spiders. There are many useful methods for obtaining genes capable of producing peptides. See, eg, Fugua, S. et al., A simple RCR method for detection and cloning low abundunt transcript, Biotechnique, Vol. 9, No. 2 (Aug. 1990); Frohman, M.A., RACE: Rapid amplification of cDAN ends, PCR protocols, ed. Innis et al., Academic Press, San Diego, CA, (1990) and US Pat. No. 4,703,008, entitled DNA Sequences Encoding Erythropoietin, which is incorporated herein by reference.

Briefly, a DNA molecule is synthesized that represents a DNA strand encoding a determined amino acid sequence or complementary to a DNA molecule encoding a determined amino acid sequence. The synthetic DNA molecule then contains a recombinant DNA molecule derived from genomic DNA of an organism such as a spider or partially comprising a DNA sequence derived from a cDNA copy of an mRNA molecule isolated from a cell or tissue of an organism such as a spider. It can be used as a probe for DNA sequence homology in cell clones. Generally, DNA molecules with 15 or more nucleotides are required for the specific identification of homologous DNA, the number requiring specific determination of at least 5 amino acids in the sequence. It will be appreciated that the number of different DNA molecules capable of encoding the determined amino acid sequence may be very large because each amino acid can be encoded by up to six specific trinucleotide DNA sequences or codons. Therefore, it is impractical to test all possible synthetic DNA probes individually, using pools of multiple such DNA molecules simultaneously as probes. The preparation of such pools referred to as degenerate probes is known in the art. Only one DNA molecule in the probe mixture will have exact sequence homology to the gene in question, but only a high degree of homology is needed so that multiple synthetic DNA molecules in the pool can specifically identify the gene. . Therefore, successful isolation of the gene in question can be achieved by synthetic DNA probe pools that do not contain all possible DNA probe sequences. In general, codons that are not used very often by the organism need not appear in the probe pool. In fact, single sequence DNA probes can be prepared by including only the DNA codon most frequently used for each amino acid by the organism, but this method is not always successful.

One technique for identifying gene sequences uses Polymerase Chain Reaction (PCR) (see US Pat. Nos. 4,683,195 and 4,683,202, incorporated herein by reference). Essentially PCR allows the generation of selected DNA sequences when the two terminal portions of the selected sequence are known. Obtain a primer or oligonucleotide probe corresponding to each end of the sequence in question. PCR is then used to synthesize the central portion of the DNA sequence.

As one method of obtaining a gene encoding a specific spider venom gene using PCR, RNA is isolated from the spider and purified. Deoxythymidylate-tailed oligonucleotides are then used as primers to reverse transcribe the spider RNA into cDNA. Then, a synthetic DNA molecule or a mixture of synthetic DNA molecules is prepared as in the degenerate probe described above, which can encode the amino-terminal amino sequence of the poison protein as measured above. This DNA mixture is used with oligonucleotides having deoxythymidylate-terminus to initiate the PCR reaction. The synthetic DNA mixture used to initiate the PCR reaction will be specifically amplified only for the target cDNA, specific to the target mRNA sequence. The product represents amplified cDNA that can be linked to any of many known cloning vectors. However, it will be appreciated that peptide classes with similar amino acid sequences may be present in spider venom, in which case mixed oligonucleotide primer sequences may be used to result in amplification of one or more related cDNAs encoding the related peptides. Since related peptides also have useful pesticidal activity, genes encoding related peptides are also within the scope of the present invention.

Finally, the resulting cDNA sequences can be cloned into appropriate vectors using conventional techniques, analyzed, and then the nucleotide sequences determined. DNA sequences encoding proteins with pesticidal effects are shown in SEQ ID NOs: 2 and 4. Direct amino acid translation of the PCR products shows that they correspond to the complete coding sequence for the mature protein.

In addition to the cDNA encoding mature DK 9.2, the upstream cDNA sequence of the DK 9.2 coding sequence was cloned and sequenced (SEQ ID NO: 7). From the translation of the complete mRNA it can be seen that DK 9.2 is synthesized as a precursor protein comprising signal peptide, propeptide and mature toxin. The function of the signal peptide is believed to be important for targeting synthetic polypeptides for secretion. Signal sequences play an important role in proper localization of newly synthesized proteins. In general, they are topogenic signals that target protein sequences attached to various destinations intracellularly or extracellularly [Blobel, G. Proc. Nat. Acad. Sci., U.S.A. 77, 1496-1500 (1980). This is particularly important for secreted proteins whose target site is extracellular. It also facilitates recombinant protein preparation because it may be easier to purify the expressed protein from the medium of the cell rather than to lyse the cells and purify from the whole cell extract. It is believed that the signal peptide encoded by the cDNA encoding the precursor protein of DK 9.2 consists of 17 amino acids of the following sequence:

Met-Lys-Val-Phe-Val-Val-Leu-Leu-Cys-Leu-Ser-Leu-Ala-

Ala-Val-Tyr-Ala

In addition to the signal peptide, translation of mRNA located upstream of the DK 9.2 coding sequence showed propeptide. The action of the propeptide sequence is unknown. The propeptide is a 21 amino acid peptide of the following sequence:

-Leu-Glu-Glu-Arg-Leu-Asp-Lys-Asp-Ala-Asp-Ile-Met-Leu-

Asp-Aer-Pro-Ala-Asp-Met-Glu-Arg-

The precursor peptide or prepro sequence may also contribute significantly to the stability, expression and folding of DK 9.2 or other recombinant protein when expressed in vivo and / or in vitro. It is also contemplated that such sequences, or portions thereof, may prove useful for the expression of other molecules, for example for gene production. As part of the present invention, data will also be provided confirming that other signals and / or propeptide sequences can be used for expression of recombinant DK 9.2.

E. Recombinant Expression

The present invention also provides a recombinant expression vector comprising a DNA sequence encoding a peptide having an insecticidal effect that can be substantially isolated from a Diegtia spider venom. The vector can express the coding sequence in the transformed cell. The invention also provides a recombinant host cell transformed or transfected with a DNA sequence encoding a peptide having a pesticidal effect that can be substantially isolated from the Diguetia spider venom in a manner that allows the host cell to express the peptide.

By providing a suitable DNA sequence encoding the protein of interest, the protein can be prepared using recombinant techniques known in the art. Such coding sequences may be obtained by recovering cDNA or genomic sequences from natural protein sources, or may be synthetically prepared using exact amino acid sequences determined from the nucleotide sequences of the genes. For the synthetic preparation of coding DNA, known codon preferences of the intended host can be utilized.

Expression systems containing promoters and essential regulatory sequences, preferably promoters and termination regulators, are readily available and known in the art for various hosts (see, eg, Sambrook et al., Molecular Colning a Laboratory Manual, Second Ed.Cold Spring Harbor Press (1989)].

Thus, the protein of interest can be produced in both prokaryotic and eukaryotic systems so that many proteins can be produced as a series of processed forms.

The most commonly used prokaryotic system is this. Although E. coli, other systems such as B. subtilis and Pseudomonas are also expected to be useful. Regulatory sequences suitable for prokaryotic systems include both lac promoters, trp promoters, hybrid promoters such as the tac promoter, and constitutive and inducible promoters including lambda phage Pl promoters. In general, foreign proteins can be prepared as fusion or mature proteins in such hosts. When the desired sequence is produced with a mature protein, the resulting sequence may be preceded by methionine, which is not necessarily effectively removed. Thus, the peptides and proteins claimed herein may be preceded by N-terminal Met when produced in bacteria. Moreover, constructs can be prepared in which the operable signal peptide precedes the coding sequence for the peptide, resulting in protein secretion. In this regard, when produced in a prokaryotic host, the signal sequence is removed upon secretion.

A wide variety of eukaryotic hosts are also available for the production of recombinant foreign proteins. As in bacteria, eukaryotic hosts may be transformed into expression systems that produce the desired protein directly, but more commonly, signal sequences are provided to secrete the protein. Eukaryotic systems have the added advantage of being able to process introns that may occur in genomic sequences encoding proteins of higher organisms. Eukaryotic systems also provide a variety of process mechanisms that cause, for example, glycosylation, oxidation or derivatization of certain amino acid residues, structural control, and the like.

Commonly used eukaryotic systems include yeast, insect cells, mammalian cells, algal cells and cells of higher plants. The list is not limiting. Terminator sequences and enhancers can be used as well as compatible and operable promoters for use in each of the above host types, such as for example the baculovirus polyhedrin promoter. As above, the promoter may be structural or inducible. For example, in mammalian systems the MTII promoter can be derived by adding heavy metal ions.

Details required for the construction of an expression system suitable for the host of interest are known in the art. The DNA encoding it for production of the recombinant protein is appropriately linked to a selected expression system, which is then transformed into a compatible host and the host is then cultured and maintained under conditions in which expression of the foreign gene occurs. The protein having the insecticidal effect of the present invention produced as described above is recovered from the culture medium, by melting the cells or from the medium according to methods known and appropriate in the art.

