US 20090087863A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0087863 A1 Mettus et al. (43) Pub. Date: Apr. 2, 2009

(54) METHOD FOR DETECTING POLYPEPTIDE now Pat. No. 7,022,897, which is a division of appli TOXC TO DIA BROTICA INSECTS cation No. 09/184,748, filed on Nov. 2, 1998, now Pat. No. 6,468,523. (76) Inventors: Anne-Marie Light Mettus, Publication Classification Feasterville, PA (US); James A. (51) Int. Cl. Baum, Doylestown, PA (US) GOIN 33/53 (2006.01) C07K 6/12 (2006.01) Correspondence Address: HOWREY LLP (52) U.S. Cl...... 435/7.2:530/389.5 C/O IP DOCKETING DEPARTMENT, 2941 (57) ABSTRACT FAIRVIEW PARK DRIVE SUTE 200 Disclosed is a novel Lepidopteran- and Coleopteran-active FALLS CHURCH, VA 22042 (US) Ö-endotoxin polypeptide, and compositions comprising the polypeptide, peptide fragments thereof, and antibodies spe (21) Appl. No.: 12/240,110 cific therefor. Also disclosed are vectors, transformed host cells, and transgenic plants that comprise nucleic acid seg (22) Filed: Sep. 29, 2008 ments encoding the polypeptide. Also disclosed are methods of identifying related polypeptides and polynucleotides, methods of making and using transgenic cells comprising the Related U.S. Application Data novel sequences of the invention, as well as methods for (62) Division of application No. 1 1/332,654, filed on Jan. controlling an insect population, Such as the Western Corn 13, 2006, now Pat. No. 7,429,454, which is a division Rootworm and Colorado potato beetle, and for conferring to of application No. 10/120.255, filed on Apr. 10, 2002, a plant population resistance to the target insect species.

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CryET70 Patent Application Publication Apr. 2, 2009 US 2009/0087863 A1

1 kb pEG1648 (Ssp Bal) (Hp B) (KSsp) HE H Hp

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pEG1657 pUC18 CryET70 US 2009/0087863 A1 Apr. 2, 2009

METHOD FOR DETECTING POLYPEPTIDE formation of a pore in the insect cells and the disruption of TOXCTO DIA BROTCA NSECTS cellular homeostasis (English and Slatin, 1992). 0006. One of the unique features of B. thuringiensis is its 1.O BACKGROUND OF THE INVENTION production of crystal proteins during sporulation which are 0001. 1.1 Field of the Invention specifically toxic to certain orders and species of insects. 0002 The present invention relates generally to the fields Many different strains of B. thuringiensis have been shown to of molecular biology. Provided are methods and composi produce insecticidal crystal proteins. Compositions including tions comprising DNA segments, and polypeptides derived B. thuringiensis strains which produce proteins having insec from bacterial species for use in insecticidal formulations and ticidal activity against lepidopteran and dipteran insects have the development of transgenic insect-resistant plants. More been commercially available and used as environmentally particularly, it concerns novel nucleic acids obtained from acceptable insecticides because they are quite toxic to the Bacillus thuringiensis that encode coleopteran- and lepi specific target insect, but are harmless to plants and other dopteran-toxic polypeptides. Various methods for making non-targeted organisms. and using these nucleic acids, DNA segments encoding Syn 0007. The mechanism of insecticidal activity of the B. thetically-modified CryET70 polypeptides, and native and thuringiensis crystal proteins has been studied extensively in synthetic polypeptide compositions are also disclosed. The the past decade. It has been shown that the crystal proteins are use of DNA segments as diagnostic probes and templates for toxic to the insect only after ingestion of the protein by the protein production, and the use of polypeptides, fusion pro insect. The alkaline pH and proteolytic enzymes in the insect teins, antibodies, and peptide fragments in various immuno logical and diagnostic applications are also disclosed, as are mid-gut solubilize the proteins, thereby allowing the release methods of making and using nucleic acid segments in the of components which are toxic to the insect. These toxic development of transgenic plant cells comprising the poly components disrupt the mid-gut cells, cause the insect to nucleotides disclosed herein. cease feeding, and, eventually, bring about insect death For 0003) 1.2. Description of the Related Art this reason, B. thuringiensis has proven to be an effective and 0004 Because crops of commercial interest are often the environmentally safe insecticide in dealing with various target of insect attack, environmentally-sensitive methods for insect pests. controlling or eradicating insect infestation are desirable in 0008. As noted by Höfte et al., (1989) the majority of many instances. This is particularly true for farmers, nursery insecticidal B. thuringiensis stains are active against insects men, growers, and commercial and residential areas which of the order Lepidoptera, i.e., caterpillar insects. Other B. seek to control insect populations using eco-friendly compo thuringiensis stains are insecticidally active against insects of sitions. The most widely used environmentally-sensitive the order Diptera, i.e., flies and mosquitoes, or against both insecticidal formulations developed in recent years have been lepidopteran and dipteran insects. In recent years, a few B. composed of microbial pesticides derived from the bacterium thuringiensis strains have been reported as producing crystal Bacillus thuringiensis. B. thuringiensis is a Gram-positive proteins that are toxic to insects of the order Coleoptera, i.e., bacterium that produces crystal proteins or inclusion bodies beetles (Krieg et al., 1983; Sick et al., 1990; Lambert et al., which are specifically toxic to certain orders and species of 1992a: 1992b). insects. Many different strains of B. thuringiensis have been shown to produce insecticidal crystal proteins. Compositions including B. thuringiensis strains which produce insecticidal 1.2.2 Genes Encoding Crystal Proteins proteins have been commercially-available and used as envi 0009. Many of the 8-endotoxins are related to various ronmentally-acceptable insecticides because they are quite degrees by similarities in their amino acid sequences. Histori toxic to the specific target insect, but are harmless to plants cally, the proteins and the genes which encode them were and other non-targeted organisms. classified based largely upon their spectrum of insecticidal activity. The review by Höfte and Whiteley (1989) discusses 12.1 8-ENDOTOXINS the genes and proteins that were identified in B. thuringiensis 0005 ö-endotoxins are used to control a wide range of prior to 1990, and sets forth the nomenclature and classifica leaf-eating caterpillars and beetles, as well as mosquitoes. tion scheme which has traditionally been applied to B. thur These proteinaceous parasporal crystals, also referred to as ingiensis genes and proteins. cry I genes encode lepidopteran insecticidal crystal proteins, crystal proteins, Bt inclusions, toxic CryI proteins. cryII genes encode Cry II proteins that are crystalline inclusions, inclusion bodies, and Bt toxins, are a toxic to both lepidopterans and dipterans. cryIII genes encode large collection of insecticidal proteins produced by B. thur coleopteran-toxic CryIII proteins, while cryIV genes encode ingiensis that are toxic upon ingestion by a susceptible insect dipteran-toxic Cry IV proteins. host. Over the past decade research on the structure and function of B. thuringiensis toxins has covered all of the 0010 Based on the degree of sequence similarity, the pro major toxin categories, and while these toxins differ in spe teins were further classified into subfamilies; more highly cific structure and function, general similarities in the struc related proteins within each family were assigned divisional ture and function are assumed. Based on the accumulated letters such as CryIA, CryIB, Cry1C, etc. Even more closely knowledge of B. thuringiensis toxins, a generalized mode of related proteins within each division were given names Such action for B. thuringiensis toxins has been created and as CryIC1, CryIC2, etc. includes: ingestion by the insect, Solubilization in the insect 0011 Recently, a new nomenclature was developed which midgut (a combination stomach and Small intestine), resis systematically classified the Cry proteins based upon amino tance to digestive enzymes sometimes with partial digestion acid sequence homology rather than upon insect target speci actually “activating the toxin, binding to the midgut cells, ficities. The classification scheme for many known toxins, not US 2009/0087863 A1 Apr. 2, 2009

including allelic variations in individual proteins, is Summa nucleic acid segment comprising at least 45 contiguous rized in Table 2 of Section 4.3. nucleotides from SEQ ID NO:1, and in one embodiment comprise the coding region from nucleotide 92 to nucleotide 1.2.3 Identification of Crystal Proteins Toxic to WCRW 2254 of SEQID NO:1. The invention also discloses compo Insects sitions and insecticidal formulations that comprise Such a polypeptide. Such composition may be a cell extract, cell 0012. The cloning and expression of the cry3Bb gene has Suspension, cell homogenate, cell lysate, cell Supernatant, been described (Donovan et al., 1992). This gene codes for a protein of 74 kDa with activity against Coleopteran insects, cell filtrate, or cell pellet of a bacteria cell that comprises a particularly the Colorado potato beetle (CPB) and the south polynucleotide that encodes such a polypeptide. Exemplary ern corn root worm (SCRW). bacterial cells that produce such a polypeptide include Bacil 0013 AB. thuringiensis strain, PS201T6, was reported to lus thuringiensis EG4140, EG1 1839, NRRL B-21885 and have activity against WCRW (Diabrotica virgifera virgifera) NRRL B-21886 cells. The composition as described in detail (U.S. Pat. No. 5,436,002, specifically incorporated herein by hereinbelow may be formulated as a powder, dust, pellet, reference). This strain also had activity against Musca domes granule, spray, emulsion, colloid, Solution, or Such like, and tica, Aedes aegypti, and Liriomyza trifoli. The Vip1A gene, may be preparable by Such conventional means as desicca which produces a vegetative, soluble, insecticidal protein, has tion, lyophilization, homogenization, extraction, filtration, been cloned and sequenced (Intl. Pat. Appl. Pub. No. WO centrifugation, sedimentation, or concentration of a culture of 96/10083, 1996). This gene produces a protein of approxi cells comprising the polypeptide. Preferably such composi mately 80 kDa with activity against WCRW and Northern tions are obtainable from cultures of Bacillus thuringiensis Corn Root Worm (NCRW). Another toxin protein with activ EG4140, EG 11839, NRRL B-21885 or NRRL B-21886 cells. ity against coleopteran insects, including WCRW, is CryIIa, In all such compositions that contain at least one such insec an 81-kDa polypeptide, the gene encoding which has been ticidal polypeptide, the polypeptide may be present in a con cloned and sequenced (Intl. Pat. Appl. Pub. No. WO centration of from about 1% to about 99% by weight. 90/13651, 1990). 0018. An exemplary insecticidal polypeptide formulation 0014) The cryET29 gene described by Rupar et al. (Intl. may be prepared by a process comprising the steps of cultur Pat. Appl. Pub. No. WO 98/13497, 1998) encodes a polypep ing Bacillus thuringiensis EG4140, EG1 1839, NRRL tide of approximately 26 kDa that has activity against B-21885 or NRRL B-21886 cells under conditions effective WCRW, SCRW, as well as activity against the larvae of the to produce the insecticidal polypeptide; and obtaining the Colorado potato beetle and the cat flea, Ctenocephalides felis. insecticidal polypeptide so produced. 0019 For example, the invention discloses and claims a method of preparing a 6-endotoxin polypeptide having insec 2O SUMMARY OF THE INVENTION ticidal activity against a coleopteran or lepidopteran insect. 0015. In sharp contrast to the prior art, the polypeptide of The method generally involves isolating from a culture of the present invention- and the novel DNA sequence that Bacillus thuringiensis EG4140, EG1 1839, NRRL B-21885 encodes it represent a new class of B. thuringiensis crystal or NRRL B-21886 cells that have been grown under appro proteins, and do not share sequence homology with any of the priate conditions, the Ö-endotoxin polypeptide produced by WCRW-active endotoxins described in the aforementioned the cells. Such polypeptides may be isolated from the cell literature. Likewise, the B. thuringiensis strains of the present culture or Supernatant or from spore Suspensions derived invention comprise novel gene sequences that express a from the cell culture and used in the native form, or may be polypeptide having insecticidal activity against both otherwise purified or concentrated as appropriate for the par coleopteran and leipdopteran insects, including WCRW. ticular application. 0016 Disclosed and claimed herein is an isolated Bacillus 0020. A method of controlling a lepidopteran or thuringiensis Ö-endotoxin polypeptide comprising at least 10 coleopteran insect population is also provided by the inven contiguous amino acids from SEQID NO:2. More preferably tion. The method generally involves contacting the popula the polypeptide comprises at least 12 to 14 contiguous amino tion with an insecticidally-effective amount of a polypeptide acids from SEQID NO:2. Still more preferably, the polypep comprising the amino acid sequence of SEQID NO:2. Such tide comprises at least 16 to 18 contiguous amino acids from methods may be used to kill or reduce the numbers of lepi SEQID NO:2, and more preferably comprises at least 20 to dopteran or coleopteran insects in a given area, or may be 30 contiguous amino acids from SEQID NO:2. In an exem prophylactically applied to an environmental area to prevent plary embodiment, the inventors have identified an insecti infestation by a susceptible insect. Preferably the insect cidally-active polypeptide comprising the 721 amino acid ingests, or is contacted with, an insecticidally-effective long sequence of SEQID NO:2. Preferably such a polypep amount of the polypeptide. tide has insecticidal activity against both coleopteran and 0021 Additionally, the invention provides a purified anti lepidopteran insects. For example, the inventors have shown body that specifically binds to the insecticidal polypeptide. that a 6-endotoxin polypeptide comprising the sequence of Also provided are methods of preparing Suchanantibody, and SEQ ID NO:2 has insecticidal activity against WCRW and methods for using the antibody to isolate, identify, character Colorado potato beetle (CPB), as well as the lepidopteran ize, and/or purify polypeptides to which Such an antibody insects Plutella xylostella and Trichoplusia ni. specifically binds. Immunological kits and immunodetection 0017 Such polypeptides preferably are encoded by a methods useful in the identification of such polypeptides and nucleic acid segment comprising at least 23 contiguous peptide fragments and/or epitopes thereof are provided in nucleotides from SEQ ID NO: 1, and more preferably are detail herein, and also represent important aspects of the encoded by a nucleic acid segment comprising at least 35 present invention. contiguous nucleotides from SEQID NO:1. Exemplary poly 0022. Such antibodies may be used to detect the presence nucleotides encoding the insecticidal polypeptide comprise a of such polypeptides in a sample, or may be used as described US 2009/0087863 A1 Apr. 2, 2009 hereinbelow in a variety of immunological methods. An host cells, transgenic cell lines, pluripotent plant cells, and exemplary method for detecting a 6-endotoxin polypeptide in transgenic plants comprising at least a first sequence region a biological sample generally involves obtaining a biological that encodes a polypeptide comprising the sequence of SEQ sample suspected of containing a 6-endotoxin polypeptide; ID NO:2 are described in detail hereinbelow. contacting the sample with an antibody that specifically binds 0028. In a further embodiment, the invention provides to the polypeptide, under conditions effective to allow the methods for preparing an insecticidal polypeptide composi formation of complexes; and detecting the complexes so tion. In exemplary embodiments, such polypeptides may be formed. formulated for use as an insecticidal agent, and may be used 0023 For such methods, the invention also provides an to control insect populations in an environment, including immunodetection kit Such a kit generally contains, in Suitable agricultural environs and the like. The formulations may be container means, an antibody that binds to the Ö-endotoxin used to kill an insect, either by topical application, or by polypeptide, and at least a first immunodetection reagent ingestion of the polypeptide composition by the insect. In Optionally, the kit may provide additional reagents or instruc certain instances, it may be desirable to formulate the tions for using the antibody in the detection of Ö-endotoxin polypeptides of the present invention for application to the polypeptides in a sample. soil, on or near plants, trees, shrubs, and the like, near live 0024 Preparation of Such Antibodies May be Achieved plants, livestock, domiciles, farm equipment, buildings, and Using the Disclosed Polypeptide as an antigen in an animal as the like. described below. Antigenic epitopes, shorter peptides, pep 0029. The present invention also provides transformed tide fusions, carrier-linked peptide fragments, and the like host cells, pluripotent plant cell populations, embryonic plant may also be generated from a whole or a portion of the tissue, plant calli, plantlets, and transgenic plants that com polypeptide sequence disclosed in SEQIDNO:2. Particularly prise a selected sequence region that encodes the insecticidal preferred peptides are those that comprise at least 10 contigu polypeptide. Such cells are preferably prokaryotic or eukary ous amino acids from the sequence disclosed in SEQ ID otic cells such as bacterial, fungal, or plant cells, with exem NO:2. plary bacterial cells including Bacillus thuringiensis, Bacil 0025. In another embodiment, the present invention also lus subtilis, Bacillus megaterium, Bacillus cereus, provides nucleic acid segments that comprise a selected Escherichia, Salmonella, Agrobacterium or Pseudomonas nucleotide sequence region that comprises the polynucleotide cells (such as Bacillus thuringiensis EG4140, EG1839, sequence of SEQID NO:1. In preferred embodiments, this NRRL B-21885 and NRRL B-21886 cells). Selected nucleotide sequence region comprises a gene that 0030 The plants and plant host cells are preferably mono encodes a polypeptide comprising at least 10 contiguous cotyledonous or dicotyledonous plant cells such as corn, amino acid residues from SEQID NO:2, and more preferably, wheat, soybean, oat, cotton, rice, rye, Sorghum, Sugarcane, comprises the amino acid sequence of SEQID NO:2. In one tomato, tobacco, kapok, flax, potato, barley, turfgrass, pas example, the gene encoding the polypeptide of SEQID NO:2 ture grass, berry, fruit, legume, vegetable, ornamental plant, comprises a coding region that extends from nucleotide 92 to shrub, cactus, succulent, and tree cell. nucleotide 2254 of SEQID NO:1. 0031 Illustrative transgenic plants of the present inven 0026. Another aspect of the invention relates to a biologi tion preferably have incorporated into their genome a selected cally-pure culture of a wild-type B. thuringiensis bacterium, polynucleotide (or “transgene'), that comprises at least a first strain EG4140, deposited on Nov. 20, 1997 with the Agricul sequence region that encodes the insecticidal polypeptide of tural Research Culture Collection, Northern Regional SEQID NO:2. Research Laboratory (NRRL) having Accession No. 0032. Likewise, a progeny (decendant, offspring, etc.) of B-21885. B. thuringiensis EG4140 is described infra in Sec any generation of Such a transgenic plant also represents an tion 5.0. B. thuringiensis EG4140 is a naturally-occurring important aspect of the invention. Preferably such progeny strain that contains a sequence region that is highly homolo comprise the selected transgene, and inherit the phenotypic gous to, and preferably identical to, a polynucleotide trait of insect resistance demonstrated by the parental plant. A sequence that encodes the 721 amino acid long polypeptide seed of any generation of all such transgenic insect-resistant sequence in SEQID NO:2. In an exemplary embodiment, the plants is also an important aspect of the invention. Preferably strain comprises a nucleotide sequence comprising the the seed will also comprise the selected transgene and will cryET70 gene disclosed in SEQID NO:1. EG4140 produces confer to the plants grown from the seed the phenotypic trait an 87-kDa insecticidal polypeptide that is related to, or iden of insect resistance. tical to, the polypeptide disclosed in SEQID NO:2. 0033. Insect resistant, crossed fertile transgenic plants 0027. A further embodiment of the invention relates to a comprising a transgene that encodes the polypeptide of SEQ vector comprising a sequence region that encodes a polypep ID NO:2 may be prepared by a method that generally involves tide comprising the amino acid sequence of SEQID NO:2, a obtaining a fertile transgenic plant that contains a chromo recombinant host cell transformed with such a recombinant Somally incorporated transgene encoding the insecticidal vector, and biologically-pure cultures of recombinant bacte polypeptide of SEQID NO:2; operably linked to a promoter ria transformed with a polynucleotide sequence that encodes active in the plant, crossing the fertile transgenic plant with a the polypeptide disclosed in SEQID NO:2. In an exemplary second plant lacking the transgene to obtain a third plant embodiment, the bacterium is B. thuringiensis EG1 1839 (de comprising the transgene; and backcrossing the third plant to posited on Nov. 20, 1997 with the NRRL and having the obtain a backcrossed fertile plant. In Such cases, the transgene accession number B-21886) described herein. Both B-21885 may be inherited through a male parent or through a female and B-21886 were deposited with the NRRL in the Patent parent. The second plant may be an inbred, and the third plant Culture Collection under the terms of the Budapest Treaty, may be a hybrid. and viability statements pursuant to International Receipt 0034. Likewise, an insect resistant hybrid, transgenic Form BP/4 were obtained. Exemplary vectors, recombinant plant may be prepared by a method that generally involves US 2009/0087863 A1 Apr. 2, 2009