Slight modifications of the primary amino acid sequence can result in a protein having substantially equal or enhanced activity compared to the peptides exemplified herein. Such modifications may be intentional, such as by site-directed mutagenesis, but may also be accidental, such as by mutations in the host producing the peptide of the invention, and all such modifications are included so long as the insecticidal effect is maintained. Mutations in a protein modify the protein's primary structure due to changes in the nucleotide sequence of the DNA encoding it (for proteins that are normally present or specifically described). These mutations specifically include allelic variants. Mutagenic changes in the primary structure of a protein result from deletions, additions or substitutions. A deletion is defined as a polypeptide that does not have one or more internal amino acid residues relative to the wild type. Additions are defined as polypeptides having one or more additional internal amino acid residues relative to the wild type. Substitution occurs when one or more amino acid residues are replaced with other residues. A protein segment is a polypeptide consisting of a primary amino acid sequence identical to a portion of the primary sequence of the protein with which the polypeptide is associated.

Preferred substitutions are those that are conservative, ie one residue is replaced with another residue of the same general type. As is well known, natural amino acids can be classified as acidic, basic, neutral and polar, or neutral and nonpolar and / or aromatic. In general, it is preferred that encoded peptides that differ from their natural form contain substituted codons for the amino acids to be substituted and for amino acids belonging to the same group.

Thus, in general 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, and the nonpolar aliphatic acid Gly , Ala, Val, Ile and Leu are conservative with respect to each other (but due to size, Gly and Ala are more closely related and Val, Ile and Leu are more closely related) and the aromatic amino acids Phe, Trp and Tyr This is interchangeable.

Pro is a nonpolar neutral amino acid, but due to its influence on the structure, it is difficult to substitute Pro or replace Pro, except when the same or similar structural results are obtained. Polar amino acids exhibiting conservative changes include Ser, Thr, Gln, Asn and less, but Met. In addition, although classified into other categories, Ala, Gly and Ser also appear to be interchangeable, and Cys may additionally belong to this group or be classified as a polar neutral amino acid. Some substitutions by codons for different classes of amino acids may also be useful.

Since recombinant materials for the proteins of the invention are provided, these proteins can be produced by recombinant techniques as well as by autoamino acid synthesizers. Due to the variety of post-translational properties provided by the various host cells, various modifications to natural proteins will also be obtained. Modified proteins differ from unmodified proteins as a result of glycosylation, amidation or lipidation patterns, or post-translational processes that alter the primary, secondary or tertiary structure of the protein, but as claimed Of course it is included within the scope of.

It should be noted that when a protein of this disclosure, such as SEQ ID NO: 1, is prepared synthetically, substitutions by amino acids not encoded by the gene may also be possible. Alternative residues include, for example, ω amino acids of the general formula H ₂ N (CH ₂ ) _n COOH, where n is 2 to 6. These are neutral, nonpolar amino acids such as sarcosine (Sar), t-butylalanine (t-BuAla), t-butylglycine (t-BuGly), N-methyl isoleucine (N-MeIle) and norleucine (Nleu). For example, pheniglycine can be substituted for Trp, Tyr or Phe aromatic neutral amino acids, citrulline (Cit) and methionine sulfoxide (MSO) are polar or neutral, cyclohexyl alanine (Cha) is neutral and nonpolar, Cysteic acid (Cya) is acidic and ornithine (Orn) is basic. Structures that confer the nature of proline residues can be obtained when one or more of these is substituted with hydroxyproline (Hyp).

F. Genetically Transgenic Plants

The present invention also provides for the expression of DNA sequences encoding peptides having a substantially isolateable insecticidal effect from the Diguetia spider venom, introduced into the germline of the plant such that it is inherited by the later generations of the plant via sexual or asexual propagation. Provided are gene transfer plants comprising a DNA sequence.

Genes encoding peptides with pesticidal effects according to the present invention can be introduced into plants by genetic engineering techniques, which are expected to be useful as a means of controlling insect pests in the production of peptides in plant cells. Therefore, it is possible to produce plants that are more resistant to insects than natural species.

The coding region for a peptide gene having a pesticidal effect that can be used to transform a plant can be the full length or partial active length of the gene. However, the gene sequence encoding the peptide must be generated and expressed as a functional peptide in the resulting plant cells. It is contemplated that genomic DNA, cDNA and synthetic DNA encoding peptides with pesticidal effects can be transformed. In addition, the gene may consist in part of cDNA clones, partly genomic clones and partly synthetic genes and various combinations thereof. In addition, the DNA encoding the gene of the peptide may comprise moieties obtained from various other species other than the source of the isolated peptide.

Moreover, peptides having an insecticidal effect can be mixed with another compound or compounds, resulting in unexpected pesticidality in transformed plants containing chimeric genes that express the compounds. These other compounds can include, for example, protease inhibitors or oral polypeptides from Bacillus thuringiensis that are orally toxic to insects. B. turingiensis proteins are thought to cause changes in the potassium permeability of insect intestinal cell membranes and to produce small pores in the membranes. Other pore-forming proteins can also be used with peptides that have pesticidal effects. Examples of such pore-forming proteins include: magenin, cecropin, atacin, methine, gramicidin S, sodium channel protein and synthetic fragments, α-toxin, apolipoprotein of Staphylococcus aureus And fragments thereof, alamethycin and various synthetic amphipathic peptides. Lectins, which bind to cell membranes to enhance endocytosis, are another class of proteins that can be used with the pesticidal effects of the invention to genetically modify plants for insect resistance.

Promoters of peptide genes are expected to be useful for expressing chimeric gene sequences, but other promoters are also expected to be useful. An effective plant promoter that may be useful is an overproduction promoter. The promoter is operably linked with the gene sequence for the peptide and should be capable of promoting the expression of the peptide so that the transformed plant has increased resistance to insect pests. Overproduction plant promoters are known that are expected to be useful in the present invention.

A chimeric gene sequence comprising a peptide gene having a pesticidal effect operably linked to a promoter can be linked to a suitable cloning vector to transform the desired plant. Generally, plasmids or viral (bacteriophage) vectors containing replication and regulatory sequences derived from species compatible with the host cell are used. Cloning vectors will typically have specific genes capable of providing phenotypic selection markers to the origin of replication as well as transformed host cells, which are typically resistant to antibiotics. Transformation vectors may be selected by said phenotypic markers after transformation in host cells.

Host cells that are expected to be useful are E. coli. Prokaryotes, including bacterial hosts such as E. coli, Salmonella ryphimurium, and Serratia marcescens, and eukaryotic hosts such as yeast or filamentous fungi.

In general, cloning vectors and host cells transformed with the vector are used to increase the number of copies of the vector. If the copy number is increased, vectors containing peptide genes can be isolated and used, for example, to introduce the gene sequences described above into plants or other host cells.

Methods for producing plants expressing foreign genes are known. For example, direct infection or co-culture by A. tumefaciens of plants, plant tissues or cells; Direct gene transfer into the plasma of exogenous DNA; Incubation with PEG; Microorganisms can be transformed by microinjection and microinjection bombardment.

Ag Biotechnology News, Vol. 7 p. 3 and 17 (Sept / Oct 1990), the transformation in tobacco by the electroporation technique described above has been identified. In this technique, plant plasma is electroporated in the presence of plasmids containing peptide gene constructs with pesticidal effect. High intensity electric shocks make the biomembrane reversibly permeable, allowing the introduction of plasmids. Electroporated plant plasma regenerates and divides the cell walls, forming plant callus. The selection of plant cells transformed with the peptides having the expressed pesticidal effect can be carried out using phenotypic markers as described above. Exogenous DNA can be in any form, for example, naked linear, circular or supercoiled DNA, DNA encapsulated in liposomes, DNA in spherooplasts, DNA in other plant plasmas, DNA may be added to the protoplast, such as DNA complexed with a salt.

All plant cells that can be transformed by Agrobacterium and the whole plant regenerated from the transformed cells also contain the present invention in order to produce transformed whole plants containing peptide genes with a metastatic pesticidal effect. Can be transformed according to. Transformation of rice is described in D. M. Raineri et al., Agrobacterium-mediated transformation of rice (Oryza sativa 1.), Biotechnology, Vol. 8, pp 33-38 (January 1990).

Another method for introducing pesticidal peptide genes into plant cells is to transform A. mutated into peptide genes having pesticidal effects. Infection of plant cells with tubemefaciens. Under appropriate conditions known in the art, transformed plant cells are grown to produce shoots, roots, and further grow into transformed plants. Peptide gene sequences having an insecticidal effect are described, for example, in A. a. Ti plasmids from tumefaciens can be introduced into appropriate plant cells. Ti plasmid is a. When infected by tumefaciens, it is delivered to plant cells and stably merges into the plant genome.

Ti plasmids contain two regions that are considered essential for the production of transformed cells. One of these, called transport DNA (T DNA), induces tumorigenesis. The other, called the viral region, is essential for tumor formation but has nothing to do with tumor maintenance. The T DNA region that is transferred to the plant genome can be increased in size by inserting the gene sequence of the enzyme without affecting its carrying capacity. Modified Ti plasmids can be used as vectors to transport the gene constructs of the present invention into appropriate plant cells by removing tumor-causing genes so that they no longer interfere.

Genetic material may also be transported into plant cells using polyethylene glycol (PEG), which forms a precipitate complex with the genetic material taken up by the cell.

Delivery of DNA into plant cells can also be accomplished by injecting into isolated protoplasts, cultured cells and tissues, and into seedlings and plant meristems. Transgenic plants and offspring therefrom are obtained by conventional methods known in the art.

Another method of introducing foreign DNA sequences into plant cells involves attaching the DNA to a particle that is forced into the plant cell using a launching device, a gene gun. As a target for this method, any plant tissue or plant organ can be used, including but not limited to embryos, apex and other meristems, shoots, somatic tissues and reproductive tissues in vivo and in vitro. Gene metastatic cells and callus are selected according to established methods. Targeted tissue is induced to form somatic embryos or to regenerate shoots to obtain transgenic plants according to procedures known in the art. The appropriate method can be selected depending on the plant species used. Transgenic maize plants have been prepared by transporting genes into embryonic cells using a high speed microinjection device [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 regard to the inserted DNA. When cells containing foreign DNA develop into vesicles or blastes, the coalesced foreign DNA will be delivered to the offspring of sexual reproduction. If the cell containing the foreign DNA is a somatic cell of a plant, the non-chimeric transgenic plant is produced by conventional asexual proliferation methods from shoot or stem cuts in vivo or in vitro according to procedures known in the art. The procedure can be selected depending on the plant species used.

After the plant cells or plants are transformed, the transformed plant cells or plants can be selected by appropriate phenotypic markers so that the peptide is expressed. These phenotypic markers include but are not limited to antibiotic resistance. Other phenotypic markers are known in the art and may be used in the present invention.

Due to a variety of different transformation systems, all plant types can in principle be transformed such that they express peptides having the pesticidal effect of the invention.

There is evidence that virtually all plants can be recovered from cultured cells or tissues, including, but not limited to, major grain crops, sugar cane, sugar beets, cotton, fruits and other trees, legumes and vegetables. It is increasing. Knowledge of whether all these plants can be transformed by Agrobacterium is currently limited. Species that are natural plant hosts for Agrobacterium may also be transformed in vitro. Monocotyledonous plants, especially cereals and grasses, are not natural hosts of Agrobacterium. Attempts to transform them with Agrobacterium have not been successful until recently, but there is now increasing evidence that certain monocotyledonous plants can be transformed by Agrobacterium. Using new experimental approaches now available, cereals and grass species can also be transformed.

Additional plants that may be transformed by Agrobacterium include Ipomoea, Passiflora, Cyclamen, Malus, Prunus, and Rosa. , Rubus, Populus, Santanion, Allium, Lilium, Nacissus, Ananas, Arachis, and Paseolus Phaseolus) and Pisum.

The regeneration process depends on the species of the plant, but generally provides a suspension of the transformed plasma containing multiple copies of the peptide gene, which generally have a pesticidal effect. The formation of embryos can then be induced from the plasma suspension to maturation and germination as natural embryos. The medium generally contains various amino acids and hormones. The buds and roots generally grow at the same time. Effective regeneration depends on the medium, genotyping and culturing process. When these three parameters are adjusted, regeneration is fully reproducible and repeatable.

Mature plants grown from transformed plant cells can produce allogeneic plants themselves. Allogeneic plants produce seeds containing genes for peptides with pesticidal effects. The seed can grow to produce a plant expressing a peptide having an insecticidal effect. For example, homologous plants can be used to develop insect resistant hybrids. In this method, an insect resistant homologous breeding system is crossed with another homologous breeding system to create a hybrid.

In diploid plants, typically one parent plant can be transformed with a peptide (toxin) gene sequence that has an insecticidal effect and the other parent plant is of wild type. After mating the parent plants, the first large hybrid (F ₁ ) will exhibit a distribution of 1/2 toxin / wild type: 1/2 toxin / wild type. The first large hybrid F ₁ generates a second large hybrid F ₂ by itself. The gene distribution of the F ₂ hybrid is 1/4 toxin / toxin: 1/2 toxin / wild type: 1/4 wild type / wild type. F ₂ hybrids with the toxin / toxin's genetic makeup are selected as insect resistant plants.

Variants herein refer to changes in the phenotype that are stable and can be inherited, including genetic variations that are sexually transmitted to plant progeny. Provided that the variant still expresses the peptide having the pesticidal effect of the present invention. A mutant herein refers to a variation that occurs as a result of an environmental condition such as irradiation or as a result of genetic variation that is meially transmitted according to well-characterized genetic laws. However, mutant plants must still express the peptides of the invention.

In general, an ideal pesticidal protein selected for expression in a transgenic plant is characterized by stability against non-target insects and vertebrates. Expression systems will be chosen such that expression levels provide pesticidal effects. Thus, the technical possibilities of obtaining these agriculturally important genetically transgenic plants are expected to provide farmers with another means to use in integrated pest management systems to reduce insect damage to crops in an environmentally safe manner. .

G. Application of Peptides as Pesticides

Peptides having a pesticidal effect of the present invention are expected to be useful for controlling invertebrate pests such as the tree of Lepidoptera by contacting the pest with an effective amount of the peptide of the present invention. For convenience, insects are the preferred pests.

Methods of controlling such pests by contacting invertebrate pests with peptides are known. Examples include synthetically encapsulating a protein to be taken orally by a pest. Recombinant hosts expressing proteins of the invention, such as Pseudomonas fluorescens, can be heat killed and subsequently applied to pests for oral ingestion and control.

Method of controlling invertebrate pests using the protein of the present invention can be used with other pest control methods, of course. For example, the above-described transgenic plants and these. E. coli can be genetically engineered to express other invertebrate toxins depending on the type of pest being controlled and other important variables.

Also provided are pesticidal compositions comprising an insecticidal effective amount of a peptide according to the invention and an agriculturally or horticulturally acceptable salt thereof in an agriculturally or horticulturally acceptable carrier.

H. Antibodies Against Peptides Having Insecticidal Effects

Another aspect of the invention is an antibody against a peptide having the pesticidal effect of the invention. In the following description, various methodologies known to those skilled in the art of immunology for detecting and purifying peptides reactive with the antibodies described herein will be mentioned.

An antibody is said to be able to bind a molecule if the molecule can be bound to the antibody by specifically reacting with the molecule. The term epitope refers to the portion of a peptide antigen that can be recognized and bound by an antibody. The antigen may have one or more epitopes. The antigen can induce the animal to produce an antibody that can bind to the epitope of the antigen. The specific response mentioned above means that the antigen is immune to its corresponding antibody in a highly selective manner and not to many other antibodies which may be caused by other antigens.

The term antibody (Ab) or monoclonal antibody (Mab) herein is meant to include not only complete molecules capable of binding antigen, but also fragments thereof (eg, Fab and F (ab ') ₂ fragments). Fab and F (ab ') ₂ fragments lack Fc fragments of complete antibodies, are removed more rapidly from circulating blood, and may have less non-specific tissue binding of insect antibodies.

Antibodies of the invention can be prepared by any of a variety of methods. Methods for preparing such antibodies are known and described in detail in the literature (see, eg, Sambrook et al., Molecular Cloning a laboratory manual, second ed. Cold Spring Harbor Press, Vol. 3, Ch. 18 (1989) )]Reference). For example, an animal can be administered to cells expressing the peptide having a pesticidal effect or a fragment thereof to induce the preparation of a serum containing a polyclonal antibody capable of binding to the peptide having a pesticidal effect. In general, peptide fragments with pesticidal effects are prepared, purified, or synthesized to contain little natural contaminants according to methods known in the art. Purified fragments or synthesized fragments or mixtures of purified natural fragments and / or synthetic fragments can be introduced into the animal to produce polyclonal antisera with greater specific activity.

Monoclonal antibodies can be prepared using known hybridoma techniques. In general, the procedure involves immunizing the animal with an antigen that has a pesticidal effect. Splenocytes of the animal are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line can be used 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 the selection are then analyzed to identify clones that secrete antibodies capable of binding to the peptide antigen with pesticidal effect.

If the peptide source is impure, only some hybridoma cells will produce antibodies that can bind to the peptide (other hybridoma cells will produce antibodies that can bind to peptide contaminants). Therefore, it may be necessary to screen for cells capable of secreting antibodies capable of binding a peptide among the hybridoma cells. The screening may involve culturing peptide (or venom) samples in the presence of monoclonal antibodies secreted from each of the specific hybridoma cell groups and secreting antibodies capable of neutralizing or alleviating the poison's ability to paralyze insects. It is preferable to carry out by identifying hybridoma cells. Once the hybridoma cells are identified, the cells can be cloned by means known in the art to generate peptide-specific monoclonal antibodies.

In order to characterize insect selective toxins (natural or recombinant) using antibody affinity chromatography, it is essential to use antibodies that can bind to peptides with pesticidal effects. Generally, the antibody is a monoclonal antibody. Once a peptide-specific monoclonal antibody is obtained, the antibody is immobilized by binding to a solid support and used to purify the peptide from natural poisons or other sources using immunoaffinity chromatography according to methods known in the art. Can be. The method allows for a high degree of purification to produce peptides that contain little natural contaminants. As used herein, a peptide is said to contain virtually no natural contaminants when naturally present in a form that lacks a compound (ie, other proteins, lipids, carbohydrates, etc.) normally associated therewith.

Once the peptide is purified, the peptide can be used to immunize an animal (eg mouse or rabbit) to induce the production of peptide-specific polyclonal antibodies.