crossing a first and a secondinbred plant, wherein one or both been isolated free of the total genomic DNAs of a particular of the first and second inbred plants comprises a chromo species. Therefore, a nucleic acid segment or polynucleotide Somally incorporated transgene that encodes the polypeptide encoding an endotoxin polypeptide refers to a nucleic acid of SEQID NO:2 operably linked to a plant expressible pro molecule that comprises at least a first crystal protein-encod moter that expresses the transgene. In illustrative embodi ing sequences yet is isolated away from, or purified free from, ments, the first and second inbred plants may be monocot total genomic DNA of the species from which the nucleic acid plants selected from the group consisting of corn, wheat, segment is obtained, which in the instant case is the genome rice, barley, oats, rye, Sorghum, turfgrass and Sugarcane. of the Gram-positive bacterial genus, Bacillus, and in particu 0035. In related embodiment, the invention also provides a lar, the species of Bacillus known as B. thuringiensis. method of preparing an insect resistant plant. The method Included within the term “nucleic acid segment, are poly generally involves contacting a recipient plant cell with a nucleotide segments and Smaller fragments of such segments, DNA composition comprising at least a first transgene that and also recombinant vectors, including, for example, plas encodes the polypeptide of SEQID NO:2 under conditions mids, cosmids, phagemids, phage, virions, baculovirses, arti permitting the uptake of the DNA composition; selecting a ficial chromosomes, viruses, and the like. Accordingly, poly recipient cell comprising a chromosomally incorporated nucleotide sequences that have between about 70% and about transgene that encodes the polypeptide; regenerating a plant 80%, or more preferably between about 81% and about 90%, from the selected cell; and identifying a fertile transgenic or even more preferably between about 91% and about 99% plant that has enhanced insect resistance relative to the cor nucleic acid sequence identity or functional equivalence to responding non-transformed plant. the polynucleotide sequence of SEQ ID NO:1 will be 0036. A method of producing transgenic seed generally sequences that are “essentially as set forth in SEQID NO:1.” involves obtaining a fertile transgenic plant comprising a Highly preferred sequences, are those which are preferably chromosomally integrated transgene that encodes a polypep about 91%, about 92%, about 93%, about 94%, about 95%, tide comprising the amino acid sequence of SEQID NO:2. about 96%, about 97%, about 98%, about 99%, or about operably linked to a promoter that expresses the transgene in 100% identical or functionally equivalent to the nucleotide a plant; and growing the plant under appropriate conditions to sequence of SEQ ID NO:1. Other preferred sequences that produce the transgenic seed. encode CryET70- or CryET70-related sequences are those 0037. A method of producing progeny of any generation which are about 81%, 82%, 83%, 84%, 85%, 86%, 87%, of an insect resistance-enhanced fertile transgenic plant is 88%, 89%, or 90% identical or functionally equivalent to the also provided by the invention. The method generally polynucleotide sequence set forth in SEQID NO:1 Likewise, involves collecting transgenic seed from a transgenic plant sequences that are about 71%, 72%, 73%, 74%, 75%, 76%, comprising a chromosomally integrated transgene that 77%, 78%, 79%, or 80% identical or functionally equivalent encodes the polypeptide of SEQID NO:2, operably linked to to the polynucleotide sequence set forth in SEQID NO: 1 are a promoter that expresses the transgene in the plant; planting also contemplated to be useful in the practice of the present the collected transgenic seed; and growing the progeny trans invention. genic plants from the seed. 0042. Similarly, a polynucleotide comprising an isolated, 0038. These methods for creating transgenic plants, prog purified, or selected gene or sequence region refers to a poly eny and seed may involve contacting the plant cell with the nucleotide which may include in addition to peptide encoding DNA composition using one of the processes well-known for sequences, certain other elements such as, regulatory plant cell transformation Such as microprojectile bombard sequences, isolated Substantially away from other naturally ment, electroporation or Agrobacterium-mediated transfor occurring genes or protein-encoding sequences. In this mation. respect, the term “gene' is used for simplicity to refer to a 0039. These and other embodiments of the present inven functional protein-, or polypeptide-encoding unit. As will be tion will be apparent to those of skill in the art from the understood by those in the art, this functional term includes following examples and claims, having benefit of the teach both genomic sequences, operator sequences and Smaller ings of the Specification herein. engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides. In certain 2.1 CryET70 Polynucleotide Segments embodiments, a nucleic acid segment will comprise at least a 0040. The present invention provides nucleic acid seg first gene that encodes a polypeptide comprising the sequence ments, that can be isolated from virtually any source, that are of SEQID NO:2. free from total genomic DNA and that encode the novel 0043. To permit expression of the gene, and translation of insecticidal polypeptides and peptide fragments thereof that the mRNA into mature polypeptide, the nucleic acid segment are disclosed herein. The polynucleotides encoding these preferably also comprises at least a first promoter operably peptides and polypeptides may encode active insecticidal linked to the gene to express the gene product in a host cell proteins, or peptide fragments, polypeptide Subunits, func transformed with this nucleic acid segment. The promoter tional domains, or the like of one or more CryET70 or may be an endogenous promoter, or alternatively, a heterolo CryET70-related crystal proteins, such as the polypeptide gous promoter selected for its ability to promote expression of disclosed in SEQID NO:2. In addition the invention encom the gene in one or more particular cell types. For example, in passes nucleic acid segments which may be synthesized the creation of transgenic plants and pluripotent plant cells entirely invitro using methods that are well-known to those of comprising a cryET70 gene, the heterologous promoter of skill in the art which encode the novel CryET70 polypeptide, choice is one that is plant-expressible, and in many instances, peptides, peptide fragments, subunits, or functional domains may preferably be a plant-expressible promoter that is tissue disclosed herein. or cell cycle-specific. The selection of plant-expressible pro 0041 As used herein, the term “nucleic acid segment” or moters is well-known to those skilled in the art of plant "polynucleotide refers to a nucleic acid molecule that has transformation, and exemplary Suitable promoters are US 2009/0087863 A1 Apr. 2, 2009

described herein. In certain embodiments, the plant-express of the coding region or may include various internal ible promoter may be selected from the group consisting of sequences, i.e., introns, which are known to occur within corn Sucrose synthetase 1, corn alcohol dehydrogenase 1, genes. corn light harvesting complex, corn heat shock protein, pea 0048. The nucleic acid segments of the present invention, small subunit RuBP carboxylase, Tiplasmid mannopine syn regardless of the length of the coding sequence itself, may be thase, Ti plasmid nopaline synthase, petunia chalcone combined with other nucleic acid sequences, such as promot isomerase, bean glycine rich protein 1, Potato patatin, lectin, ers, polyadenylation signals, additional restriction enzyme CaMV 35S, and the S-E9 small subunit RUBP carboxylase sites, multiple cloning sites, other coding segments, and the promoter. like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost 0044) “Isolated substantially away from other coding any length may be employed, with the total length preferably sequences' means that the gene of interest, in this case, a gene being limited by the ease of preparation and use in the encoding a bacterial crystal protein, forms the significant part intended recombinant nucleic acid protocol. For example, of the coding region of the DNA segment, and that the DNA nucleic acid fragments may be prepared that include a short segment does not contain large portions of naturally-occur contiguous stretch encoding the peptide sequence disclosed ring coding DNA, such as large chromosomal fragments or in SEQID NO:2, or that are identical to or complementary to other functional genes or operon coding regions. Of course, nucleic acid sequences which encode the peptides disclosed this refers to the DNA segment as originally isolated, and in SEQID NO:2, and particularly those nucleic acid segments does not exclude genes, recombinant genes, synthetic linkers, disclosed in SEQ ID NO: 1. For example, nucleic acid or coding regions later added to the segment by the hand of sequences such as about 23 nucleotides, and that are up to a. about 10,000, about 5,000, about 3,000, about 2,000, about 0045. In particular embodiments, the invention concerns 1,000, about 500, about 200, about 100, about 50, and about isolated polynucleotides (such as DNAs, RNAs, antisense 23 or so base pairs in length (including all intermediate DNAs, antisense RNAs, ribozymes, and PNAs) and recom lengths) that comprise a contiguous nucleotide sequence binant vectors comprising polynucleotide sequences that from SEQID NO:1, or those that encode a contiguous amino encode one or more polypeptides comprise all or at least 10 acid sequence from SEQ ID NO:2 are contemplated to be contiguous contiguous amino acids from SEQID NO:2. particularly useful. 0049. In one embodiment, the invention also provides an 0046. The term “a sequence essentially as set forth in SEQ isolated nucleic acid segment characterized as: (i) a nucleic ID NO:2 means that the sequence substantially corresponds acid segment comprising a sequence region that consists of at to a portion of the sequence of SEQID NO:2 and has rela least 23 contiguous nucleotides that have the same sequence tively few amino acids that are not identical to, or a biologi as, or are complementary to, 23 contiguous nucleotides of cally functional equivalent of the amino acids of any of these SEQ ID NO:1; or (ii) a nucleic acid segment of from 23 to sequences. The term “biologically functional equivalent” is about 2344 nucleotides in length that hybridizes to the nucleic well understood in the art and is further defined in detail acid segment of SEQ ID NO:1; or the complement thereof, herein (e.g. see Illustrative Embodiments). Accordingly, understandard high-stringency hybridization conditions. sequences that have between about 70% and about 80%, or 0050. It will be readily understood that “intermediate more preferably between about 81% and about 90%, or even lengths, in the context of polynucleotide sequences, or more preferably between about 91% and about 99% amino nucleic acid segments, or primer or probes specific for the acid sequence identity or functional equivalence to the amino disclosed gene, means any length between the quoted ranges, acid sequence of SEQ ID NO:2 will be sequences that are such as from about 24, 25, 26, 27, 28, 29, etc.; 30, 31, 32,33, “essentially as set forth in SEQID NO:2. Highly preferred 34, 35, 36, 37, 38, 39, etc.; 40, 41, 42, 43, 44, 45, 46, 47, 48, sequences, are those which are preferably about 91%, about 49, 50,51,52,53,54, 55,56, 57,58, 59,60, 65,70, 75,80, 85, 92%, about 93%, about 94%, about 95%, about 96%, about 90, 95, etc.; 100, 101, 102, 103,104, etc.; 110, 120, 125, 130, 97%, about 98%, about 99%, or about 100% identical or 135, 140,145, 150, 155, 160, 165, 170, 180, 190, etc.; includ functionally equivalent to the amino acid sequence of SEQID ing all integers in the ranges of from about 200-500: 500-1, NO:2. Other preferred sequences are those which are about 000; 1,000-2,000; 2,000-3,000; 3,000-5,000; and up to and 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% including sequences of about 10,000 or so nucleotides and the identical or functionally equivalent to the amino acid like. sequence of SEQ ID NO:2. Likewise, sequences that are 0051. Likewise, it will be readily understood that “inter about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or mediate lengths, in the context of polypeptides or peptides, 80% identical or functionally equivalent to the polypeptide means any length between the quoted ranges of contiguous sequence set forth in SEQID NO:2 are also contemplated to amino acids. For example, when considering CryET70-de be useful in the practice of the present invention. rived peptides, all lengths between about 10 and about 100 0047. It will also be understood that amino acid and contiguous amino acid sequences are contemplated to be nucleic acid sequences may include additional residues. Such useful in particular embodiments disclosed herein. For as additional N- or C-terminal amino acids or 5' or 3' example, peptides comprising contiguous amino acid sequences, and yet still be essentially as set forth in one of the sequences having about 10, about 11, about 12, about 13, sequences disclosed herein, so long as the sequence meets the about 14, about 15, about 16, about 17, about 18, about 19, criteria set forth above, including the maintenance of biologi about 20, about 21, about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, cal protein activity where protein expression is concerned. 32,33,34, 35,36, 37,38,39, 40, 41, 42, 43,44, 45,46, 47, 48, The addition of terminal sequences particularly applies to 49, 50, 51, 52,53,54, 55,56, 57,58, 59, 60, 65, etc., 70, 75, nucleic acid sequences that may, for example, include various etc., 80, 85, etc., 90, 95, etc., and even those peptides com non-coding sequences flanking either of the 5' or 3' portions prising at least about 96.97, 98, 99, 100, 101, 102, 103, and US 2009/0087863 A1 Apr. 2, 2009