DNA probes of suitable size, generally 10-50 nucleotides, can be derived from DNA sequences encoding peptides with a pesticidal effect that can be substantially isolated from the Diguetia spider venom. The probe encodes a peptide having a pesticidal effect that can be substantially isolated from the Diguetia spider poison by contacting the DNA encoding the poison with the DNA probe and detecting the probe bound to the DNA by methods known in the art. It can be used to detect the presence of DNA.

I. Insecticidal Microorganisms

Peptides with pesticidal effects are expected to be useful for enhancing or enhancing the toxicity of microorganisms such as baculovirus and hybrid bacteria, alone or in combination with other insect toxins.

Heliothis virescens (cotton beetle), Orgyia pseudotsugata (Douglas fir venom), Lymantria dispar (cicada moth), Autographa California (Autographa) A variety of baculoviruses in some countries, including infections of californica (Puganus zavalle), Neodiprion sertifer (European pine flies), and Laspeyresia pomonella (moths) It is approved and used as a pesticide. The introduction of at least one insect-selective toxin into the genome is expected to significantly increase the potency of the insecticide.

Recombinant expression vectors that are expected to be particularly suitable for use in the present invention are baculovirus expression vectors of the type disclosed in US Pat. No. 4,879,236, which is incorporated herein by reference in its entirety (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 has been described in US Pat. No. 4,879,236 and in Miller et al., Science, 219, 715-721 (1983). It is expected to be useful for cyste that can be cloned into baculoviruses such as autographer californica (AcMNPV) expression vectors as shown. The recombinant expression vector virus can then be applied to the plant or animal that the insect is a pest, and when the virus is ingested by the pest insect, the recombinant virus will invade cells of the insect barrier and begin replication. Upon replication, genes for proteins with insecticidal effects will be expressed, causing insects to become incapacitated or killed within a shorter period of time than when insects ingested the wild type AcMNPV virus.

In addition, hybrid viruses that are expected to be useful are taught in EP 0 340 948. Hybrid viruses expressing the DNA of the present invention are expected to provide viruses with a modified insect host range. For example, the fusion protein may be expressed as a single polypeptide of a hybrid gene consisting of the DNA of the present invention and a specific insect gut cell recognition protein gene that induces a peptide having an expressed pesticidal effect as a host insect target.

Various prokaryotic and eukaryotic microorganisms can be transformed by the method taught in EP 0 325 400 to express hybrid toxin genes encoding proteins with pesticidal effects.

Hybrid bacterial cells comprising plasmids with genes encoding proteins of the invention are expected to be useful in the methods of the invention. Insects are controlled by applying the hybrids to insects (see, eg, US Pat. No. 4,797,279, incorporated herein by reference in its entirety).

Other examples of using suitable baculoviruses prior to use in the present invention include Tomski et al., Insect paralysis by baculovirus-mediated expression of a miteneurotoxin gene, Nature, 352: 82-85 (1991) and Stewart et al. , Construction of an improved baculovirus insecticide containing an insect-specific toxin gene, Nature, 352: 85-88 (1991); McCutchen, et al., Development of a recombinant Baculovirus expressing an insect selective Neurotoxin: Potential for Pest Control, Biotechnology, 9: 848-851 (1991).

J. Cross-Hybrid Formation: DNA Sequence as Probe for Related Compound

DNA probes of suitable size can be derived from the DNA sequences of the present invention. The probe can be used to hybridize with nucleic acids from other sources to detect the presence of nucleic acids encoding peptides having the pesticidal effect of the present invention. Screening with signal sequences, fragments of cDNA or even oligonucleotide probes encoding whole cDNAs under reduced stringency conditions allows access to other active peptides having functional homology to the class of toxin molecules described herein. Sources of nucleic acid suitable for cross-hybridization with nucleotide probes generated from the DNA sequences of the present invention include, but are not limited to, spiders of the same genus, spiders of the related genus, and habitats of the same genus, but not limited to the same genus. .

K. Identification of Promoter Sequences

Another aspect of the invention provides a promoter sequence that regulates the synthesis of DK 9.2 in a spider. Genome libraries were screened using mature toxin cDNA as a probe to investigate the makeup of the gene for DK 9.2. The presence of putative promoters can be seen from analysis of genomic DNA upstream of the electron initiation point (presumably at the 5 'end of the cDNA). The DNA sequence for the 421 base pairs of the promoter region is shown in SEQ ID NO: 8. The putative promoter generally contains many essential regulatory signals present in the region immediately upstream of the transcription initiation site (see McCnight et al., 1982. Transcriptional Control Signals of an Eucaryotic Protein—for an overview of promoter regulatory signals). Coding Gene.Science 217: 316). Standard TATA boxes appear at the −30 position from the proposed transcription initiation site and have been shown to be important for regulating the starting point of RNA synthesis. There are also two distal palindrome sequences containing consensus CAAT boxes that are thought to be important for the binding of RNA polymerase II enzyme to one sequence. The promoter or region of this promoter may be a plant, animal or bacterial cell. In, for example, it is expected to have utility in the transcription and expression of various genes in recombinant constructs.

The following examples are presented to illustrate certain compositions and methods that fall within the scope of the invention but are not intended to limit the scope of the invention.

Substances and Methods

Spiders: Spiders [Spider Pharm, Inc. of Black Canyon City, AZ, and was provided with species identification. The poison of the Diguetia canities spider was electrically squeezed using a method of protection to prevent poisoning by backflow or hemolymph.

Toxin Purification: The crude venom solution (stored at −80 ° C.) was thawed, thoroughly mixed and dissolved in 0.1% trifluoroacetic acid (TFA) before chromatography. Crude venom was fractionated by reverse phase liquid chromatography (RPLC) incorporating a Beckman System Gold 126 solvent and a 168 photodiode-array detector modulus. Acetone nitrile or isopropanol was used with TFA as ion pairing reagent. The following columns were used for purification along with guard columns of the same matrix: Dynamax 300 A RP C ₁₈ column (25 cm x 4.6 mm inner diameter, 12 μm particle size), Vydac 300 AC ₁₈ Analytical columns (25 cm x 4.6 mm inner diameter, 5 μm particle size) and Bidq 300 AC ₁₈ semi-preparative columns (25 cm x 10 mm inner diameter, 5 μm particle size). Peak detection was performed by observing at 220 nm and collecting fractions using a GILSON 208 microfraction collector. All fractions were fractionated and lyophilized and stored at -80 ° C.

Fast Atomic Bombardment Mass Spectroscopy (FAB-MS): Mass spectra were measured on a VG instrument ZAB-SE mass spectrometer at Illinois State University under standard FAB conditions (xenon gas, resolution 1000, acceleration voltage 8 kV, light source temperature 40 ° C.). Samples (about 1 μg) were analyzed in 4 μl thioglycerol containing 25% aqueous TFA (1.0%).

Example 1

Insecticidal from Diguetia caninesis

Initial Fractionation and Identification of Peptide-Containing Fractions

As described above, freeze-dried whole venom (25 μl), obtained from Diguetia canines, was dissolved in 0.1% trifluoroacetic acid (TFA) and eluted at a flow rate of 3.5 mL / min. Fractionation was performed by reversed phase liquid chromatography on a pre-drug RP C ₁₈ column. Aqueous 0.1% TFA: A linear gradient eluent starting with a 85:15 mixture of 50% acetonitrile, 0.1% TFA and ending in a 50:50 mixture after 180 minutes was used.

Each fraction was lyophilized from the eluate and then lyophilized from water and concentrated. The residue was stored at -80 ° C. Each fraction was dissolved in 25 μl buffered saline solution to investigate pesticidality. Insecticidal activity of several fractions was confirmed by testing against tobacco budworm (Heliotis viresense) TBW (Table 1).

Five TBW larvae, individually for each test fraction, were injected with 3 μl of test urea using a 50 μl Hamilton syringe equipped with 33 gauge needles and a PB 600 repeat microdispenser. Infusions were performed by inserting the needle into the lateral centerline of the abdomen (near one of the full widths) at a low angle to avoid damage to the internal organs. After injection, each insect was placed in a separate container containing artificial food. Observed periodically. Paralysis was observed 24 hours after infusion. An average weight of 0.3 g was used for the tobacco budworm when calculating the dose. Only one set of control insects was injected with buffered saline solution.

Paralysis progressed slowly and progressed with periodic marked muscle spasms. In severe cases, the spasms started within 15-30 minutes after injection and gradually increased strongly until the larvae were completely incapacitated. Sometimes the tremor continued for more than 48 hours after injection. This sometimes occurred even when the dose was less than lethal and the larva eventually recovered. Severity of tremor was the most reliable indicator of toxicity, and liver larvae did not recover until full paralysis and / or body contraction.

Paralysis is defined as the inability of an insect to return to itself when it is turned to its side or back.

** Whole Venom Equivalent (WVE), readback equivalent is usually the amount of any toxin present in 1 μl of exploited readout and 5WVE from 25 μl isolate is 20% of recovered residue.

Example 2

Purification of Diguetia cantinez fraction 9

Observe the main component of TBW active fraction 9 at 220 nm and add once via Bidq RP C ₁₈ (25 cm x 10 mm inner diameter) using a linear (1.0% / min) solvent gradient of isopropanol / 0.1% TFA at 3.5 ml / min. Purified to homogeneity by chromatography (one visible band at a molecular weight ranging from approximately 6,500 Daltons, respectively, by SDS-PAGE electrophoresis).

Peaks were detected and fractions were collected as described in Example 1. Two fractions were collected: the first eluted at 21.81 minutes (fraction 9.1) and the second fraction eluted at 22.39 minutes (fraction 9.2).

Fractions 9.1 and 9.2 were lyophilized from the eluate and then concentrated by lyophilization from water to leave residues 9.1 and 9.23, respectively. The purity of the lyophilized fractions was estimated to be at least 99%. Residue 9.2 obtained from 25 μl of readout was evaluated to contain about 6 μl of pure protein. Five insects each were injected with 6.3 μg of residue in buffered saline solution and five insects with buffered saline solution only for control and residue 9.2 was tested for insecticidal activity against tobacco budworm as described in Example 1. Tested. Insects were examined 24 and 48 hours later. After 24 hours of reading, all insects treated with residue 9.2 solution were paralyzed and subsequently killed while the control insects were normal. No change was observed at 48 hours.

Analysis of the polypeptide by high speed atomic bombardment mass spectrometry showed a molecular weight of 6371 ± 2. N-terminal amino acid sequence analysis confirmed the partial amino acid sequence for residue 9.2 substantially as shown in SEQ ID NO: 1, amino acids 1-33.

H. The LD ₅₀ of Diguetia Toxin 9.2 in Byresense (TBW) was approximately 10 nmol / g. The symptoms were similar to those associated with the administration of protoxins as described in Example 1.

Example 3

Purification of Diguetia cantinez fraction 11

In a similar manner as in Example 2, fraction 11 isolated from Diguetia cantinese readout as in Example 1 was further purified by reverse phase liquid chromatography to yield one fraction. This fraction was lyophilized from the eluate and then lyophilized from water to concentrate to obtain a residue, which was designated as residue 11. This residue was tested for pesticidal activity against tobacco budworm at 5.0 WVE / g as outlined in Example 2. After 24 hours, 80% of insects treated with residue 11 showed paralysis, and after 48 hours, 60% of insects treated with the peptides showed paralysis. Control insects appeared normal.

Analysis of the polypeptide by high speed atomic bombardment mass spectrometry showed a molecular weight of 6740 ± 2. N-terminal amino acid sequence analysis confirmed the partial amino acid sequence for residue 11 substantially as shown in SEQ ID NO: 3, amino acids 1 to 29.

Example 4

Purification of Diguetia Kenities Fraction 12

In a similar manner to Example 2, fraction 12, isolated from Diguetia caninesis monologue as in Example 1, was further purified by reverse phase liquid chromatography to yield one fraction. This fraction was lyophilized from the eluate and then lyophilized from water to concentrate to give a residue, which was represented as residue 12. This residue was tested for pesticidal activity against tobacco budworms as outlined in Example 2. After 24 hours, 100% of insects treated with residue 12 showed paralysis, and after 48 hours, 80% of insects treated with residue 12 showed paralysis. Control insects showed normal.

Analysis of the polypeptide by high speed atom bombardment spectroscopy showed a molecular weight of 7080 ± 2. N-terminal amino acid sequence analysis confirmed the test partial amino acid sequence for residue 12 as shown in SEQ ID NO: 5.

Limited material supply did not accurately calibrate the toxicity, resulting in residue 9.2 being tested at 39% and 67% higher doses (nmol / g) than residues 11 and 12, respectively. The doses chosen ranged from minimum lethal doses to ineffective levels but were not achieved in all cases. Nevertheless, the results showed that these peptides had quite similar toxicity. Residue 9.2 may be slightly more effective than others, but perhaps the difference is less than factor 3. The minimum 100% lethal dose for the peptide (residues 9.2, 11 and 12) appears to be 3.0 to 5.0 nmol / g. This compares well with commercial insecticides, for example, Teflubenzuron has a local LD ₅₀ (SAW) of 1.0 nmol / g.

Example 5

Isolation of Coding Genes for Residues 9.2 and 11 Isolated from Diguetia Canetis Poison

Step 1: RNA Isolation

Spiders were harvested from external sources and identified as Diguetia canines. Living spiders were frozen and the head was removed under liquid nitrogen. RNA was extracted from the head and chest using the protocol of Chomczynski and Sacchi, Analytical Biochemistry, 162, 156 (1987). Oligo d (T) cellulose (Sweden, Pharmacia LKB) chromatography was used to purify polyadenylation messenger RNA (mRNA).

Step 2: cDNA Synthesis

The messenger RNA was reverse transcribed into cDNA using murine leukemia virus reverse transcriptase (Bethesda Reserch Laboratories, Midland) according to the manufacturer's protocol. 20 μl of reaction mixture was prepared as enzyme buffer, 50 ng of mRNA, 2 units of RNase H, 30 ng of d (T) Not I as supplied in cDNA Synthesis Kit (Boehringer Mannheim, Ind.). Primer (Promega, Madison, Wisconsin), 1 mM of each deoxynucleoside triphosphate and 100 μl of reverse transcriptase. The reaction mixture was incubated at 37 ° C. for 1 hour and continued at 42 ° C. for 10 minutes. The reaction mixture was ethanol precipitated and resuspended in 20 μl of water.

Step 3: Primer Synthesis

A degenerate primer DNA sequence mixture capable of encoding amino acid residues 1 to 8 according to SEQ ID NO: 1 was designed using some Drosphila codon affinity to reduce the degree of degeneration. The primers were synthesized by the Howard Hughes Medical Institute contract facility at Utah State University.

Step 4: amplification

Primer-induced enzymatic amplification of DNA by thermostable DNA polymerase was initially described in Saikki et al., Science, 239-487 (1988). For application to the present invention, 5 μl of Digutia thoracic cDNA was used as a template for the polymerase chain reaction including the reagent contained in the GeneAmp ^™ DNA Amplification Kit (Perkin Elmer Cetus, Calif.). . The amplification reaction included 2 μM concentration of sense and antisense primers, 100 μM of each deoxynucleotide triphosphate and 4 units of thermostable recombinant Taq I polymerase. The reaction was carried out in a DNA thermal cycler manufactured by Perkin Elmer Setters. Selective amplification of genes encoding residue 9.2 from the toxin class associated with similar NH ₂ -terminal amino acid sequences was achieved using stringent annealing temperatures (58 ° C.). The conditions amplified a single DNA about 275 base pairs in length as measured by agarose gel electrophoresis. Using less stringent annealing conditions, five or more amplification products with sizes ranging from about 200 to 360 base pairs could be detected by agarose gel electrophoresis. The various PCR products obtained under lower stringency will encode polypeptides involved in pesticidal activity against amino acid sequence, approximate size and residue 9.2. The related polypeptide is expected to include residues 11 and 12 since the N-terminal sequences of the polypeptides are very similar to each other and to residue 9.2 as shown in SEQ ID NOs 1, 3 and 5.

Step 5: Cloning of PCR Product

Centricon-100 (Amicon) molecular size separation unit was used to purify PCR products obtained from both reactions under high stringency and low stringency to remove unincorporated primers. The retained product was then cleaved with restriction enzyme Not I (MBR, Wisconsin Milwaukee) which cleaved within the primer downstream (3 ′ termini) to produce an adhesive terminus. The vector pKS (Stratagene, La Jolla, Calif.) Was double cut with EcoR V (US Biochemical) and Not I to create a site specific for directional cloning. The vector and the insert were linked to transform the complex DH5αF '. Colony lifts were screened with ³² P labeled internal probes, and candidate colonies were further characterized by DNA sequencing of mini-preliminary DNA (Sequenase Version 2.0 from US Biochemical) using internal probes as primers.

The full DNA sequence of the two clones' cDNA inserts is shown in SEQ ID NOs: 2 and 4. Only the former were obtained from clones derived from stringent PCR reactions, while clones obtained from low stringency PCR reaction products found both types of cDNA inserts. The amino acid sequence of the polypeptide encoded by SEQ ID NO 2 is shown in SEQ ID NO 1. This polypeptide has the same N-terminal sequence as determined for residue 9.2. The calculated molecular weight of the polypeptide is 6377.9 Daltons. Assuming that all cysteines in the natural polypeptide are in the intramolecular disulfide bond state (cystine), the calculated molecular weight was 6369.7 Daltons.

Therefore, the polypeptide appears to be identical to the polypeptide isolated from residue 9.2, which shows a molecular weight of 6371 ± 2 by mass spectrometry. The amino acid sequence of the polypeptide encoded by SEQ ID NO: 4 is shown in SEQ ID NO: 3. The polypeptide has the same N-terminal sequence as determined for residue 11. Therefore, the polypeptide appears to be the same polypeptide isolated on residue 11.

Example 6

Mammal Toxicity

As described herein, 5 μl of readout obtained from D. kenitis was lethal to mice by intraperitoneal injection (IP) in three separate tests. Treated mice were initially indistinguishable from saline controls. About 10-15 minutes after infusion, treated mice were moderately hyperactive and showed a unique hopping gait followed by short uncoordination, shortness of breath and convulsions. He died within two minutes of the onset of initial symptoms.