104, or more contiguous amino acids from SEQID NO:2 are polypeptides may be identified that are highly homologous to explicitly considered to fall within the scope of the present (or even identical to) this sequence, but which may have been invention. isolated from different organisms or sources, or alternatively, 0052 Furthermore, it will also be readily understood by may even have been synthesized entirely, or partially denovo. one of skill in the art, that “intermediate lengths, in the AS Such, all polypeptide sequences, whether naturally-occur context of larger CryET70 and CryET70-related polypep ring, or artificially-created, that are structurally homologous tides, means any length between the quoted ranges of con to the primary amino acid sequence of SEQID NO:2 and that tiguous amino acids that comprise Such a polypeptide. For have similar insecticidal activity against the target insects example, when considering the polypeptides of the present disclosed herein are considered to fall within the scope of this invention, all lengths between about 100 and about 730 con disclosure. Likewise, all polynucleotide sequences, whether tiguous amino acid sequences are contemplated to be useful naturally-occurring, or artificially-created, that are structur in particular embodiments disclosed herein. For example, ally homologous to the nucleotide sequence of SEQID NO:1, polypeptides comprising a contiguous amino acid sequence or that encodes a polypeptide that is homologous, and bio having at least about 100, about 101, about 102, 103, 104. logically-functionally equivalent to the amino acid sequence 105, 106, 107, 108, 109, 110, 115, 120, 125, 130, 135, 140, disclosed in SEQID NO:2 are also considered to fall within 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, etc., the scope of this disclosure. 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 220, 0055. If desired, one may also preparefusion proteins and 230, 240, 250, 260, 270, 280, 290, etc., 300, 310,320, 330, peptides, e.g., where the peptide-coding regions are aligned 340, 350, 360, 370, 380,390, 400, etc., 410, 430, 450, 470, within the same expression unit with other proteins or pep 490, etc., 500,525,550, 575,600, 650,674, etc., 700, etc., and tides having desired functions, such as for purification or even those polypeptides that comprise at least about 721 or immunodetection purposes (e.g., proteins that may be puri more amino acids are explicitly considered to fall within the fied by affinity chromatography and enzyme label coding scope of the present invention. Particularly in the case of regions, respectively). fusion proteins comprising a whole or a portion of the amino 0056 Recombinant vectors form further aspects of the acid sequence of SEQ ID NO:2, longer polypeptide present invention. Particularly useful vectors are contem sequences may be preferred, including sequences that com plated to be those vectors in which the coding portion of the prise about 703,740, 750, 760, 770, 780, 790, or even about DNA segment, whether encoding a full-length insecticidal 800 or greater amino acids in length. protein or Smaller peptide, is positioned under the control of 0053. It will also be understood that this invention is not a promoter. The promoter may be in the form of the promoter limited to the particular nucleic acid sequences which encode that is naturally associated with a gene encoding peptides of peptides of the present invention, or which encode the amino the present invention, as may be obtained by isolating the 5' acid sequence of SEQID NO:2, including the DNA sequence non-coding sequences located upstream of the coding seg which is particularly disclosed in SEQID NO: 1. Recombi ment or exon, for example, using recombinant cloning and/or nant vectors and isolated DNA segments may therefore vari PCRTM technology, in connection with the compositions dis ously include the polypeptide-coding regions themselves, closed herein. In many cases, the promoter may be the native coding regions bearing selected alterations or modifications CryET70 promoter, or alternatively, a heterologous promoter, in the basic coding region, or they may encode larger Such as those of bacterial origin (including promoters from polypeptides that nevertheless include these peptide-coding other crystal proteins), fungal origin, viral, phage or regions or may encode biologically functional equivalent pro phagemid origin (including promoters such as CaMV35, and teins or peptides that have variant no acids sequences. its derivatives, T3, T7, W, and (p promoters and the like), or 0054 The DNA segments of the present invention encom plant origin (including constitutive, inducible, and/or tissue pass biologically-functional, equivalent peptides. Such specific promoters and the like). sequences may arise as a consequence of codon degeneracy 2.2 Nucleic Acid Segments as Hybridization Probes and functional equivalency that are known to occur naturally and Primers within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally-equivalent proteins or peptides 0057. In addition to their use in directing the expression of may be created via the application of recombinant DNA tech crystal proteins or peptides of the present invention, the nology, in which changes in the protein structure may be nucleic acid sequences described herein also have a variety of engineered, based on considerations of the properties of the other uses. For example, they have utility as probes or primers amino acids being exchanged. Changes designed by man may in nucleic acid hybridization embodiments. The invention be introduced through the application of site-directed provides a method for detecting a nucleic acid sequence mutagenesis techniques, e.g., to introduce improvements to encoding a 6-endotoxin polypeptide. The method generally the antigenicity of the protein or to test mutants in order to involves obtaining sample nucleic acids Suspected of encod examine activity at the molecular level. Alternatively, native, ing a 6-endotoxin polypeptide; contacting the sample nucleic as yet-unknown or as yet unidentified polynucleotides and/or acids with an isolated nucleic acid segment comprising at polypeptides structurally and/or functionally-related to the least 23 contiguous nucleotides from SEQ ID NO:1, under sequences disclosed herein may also be identified that fall conditions effective to allow hybridization of substantially within the scope of the present invention. Such polynucle complementary nucleic acids; and detecting the hybridized otides are those polynucleotides that encode a polypeptide complementary nucleic acids thus formed. structurally and/or functionally similar or identical to, the 0058. In the practice of such a method, it is contemplated polypeptide characterized herein as a “CryET70 polynucle that nucleic acid segments that comprise a sequence region otide. Since the designation “CryET70' is an arbitrary name that consists of at least about a 23 nucleotide long contiguous chosen to readily identify polypeptides comprising the amino sequence that has the same sequence as, or is complementary acid sequence of SEQID NO:2, it is likely that many other to, an about 23 nucleotide long contiguous nucleic acid seg US 2009/0087863 A1 Apr. 2, 2009

ment of SEQ ID NO: 1 will find particular utility. Longer NaCl at temperatures of about 50° C. to about 70° C. Such contiguous identical or complementary sequences, e.g., those selective conditions tolerate little, if any, mismatch between of about 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, the probe and the template or target strand, and would be 70, 75, 80, 85,90, 95, 100, 150, 200, 250, 300,350, 400, 450, particularly Suitable for isolating crystal protein-encoding 500, 550, 600, 650, 700, 750, 800, 850,900,950, 1000, 1100, DNA segments. Detection of DNA segments via hybridiza 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, tion is well-knownto those of skill in the art, and the teachings 2200,2300, bp, etc. (including all intermediate lengths and up of U.S. Pat. Nos. 4,965,188 and 5,176,995 (each incorporated to and including the full-length sequence of about 2344 base herein by reference) are exemplary of the methods of hybrid pairs encoding the CryET70 polypeptide will also be of use in ization analyses. Teachings Such as those found in the texts of certain embodiments. Maloy et al., 1990; Maloy 1994; Segal, 1976; Prokop, 1991; 0059 Also provided is a nucleic acid detection kit com and Kuby, 1994, are particularly relevant. prising, in Suitable container means, at least a first nucleic 0063 Of course, for some applications, for example, acid segment comprising at least 23 contiguous nucleotides where one desires to prepare mutants employing a mutant from SEQID NO: 1, and at least a first detection reagent. The primer Strand hybridized to an underlying template or where ability of such nucleic acid probes to specifically hybridize to one seeks to isolate crystal protein-encoding sequences from crystal protein-encoding sequences will enable them to be of related species, functional equivalents, or the like, less strin use in detecting the presence of complementary sequences in gent hybridization conditions will typically be needed in a given sample. However, other uses are envisioned, includ order to allow formation of the heteroduplex. In these circum ing the use of the sequence information for the preparation of stances, one may desire to employ “low stringency” or mutant species primers, or primers for use in preparing other “reduced stringency hybridization conditions such as those genetic constructions. employing from about 0.15 M to about 0.9 M salt, at tem 0060 Nucleic acid molecules having sequence regions peratures ranging from about 20° C. to about 55° C. Cross consisting of contiguous nucleotide stretches of about 23 to hybridizing species can thereby be readily identified as posi about 50, or even up to and including sequences of about tively hybridizing signals with respect to control 100-200 nucleotides or so, identical Or complementary to the hybridizations. In any case, it is generally appreciated that DNA sequence of SEQ ID NO:1, are particularly contem conditions can be rendered more stringent by the addition of plated as hybridization probes for use in, e.g., Southern and increasing amounts of formamide, which serves to destabilize Northern blotting. Intermediate-sized fragments will also the hybrid duplex in the same manner as increased tempera generally find use in hybridization embodiments, wherein the ture. Thus, hybridization conditions can be readily manipu length of the contiguous complementary region may be var lated, and thus will generally be a method of choice depend ied, such as between about 25-30, or between about 30 and ing on the desired results. Regardless of what particular about 40 or so nucleotides, but larger contiguous complemen combination of salts (such as NaCl or NaCitrate and the like), tarity stretches may be used, such as those from about 200 to organic buffers (including eg, formamide and the like), and about 300, or from about 300 to about 400 or 500 or so incubation or washing temperatures are employed, the skilled nucleotides in length, according to the length complementary artisan will readily be able to employ hybridization condi sequences one wishes to detect. It is even possible that longer tions that are “high.” “medium, or “low” stringency, and will contiguous sequence regions may be utilized including those be able to interpret the results from hybridization analyses sequences comprising at least about 600, 700, 800, 900, 1000, using such conditions to determine the relative homology of 1100, 1200, 1300, 1400, 1500, or more contiguous nucle a target nucleic acid sequence to that of the particular novel otides from SEQID NO:1. polynucleotide probe sequence employed from SEQID NO: 0061. Of course, fragments may also be obtained by other 1 techniques such as, e.g., by mechanical shearing or by restric 0064. In certain embodiments, it will be advantageous to tion enzyme digestion. Small nucleic acid segments or frag employ nucleic acid sequences of the present invention in ments may be readily prepared by, for example, directly syn combination with an appropriate means, such as a label, for thesizing the fragment by chemical means, as is commonly determining hybridization. A wide variety of appropriate practiced using an automated oligonucleotide synthesizer. indicator means are known in the art, including fluorescent, Also, fragments may be obtained by application of nucleic radioactive, enzymatic or other ligands. Such as avidin/biotin, acid reproduction technology, such as the PCRTM technology which are capable of giving a detectable signal. In preferred of U.S. Pat. Nos. 4,683,195 and 4,683.202(each incorporated embodiments, one will likely desire to employ a fluorescent herein by reference), by introducing selected sequences into label or an enzyme tag, Such as urease, alkaline phosphatase recombinant vectors for recombinant production, and by or peroxidase, instead of radioactive or other environmentally other recombinant DNA techniques generally known to those undesirable reagents. In the case of enzyme tags, colorimetric of skill in the art of molecular biology. indicator Substrates are known that can be employed to pro 0062 Accordingly, the nucleotide sequences of the inven vide a means visible to the human eye or spectrophotometri tion may be used for their ability to selectively form duplex cally, to identify specific hybridization with complementary molecules with complementary stretches of DNA fragments. nucleic acid-containing samples. Depending on the application envisioned, one will desire to 0065. In general, it is envisioned that the hybridization employ varying conditions of hybridization to achieve vary probes described herein will be useful both as reagents in ing degrees of selectivity of probe towards target sequence. Solution hybridization as well as in embodiments employing For applications requiring high selectivity, one will typically a solid phase. In embodiments involving a solid phase, the test desire to employ relatively stringent conditions to form the DNA (or RNA) is adsorbed or otherwise affixed to a selected hybrids. "High Stringency hybridization conditions, e.g., matrix or Surface. This fixed, single-stranded nucleic acid is typically employ relatively low salt and/or high temperature then subjected to specific hybridization with selected probes conditions, such as provided by about 0.02 M to about 0.15 M under desired conditions. The selected conditions will depend US 2009/0087863 A1 Apr. 2, 2009 on the particular circumstances based on the particular crite Scription-terminating region, whereby the promoter is ria required (depending, for example, on the G+C content, capable of driving the transcription of the coding region in the type of target nucleic acid, source of nucleic acid, size of cell, and hence providing the cell the ability to produce the hybridization probe, etc.). Following washing of the hybrid polypeptide in vivo. Alternatively, in instances where it is ized surface So as to remove nonspecifically bound probe desirable to control, regulate, or decrease the amount of a molecules, specific hybridization is detected, or even quanti particular recombinant crystal protein expressed in a particu tated, by means of the label. lar transgenic cell, the invention also provides for the expres sion of crystal protein antisense mRNA. The use of antisense 2.3 Vectors and Methods for Recombinant mRNA as a means of controlling or decreasing the amount of Expression of Cry70 and Cry70-Related a given protein of interest in a cell is well-known in the art. Polypeptides 0069. Another aspect of the invention comprises trans genic plants which express a gene, gene segment, or sequence 0066. In other embodiments, it is contemplated that cer tain advantages will begained by positioning the coding DNA region that encodes at least one or more of the novel polypep segment under the control of a recombinant, or heterologous, tide compositions disclosed herein. As used herein, the term promoter. As used herein, a recombinant or heterologous “transgenic plant' is intended to refer to a plant that has promoter is intended to refer to a promoter that is not nor incorporated DNA sequences, including but not limited to mally associated with a DNA segment encoding a crystal genes which are perhaps not normally present, DNA protein or peptide in its natural environment. Such promoters sequences not normally transcribed into RNA or translated may include promoters normally associated with other genes, into a protein (“expressed”), or any other genes or DNA and/or promoters isolated from any bacterial, viral, eukary sequences which one desires to introduce into the non-trans otic, or plant cell. Naturally, it will be important to employ a formed plant. Such as genes which may normally be present in promoter that effectively directs the expression of the DNA the non-transformed plant but which one desires to either segment in the cell type, organism, or even animal, chosen for genetically engineer or to have altered expression. expression. The use of promoter and cell type combinations 0070. It is contemplated that in some instances the genome for protein expression is generally known to those of skill in of a transgenic plant of the present invention will have been the art of molecular biology, for example, see Sambrooketal, augmented through the stable introduction of one or more 1989. The promoters employed may be constitutive, or induc transgenes, either native, synthetically modified, or mutated, ible, and can be used under the appropriate conditions to that encodes an insecticidal polypeptide that is identical to, or direct high level expression of the introduced DNA segment, highly homologous to the polypeptide disclosed in SEQ ID Such as is advantageous in the large-scale production of NO:2. In some instances, more than one transgene will be recombinant proteins or peptides. Appropriate promoter sys incorporated into the genome of the transformed host plant tems contemplated for use in high-level expression include, cell. Such is the case when more than one crystal protein but are not limited to, the Pichia expression vector system encoding DNA segment is incorporated into the genome of (Pharmacia LKB Biotechnology). Such a plant. In certain situations, it may be desirable to have 0067. In connection with expression embodiments to pre one, two, three, four, or even more B. thuringiensis crystal pare recombinant proteins and peptides, it is contemplated proteins (either native or recombinantly-engineered) incor that longer DNA segments will most often be used, with DNA porated and stably expressed in the transformed transgenic segments encoding the entire peptide sequence being most plant. Alternatively, a second transgene may be introduced preferred. However, it will be appreciated that the use of into the plant cell to confer additional phenotypic traits to the shorter DNA segments to direct the expression of crystal plant. Such transgenes may confer resistance to one or more peptides or epitopic core regions, such as may be used to insects, bacteria, fingi, viruses, nematodes, or other patho generate anti-crystal protein antibodies, also falls within the genS. Scope of the invention. DNA segments that encode peptide 0071. A preferred gene which may beintroduced includes, antigens from about 8 to about 50 amino acids in length, or for example, a crystal protein-encoding DNA sequence from more preferably, from about 8 to about 30 amino acids in bacterial origin, and particularly one or more of those length, or even more preferably, from about 8 to about 20 described herein which are obtained from Bacillus spp. amino acids in length are contemplated to be particularly Highly preferred nucleic acid sequences are those obtained useful. Such peptide epitopes may be amino acid sequences from B. thuringiensis, or any of those sequences which have which comprise a contiguous amino acid sequence from SEQ been genetically engineered to decrease or increase the insec ID NO:2. ticidal activity of the crystal protein in such a transformed host cell. 2.4 Transgenic Plants Expressing CryET70 0072 Means for transforming a plant cell and the prepa Polypeptides ration of pluripotent plant cells, and regeneration of a trans genic cell line from a transformed cell, cell culture, embryo, 0068. In yet another aspect, the present invention provides or callus tissue are well-known in the art, and are discussed methods for producing a transgenic plant that expresses a herein. Vectors, (including plasmids, cosmids, phage, selected nucleic acid segment comprising a sequence region phagemids, baculovirus, viruses, virions, BACs bacterial that encodes the novel endotoxin polypeptides of the present artificial chromosomes.YACs eastartificial chromosomes) invention. The process of producing transgenic plants is well comprising at least a first nucleic acid segment encoding an known in the art. In general, the method comprises transform insecticidal polypeptide for use in transforming Such cells ing a Suitable plant host cell with a DNA segment that con will, of course, generally comprise either the operons, genes, tains a promoter operatively linked to a coding region that or gene-derived sequences of the present invention, either encodes one or more CryET70 polypeptides. Such a coding native, or synthetically-derived, and particularly those encod region is generally operatively linked to at least a first tran ing the disclosed crystal proteins. These nucleic acid con US 2009/0087863 A1 Apr. 2, 2009