Residues 9.2, 11 and 12 were injected intraperitoneally into mice at doses of approximately 4.2, 0.9 and 1.2 mg / kg, respectively, with no effect within 24 hours for all three peptides.

The residue 9.2 was injected intraventricularly into four mice (28 g) at a dose of about 30 μg (about 1 mg / kg) per animal. No effect was seen until 48 hours after injection. Another mouse was intraperitoneally injected with about 125 μg (4.2 mg / kg) of residue 9.2 and no effect was seen.

In order to characterize the vertebrate toxicity of the venom, reverse phase fractions of venom were collected and tested in mice (Figure 1). Fraction 2 included components with tobacco budworm (TBW) activity. At doses equivalent to 25 μl of readout (WVE), intraperitoneal injection of fractions 1 and 2 had no effect, and fraction 3 had the same effect as observed by readonly injection. These results suggest a clear distinction between the pesticidal effect and the vertebrate (toxic) activity present in the components of the Diguetia canines.

Example 7

Electrophysiological data

DK 9.2 was tested at 1 μM for synaptic transmission (induced population spikes) at Sharper flanking-CA1 vertebral cell synapses in the hippocampus slices of rats. The data in FIG. 2 shows time-averaged population spike records for (a) 5 minutes before DK 9.2 addition and (b) 15 to 20 minutes after DK 9.2 addition. The recordings can overlap, indicating that at this concentration DK 9.2 has no activity in the rat CNS that can be detected by the assay. The assay can detect various 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. Liss, Inc., New York, 1986]. In particular, molecules that prevent inactivation of sodium channels, such as certain scorpion toxins, result in significant expansion of the last phase of the population spike reaction. [Kandea M, Myama Y, Ikemoto Y and Akaike N., Scorpion toxin prolongs an inactivation phase of the voltage-dependent sodium current in rat isolated single hippocampal neurons, Brain Res, 487: 192-195, 1989; Alan L. Mueller, Natural Product Sciences, Inc., unpublished observations]. In contrast, DK 9.2 is active in insect preparations (housefly) at concentrations 100 times lower and in a manner that suggests preventing inactivation of insect sodium channels.

Example 8

Upstream cDNA sequence encoding the precursor of DK 9.2

To obtain an upstream sequence of cDNA encoding DK 9.2, internal oligonucleotides corresponding to nucleic acid residues 159-178 on the antisense strand of the DNA sequence shown in SEQ ID NO: 2 were synthesized. The Eco RI restriction site is at the 5 'end of the prai. 10 [mu] l of single stranded poison cDNA was attached to the deoxyguanosine residue at the 3 'end using an enzyme, terminal deoxynucleotide transferase (Betesda Research Laboratories). 20 μl of the reaction containing 14 units of enzyme and 500 μM of dGTP was incubated at 37 ° C. for 15 minutes. Samples were ethanol precipitated and resuspended in 20 μl H ₂ O.

DNA sequences upstream of the gene specific primers were amplified using fixed PCR techniques similar to those used for downstream / mature toxin cDNA sequences. The amplification reaction included a 2 μM concentration of sensor (d (C) terminal primer) and antisense primer, 100 μM of each deoxynucleotide triphosphate and 4 units of thermostable recombinant Taq polymerase. The temperature profile was as follows: 2 min at 94 ° C., 2 min at 37 ° C., 1 min at 37 ° C., and after repeating the cycle twice, the program was inserted at the second step with an imagined annealing temperature of 54 ° C. Were switched to the same profile. The cycle was repeated 32 times.

Immobilized PCR gave 380 bp fragments as evident on 4% agarose gels in the presence of ethidium bromide. This reaction product is Large (Clenau) fragments of E. coli DNA polymerase I (Molecular Biology Resoruces, Madison, Wisconsin) were charged at the end and precipitated by addition of ethanol. The product was resuspended and digested with restriction enzyme EcoRI. The cleaved sections were kinated with enzyme T4 kinase in the presence of 1 mM ATP and subsequently linked to EcoRI and Eco RV cleaved pBluescriptsKS vectors. Subclones were analyzed by double-stranded DNA sequencing using Cyquinase 2.0 (USB).

Translation of the cDNA sequence contained in the upstream region to mature peptide toxins revealed that DK 9.2 is synthesized as a precursor protein. The upstream cDNA sequence is shown in SEQ ID NO: 6. The encoded precursor protein consists of a signal sequence and a propeptide region (SEQ ID NO: 7), which are removed to provide a mature, peptide toxin that is isolated from spider venom. The signal sequence and propeptide sequence are believed to be essential for the production and secretion of DK 9.2 in spiders.

Example 9

Recombinant baculovirus production

Rossi, et al., J. Biol, Chem. 275: 9226 (1982)] using the method of repidoterra signal sequence from two synthetic oligonucleotides [Jones et al., Molecular Cloning Regulation and Complete Sequence of a Hemocyanin-Related Juvenil Hormone-Supressible Proteim From Insect Hemolymphs, J. Biol Chem. 265: 8596 (1990). Two 48-mers were purified by ion exchange chromatography. These two oligonucleotides share 11 base pair complementarity sequences at their 3 'ends. When the sequence was annealed in the presence of four deoxyribonucleoside triphosphates and a Klenow fragment of DNA polymerase I, a double-stranded product was synthesized. Hydrolysis phosphate chromatography was used to purify the reaction product and the double-stranded DNA molecules were cleaved with Aat II, a restriction enzyme suitable for inserting the upstream sequence of DK 9.2 cDNA cloned into pKS-DK9c. Subclones were screened for insertion of signal sequences and examined by DNA sequence analysis.

DNA sequencing confirmed in-rfame fusion between two cDNA sequences. The entire synthetic gene construct was cut and processed to clone into the NheI site of the baculovirus transport vector, pBlueBac (Vialard, J., et al., J. Virology 64: 3-50 (1990)). Subclones were sequenced to confirm correct insertion of the constructs. DNA sequencing of the plasmid WR9 confirmed the insertion of the synthetic caterspider gene (pre Dk 9.2) in the baculovirus transport vector pBlueBac (Figure 3). Insertion of the recombinant gene of the invention into the baculovirus genome is carried out by co-expression of β-galactosidase and can be detected by color change upon growth on the indicated medium, thus the use of the pBlueBac vector speeds the screening process. Let's do it.

Recombinant baculoviruses encoding preDK 9.2 can be used to describe the Spodoptera frugiperda strain Sf9 (ATCC # CRL1711) cells in A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas. Agricultural Experiment Bulletin No. 1555, 1988] was used to transfect with a mixture of 1 μg AcMNPV viral DNA and 2 μg plasmid DNA. On day 4 after transfection, dilutions of the cell supernatants formed plaques on 100 mm plates covered with agarose containing 5 × 10 ⁶ Sf9 cells and containing Bluo-gal (Gibso BRL, Gatorsburg, Midland) as substrate. It was. Within 5 to 6 days, the recombinant could be detected by its light blue color. Plaques were taken using a Pasteur pipette and eluted in 1 ml of medium. The eluate was used to reinfect Sf9 cells inoculated into T-25 flasks. On day 3 post infection, a small amount of supernatant was collected from six different isolates and used to prepare viral DNA. PCR amplification using virus specific primers from the region around the polyhedrin gene confirmed that 5 out of 6 virus isolates contained inserts of appropriate size and no wildtype contaminants. Sources of titrated recombinant virus were then prepared for in vivo and in vitro testing.

Example 10

Biological Activity of Recombinant DK 9.2

The biological activity of the recombinant peptide residue 9.2 (DK 9.2), prepared as in Example 8, was tested in the TBW larvae of the insect stage. Doses were calculated based on A ₂₈₀ of the test solution. At a dose of 16 μg / g, significant muscle spasm was induced within 2 hours after injection with the recombinant material. Half of these larvae (3 out of 6) were paralyzed and severely contracted after 48 hours, while the other three larvae still showed mild to moderate muscle spasms. A dose of 8 μg / g also paralyzed three out of six larvae, whereas a dose of 5.2 μg / g paralyzed two out of six larvae. From the results, it was confirmed that recombinant DK 9.2 has bioactivity.

Example 11

Recombinant Baculovirus-In Vivo Testing

A. Biological Activity of DK 9.2 Recombinant Nucleus Polygonal Virus (vAcDK9.2)

(1) Titration of virus preparation

Viral stocks were titrated by plaque assay [Luria at al., General Virology, 1987, pp. 21-32; John Wiley and Sons, New York. Titers are expressed in plaque forming units (PFUs) per unit volume, while doses are expressed in PFU / larvae. One PFU is the functional equivalent of one mature virion (virus) in a formulation where all virions can successfully infect one host cell (see above, Luria et al.). For example, 10 ³ host cells can be infected with each μl of viral preparation containing 10 ⁶ PFU / ml (ie, 10 ³ PFU / μl).

(2) bioassay

The biological activity of DK 9.2 recombinant nuclear hyperhidrosis virus (rNPV), vAcDK 9.2, was tested in a series of dose response assays. In this assay, TBW larvae (average weight about 250 mg) were injected with various doses of vAcDK 9.2 or tissue culture medium (n = 10) in tissue culture medium. As in the toxin injection experiment described in Example 1, the treated larvae were maintained and periodically observed while feeding in each container. The result of the two virus injection assays is illustrated in Table II. At doses above 5000 PFU / larvae, typical symptoms of DK 9.2 toxicity (muscle tremor and convulsions) began appearing about 24 hours after infection and at least half of the test insects became incapacitated (ie, paralyzed or partially paralyzed) within 48 hours. ). The lowest dose tested, 50 PFU / larvae, induced tremor and convulsions within 48 and incapacitated at least 70% of the larvae within 96-120 hours.