structs can further include structures such as promoters, accordance with the present invention, may be employed to enhancers, polylinkers, introns, terminators, or even gene detect crystal proteins or crystal protein-related epitope-con sequences which have positively- or negatively-regulating taining peptides. In general, these methods will include first activity upon the cloned Ö-endotoxin gene as desired. The obtaining a sample Suspected of containing Such a protein, DNA segment or gene may encode either a native or modified peptide or antibody, contacting the sample with an antibody crystal protein, which will be expressed in the resultant or peptide in accordance with the present invention, as the recombinant cells, and/or which will confer to a transgenic case may be, under conditions effective to allow the forma plant comprising Such a segment, an improved phenotype (in tion of an immunocomplex, and then detecting the presence this case, increased resistance to insect attack, infestation, or of the immunocomplex. colonization). 0078. In general, the detection of immunocomplex forma 0073. The preparation of a transgenic plant that comprises tion is quite well known in the art and may be achieved at least one polynucleotide sequence encoding a CryET70 or through the application of numerous approaches. For CryET70-derived polypeptide for the purpose of increasing example, the present invention contemplates the application or enhancing the resistance of such a plant to attack by a target of ELISA, RIA, immunoblot (e.g., dot blot), indirect immu insect represents an important aspect of the invention. In nofluorescence techniques and the like. Generally, immuno particular, the inventors describe herein the preparation of complex formation will be detected through the use of a label, insect-resistant monocotyledonous or dicotyledonous plants, Such as a radiolabel or an enzyme tag (such as alkaline phos by incorporating into Such a plant, a transgenic DNA segment phatase, horseradish peroxidase, or the like). Of course, one encoding one or more CryET70 polypeptides which are toxic may find additional advantages through the use of a second to a coleopteran or lepidopteran insect. ary binding ligand Such as a second antibody or a biotin/ 0074. In a related aspect, the present invention also avidin ligand binding arrangement, as is known in the art. encompasses a seed produced by the transformed plant, a 007.9 For assaying purposes, it is proposed that virtually progeny from Such seed, and a seed produced by the progeny any sample Suspected of comprising eithera crystal protein or of the original transgenic plant, produced in accordance with peptide or a crystal protein-related peptide orantibody sought the above process. Such progeny and seeds will have a crystal to be detected, as the case may be, may be employed. It is protein-encoding transgene stably incorporated into their contemplated that such embodiments may have application in genome, and Such progeny plants will inherit the traits the tittering of antigen orantibody samples, in the selection of afforded by the introduction of a stable transgene in Mende hybridomas, and the like. In related embodiments, the present lian fashion. All Such transgenic plants having incorporated invention contemplates the preparation of kits that may be into their genome transgenic DNA segments encoding one or employed to detect the presence of crystal proteins or related more CryET70 crystal proteins or polypeptides are aspects of peptides and/or antibodies in a sample. Samples may include this invention. As well-known to those of skill in the art, a cells, cell Supernatants, cell Suspensions, cell extracts, progeny of a plant is understood to mean any offspring or any enzyme fractions, protein extracts, or other cell-free compo descendant from Such a plant. sitions suspected of containing crystal proteins or peptides. Generally speaking, kits in accordance with the present 2.5 Crystal Protein Screening and Detection Kits invention will include a suitable crystal protein, peptide oran antibody directed against Such a protein or peptide, together 0075. The present invention contemplates methods and with an immunodetection reagent and a means for containing kits for screening samples Suspected of containing crystal the antibody or antigen and reagent. The immunodetection protein polypeptides or crystal protein-related polypeptides, reagent will typically comprise a label associated with the or cells producing Such polypeptides. A kit may contain one antibody or antigen, or associated with a secondary binding or more antibodies specific for the CryET70 amino acid ligand. Exemplary ligands might include a secondary anti sequence shown in SEQID NO:2, or one or more antibodies specific for a peptide derived from the sequence shown in body directed against the first antibody or antigen or a biotin SEQID NO:2, and may also contain reagent(s) for detecting or avidin (or Streptavidin) ligand having an associated label. an interaction between a sample and an antibody of the Of course, as noted above, a number of exemplary labels are present invention. The provided reagent(s) can be radio-, known in the art and all Such labels may be employed in fluorescently- or enzymatically-labeled. The kit can contain a connection with the present invention. known radiolabeled agent capable of binding or interacting 0080. The container will generally include a vial into with a nucleic acid or antibody of the present invention. which the antibody, antigen or detection reagent may be 0076. The reagent(s) of the kit can be provided as a liquid placed, and preferably suitably aliquotted. The kits of the Solution, attached to a solid Support or as a dried powder. present invention Will also typically include a means for Preferably, when the reagent(s) are provided in a liquid solu containing the antibody, antigen, and reagent containers in tion, the liquid Solution is an aqueous solution. Preferably, close confinement for commercial sale. Such containers may when the reagent(s) provided are attached to a solid Support, include injection or blow-molded plastic containers into the Solid Support can be chromatograph media, a test plate which the desired vials are retained. having a plurality of wells, or a microscope slide. When the reagent(s) provided are a dry powder, the powder can be 2.6 Insecticidal Compositions and Methods of Use reconstituted by the addition of a suitable solvent, that may be I0081. The inventors contemplate that the polypeptide provided. compositions disclosed herein will find particular utility as 0077. In still flirter embodiments, the present invention insecticides for topical and/or systemic application to field concerns immunodetection methods and associated kits. It is crops, grasses, fruits and vegetables, lawns, trees, and/or proposed that the crystal proteins or peptides of the present ornamental plants. Alternatively, the polypeptides disclosed invention may be employed to detect antibodies having reac herein may be formulated as a spray, dust, powder, or other tivity therewith, or, alternatively, antibodies prepared in aqueous, atomized or aerosol for killing an insect, or control US 2009/0087863 A1 Apr. 2, 2009

ling an insect population. The polypeptide compositions dis Pseudomonas spp. cells transformed with a DNA segment closed herein may be used prophylactically, or alternatively, disclosed herein and expressing the crystal protein are also may be administered to an environment once target insects, contemplated to be useful. such as WCRW, have been identified in the particular envi ronment to be treated. 2.6.3 Powders, Dusts, and Spore Formulations 0082) Regardless of the method of application, the amount I0086. In a third important embodiment, the bioinsecticide of the active polypeptide component(s) is applied at an insec composition comprises a wettable powder, dust, spore crystal ticidally-effective amount, which will vary depending on formulation, cell pellet, or colloidal concentrate. This powder Such factors as, for example, the specific target insects to be comprises bacterial cells which expresses a novel crystal controlled, the specific environment, location, plant, crop, or protein disclosed herein. Preferred bacterial cells are B. thu agricultural site to be treated, the environmental conditions, ringiensis NRRL B-21885 or NRRL B-21886 cells, however, and the method, rate, concentration, stability, and quantity of bacterial cells such as those of other strains of B. thuringien application of the insecticidally-active polypeptide composi sis, or cells of stains of bacteria Such as B. megaterium, B. tion. The formulations may also vary with respect to climatic subtilis, B. cereus, E coli, Salmonella spp., Agrobacterium conditions, environmental considerations, and/or frequency spp., or Pseudomonas spp. and the like, may also be trans of application and/or severity of insect infestation. formed with one or more nucleic acid segments as disclosed herein. Such transformed or recombinant bacterial cells will 0083. The insecticide compositions described may be preferably express at least one polypeptide comprising an at made by formulating either the bacterial cell, crystal and/or least 10 contiguous amino acid sequence from SEQID NO:2. spore Suspension, or isolated protein component with the and will produce a polypeptide that has insectical activity desired agriculturally-acceptable carrier. The compositions against a target insect. Such dry forms of the insecticidal may be formulated prior to administration in an appropriate compositions may be formulated to dissolve immediately means such as lyophilized, freeze-dried, desiccated, or in an upon wetting, or alternatively, dissolve in a controlled-re aqueous carrier, medium or Suitable diluent, such as saline or lease, Sustained-release, or other time-dependent manner. other buffer. The formulated compositions may be in the form Such compositions may be applied to, or ingested by, the ofa dust or granular material, or a suspension in oil (vegetable target insect, and as such, may be used to control the numbers or mineral), or water or oil/water emulsions, or as a wettable of insects, or the spread of Such insects in a given environ powder, or in combination with any other carrier material ment. suitable for agricultural application. Suitable agricultural car riers can be solid or liquid and are well known in the art. The 2.6.4 Aqueous Suspensions and Bacterial Cell Filtrates or term 'agriculturally-acceptable carrier covers all adjuvants, Lysates inert components, dispersants, Surfactants, tackifiers, bind I0087. In a fourth important embodiment, the bioinsecti ers, etc. that are ordinarily used in insecticide formulation cide composition comprises an aqueous Suspension of bacte technology; these are well known to those skilled in insecti rial cells or an aqueous Suspension of parasporal crystals, or cide formulation. The formulations may be mixed with one or an aqueous Suspension of bacterial cell lysates or filtrates, more Solidor liquid adjuvants and prepared by various means, etc., such as those described above which express the crystal e.g., by homogeneously mixing, blending and/or grinding the protein. Such aqueous Suspensions may be provided as a insecticidal composition with Suitable adjuvants using con concentrated Stock solution which is diluted prior to applica ventional formulation techniques. tion, or alternatively, as a diluted solution ready-to-apply. I0088 For these methods involving application of bacterial 2.6.1 Oil Flowable Suspensions cells, the cellular host containing the crystal protein gene(s) may be grown in any convenient nutrient medium, where the 0084. In a preferred embodiment, the bioinsecticide com DNA construct provides a selective advantage, providing for position comprises an oil flowable Suspension of bacterial a selective medium so that substantially all or all of the cells cells which expresses the novel crystal protein disclosed retain the B. thuringiensis gene. These cells may then be herein. Preferably the cells are B. thuringiensis NRRL harvested in accordance with conventional ways. Alterna B-21885 or NRRL B-21886 cells, however, any such bacte tively, the cells can be treated prior to harvesting. rial host cell expressing the novel nucleic acid segments dis I0089. When the insecticidal compositions comprise intact closed herein and producing a polypeptide comprising the B. thuringiensis cells expressing the protein of interest, Such amino acid sequence of SEQID NO:2 is contemplated to be bacteria may beformulated in a variety of ways. They may be useful. Exemplary bacterial species include those such as B. employed as wettable powders, granules or dusts, by mixing thuringiensis, B. megaterium, B. subtilis, B. cereus, E. coli, with various inert materials, such as inorganic minerals (phyl Salmonella spp., Agrobacterium spp., or Pseudomonas spp. losilicates, carbonates, Sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut 2.6.2 Water-Dispersible Granules shells, and the like). The formulations may include spreader Sticker adjuvants, stabilizing agents, other pesticidal addi 0085. In another important embodiment, the bioinsecti tives, or Surfactants. Liquid formulations may be acqueous cide composition comprises a water dispersible granule. This based or non-aqueous and employed as foams, Suspensions, granule comprises bacterial cells which expresses a novel emulsifiable concentrates, or the like. The ingredients may crystal protein disclosed herein. Preferred bacterial cells are include rheological agents, surfactants, emulsifiers, dispers B. thuringiensis NRRL B-21885 or NRRL B-21886 cells, ants, or polymers. however, bacteria such as B. megaterium, B. subtilis, B. 0090 Alternatively, the novel insecticidal polypeptides cereus, E. coli, Salmonella spp., Agrobacterium spp., or may be prepared by native or recombinant bacterial expres US 2009/0087863 A1 Apr. 2, 2009

sion systems in vitro and isolated for Subsequent field appli 0096. The concentration of insecticidal composition cation. Such protein may be either in crude cell lysates, Sus which is used for environmental, systemic, topical, or foliar pensions, colloids, etc., or alternatively may be purified, application will vary widely depending upon the nature of the particular formulation, means of application, environmental refined, buffered, and/or further processed, before formulat conditions, and degree of biocidal activity. Typically, the ing in an active biocidal formulation. Likewise, under certain bioinsecticidal composition will be present in the applied circumstances, it may be desirable to isolate crystals and/or formulation at a concentration of at least about 1% by weight spores from bacterial cultures expressing the crystal protein and may be up to and including about 99% by weight. Dry and apply solutions, Suspensions, or colloidal preparations of formulations of the polypeptide compositions may be from Such crystals and/or spores as the active bioinsecticidal com about 1% to about 99% or more by weight of the protein position. composition, while liquid formulations may generally com prise from about 1% to about 99% or more of the active 2.6.5 Multifunctional Formulations ingredient by weight. As such, a variety of formulations are preparable, including those formulations that comprise from 0091. In certain embodiments, when the control of mul about 5% to about 95% or more by weight of the insecticidal tiple insect species is desired, the insecticidal formulations polypeptide, including those formulations that comprise from described herein may also further comprise one or more about 10% to about 90% or more by weight of the insecticidal chemical pesticides, (such as chemical pesticides, nemato polypeptide. Naturally, compositions comprising from about cides, fungicides, Virucides, microbicides, amoebicides, 15% to about 85% or more by weight of the insecticidal insecticides, etc.), and/or one or more Ö-endotoxin polypep polypeptide, and formulations comprising from about 20% to tides having the same, or different insecticidal activities or about 80% or more by weight of the insecticidal polypeptide insecticidal specificities, as the insecticidal polypeptide iden are also considered to fall within the scope of the present tified in SEQ ID NO:2. The insecticidal polypeptides may disclosure. 0097. In the case of compositions in which intact bacterial also be used in conjunction with other treatments such as cells that contain the insecticidal polypeptide are included, fertilizers, weed killers, cryoprotectants, Surfactants, deter preparations will generally contain from about 10 to about gents, insecticidal soaps, dormant oils, polymers, and/or 10 cells/mg, although in certain embodiments it may be time-release or biodegradable carrier formulations that per desirable to utilize formulations comprising from about 10 to mit long-term dosing of a target area following a single appli about 10" cells/mg, or when more concentrated formulations cation of the formulation. Likewise the formulations may be are desired, compositions comprising from about 10 to about prepared into edible “baits” or fashioned into insect “traps to 10' or 10' cells/mg may also be formulated. Alternatively, permit feeding or ingestion by a target insect of the insecti cell pastes, spore concentrates, or crystal protein Suspension cidal formulation. concentrates may be prepared that contain the equivalent of 0092. The insecticidal compositions of the invention may from about 10' to 10" cells/mg of the active polypeptide, also be used in consecutive or simultaneous application to an and Such concentrates may be diluted prior to application. environmental site singly or in combination with one or more 0098. The insecticidal formulation described above may additional insecticides, pesticides, chemicals, fertilizers, or be administered to a particular plant or target area in one or other compounds. more applications as needed, with a typical field application rate per hectare ranging on the order of from about 50 g/hect are to about 500 g/hectare of active ingredient, or alterna 2.6.6 Application Methods and Effective Rates tively, from about 500 g/hectare to about 1000 g/hectare may 0093. The insecticidal compositions of the invention are be utilized. In certain instances, it may even be desirable to applied to the environment of the target insect, typically onto apply the insecticidal formulation to a target area at an appli the foliage of the plant or crop to be protected, by conven cation rate of from about 1000 g/hectare to about 5000 g/hect are or more of active ingredient In fact, all application rates in tional methods, preferably by spraying. The strength and the range of from about 50 g of active polypeptide per hectare duration of insecticidal application will be set with regard to to about 10,000 g/hectare are contemplated to be useful in the conditions specific to the particular pest(s), crop(s) to be management, control, and killing, of target insect pests using treated and particular environmental conditions. The propor such insecticidal formulations. As such, rates of about 100 tional ratio of active ingredient to carrier will naturally g/hectare, about 200 g/hectare, about 300 g/hectare, about depend on the chemical nature, solubility, and stability of the 400 g/hectare, about 500 g/hectare, about 600 g/hectare, insecticidal composition, as well as the particular formulation about 700 g/hectare, about 800 g/hectare, about 900 g/hect contemplated. are, about 1 kg/hectare, about 1.1 kg/hectare, about 1.2 0094. Other application techniques, including dusting, kg/hectare, about 1.3 kg/hectare, about 1.4 kg/hectare, about sprinkling, Soil soaking, soil injection, seed coating, seedling 1-5 kg/hectare, about 1.6 kg/hectare, about 1.7 kg/hectare, coating, foliar spraying, aerating, misting, atomizing, fumi about 1.8 kg/hectare, about 1.9 kg/hectare, about 2.0 kg/hect gating, aerosolizing, and the like, are also feasible and may be are, about 2.5 kg/hectare, about 3.0 kg/hectare, about 3.5 required under certain circumstances such as e.g., insects that kg/hectare, about 4.0 kg/hectare, about 4.5 kg/hectare, about 6.0 kg/hectare, about 7.0 kg/hectare, about 8.0 kg/hectare, cause root or stalk infestation, or for application to delicate about 8.5 kg/hectare, about 9.0 kg/hectare, and even up to and Vegetation or ornamental plants. These application proce including about 10.0 kg/hectare or greater of active polypep dures are also well-known to those of skill in the art. tide may be utilized in certain agricultural, industrial, and 0095. The insecticidal compositions of the present inven domestic applications of the pesticidal formulations tion may also be formulated for preventative or prophylactic described hereinabove. application to an area, and may in certain circumstances be applied to pets, livestock, animal bedding, or in and around 2.7 Epitopic Core Sequences farm equipment, barns, domiciles, or agricultural or indus 0099. The present invention is also directed to protein or trial facilities, and the like. peptide compositions, free from total cells and otherpeptides, US 2009/0087863 A1 Apr. 2, 2009

which comprise a purified peptide which incorporates an peptides that exhibit an attractive force towards each other. epitope that is immunologically cross-reactive with one or Thus, certain epitope core sequences of the present invention more antibodies that are specific for the disclosed polypeptide may be operationally defined in terms of their ability to com sequences. In particular, the invention concerns epitopic core pete with or perhaps displace the binding of the desired pro sequences derived from CryET70 and CryET70-related tein antigen with the corresponding protein-directed antisera. polypeptides. 0105. In general, the size of the polypeptide antigen is not 0100. As used herein, the term “incorporating an epitope believed to be particularly crucial, so long as it is at least large (s) that is immunologically cross-reactive with one or more enough to carry the identified core sequence or sequences. antibodies that are specific for the disclosed polypeptide The Smallest useful core sequence anticipated by the present sequence' is intended to refer to a peptide or protein antigen disclosure would generally be on the order of about 8 amino which includes a primary, secondary or terry structure similar acids in length, with sequences on the order of 10 to 20 being to an epitope located within the disclosed polypeptide. The more preferred. Thus, this size will generally correspond to level of similarity will generally be to such a degree that the Smallest peptide antigens prepared in accordance with the monoclonal or polyclonal antibodies directed against the invention. However, the size of the antigen may be larger crystal protein or polypeptide will also bind to, react with, or where desired, so long as it contains a basic epitopic core otherwise recognize, the cross-reactive peptide or protein Sequence. antigen. Various immunoassay methods may be employed in 0106 The identification of epitopic core sequences is conjunction with Such antibodies, such as, for example, West known to those of skill in the art, for example, as described in ern blotting, ELISA, RIA, and the like, all of which are known U.S. Pat. No. 4,554,101, incorporated herein by reference, to those of skill in the art. which teaches the identification and preparation of epitopes 0101 The identification of immunodominant epitopes, from amino acid sequences on the basis of hydrophilicity. and/or their functional equivalents, Suitable for use in vac Moreover, numerous computer programs are available foruse cines is a relatively straightforward matter. For example, one in predicting antigenic portions of proteins (see e.g., Jameson may employ the methods of Hopp, as taught in U.S. Pat. No. and Wolf, 1988: Wolf et al., 1988). Computerized peptide 4.554,101, incorporated herein by reference, which teaches sequence analysis programs (e.g., DNAStar R Software, the identification and preparation of epitopes from amino acid DNAStar, Inc., Madison, Wis.) may also be useful in design sequences on the basis of hydrophilicity. The methods ing synthetic peptides in accordance with the present disclo described in several other papers, and software programs SUC. based thereon, can also be used to identify epitopic core 0107 Syntheses of epitopic sequences, or peptides which sequences (see, for example, Jameson and Wolf, 1988; Wolf include an antigenic epitope within their sequence, are readily et al., 1988: U.S. Pat. No. 4,554,101). The amino acid achieved using conventional synthetic techniques such as the sequence of these "epitopic core sequences' may then be Solid phase method (e.g., through the use of commercially readily incorporated into peptides, either through the appli available peptide synthesizer Such as an Applied Biosystems cation of peptide synthesis or recombinant technology. Model 430A Peptide Synthesizer). Peptide antigens synthe 0102 Preferred peptides for use in accordance with the present invention will generally be on the order of about 8 to sized in this manner may then be aliquotted in predetermined about 20 amino acids in length, and more preferably about 8 amounts and stored in conventional manners, such as in aque to about 15 amino acids in length. It is proposed that shorter ous Solutions or, even more preferably, in a powder or lyo antigenic crystal protein-derived peptides will provide advan philized State pending use. tages in certain circumstances, for example, in the prepara 0108. In general, due to the relative stability of peptides, tion of immunologic detection assays. Exemplary advantages they may be readily stored in aqueous Solutions for fairly long include the ease of preparation and purification, the relatively periods of time if desired, e.g., up to six months or more, in low cost and improved reproducibility of production, and virtually any aqueous Solution without appreciable degrada advantageous biodistribution. tion or loss of antigenic activity. However, where extended 0103. It is proposed that particular advantages of the aqueous storage is contemplated it will generally be desirable present invention may be realized through the preparation of to include agents including buffers such as Tris or phosphate synthetic peptides which include modified and/or extended buffers to maintain a pH of about 7.0 to about 7.5. Moreover, epitopic/immunogenic core sequences which result in a "uni it may be desirable to include agents which will inhibit micro Versal epitopic peptide directed to crystal proteins, and in bial growth, such as sodium azide or Merthiolate. For particular CryET70 and related sequences. These epitopic extended storage in an aqueous state it will be desirable to core sequences are identified herein in particular aspects as store the solutions at about 4°C., or more preferably, frozen. hydrophilic regions of the particular polypeptide antigen. It is Of course, where the peptides are stored in a lyophilized or proposed that these regions represent those which are most powdered State, they may be stored virtually indefinitely, e.g., likely to promote T-cell or B-cell stimulation, and, hence, in metered aliquots that may be rehydrated with a predeter elicit specific antibody production. mined amount of water (preferably distilled) or buffer prior to 0104. An epitopic core sequence, as used herein, is a rela SC. tively short stretch of amino acids that is “complementary’ to, and therefore will bind, antigen binding sites on the crystal 2.8 Definitions protein-directed antibodies disclosed herein. Additionally or alternatively, an epitopic core sequence is one that will elicit 0109 The following words and phrases have the meanings antibodies that are cross-reactive with antibodies directed set forth below. against the peptide compositions of the present invention. It 0110 A, an: In keeping with long-standing patent tradi will be understood that in the context of the present disclo tion, “a” or “an used throughout this disclosure is intended to Sure, the term “complementary refers to amino acids or mean “one or more. US 2009/0087863 A1 Apr. 2, 2009

0111 Comprising, comprises: In keeping with long polynucleotides into a host cell, thereby generating a “trans standing patent tradition, "comprising and "comprises' used formed’ or “recombinant host cell. throughout this disclosure is intended to mean “including, but not limited to.” 3.O BRIEF DESCRIPTION OF THE DRAWINGS 0112 Expression: The combination of intracellular pro I0121 The drawings form part of the present specification cesses, including transcription and translation undergone by a and are included to further demonstrate certain aspects of the coding DNA molecule Such as a structural gene to produce a present invention. The invention may be better understood by polypeptide. reference to one or more of these drawings in combination 0113 Promoter: A recognition site on a DNA sequence or with the detailed description of specific embodiments pre group of DNA sequences that provide an expression control sented herein. element for a structural gene and to which RNA polymerase (0.122 FIG. 1. Structural maps of the cryET70 plasmids specifically binds and initiates RNA synthesis (transcription) pEG1648 and pEG 1657. An approximately 8.5 kb DNA frag of that gene. ment, obtained from a partial MboI digest of EG4140 DNA, was cloned into the unique BamHI site of shuttle vector 0114 Regeneration: The process of growing a plant from pHT315 to generate the cryET70 plasmidpEG1648. A 5.8 kb a plant cell (e.g., plant protoplast or explant). HindIII-EcoRI fragment containing the cryET70 gene was 0115 Structural gene: A gene that is expressed to produce isolated from plasmid plG1648 and inserted into puC18 to a polypeptide. yield plasmid pEG 1657. Designations: ori 1030 (solid box) 011.6 Transformation: A process of introducing an exog =B. thuringiensis plasmid replication origin; ery (open box) enous DNA sequence (e.g., a vector, a recombinant DNA =erythromycin resistance gene; puC18 and puC19 (shaded molecule) into a cell or protoplast in which that exogenous boxes)=E. coli cloning vector fragments; solid line-cryET70 DNA is incorporated into a chromosome or is capable of DNA insert. Restriction site abbreviations: B=BamHI, Bal-Ball, E=EcoRI, H=HindIII, Hip-HpaI, K-KpnI, autonomous replication. Mb=MboI. 0117 Transformed cell: A cell whose DNA has been altered by the introduction of an exogenous DNA molecule 4.O DESCRIPTION OF ILLUSTRATIVE into that cell. EMBODIMENTS 0118 Transgenic cell: Any cell derived from or regener ated from a transformed cellor derived from a transgenic cell. 4.1 SOME Advantages of the Invention Exemplary transgenic cells include plant calli derived from a I0123. The present invention provides a novel 8-endotoxin, transformed plant cell and particular cells such as leaf, root, designated CryET70, which is highly toxic to WCRW. This stem, e.g., Somatic cells, or reproductive (germ) cells protein has an amino acid sequence which is unrelated to obtained from a transgenic plant. other Ö-endotoxins that are toxic to coleopteran insects. 0119 Transgenic plant: A plant or a progeny of any gen CryET70 represents a new class of coleopteran active insec eration of the plant that was derived from a transformed plant ticidal crystal proteins. Unlike other WCRW toxic insecti cell or protoplast, wherein the plant nucleic acids contains an cidal crystal proteins from B. thuringiensis, CryET70 does exogenous selected nucleic acid sequence region not origi not have significant toxicity to SCRW or CPB. The only nally present in a native, non-transgenic plant of the same known protein that is related to CryET70 is Cry22, an insec strain The terms “transgenic plant and “transformed plant” ticidal crystal protein that is reported to be toxic only to have sometimes been used in the art as synonymous terms to hymenopteran insects (GenBank Accession No. 134547). define a plant whose DNA contains an exogenous DNA mol ecule. However, it is thought more scientifically correct to 4.2 Insect Pests refer to a regenerated plant or callus obtained from a trans 0.124. Almost all field crops, plants, and commercial farm formed plant cell or protoplast or from transformed pluripo ing areas are susceptible to attack by one or more insect pests. tent plant cells as being a transgenic plant. Preferably, trans Particularly problematic are the lepidopteran and coleopteran genic plants of the present invention include those plants that pests identified in Table 1. For example, vegetable and cole comprise at least a first selected polynucleotide that encodes crops such as artichokes, kohlrabi, arugula, leeks, asparagus, an insecticidal polypeptide. This selected polynucleotide is lentils, beans, lettuce (e.g., head, leaf, romaine), beets, bok preferably a 6-endotoxin coding region (or gene) operably choy, malanga, broccoli, melons (e.g., muskmelon, water linked to at least a first promoter that expresses the coding melon, crenshaw, honeydew, cantaloupe), brussels sprouts, region to produce the insecticidal polypeptide in the trans cabbage, cardoni, carrots, napa, cauliflower, okra, onions, genic plant. Preferably, the transgenic plants of the present celery, parsley, chick peas, parsnips, chicory, peas, Chinese invention that produce the encoded polypeptide demonstrate cabbage, peppers, collards, potatoes, cucumber, pumpkins, a phenotype of improved resistance to target insect pests. cucurbits, radishes, dry bulb onions, rutabaga, eggplant, Sal Such transgenic plants, their progeny, descendants, and seed Sify, escarole, shallots, endive, soybean, garlic, spinach, from any Such generation are preferably insect resistant green onions, squash, greens, Sugar beets, Sweet potatoes, plants. turnip, Swiss chard, horseradish, tomatoes, kale, turnips, and 0120 Vector: A nucleic acid molecule capable of replica a variety of spices are sensitive to infestation by one or more tion in a host cell and/or to which another nucleic acid seg of the following insect pests: alfalfa looper, armyworm, beet ment can be operatively linked so as to bring about replication armyworm, artichoke plume moth, cabbage budworm, cab of the attached segment. Plasmids, phage, phagemids, and bage looper, cabbage webworm, corn earworm, celery leaf cosmids are all exemplary vectors. In many embodiments, eater, cross-striped cabbageworm, european corn borer, dia vectors are used as a vehicle to introduce one or more selected mondback moth, green cloverworm, imported cabbageworm, US 2009/0087863 A1 Apr. 2, 2009 14 melonworm, omnivorous leafroller, pickleworm, rindworm I0127 Field crops such as canola/rape seed, evening prim complex, Saltmarsh caterpillar, soybean looper, tobacco bud rose, meadow foam, corn (field, Sweet, popcorn), cotton, worm, tomato fuitworm, tomato hornworm, tomato pin hops, jojoba, peanuts, rice, safflower, Small grains (barley, worm, Velvetbean caterpillar, and yellowstriped armyworm. oats, rye, wheat, etc.), Sorghum, soybeans, Sunflowers, and 0.125. Likewise, pasture and hay crops such as alfalfa, tobacco are often targets for infestation by insects including pasture grasses and silage are often attacked by Such pests as armyworm, asian and other corn borers, banded Sunflower armyworm, beef armyworm, alfalfa caterpillar, European moth, beet armyworm, bollworm, cabbage looper, corn root skipper, a variety of loopers and webworms, as well as yel worm (including Southern and western varieties), cotton leaf lowstriped armyworms. perforator, diamondback moth, european corn borer, green 0126 Fruit and vine crops such as apples, apricots, cher cloverworm, headmoth, headworm, imported cabbageworm, ries, nectarines, peaches, pears, plums, prunes, quince loopers (including Anacamptodes spp.), obliquebanded lea almonds, chestnuts, filberts, pecans, pistachios, walnuts, cit froller, omnivorous leaftier, podworm, podworm, Saltmarsh rus, blackberries, blueberries, boysenberries, cranberries, caterpillar, Southwestern corn borer, soybean looper, spotted currants, loganbernies, raspberries, Strawberries, grapes, avo cutworm, Sunflower moth, tobacco budworm, tobacco horn cados, bananas, kiwi, persimmons, pomegranate, pineapple, worm, velvetbean caterpillar, tropical fruits are often susceptible to attack and defoliation I0128 Bedding plants, flowers, ornamentals, vegetables by achema sphinx moth, amorbia, armyworm, citrus cut and container stock are frequently fed upon by a host of insect worm, banana skipper, blackheaded fireworm, blueberry lea pests such as armyworm, azalea moth, beet armyworm, dia froller, cankerworm, cherry fruitworm, citrus cutworm, cran mondback moth, ello mot (hornworm), Florida fern caterpil berry girdler, eastern tent caterpillar, fall webworm, fall lar, Io moth, loopers, oleander moth, omnivorous leafroller, webworm, filbert leafroller, filbert webworm, fruit tree lea omnivorous looper, and tobacco budworm. froller, grape berry moth, grape leaffolder, grapeleaf skel I0129. Forests, Suit, ornamental, and nut-bearing trees, as etonizer, green fruitworm, gummoSoS-batrachedra commo well as shrubs and other nursery stock are often susceptible to sae, gypsy moth, hickory shuckworm, hornworms, loopers, attack from diverse insects such as bagworm, blackheaded navel orangeworm, obliquebanded leafroller, omnivorous budworm, browntail moth, California oakworm, douglas fir leafroller, omnivorous looper, orange tortrix, orangedog, ori tussock moth, elm spanworm, fall webworm, fruittree leafrol ental fruit moth, pandemis leafroller, peach twig borer, pecan ler, greenstriped mapleworm, gypsy moth, jack pine bud nut casebearer, redbanded leafroller, redhumped caterpillar, worm, mimosa webworm, pine butterfly, redhumped cater roughskinned cutworm, Saltmarsh caterpillar, spanworm, tent pillar, saddleback caterpillar, saddle prominent caterpillar, caterpillar, thecla-thecla basillides, tobacco budworm, tortrix spring and fall cankerworm, spruce budworm, tent caterpillar, moth, tufted apple budmoth, variegated leafroller, walnut cat tortrix, and western tussock moth. Likewise, turfgrasses are erpillar, western tent caterpillar, and yellowstriped army often attacked by pests such as armyworm, Sod webworm, WO. and tropical sod webworm.