B. Biological Activity of vAcDK 9.2 Against Wild Type NPV

Further studies were conducted to determine the efficiency of vAcDK 9.2 compared to the parental wild type virus Autographer California NPV (wt-AcMNPV). The purpose of the assay was to determine whether larvae infected with vAcDK 9.2 were neutralized in less time than larvae infected with the same dose of wt-AcMNPV.

(1) Injection analysis

The biological activities of vAcDK 9.2 and wt-AcMNPV were compared in a series of infusion assays. 5x10 ⁵ PFU / larvae of Tobacco Budworm (Heliotis viresense) larvae, cabbage larvae (Trichoplusia ni) larvae and beet giant worms (Spodoptera exigua larvae) VAcDK 9.2 or wt-AcMNPV was injected and control larvae were injected with tissue culture medium The results summarized in Table III demonstrate that vAcDK 9.2 is substantially more effective than wt-AcMNPV in all three species.

(2) feeding analysis

To test the activity of vAcDK 9.2 by ingestion, it was necessary to generate polyhedrin-pol-recombinants in the oral infection form. This is a method reported by other researchers for the preparation of oral infection pol-recombinant NPV (see, eg, Kuroda et al., 1989, J. Virology 63 (4): 1677-1685, and Price et al., 1989 Proc). Natl.Acad. Sci. 86: 1453-1456), vAcDK 9.2 was co-occlusion with wt-AcMNPV. SF-9 cells were simultaneously infected with vAcDK 9.2 and wt-AcMNPV under a muiplicity of infection (MOI) of 10 and 2, respectively. At the same time, the second group of SF-9 cells were infected with wt-AcMNPV alone (MOI = 2). Five days after infection, inclusion bodies were harvested by cell disruption and time-depth separation (eg, Wood, 1980, Virology 104: 392-399). Inclusion bodies were counted on a hemacyto meter pigment meter. Cells co-infected with vAcDK 9.2 and wt-AcMNPV produced smaller and smaller inclusion bodies than cells infected with wt-AcMNPV alone. The yield of polyhedral inclusion bodies (Polyhedral Inclusion Bodies (PIB)) from mixed infection was 4.6 PIB / cell, while the yield from pure wt-AcMNPV infection was 33.3 PIB / cell.

To measure the biological activity of co-occluded vAcDK 9.2 compared to wt-AcMNPV, food incorporation assays were used. Mixed or wild type PIB was incorporated into non-agar primary insect food at a concentration of 10 ⁵ PIB / g of food. The food was dispensed into small containers which were subsequently infected with newborn TBW larvae (1 per container, n = 20). Control larvae were fed the same amount of untreated food. Larvae were randomly fed and periodically monitored for progress. The results are shown in Table II. There was no effect during the first 48 hours, but after 72 hours more than 50% of the larvae fed the mixed PIB were paralyzed. Wild type virus was not affected at this time. After 96 hours, more than 90% of the recombinant-treated larvae were paralyzed while only 10% of the wild-type treated larvae died or died. After 120 hours, 75% of the larvae treated with wt-AcMNPV were dying while 100% of the recombinant-treated larvae died. Thus, as in the infusion assay, the vAcDK 9.2 treated larvae were rendered useless in a significantly shorter time than the larvae treated with wt-AcMNPV.

1. Virus was injected at ⁵ × 10 ⁵ PFU / larvae, and control was injected with the same volume of tissue culture medium.

2. n = 8; An average mass of about 300 mg per larvae.

3. Two of the eight larvae appeared to have died from physical shock due to infusion and were excluded from further analysis.

4. Mortality from wt-AcMNPV was 100% on day 9.

5. n = 10; An average mass of about 165 mg per larva.

6. n = 20 for vAcDK 9.2; n = 10 for wt-AcMNPV and control; An average mass of about 200 mg per larva.

Example 12

Promoters for genes encoding DK 9.2

To regulate the regulatory elements of the gene encoding DK 9.2, genome libraries were constructed. Herman and Frichauf [Medthods in Enzymology, Vol. 152, Academic Press, Inc. 1987, pp. DNA was prepared in spiders using a protocol adapted from 180-183. This DNA was partially digested with Sau 3A and centrifuged using a TLS-55 rotor (Beckman Co., Ltd) at a concentration of 10% to 38% sucrose for 16 hours at 25,000 rpm. Fractionated. DNA fractions of 35 to 45 kb were collected and prepared for insertion into Xho I cleaved cosmid vectors using the partial filling method. One quarter of the ligation mixture was packaged into phage particles using Gigapak gold ^™ (stratazine, La Jolla, Calif.), And an equivalent of at least 3 × ^{10 9} bp of spider DNA was obtained after transformation. Libraries were plated on 25 150 mm petri folates and lifted on nylon filters to probe with radiolabeled DNA.

Colonine lifts of the Diegutia genome library were sequentially screened with radiolabeled cDNA encoding DK 9.2, and end-labeled oligonucleotides encoding amino-terminal, carboxy terminus and internal probes. One cosmid cDK2 was hybridized with all probes. Southern blots of Eco R1 cleaved cosmid DNA showed that the 3.0 kb fragment hybridized with both internal and C-terminal probes. Double stranded sequencing of cosmid cDK2 using the three primers identified above confirmed the isolation of the gene for DK 9.2, with amino terminal oligonucleotides (high homology to all Diegtea pesticidal peptides). Is primed at several sites on the cosmid DNA, suggesting the possibility that another (or other) gene of this class may exist on the same contiguous portion of the DNA.

To examine the promoter region of the gene for DK 9.2, oligonucleotides corresponding to sense stranded pre / signal sequences were synthesized. PCR amplification between the primers and the gene-specific primers of the present invention led to mapping of signal sequences up to 3,000 bp upstream of the amino terminal exon. The primers on the antisense strand were then sequenced to genomic DNA in the region immediately above the 5 'end of the cDNA encoding the signal peptide.

Another intron at bp-11 was suggested from the initiating methionine codon from DNA sequencing upstream of the signal sequence on genomic DNA. Oligonucleotide primers corresponding to the 5 'region of precursor DK 9.2 cDNA were synthesized and used to prime PCR reactions to determine how large intermediate sequences exist between transcription initiation sites and translation initiation sites. The 1000 bp amplification product demonstrated the presence of introns and allowed the size to be assessed. DNA sequence analysis of genomic DNA upstream of the transcription start point (presumably corresponding to the 5 'end of the cDNA) indicates the presence of a promoter. The DNA sequence of the promoter region is shown in SEQ ID NO: 8. The putative promoter generally contains many essential regulatory signals present in the region just upstream of the translation initiation site among other eukaryotic promoters. The promoter, or part of the promoter, can be used for transcription / translation of other eukaryotic genes in bacteria, viruses, plants or animals.

Example 13

Neurophysiological studies related to the mode of action of DK 9.2

Neurophysiology studies were conducted to elucidate the mode of action of DK 9.2. Records obtained from the neuromuscular junctions and peripheral nerves of maggots indicate that DK 9.2 induces repetitive excitatory discharge in nerves sensitive to blocking by tetrodotoxin. Thus, the site of action of the toxin is probably the voltage-sensitive sodium channel of the neural membrane. Further studies have shown that DK 9.2 consistently excites peripheral nerves at critical concentrations of 10 nM. In addition, DK 9.2 is at least 50 times more potent than mammalian toxin 4 of the scorpion Leiurus questriatus.

The experimental animals used in this study were the third larvae of housefly, Musca domestica. Larvae were fixed with insect pins and opened to the dorsal side by a central sagittal incision. The body was pinned flat and the viscera were removed to expose body wall muscle tissue. Where peripheral nerves come from the brain, the peripheral nerves are cut and the brain removed. The preparation was immersed in insect saline (pH = 7.2) consisting of NaCl (140 mM), KCl (5 mM), CaCl ₂ (0.75 mM), MgCl ₂ (4 mM), NaHCO ₃ (5 mM) and HEPES (5 mM).

Neuromuscular delivery was recorded by attaching a stimulus suction electrode to any convenient nerve trunk. The stimulation threshold was slowly raised until contractile fibers of muscle 6 or 7 were observed. The fibers were then pierced with recording intracellular microelectrodes connected to intracellular preamplifiers used to investigate Excitatory Post Synaptic Potentials (EPS) in response to nerve stimulation.

To record the rising electrical activity in peripheral nerve fibers, a suction electrode was attached to the AC preamplifier. The signal was amplified 100 times and the effluent was filtered at 0.3 and 1 kHz. All records were digitalized on a MacLab computerized metrology system for display and analysis.

Initial experiments on the action of DK 9.2 used neuromuscular junction preparations. A single stimulus applied to the peripheral nerve caused a single EPSP in the wall muscle. In the presence of DK 9.2, a single stimulus resulted in a high frequency discharge of the EPSP. In addition to the stimulus-induced discharge, a spontaneous excited discharge was also seen, which lasted for several minutes. The duration of excitation was typically more than 1 second and the frequency of the excited EPSP was higher than 30 Hz. The presence of excitatory discharges resulted in violent contractions in body wall muscle tissue, which made it difficult to maintain intracellular recordings as the electrodes bounced off the muscles during excitation-initiated contractions. Therefore, most of the experiments were performed in saline containing a high concentration of sucrose (300mM), which suppresses shrinkage but does not affect electrical activity. Similar results were obtained using standard or high concentration sucrose saline.

The excitatory discharges observed after DK 9.2 treatment were similar to those observed in the presence of scorpion α toxin, a protein known to interact with the low pressure-sensitive sodium channels of the neural membrane. Thus, the excitation discharge must be blocked by a specific sodium channel blocker, tetrodotoxin (TTX). The ability of TTX to prevent or reverse DK 9.2-induced excitability is shown in FIG. 5. In FIG. 5A, EPSP was rapidly blocked by 1 μM TTX, making the formulation insensitive to subsequent treatment with 100 nM DK 9.2. Similar experiments are shown in Figure 5B, where excitation is induced by DK 9.2 and reversed by subsequent TTX application. In this experiment, when the electrode pulled out of the muscle by the onset of excitement and attempted to construct the recording again, an impalement cartifact was produced. Once an appropriate recording site was found, excitement was observed for about 2 minutes and the formulation showed a stop of activity within a few seconds after TTX treatment.

To solve the problems associated with intracellular recording from contractile muscle, the experiment was repeated using the recording of cells from maggot peripheral nerves. These recordings consist of ascending sensory nerve activity with spontaneous reverse activation of motor fibers. At 70 nM DK 9.2 is not affected by subsequent treatment with saline, but increases neuronal discharge, which is rapidly blocked by 0.4 μM TTX. In addition, application of DK 9.2 after applying TTX prevents the appearance of excitement.

Another study was conducted to measure the sensitivity of insect nerves to DK 9.2 and compare their effects with those of standard scorpion toxins. DK 9.2 was tested on peripheral nerve agents starting at 1 nM and increasing the concentration every 5 minutes or until an increase in activity was observed. In this formulation, 1 nM and 5 nM DK 9.2 were inactive, but 10 nM induced activity after a short delay. The results from the five neuronal agents used to determine the effective critical concentration of DK 9.2 on insect nerves are summarized in Table IV.

In the last experimental group, the sensitivity of insect nerves to toxin 4 (LqTX 4) of the scorpion Laureus quinqueriatus was measured in a similar manner. There was no effect when the formulation (n = 4) was exposed to LqTX 4 at a concentration of 500 nM or less. This is consistent with previous studies in which LqTX 4 was found to be specific for sodium channels in mammals. Subsequent stimulation of the agent with DK 9.2 required 10-30 nM to elicit a response. A slight increase in the effective critical concentration of DK 9.2 is probably due to a weak blocking action by inactive LqTX 4. These experiments demonstrate that DK 9.2 is at least 50 times more active than LqTX4 on insect neurons.

Sequence Listing

(1) General Information:

(i) Applicant: Karen J. Karen J. Krapcho; Bradford Carr VanWagenen; second. egg. Hunter Jackson

(ii) Name of the Invention: Peptides with Insecticidal Effect

(iii) SEQ ID NO: 8

(iv) communication address:

(A) Recipients: Woodcock, Washburn, Kurtz, Mackiewicz and Norris

(B) Street: One Liberty Place-46th Floor

(C) City: Philadelphia

Note (D): PA

(E) Country: United States

(F) Zip code: 19103

(v) computer readable:

(A) Media type: diskette, 3.5 inches, 1.44 Mb storage

(B) Computer: IBM PS / 2

(C) working system: PC-DOS

(D) Software: WordPerfect 5.0

(vi) Current application data:

(A) Application number:

(B) filing date:

(C) Classification:

(vii) Priority data:

(A) Application number: 662,373

(B) Application date: March 1, 1991

(viii) Agent Information:

(A) Name: John W. John W. Caldwell, Esq.

(B) Registration Number: 28,937

(C) Reference / Doc.No .: FMC-0054

(ix) Communications Information:

(A) Phone: (215) 568-3100

(B) Fax: (215)568-3439

(2) Information about SEQ ID NO: 1:

(i) sequence properties:

(A) Length: 56 amino acids

(B) type; amino acid

(D) Topology: Unknown

(xi) Sequence Initiation: SEQ ID NO: 1:

(2) Information for SEQ ID NO: 2:

(i) sequence properties:

(A) Length: 275 base pairs

(B) Type: nucleic acid

(C) strand: double

(D) Topology: Unknown

(xi) Sequence Initiation: SEQ ID NO: 2

(2) Information about sequence identification number 3:

(i) sequence properties:

(A) Length: 58 amino acids

(B) Type: Amino Acid

(D) Topology: Unknown

(xi) sequence initiation: SEQ ID NO: 3

(2) Information about SEQ ID NO: 4:

(i) sequence properties:

(A) Length: 204 base pairs

(B) Type: nucleic acid

(C) strand: double

(D) Topology: Unknown

(xi) Sequence Initiation: SEQ ID NO 4:

(2) Information about SEQ ID NO:

(i) sequence properties:

(A) Length: 62 amino acids

(B) Type: Amino Acid

(D) Topology: Unknown

(xi) Sequence Initiation: SEQ ID NO 5:

(2) Information about SEQ ID NO 6:

(i) sequence properties:

(A) Length: 359 base pairs

(B) Type: nucleic acid

(C) strand: double

(D) Topology: Unknown

(xi) Sequence Initiation: SEQ ID NO 6:

(2) information about sequence identification number 7

(i) sequence properties:

(A) Length: 38 amino acids

(B) Type: Amino Acid

(C) strands:

(D) Topology: Unknown

(xi) sequence initiation: SEQ ID NO: 7:

(2) Information about SEQ ID NO: 8:

(i) sequence properties:

(A) Length: 421 base pairs

(B) Type: nucleic acid

(C) strand: double

(D) Topology: Unknown

(xi) sequence initiation: SEQ ID NO: 8

Claims (16)

Peptides having agriculturally or horticulturally acceptable salts thereof having an insecticidal effect isolated from the purified Diguetia spider poison, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3 and 5. And The peptide of claim 1, wherein said spider venom is obtained from Diguetia cantines. Encodes a peptide having an insecticidal effect that is isolateable from a Diguetia spider venom having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3 and 5 and selected from the group consisting of SEQ ID NOs: 2, 4 and 6 An isolated DNA sequence, characterized by the DNA sequence being. Consisting of SEQ ID NOs: 2, 4, and 6, which encode a peptide having an insecticidal effect that is isolateable from a Diguetia spider poison having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, and 5 in transformed cells A recombinant expression vector comprising a DNA sequence selected from the group consisting of and capable of expressing it. 5. The recombinant expression vector of claim 4, wherein said spider venom is obtained from Diguetia kenities. SEQ ID NOs: 2, 4, and 6, which encode a peptide having an insecticidal effect that is isolable from a Diguetia spider poison having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, and 5 in a host or host insect cell A recombinant baculovirus expression vector capable of expressing a DNA sequence having a sequence selected from the group consisting of: Identifying a sequence encoding the peptide in such a way that the host cell expresses a peptide having an isolated pesticidal effect from the Diguetia spider poison having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3 and 5 A recombinant host cell transformed or transfected with a DNA sequence characterized by a sequence selected from the group consisting of numbers 2, 4 and 6. 8. The recombinant host cell of claim 7, wherein said spider venom is obtained from Diguetia cantines. (a) encodes a peptide having an insecticidal effect that is isolateable from a Diguetia spider poison having an amino acid sequence selected from the group consisting of transformed or transfected recombinant expression vector sequence sequences 1,3 and 5 in a recombinant host cell (B) cultivating a recombinant host cell having a DNA sequence having a sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6, wherein said coding sequence can be expressed in a transformed cell, (b) Recovering the peptide having the pesticidal effect from the recombinant host cell culture, method of producing a peptide having an insecticidal effect. Peptides having an insecticidal effect which is insulated from Diguetia spider poisons having an amino acid sequence selected from the group consisting of an effective amount of sequence identification numbers 1, 3 and 5 or agriculturally or horticulturally acceptable thereof A method of controlling invertebrate pests, characterized by contact with salts. The method of claim 10, wherein said spider venom is obtained from Diguetia cantines. The method of claim 10 wherein the pest is an insect. The method of claim 10, wherein the pest is Redodorftera. A pesticidal composition characterized by a pesticidal effective amount of the peptide according to claim 1 and an agriculturally or horticulturally acceptable salt thereof in an agriculturally or horticulturally acceptable carrier. Selected from the group consisting of SEQ ID NOs: 2, 4, and 6, which encode a peptide having an insecticidal effect that is isolateable from a Diguetia spider poison having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, and 5 DNA probes derived from DNA sequences. (a) obtaining nucleic acid from a spider, (b) contacting the hexane with the DNA probe of claim 15, and (c) detecting the probe bound to the nucleic acid. A method for detecting the presence of a nucleic acid encoding a peptide having a possible pesticidal effect.