TABLE 1

TAXONOMY OF COLEOPTER ANPESTS IN THE SUBORDERS ARCHOSTEMATA AND POLYPHAGA Super Infraorder family Family Subfamily Tribe Genus Species Cupedidae (reticulated Priacna P Serraia beetles) Bostrichiformia Dermestidae (skin and Attagentis A. pelio larder beetles) Chrysomeliformia Cerambycidae (long Agapanthia Agapanthia sp. horned beetles) Lepturinae Leptura Leptura sp. (flower long-horned beetle) Rhagium Rhagium sp. Megacyllene M. robiniae Prioninae Derobrachus D. geminatus Tetraopes T. tetropthalmus Chrysomelidae (leaf Chlamisinae Exena E. neglecta beetles) Chrysomelinae Chrysomelini Chrysomela C. tremula, Chrysomela sp. Oreina O. cacaiae Doryphorini Chrysoline Chrysolina sp. Leptinotarsa L. decemlineata (Colorado potato beetle) Gonioctenini Goniocietna G. fornicata, G. holdausi, G. intermedia, G. interposita, G. kamikawai, G. innaeana, G. nigroplagiata, G. Occidentalis, G. olivacea, G. pallida, G. quin quieptinctata, G. rubripennis, G. rufipes, G. treaecim-maculata, G. variabilis, G. vimina is US 2009/0087863 A1 Apr. 2, 2009 15

TABLE 1-continued

TAXONOMY OF COLEOPTER ANPESTS IN THE SUBORDERS ARCHOSTEMATA AND POLYPHAGA Super Infraorder family Family Subfamily Tribe Genus Species Timarchini Timarcha Timarchasp. Criocerinae Ouiema Outlema sp. Galerucinae Galerucini Monoxia M. inornata, Monoxia sp. Ophraeila O. arctica, O. artemisiae, O. bilineata, O. communa, O. conferta, O. cribrata, O. notata, O. notitiata, O. nuda, O. pilosa, O. Sexvittata, O. Siobodkini Luperini Cerotona C. trifurcata Diabrotica D. barberi (northern corn rootworm), D. undecimptinctata, (Southern corn rootworm), D. virgifera (western corn rootworm) unclassified Lachnaia Lachnaia sp. Chrysomeidae Epitrix E. cucumenis (Harris) (potato flea beetle), E. fiscala (eggplant flea beetle) Curculionidae (weevils) Curculioninae Anthonomis A. grandis (boll weevil) Entiminae Naupactini Aranigits A. Conirostris, A. globocatius, A. intermedius, A. pianioculus, A. tesselaints Otiorhynchus Otiorhynchus sp. Phyllobiini DiaPrepes D. abbreviata Phyllobius Phyllobius sp. Galapaganus G. galapagoensis Hyperinae Hypera H. brunneipennis (Egyptian alfalfa weevil), H. postica (alfalfa weevil), H. punctata (clover leaf weevil) Molytinae Pissodes P. afinis, F2 memorensi, PSchwarzi, P Strobi, P. termina is Rhynchophorinae Sitophilini Sitophilus S. granarius (granary weevil), S. zeanais (maize weevil) Nemonychidae Lebanorhinus L. Sticcinits Scolytidae Ips I. actiminaiti, I. amitiniis, I. Cembrae, I. duplicatii, I. mannsfeldi, I. Sexaeniatus, I. typographits Orthotomicus O. erostis Tonicus T. minor Cuculiformia Coccinellidae (ladybird Epilachna E. borealis (squash ladybird beetles) beetle), E. varivstis (Mexican bean beetle) Cuculidae (flat bark Cryptolestes C. ferruginetis beetles) Oryzaephilus O. Surinamensis (saw-toothed (grain grain beetle) beetles) Lagriidae (long-joined Lagria Lagria sp. beetles) Meloidae (blister beetles) Epicalita E. funebris Meloe M. proscarabaeus Rhipiphoridae Rhipiphorus R. fasciatus Tenebrionidae (darkling Alphitobius A. diaperint is ground beetles) (lesser mealworm) Hegeter H. amaroides, H. brevicois, H. CoStipennis, H. fernandezi, H. glaber, H. gomerensis, H. gran canariensis, H. impressus, H. intercedens, H. iaiterais, H. picifions, H. politiis, H. Subrotundatiis, H. tentii punctatut, H. transversus, H. webbianus US 2009/0087863 A1 Apr. 2, 2009 16

TABLE 1-continued

TAXONOMY OF COLEOPTER ANPESTS IN THE SUBORDERS ARCHOSTEMATA AND POLYPHAGA

Super Infraorder family Family Subfamily Tribe Genus Species Misolampus M. got idoti Paiorus Pficicola, Pratzeburgi (small eyed flour beetle), P subdepressus (depressed flour beetle) Pineia P. baetica, P. canariensis, Pcriba, Peievate, Pestevezi, Pfernan-deziopezi, P. grandis, Pgranulicoli, Pintegra, Pinterjecta, P. laevigata, Plutaria, Praduia, P. sparsa, P. varioiosa Tenebrio T. molitor (yellow mealworm), T. obscurus (dark mealworm) Tentyria T. Schaumi Triboium T. brevicornis, T. castaneum (red flour beetle), T. confusum (confused flour beetle), T. freemani, T. madens Zophobas Z. atratus Z. rugipes Elateriformia Elateroidea Octinodes Octinodes sp. Pyrophorus P plagio-phthalamus Scarabaeiformia Lucanidae (Stag beetles) Dorctiis D. paralleio-pipedits Lticantis L. Cervus Scarabaeidae (lamellicorn Allomyrina A. dichotoma beetles) Cetoniinae Pachnoda P marginata (flower beetle) Dynastinae Xvioryctes X. faints Geotrupinae Geotripes G. Stercorostis (earth-boring dung beetles) Melonlonthiinae Costelytra C. zealandica (chafers) Holotrichia H. diomphalia Meioioniha M. melolontha (cockchafer) Odontria O. Striata O. variegata Prodontria P. bicolorata, P. capito, Piewisii, Ptarsis, P. modesta, Pipinguis, P. praeiatella, P. truncata, Prodontria sp. Scythrodes S. squalidus Rutelinae Popiilia Piaponica (Japanese beetle) (shining leaf chafers) Scarabaeinae Copris C. lunaris (black dung beetle) Scarabaeus Scarabaei is sp. (Scarab) Staphyliniformia Hydrophilidae Cercyon Cercyon sp. Silphidae Nicrophorus N. americantis, N. marginiatus, N. orbicois, N. tomentosus Staphyllinidae (rove Carpelimits Carpelimits sp. beetles) Piedits P. mesomeini is Tachyporus Tachyporus sp. Xanthoints Xantholin its sp.

US 2009/0087863 A1 Apr. 2, 2009

4.4 Probes and Primers regulated by that promoter. Means for operatively linking a promoter to a coding region are well known in the art. 0131. In another aspect, DNA sequence information pro 0.136. In a preferred embodiment, the recombinant expres vided by the invention allows for the preparation of relatively sion of DNAs encoding the crystal proteins of the present short DNA (or RNA) sequences having the ability to specifi invention is preferable in a Bacillus host cell. Preferred host cally hybridize to gene sequences of the selected polynucle cells include B. thuringiensis, B. megaterium, B. subtilis, and otides disclosed herein. In these aspects, nucleic acid probes related bacilli, with B. thurigiensis host cells being highly of an appropriate length are prepared based on a consider preferred Promoters that function in bacteria are well-known ation of a selected crystal protein-encoding gene sequence, in the art. An exemplary and preferred promoter for the Bacil e.g., a sequence such as that shown in SEQ ID NO: 1. The lus-derived crystal proteins include any of the known crystal ability of such DNAs and nucleic acid probes to specifically protein gene promoters, including the cryET70 gene pro hybridize to a crystal protein-encoding gene sequence lends moter itself. Alternatively, mutagenized or recombinant pro them particular utility in a variety of embodiments. Most moters may be engineered by the hand of man and used to importantly, the probes may be used in a variety of assays for promote expression of the novel gene segments disclosed detecting the presence of complementary sequences in a herein. given sample. 0.137 In an alternate embodiment, the recombinant 0.132. In certain embodiments, it is advantageous to use expression of DNAs encoding the crystal proteins of the oligonucleotide primers. The sequence of Such primers is present invention is performed using a transformed Gram designed using a polynucleotide of the present invention for negative bacterium such as an E. coli or Pseudomonas spp. use in detecting, amplifying or mutating a defined segment of host cell. Promoters which function in high-level expression a crystal protein gene from B. thuringiensis using PCRTM of target polypeptides in E. coli and other Gram-negative host technology. Segments of related crystal protein genes from cells are also well-known in the art. other species may also be amplified by PCRTM using such 0.138. Where an expression vector of the present invention primers. is to be used to transform a plant, a promoter is selected that 0133) To provide certain of the advantages in accordance has the ability to drive expression in plants. Promoters that with the present invention, a preferred nucleic acid sequence function in plants are also well known in the art. Useful in employed for hybridization studies or assays includes expressing the polypeptide in plants are promoters that are sequences that are complementary to at least an about 23 to inducible, constitutive, temporally-regulated, spatially-regu about 40 or so long nucleotide stretch of a crystal protein lated, and spatio-temporally regulated, including those that encoding sequence, such as that shown in SEQID NO:1. A are native, semi-synthetic, and synthetic (Chan et al., 1989). size of at least about 14 or 15 or so nucleotides in length helps 0.139. A promoter is also selected for its ability to direct the to ensure that the fragment will be of sufficient length to form transformed plant cell's or transgenic plant's transcriptional a duplex molecule that is both stable and selective. Molecules activity to the coding region. Structural genes can be driven having complementary sequences over stretches greater than by a variety of promoters in plant tissues. Promoters can be about 23 or so bases in length are generally preferred, though, near-constitutive, such as the CaMV 35S promoter, or tissue in order to increase stability and selectivity of the hybrid, and specific or developmentally specific promoters affecting thereby improve the quality and degree of specific hybrid dicots or monocots. molecules obtained. One will generally prefer to design 0140. Where the promoter is a near-constitutive promoter nucleic acid molecules having gene-complementary stretches such as CaMV 35S, increases in polypeptide expression are of about 14 to about 20 nucleotides, or even longer where found in a variety of transformed plant tissues (e.g., callus, desired Such fragments may be readily prepared by, for leaf, seed and root). Alternatively, the effects of transforma example, directly synthesizing the fragment by chemical tion can be directed to specific plant tissues by using plant means, by application of nucleic acid reproduction technol integrating vectors containing a tissue-specific promoter. ogy, such as the PCRTM technology of U.S. Pat. Nos. 4,683, 0.141. An exemplary tissue-specific promoter is the lectin 195, and 4,683.202, specifically incorporated herein by ref promoter, which is specific for seed tissue. The Lectin protein erence, or by excising selected DNA fragments from in Soybean seeds is encoded by a single gene (Lel) that is only recombinant plasmids containing appropriate inserts and expressed during seed maturation and accounts for about 2 to suitable restriction sites. about 5% of total seed mRNA. The lectin gene and seed specific promoter have been fully characterized and used to 4.5 Expression Vectors direct seed specific expression in transgenic tobacco plants (Vodkinet al., 1983; Lindstrom et al., 1990.) 0134. The present invention contemplates a polynucle 0142. An expression vector containing a coding region otide of the present invention comprised within one or more that encodes a polypeptide of interest is engineered to be expression vectors. Thus, in one embodiment an expression under control of the lectin promoter and that vector is intro vector comprises a nucleic acid segment containing a duced into plants using, for example, a protoplast transfor cryET70 gene operably linked to a promoter which expresses mation method (Dhir et al., 1991a). The expression of the the gene. Additionally, the coding region may also be oper polypeptide is directed specifically to the seeds of the trans ably linked to a transcription-terminating region, whereby the genic plant. promoter drives the transcription of the coding region, and the 0.143 A transgenic plant of the present invention produced transcription-terminating region halts transcription at Some from a plant cell transformed with a tissue specific promoter point 3' of the coding region. can be crossed with a second transgenic plant developed from 0135. As used herein, the term “operatively linked' means a plant cell transformed with a different tissue specific pro that a promoter is connected to an coding region in Sucha way moter to produce a hybrid transgenic plant that shows the that the transcription of that coding region is controlled and effects of transformation in more than one specific tissue. US 2009/0087863 A1 Apr. 2, 2009

0144 Exemplary tissue-specific promoters are corn are then joined by hydrogen bonding between the comple sucrose synthetase 1 (Yang et al., 1990), corn alcohol dehy mentary homopolymeric tails to form recombinant DNA drogenase 1 (Vogel et al., 1989), corn light harvesting com molecules. plex (Simpson, 1986), corn heat shock protein (Odell et al., 0151. A coding region that encodes a polypeptide having 1985), pea small subunit RUBP carboxylase (Poulsen et al., the ability to confer insecticidal activity to a cell is preferably 1986; Cashmore etal, 1983), Tiplasmid mannopine synthase a CryET70 B. thuringiensis crystal protein-encoding gene. In (Langridge et al., 1989), Tiplasmid nopaline synthase (Lan preferred embodiments, such a polypeptide has the amino gridge et al., 1989), petunia chalcone isomerase (van Tunenet acid residue sequence of SEQ ID NO:2, or a functional al., 1988), bean glycine rich protein 1 Keller et al., 1989), equivalent thereof. In accordance with Such embodiments, a CaMV 35S transcript (Odell et al., 1985) and Potato patatin coding region comprising the DNA sequence of SEQ ID (Wenzleretal, 1989). Preferred promoters are the cauliflower NO:1 is also preferred mosaic virus (CaMV 35S) promoter and the S-E9 small sub unit RuBP carboxylase promoter. 4.6 Characteristic of the CryET70 Polypeptide 0145 The choice of which expression vector and ulti Isolated from EG4140 mately to which promoter a polypeptide coding region is operatively linked depends directly on the functional proper 0152 The present invention provides a novel polypeptide ties desired, e.g., the location and timing of protein expres that defines a whole or a portion of a B. thuringiensis sion, and the host cell to be transformed. These are well CryET70 crystal protein. known limitations inherent in the art of constructing recom 0153. In a preferred embodiment, the invention discloses binant DNA molecules. However, a vector useful in practic and claims an isolated and purified CryET70 polypeptide. ing the present invention is capable of directing the expres The CryET70 polypeptide isolated from EG4140 comprises a sion of the polypeptide coding region to which it is -amino acid sequence, and has a calculated molecular mass operatively linked. of approximately 87,000 Da CryET70 has a calculated iso electric constant (p) equal to . The amino acid com 0146 Typical vectors useful for expression of genes in position of the CryET70 polypeptide is given in Table 3. higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacte TABLE 3 rium tumefaciens described (Rogers et al., 1987). However, several other plant integrating vector systems are known to AMINOACID COMPOSITION OF CRYETTO function in plants including pCaMVCN transfer control vec tor described (Fromm et al., 1985). pCaMVCN (available Amino Acid # Residues % Total from Pharmacia, Piscataway, N.J.) includes the cauliflower Ala Arg mosaic virus CaMV 35S promoter. ASn 0147 In preferred embodiments, the vector used to Asp express the polypeptide includes a selection marker that is Cys Gln effective in a plant cell, preferably a drug resistance selection Glu marker. One preferred drug resistance marker is the gene Gly whose expression results in kanamycin resistance; i.e., the His chimeric gene containing the nopaline synthase promoter, Ile Leu Tn5 neomycin phosphotransferase II (nptII) and nopaline Lys synthase 3' non-translated region described (Rogers et al., Met 1988). Phe Pro 0148 RNA polymerase transcribes a coding DNA Ser sequence through a site where polyadenylation occurs. Typi Thr cally, DNA sequences located a few hundred base pairs down Tro stream of the polyadenylation site serve to terminate tran Tyr Wall scription. Those DNA sequences are referred to herein as Acidic (Asp + Glu) transcription-termination regions. Those regions are required Basic (Arg + Lys) for efficient polyadenylation of transcribed messenger RNA Aromatic (Phe + Trp + Tyr) (mRNA). Hydrophobic (Aromatic + Ile + Leu + Met + Val) 0149 Means for preparing expression vectors are well known in the art. Expression (transformation vectors) used to transform plants and methods of making those vectors are 4.7 Nomenclature of the Novel Proteins described in U.S. Pat. Nos. 4,971,908, 4,940,835, 4,769,061 and 4,757,011, the disclosures of which are specifically incor 0154 The inventors have arbitrarily assigned the designa porated herein by reference in their entirety. Those vectors tion CryET70 to the novel protein of the invention. Likewise, can be modified to include a coding sequence in accordance the arbitrary designation of cryET70 has been assigned to the with the present invention. novel nucleic acid sequence which encodes this polypeptide. 0150. A variety of methods has been developed to opera Formal assignment of gene and protein designations based on tively inserta DNA segment into a vector via complementary the revised nomenclature of crystal protein endotoxins will be cohesive termini or blunt ends. For instance, complementary assigned by a committee on the nomenclature of B. thuring homopolymer tracts can be added to the DNA segment to be iensis, formed to systematically classify B. thuringiensis inserted and to the vector DNA. The vector and DNA segment crystal proteins. The inventors contemplate that the arbitrarily US 2009/0087863 A1 Apr. 2, 2009 20 assigned designations of the present invention will be Super mediated transfection and protoplast fusion, frequently gives seded by the official nomenclature assigned to these rise to cell lines that carry one, or at most a few, integrated Sequences. copies of the foreign DNA. 0160 The introduction of DNA by means of electropora tion, is well-known to those of skill in the art. In this method, 4.8 Transformed Host Cells and Transgenic Plants certain cell wall-degrading enzymes, such as pectin-degrad ing enzymes, are employed to render the target recipient cells 0155 Methods and compositions for transforming a bac more Susceptible to transformation by electroporation than terium, a yeast cell, a plant cell, or an entire plant with one or untreated cells. Alternatively, recipient cells are made more more expression vectors comprising a crystal protein-encod Susceptible to transformation, by mechanical wounding. To ing gene segment are further aspects of this disclosure. A effect transformation by electroporation one may employ transgenic bacterium, yeast cell, plant cell or plant derived either friable tissues such as a Suspension culture of cells, or from Such a transformation process or the progeny and seeds embryogenic callus, or alternatively, one may transform from Such a transgenic plant are also further embodiments of immature embryos or other organized tissues directly. One the invention. would partially degrade the cell walls of the chosen cells by 0156 Means for transforming bacteria and yeast cells are exposing them to pectin-degrading enzymes (pectolyases) or well known in the art. Typically, means of transformation are mechanically wounding in a controlled manner. Such cells similar to those well known means used to transform other would then be recipient to DNA transfer by electroporation, bacteria or yeast Such as E. coli or Saccharomyces cerevisiae. which may be carried out at this stage, and transformed cells Methods for DNA transformation of plant cells include Agro then identified by a Suitable selection or screening protocol bacterium-mediated plant transformation, protoplast trans dependent on the nature of the newly incorporated DNA. formation, gene transfer into pollen, injection into reproduc tive organs, injection into immature embryos and particle 4.8.2 Microprojectile Bombardment bombardment. Each of these methods has distinct advantages 0.161. A further advantageous method for delivering trans and disadvantages. Thus, one particular method of introduc forming DNA segments to plant cells is microprojectile bom ing genes into a particular plant strain may not necessarily be bardment. In this method, particles may be coated with the most effective for another plant strain, but it is well known nucleic acids and delivered into cells by a propelling force. which methods are useful for a particular plant stain. Exemplary particles include those comprised of tungsten, O157. There are many methods for introducing transform gold, platinum, and the like. ing DNA segments into cells, but not all are suitable for 0162 An advantage of microprojectile bombardment, in delivering DNA to plant cells. Suitable methods are believed addition to it being an effective means of reproducibly stably to include virtually any method by which DNA can be intro transforming monocots, is that neither the isolation of proto duced into a cell. Such as by Agrobacterium infection, direct plasts (Cristou et al., 1988) nor the susceptibility to Agrobac delivery of DNA such as, for example, by PEG-mediated terium infection is required. An illustrative embodiment of a transformation of protoplasts (Omirulleh et al., 1993), by des method for delivering DNA into maize cells by acceleration is iccation/inhibition-mediated DNA uptake, by electropora a Biolistics Particle Delivery System, which can be used to tion, by agitation with silicon carbide fibers, by acceleration propel particles coated with DNA or cells through a screen, of DNA coated particles, etc. In certain embodiments, accel Such as a stainless Steel or Nytex screen, onto a filter Surface eration methods are preferred and include, for example, covered with corn cells cultured in Suspension. The screen microprojectile bombardment and the like. disperses the particles so that they are not delivered to the 0158 Technology for introduction of DNA into cells is recipient cells in large aggregates. It is believed that a screen well-known to those of skill in the art. Four general methods intervening between the projectile apparatus and the cells to for delivering a gene into cells have been described: (1) be bombarded reduces the size of projectiles aggregate and chemical methods (Graham and van der Eb. 1973; Zatloukal may contribute to a higher frequency of transformation by et al., 1992); (2) physical methods such as microinjection reducing damage inflicted on the recipient cells by projectiles (Capecchi, 1980), electroporation (Wong and Neumann, that are too large. 1982: Fromm et al., 1985; U.S. Pat. No. 5,384.253) and the 0163 For the bombardment, cells in suspension are pref gene gun (Johnston and Tang, 1994: Fynan et al., 1993); (3) erably concentrated on filters or solid culture medium. Alter viral vectors (Clapp, 1993: Lu et al., 1993; Eglitis and Ander natively, immature embryos or other target cells may be son, 1988; Eglitis et al., 1988); and (4) receptor-mediated arranged on solid culture medium. The cells to be bombarded mechanisms (Curiel et al., 1991; 1992; Wagner et al., 1992). are positioned at an appropriate distance below the macro projectile stopping plate. If desired, one or more screens are 4.8.1 Electroporation also positioned between the acceleration device and the cells to be bombarded. Through the use of techniques set forth 0159. The application of brief, high-voltage electric herein one may obtain up to 1000 or more foci of cells tran pulses to a variety of animal and plant cells leads to the siently expressing a marker gene. The number of cells in a formation of nanometer-sized pores in the plasma membrane focus which express the exogenous gene product 48 hr post DNA is taken directly into the cell cytoplasm either through bombardment often range from 1 to 10 and average 1 to 3. these pores or as a consequence of the redistribution of mem 0164. In bombardment transformation, one may optimize brane components that accompanies closure of the pores. the prebombardment culturing conditions and the bombard Electroporation can be extremely efficient and can be used ment parameters to yield the maximum numbers of stable both for transient expression of clones genes and for estab transformants. Both the physical and biological parameters lishment of cell lines that carry integrated copies of the gene for bombardment are important in this technology. Physical of interest. Electroporation, in contrast to calcium phosphate factors are those that involve manipulating the DNA/micro US 2009/0087863 A1 Apr. 2, 2009

projectile precipitate or those that affect the flight and veloc rice, corn, and wheat must usually be transformed using alter ity of either the macro- or microprojectiles. Biological factors native methods. However, as mentioned above, the transfor include all steps involved in manipulation of cells before and mation of asparagus using Agrobacterium can also be immediately after bombardment, the osmotic adjustment of achieved (see, for example, Bytebier et al., 1987). target cells to help alleviate the trauma associated with bom 0169. A transgenic plant formed using Agrobacterium bardment, and also the nature of the transforming DNA, such transformation methods typically contains a single gene on as linearized DNA or intact supercoiled plasmids. It is one chromosome. Such transgenic plants can be referred to as believed that pre-bombardment manipulations are especially being heterozygous for the added gene. However, inasmuch important for Successful transformation of immature as use of the word "heterozygous' usually implies the pres embryos. ence of a complementary gene at the same locus of the second 0.165 Accordingly, it is contemplated that one may wish to chromosome of a pair of chromosomes, and there is no such adjust various of the bombardment parameters in Small scale gene in a plant containing one added gene as here, it is studies to fully optimize the conditions. One may particularly believed that a more accurate name for Such a plant is an wish to adjust physical parameters such as gap distance, flight independent segregant, because the added, exogenous gene distance, tissue distance, and helium pressure. One may also segregates independently during mitosis and meiosis. minimize the trauma reduction factors (TRFs) by modifying 0170 More preferred is a transgenic plant that is homozy conditions which influence the physiological state of the gous for the added structural gene; i.e., a transgenic plant that recipient cells and which may therefore influence transfor contains two added genes, one gene at the same locus on each mation- and integration efficiencies. For example, the chromosome of a chromosome pair. A homozygous trans osmotic state, tissue hydration and the Subculture stage or cell genic plant can be obtained by sexually mating (selfing) an cycle of the recipient cells may be adjusted for optimum independent segregant transgenic plant that contains a single transformation. The execution of other routine adjustments added gene, germinating some of the seed produced and will be known to those of skill in the art in light of the present analyzing the resulting plants produced for enhanced car disclosure. boxylase activity relative to a control (native, non-transgenic) or an independent segregant transgenic plant. 4.8.3 Agrobacterium-Mediated Transfer 0171 It is to be understood that two different transgenic 0166 Agrobacterium-mediated transfer is a widely appli plants can also be mated to produce offspring that contain two cable system for introducing genes into plant cells because independently segregating added, exogenous genes. Selfing the DNA can be introduced into whole plant tissues, thereby of appropriate progeny can produce plants that are homozy bypassing the need for regeneration of an intact plant from a gous for both added, exogenous genes that encode a polypep protoplast. The use of Agrobacterium-mediated plant inte tide of interest. Back-crossing to a parental plant and out grating vectors to introduce DNA into plant cells is well crossing with a non-transgenic plant are also contemplated. known in the art. See, for example, the methods described (Fraley et al., 1985; Rogers et al., 1987). Further, the integra 4.8.4 Other Transformation Methods tion of the Ti-DNA is a relatively precise process resulting in 0172 Transformation of plant protoplasts can be achieved few rearrangements. The region of DNA to be transferred is using methods based on calcium phosphate precipitation, defined by the border sequences, and intervening DNA is polyethylene glycol treatment, electroporation, and combina usually inserted into the plant genome as described (Spiel tions of these treatments (see, e.g., Potrykus et al., 1985; Lorz mann et al., 1986). et al., 1985; Fromm et al., 1986: Uchimiya et al., 1986; Callis 0167 Modern Agrobacterium transformation vectors are et al., 1987; Marcotte et al., 1988). capable of replication in E. coli as well as Agrobacterium, 0173 Application of these systems to different plant stains allowing for convenient manipulations as described (Klee et depends upon the ability to regenerate that particular plant al., 1985). Moreover, recent technological advances in vec strain from protoplasts. Illustrative methods for the regenera tors for Agrobacterium-mediated gene transfer have tion of cereals from protoplasts are described (Fujimura et al., improved the arrangement of genes and restriction sites in the 1985; Toriyama et al., 1986; Yamada et al., 1986; Abdullah et vectors to facilitate construction of vectors capable of al., 1986). expressing various polypeptide coding genes. The vectors 0.174. To transform plant strains that cannot be success described (Rogers et al., 1987), have convenient multi-linker fully regenerated from protoplasts, other ways to introduce regions flanked by a promoter and a polyadenylation site for DNA into intact cells or tissues can be utilized. For example, direct expression of inserted polypeptide coding genes and regeneration of cereals from immature embryos or explants are suitable for present purposes. In addition, Agrobacterium can be effected as described (Vasil, 1988). In addition, “par containing both armed and disarmed Tigenes can be used for ticle gun' or high-velocity microprojectile technology can be the transformations. In those plant strains where Agrobacte utilized (Vasil, 1992). rium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene 0.175. Using that latter technology, DNA is carried through transfer. the cell wall and into the cytoplasm on the surface of small 0168 Agrobacterium-mediated transformation of leaf metal particles as described (Klein et al., 1987; Klein et al., disks and other tissues such as cotyledons and hypocotyls 1988; McCabe et al., 1988). The metal particles penetrate appears to be limited to plants that Agrobacterium naturally through several layers of cells and thus allow the transforma infects. Agrobacterium-mediated transformation is most effi tion of cells within tissue explants. cient in dicotyledonous plants. Few monocots appear to be natural hosts for Agrobacterium, although transgenic plants 4.8.5 Gene Expression in Plants have been produced in asparagus using Agrobacterium vec 0176 Although great progress has been made in recent tors. Therefore, commercially important cereal grams such as years with respect to preparation of transgenic plants which US 2009/0087863 A1 Apr. 2, 2009 22 express bacterial proteins such as B. thuringiensis crystal sequence. However, the number of ATTTA sequences and/or proteins, the results of expressing native bacterial genes in the sequence context in which they occur also appear to be plants are often disappointing. Unlike microbial genetics, important in determining whether they function as destabi little was known by early plant geneticists about the factors lizing sequences. Shaw and Kamen showed that a trimer of which affected heterologous expression of foreign genes in ATTTA had much less effect than a pentamer on mRNA plants. In recent years, however, several potential factors have stability and a dimer or a monomer had no effect on stability been implicated as responsible in varying degrees for the level (Shaw and Kamen, 1987). Note that multimers of ATTTA of protein expression from a particular coding sequence. For Such as a pentamer automatically create an A+T rich region. example, Scientists now know that maintaining a significant This was shown to be a cytoplasmic effect, not nuclear. In level of a particular mRNA in the cell is indeed a critical other unstable mRNAs, the ATTTA sequence may be present factor. Unfortunately, the causes for low steady state levels of in only a single copy, but it is often contained in an A+T rich mRNA encoding foreign proteins are many. First, full length region. From the animal cell data collected to date, it appears RNA synthesis may not occurata high frequency. This could, that ATTTA at least in some contexts is important in stability, for example, becaused by the premature termination of RNA but it is not yet possible to predict which occurrences of during transcription or due to unexpected mRNA processing ATTTA are destabiling elements or whether any of these during transcription. Second, full length RNA may be pro effects are likely to be seen in plants. duced in the plant cell, but then processed (splicing, polyA 0179 Some studies on mRNA degradation in animal cells addition) in the nucleus in a fashion that creates a nonfunc also indicate that RNA degradation may begin in Some cases tional mRNA. If the RNA is not properly synthesized, termi with nucleolytic attack in A+T rich regions. It is not clear if nated and polyadenylated, it cannot move to the cytoplasm for these cleavages occur at ATTTA sequences. There are also translation. Similarly, in the cytoplasm, if mRNAs have examples of mRNAs that have differential stability depend reduced half lives (which are determined by their primary or ing on the cell type in which they are expressed or on the stage secondary sequence) insufficient protein product will be pro within the cell cycle at which they are expressed. For duced. In addition, there is an effect, whose magnitude is example, histone mRNAs are stable during DNA synthesis uncertain, of translational efficiency on mRNA half-life. In but unstable if DNA synthesis is disrupted. The 3' end of some addition, every RNA molecule folds into a particular struc histone mRNAs seems to be responsible for this effect (Pan ture, or perhaps family of structures, which is determined by dey and Marzluff, 1987). It does not appear to be mediated by its sequence. The particular structure of any RNA might lead ATTTA, nor is it clear what controls the differential stability to greater or lesser stability in the cytoplasm. Structure perse of this mRNA. Another example is the differential stability of is probably also a determinant of mRNA processing in the IgG mRNA in B lymphocytes during B cell maturation (Gen nucleus. Unfortunately, it is impossible to predict, and nearly ovese and Milcarek, 1988). A final example is the instability impossible to determine, the structure of any RNA (except for of a mutant B-thallesemic globin mRNA. In bone marrow tRNA) in vitro or in vivo. However, it is likely that dramati cells, where this gene is normally expressed, the mutant cally changing the sequence of an RNA will have a large mRNA is unstable, while the wild-type mRNA is stable. effect on its folded structure It is likely that structure perse or When the mutant gene is expressed in HeLa or L cells in vitro, particular structural features also have a role in determining the mutant mRNA shows no instability. These examples all RNA stability. provide evidence that mRNA stability can be mediated by cell 0177. To overcome these limitations in foreign gene type or cell cycle specific factors. Furthermore this type of expression, researchers have identified particular sequences instability is not yet associated with specific sequences. and signals in RNAS that have the potential for having a Given these uncertainties, it is not possible to predict which specific effect on RNA stability. In certain embodiments of RNAs are likely to be unstable in a given cell. In addition, the invention, therefore, there is a desire to optimize expres even the ATTTA motif may act differentially depending on sion of the disclosed nucleic acid segments in planta. One the nature of the cell in which the RNA is present. Shaw and particular method of doing so, is by alteration of the bacterial Kamen (1987) have reported that activation of protein kinase gene to remove sequences or motifs which decrease expres C can block degradation mediated by ATTTA. sion in a transformed plant cell. The process of engineering a 0180. The addition of a polyadenylate string to the 3' end coding sequence for optimal expression in planta is often is common to most eukaryotic mRNAS, both plant and ani referred to as “plantizing a DNA sequence. mal. The currently accepted view of polyA addition is that the 0.178 Particularly problematic sequences are those which nascent transcript extends beyond the mature 3' terminus. are A+Trich Unfortunately, since B. thuringiensis has an A+T Contained within this transcript are signals for polyadenyla rich genome, native crystal protein gene sequences must tion and proper 3' end formation. This processing at the 3' end often be modified for optimal expression in a plant. The involves cleavage of the mRNA and addition of polyA to the sequence motif ATTTA (or AUUUA as it appears in RNA) has mature 3' end. By searching for consensus sequences near the been implicated as a destabilizing sequence in mammalian polyA tract in both plant and animal mRNAs, it has been cell mRNA (Shaw and Kamen, 1986). Many short lived possible to identify consensus sequences that apparently are mRNAs have A+T rich 3' untranslated regions, and these involved in polyA addition and 3' end cleavage. The same regions often have the ATTTA sequence, sometimes present consensus sequences seem to be important to both of these in multiple copies or as multimers (e.g., ATTTATTTA . . . ). processes. These signals are typically a variation on the Shaw and Kamen showed that the transfer of the 3' end of an sequence AATAAA. In animal cells, some variants of this unstable mRNA to a stable RNA (globin or VA1) decreased sequence that are functional have been identified; in plant the stable RNA's half life dramatically. They further showed cells there seems to be an extended range of functional that a pentamer of ATTTA had a profound destabilizing effect sequences (Wickens and Stephenson, 1984; Dean et al., on a stable message, and that this signal could exert its effect 1986). Because all of these consensus sequences are varia whether it was located at the 3' end or within the coding tions on AATAAA, they all are A+T rich sequences. This US 2009/0087863 A1 Apr. 2, 2009 sequence is typically found 15 to 20 bp before the polyA tract in a mature mRNA. Studies in animal cells indicate that this TABLE 4 - continued sequence is involved in both polyA addition and 3' matura tion. Site directed mutations in this sequence can disrupt these POLYADENYLATION SITES IN PLANT GENES functions (Conway and Wickens, 1988; Wickens et al., 1987). P6A. ATAAAA However, it has also been observed that sequences up to 50 to 100 bp 3' to the putative polyA signal are also required; i.e., a P7A ATGAAA gene that has a normal AATAAA but has been replaced or disrupted downstream does not get properly polyadenylated P8A AAGCAT (Gil and Proudfoot, 1984; Sadofsky and Alwine, 1984; P9A ATTAAT McDevitt et al., 1984). That is, the polyA signal itself is not Sufficient for complete and proper processing. It is not yet P1 OA ATACAT known what specific downstream sequences are required in P11A AAAATA addition to the polyA signal, or if there is a specific sequence that has this function. Therefore, sequence analysis can only P12A ATTAAA Minor animal site identify potential polyA signals. P13A AATTAA 0181. In naturally occurring mRNAs that are normally polyadenylated, it has been observed that disruption of this P14A. AATACA process, either by altering the polyA signal or other sequences in the mRNA, profound effects can be obtained in the level of P15A CATAAA functional mRNA. This has been observed in several natu rally occurring mRNAS, with results that are gene-specific so 0.184 The present invention provides a method for prepar far. ing synthetic plant genes which genes express their protein 0182. It has been shown that in natural mRNAs proper product at levels significantly higher than the wild-type genes polyadenylation is important in mRNA accumulation, and which were commonly employed in plant transformation that disruption of this process can effect mRNA levels sig heretofore. In another aspect, the present invention also pro nificantly. However, insufficient knowledge exists to predict vides novel synthetic plant genes which encode non-plant the effect of changes in a normal gene. In a heterologous gene, proteins. it is even harder to predict the consequences. However, it is 0185. As described above, the expression of native B. thu possible that the putative sites identified are dysfunctional. ringiensis genes in plants is often problematic. The nature of That is, these sites may not act as proper polyA sites, but the coding sequences of B. thuringiensis genes distinguishes instead function as aberrant sites that give rise to unstable them from plant genes as well as many other heterologous mRNAS. genes expressed in plants. In particular, B. thuringiensis 0183 In animal cell systems, AATAAA is by far the most genes are very rich (~62%) in adenine (A) and thymine (T) common signal identified in mRNAS upstream of the polyA, while plant genes and most other bacterial genes which have but at least four variants have also been found (Wickens and been expressed in plants are on the order of 45-55% A+T. Stephenson, 1984). In plants, not nearly so much analysis has 0186. Due to the degeneracy of the genetic code and the been done, but it is clear that multiple sequences similar to limited number of codon choices for any amino acid, most of AATAAA can be used The plant sites in Table 4 called major the “excess' A+T of the structural coding sequences of some or minor refer only to the study of Dean et al. (1986) which Bacillus species are found in the third position of the codons. analyzed only three types of plant gene. The designation of That is, genes of some Bacillus species have A or T as the third polyadenylation sites as major or minor refers only to the nucleotide in many codons. Thus A+T content in part can frequency of their occurrence as functional sites in naturally determine codon usage bias. In addition, it is clear that genes occurring genes that have been analyzed. In the case of plants evolve for maximum function in the organism in which they this is a very limited database. It is hard to predict with any evolve. This means that particular nucleotide sequences certainty that a site designated major or minor is more or less found in a gene from one organism, where they may play no likely to function partially or completely when found in a role except to code for a particular stretch of amino acids, heterologous gene Such as those encoding the crystal proteins have the potential to be recognized as gene control elements of the present invention. in another organism (such as transcriptional promoters or terminators, polyA addition sites, intron splice sites, or spe TABLE 4 cific mRNA degradation signals). It is perhaps Surprising that Such misread signals are not a more common feature of het POLYADENYLATION SITES IN PLANT GENES erologous gene expression, but this can be explained in part by the relatively homogeneous A+T content (-50%) of many PA AATAAA Major consensus site organisms. This A+T content plus the nature of the genetic P1A AATAAT Major plant site code put clear constraints on the likelihood of occurrence of any particular oligonucleotide sequence. Thus, a gene from E. P2A AACCAA Minor plant site coli with a 50% A+T content is much less likely to contain any P3A ATATAA particular A+T rich segment than a gene from B. thuringien Sis. P4A AATCAA 0187. Typically, to obtain high-level expression of the PSA ATACTA Ö-endotoxin genes in plants, existing structural coding sequence (“structural gene') which codes for the 8-endotoxin are modified by removal of ATTTA sequences and putative US 2009/0087863 A1 Apr. 2, 2009 24 polyadenylation signals by site directed mutagenesis of the ranging from about 18 to about 100 bases have been utilized. DNA comprising the structural gene. It is most preferred that In most cases, a minimum of about 5 to about 8 base pairs of substantially all the polyadenylation signals and ATTTA homology to the template DNA on both ends of the synthe sequences are removed although enhanced expression levels sized fragment are maintained to insure proper hybridization are observed with only partial removal of either of the above identified sequences. Alternately if a synthetic gene is pre of the primer to the template. The oligonucleotides should pared which codes for the expression of the subject protein, avoid sequences longer than five base pairs A+T or G+C. codons are selected to avoid the ATTTA sequence and puta Codons used in the replacement of wild-type codons should tive polyadenylation signals. For purposes of the present preferably avoid the TA or CG doublet wherever possible. invention putative polyadenylation signals include, but are Codons are selected from a plant preferred codon table (such not necessarily limited to, AATAAA, AATAAT. AACCAA, as Table 5 below) so as to avoid codons which are rarely found ATATAAAATCAA, ATACTA, ATAAAA, ATGAAAAAG in plant genomes, and efforts should be made to select codons CAT, ATTAAT, ATACAT. AAAATA, ATTAAA AATTAA, to preferably adjust the G+C content to about 50%. AATACA and CATAAA. In replacing the ATTTA sequences and polyadenylation signals, codons are preferably utilized TABLE 5 which avoid the codons which are rarely found in plant genomes. PREFERRED CODON USAGE IN PLANTS 0188 The selected DNA sequence is scanned to identify regions with greater than four consecutive adenine (A) or Amino Acid Codon Percent Usage in Plants thymine (T) nucleotides. The A+T regions are scanned for ARG CGA 7 potential plant polyadenylation signals. Although the absence CGC 11 CGG 5 of five or more consecutive A or T nucleotides eliminates CGU 25 most plant polyadenylation signals, if there are more than one AGA 29 of the minor polyadenylation signals identified within ten AGG 23 nucleotides of each other, then the nucleotide sequence of this LEU CUA. 8 region is preferably altered to remove these signals while CUC 2O maintaining the original encoded amino acid sequence. CUG 10 0189 The second step is to consider the about 15 to about CUU 28 30 or so nucleotide residues surrounding the A+Trich region UUA 5 identified in step one. If the A+T content of the surrounding UUG 3 O region is less than 80%, the region should be examined for SER UCA 14 polyadenylation signals. Alteration of the region based on UCC 26 polyadenylation signals is dependent upon (1) the number of UCG 3 UCU 21 polyadenylation signals present and (2) presence of a major AGC 21 plant polyadenylation signal. AGU 15 0190. The extended region is examined for the presence of plant polyadenylation signals. The polyadenylation signals THR ACA 21 ACC 41 are removed by site-directed mutagenesis of the DNA ACG 7 sequence. The extended region is also examined for multiple ACU 31 copies of the ATTTA sequence which are also removed by mutagenesis. PRO CCA 45 CCC 19 0191 It is also preferred that regions comprising many CCG 9 consecutive A+T bases or CCU 26 0.192 G+C bases are disrupted since these regions are ALA GCA 23 predicted to have a higher likelihood to form hairpinstructure GCC 32 due to self-complementarity. Therefore, insertion of hetero GCG 3 geneous base pairs would reduce the likelihood of self GCU 41 complementary secondary structure formation which are GLY GGA 32 known to inhibit transcription and/or translation in some GGC 2O organisms. In most cases, the adverse effects may be mini GGG 11 mized by using sequences which do not contain more than GGU 37 five consecutive A+T or G+C. ILE AUA. 12 AUC 45 4.8.6. Synthetic Oligonucleotides for Mutagenesis AUU 43

0193 When oligonucleotides are used in the mutagenesis, WAL GUA. 9 it is desirable to maintain the properamino acid sequence and GUC 2O reading frame, without introducing common restriction sites GUG 28 such as BglII, HindIII, SacI, KpnI, EcoRI, NcoI, PstI and Sall GUU 43 into the modified gene. These restriction sites are found in poly-linkerinsertion sites of many cloning vectors. Ofcourse, LYS AAA 36 the introduction of new polyadenylation signals, ATTTA AAG 64 sequences or consecutive stretches of more than five A+T or ASN AAC 72 G+C, should also be avoided. The preferred size for the oli AAU 28 gonucleotides is about 40 to about 50 bases, but fragments US 2009/0087863 A1 Apr. 2, 2009 25

et al., 1991; Vasil et al., 1992) to deliver the DNA coated on TABLE 5- continued microprojectiles into the recipient cells. Transgenic plants are then regenerated from transformed embryonic calli that PREFERRED CODON USAGE IN PLANTS express the insecticidal proteins. Amino Acid Codon Percent Usage in Plants 0198 The formation of transgenic plants may also be accomplished using other methods of cell transformation GLN CAA 64 which are known in the art such as Agrobacterium—mediated CAG 36 DNA transfer (Fraley et al., 1983). Alternatively, DNA can be introduced into plants by direct DNA transfer into pollen HIS CAC 65 (Zhou et al., 1983; Hess, 1987; Luo et al., 1988), by injection CAU 35 of the DNA into reproductive organs of a plant, or by direct GLU GAA 48 injection of DNA into the cells of immature embryos fol GAG 52 lowed by the rehydration of desiccated embryos (Neuhaus et al, 1987: Benbrook et al., 1986). ASP GAC 48 0199 The regeneration, development, and cultivation of GAU 52 plants from single plant protoplast transformants or from TYR UAC 68 various transformed explants is well known in the art (Weiss UAU 32 bach and Weissbach, 1988). This regeneration and growth CYS UGC 78 process typically includes the steps of selection of trans UGU 22 formed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted PHE UUC 56 plantlet stage. Transgenic embryos and seeds are similarly UUU 44 regenerated. The resulting transgenic rooted shoots are there MET AUG 1OO after planted in an appropriate plant growth medium such as soil. TRP UGG 1OO 0200. The development or regeneration of plants contain ing the foreign, exogenous gene that encodes a polypeptide of 0194 Regions with many consecutive A+T bases or G+C interest introduced by Agrobacterium from leaf explants can bases are predicted to have a higher likelihood to form hairpin be achieved by methods well known in the art such as structures due to self-complementarity. Disruption of these described (Horsch et al., 1985). In this procedure, transfor regions by the insertion of heterogeneous base pairs is pre mants are cultured in the presence of a selection agent and in ferred and should reduce the likelihood of the formation of a medium that induces the regeneration of shoots in the plant self-complementary secondary structures such as hairpins strain being transformed as described (Fraley et al., 1983). which are known in Some organisms to inhibit transcription 0201 This procedure typically produces shoots within (transcriptional terminators) and translation (attenuators). two to four months and those shoots are then transferred to an 0.195 Alternatively, a completely synthetic gene for a appropriate root-inducing medium containing the selective given amino acid sequence can be prepared, with regions of agent and an antibiotic to prevent bacterial growth. Shoots five or more consecutive A+T or G+C nucleotides being that rooted in the presence of the selective agent to form avoided. Codons are selected avoiding the TA and CG dou plantlets are then transplanted to soil or other media to allow blets in codons whenever possible. Codon usage can be nor the production of roots. These procedures vary depending malized against a plant preferred codon usage table (such as upon the particular plant Strain employed, such variations Table 5) and the G+C content preferably adjusted to about being well known in the art. 50%. The resulting sequence should be examined to ensure 0202 Preferably, the regenerated plants are self-polli that there are minimal putative plant polyadenylation signals nated to provide homozygous transgenic plants, as discussed and ATTTA sequences. Restriction sites found in commonly before. Otherwise, pollen obtained from the regenerated used cloning vectors are also, preferably avoided. However, plants is crossed to seed-grown plants of agronomically placement of several unique restriction sites throughout the important, preferably inbred lines. Conversely, pollen from gene is useful for analysis of gene expression or construction plants of those importantlines is used to pollinate regenerated of gene variants. plants. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known 4.9 Methods for Producing Insect-Resistant to one skilled in the art. Transgenic Plants 0203 A transgenic plant of this invention thus has an increased amount of a coding region (e.g., a gene) that 0196. By transforming a suitable host cell, such as a plant encodes a polypeptide in accordance with SEQ ID NO:2. A cell, with a recombinant cryET70 gene-containing segment, preferred transgenic plant is an independent segregant and the expression of the encoded crystal protein (i.e. a bacterial can transmit that gene and its activity to its progeny. A more crystal protein or polypeptide having insecticidal activity preferred transgenic plant is homozygous for that gene, and against Coleopterans) can result in the formation of insect transmits that gene to all of its offspring on sexual mating. resistant plants. Seed from a transgenic plant may be grown in the field or 0.197 By way of example, one may utilize an expression greenhouse, and resulting sexually mature transgenic plants vector containing a coding region for a B. thuringiensis crys are self-pollinated to generate true breeding plants. The prog tal protein and an appropriate selectable marker to transform eny from these plants become true breeding lines that are a suspension of embryonic plant cells, such as wheat or corn evaluated for, by way of example, increased insecticidal cells using a method Such as particle bombardment (Maddock capacity against coleopteran insects, preferably in the field, US 2009/0087863 A1 Apr. 2, 2009 26 under a range of environmental conditions. The inventors provided herein. For example, antibodies to the 8-endotoxins contemplate that the present invention will find particular disclosed and claimed herein can be used to identify and utility in the creation of transgenic plants of commercial isolate other ö-endotoxins from a mixture of proteins. Spe interest including various turf and pasture grasses, rye, wheat, cifically, antibodies may be raised to the portions of the 8-en corn, kapok, flax, rice, barley, oats, Sugarcane, cotton, tomato, dotoxins which are most constant and most distinct from potato, soybeans and other legumes, tobacco, Sorghum, as other B. thuringiensis 8-endotoxins. These antibodies can well as a variety of ornamental plants including cacti and then be used to specifically identify equivalent 8-endotoxins Succulents, fruits, berries, vegetables, and also a number of with the characteristic insecticidal activity by immunopre nut- and fruit-bearing trees and plants. cipitation, enzyme linked immunoassay (ELISA), or Western 0204 Transgenic plants comprising one or more blotting. trangenes that encode a polypeptide in accordance with SEQ 0208 A further method for identifying the 8-endotoxins ID NO:2 will preferably exhibit a phenotype of improved or and genes of the Subject invention is through the use of oli enhanced insect resistance to the target coleopteran and lepi gonucleotide probes. These probes are nucleotide sequences dopteran insects as described herein. These plants will pref having a detectable label. As is well known in the art, if the erably provide transgenic seeds, which will be used to create probe molecule and nucleic acid sample hybridize by forming lineages of transgenic plants (i.e. progeny or advanced gen a strong bond between the two molecules, it can be reason erations of the original transgenic plant) that may be used to ably assumed that the probe and sample are essentially iden produce seed, or used as animal or human foodstuffs, or to tical. The probe's detectable label provides a means for deter produce fibers, oil, fruit, grains, or other commercially-im mining in a known manner whether hybridization has portant plant products or plant-derived components. In Such occurred. Such a probe analysis provides a rapid method for instances, the progeny and seed obtained from any generation identifying formicidal 6-endotoxin genes of the Subject of the transformed plants will contain the selected chromo invention. Somally-integrated transgene that encodes the Ö-endotoxin of 0209. The nucleotide segments which are used as probes the present invention. The transgenic plants of the present according to the invention can be synthesized by use of DNA invention may be crossed to produce hybrid or inbred lines synthesizers using standard procedures. In the use of the with one or more plants that have desirable properties. In nucleotide segments as probes, the particular probe is labeled certain circumstances, it may also be desirable to create trans with any suitable label known to those skilled in the art, genic plants, seed, and progeny that contain one or more including radioactive and non-radioactive labels. Typical additional transgenes incorporated into their genome in addi radioactive labels include 'P, 'I, S, or the like. A probe tion to the transgene encoding the polypeptide of the inven labeled with a radioactive isotope can be constructed from a tion. For example, the transgenic plants may contain a second nucleotide sequence complementary to the DNA sample by a gene encoding the same, or a different insect-resistance conventional nick translation reaction, using a DNase and polypeptide, or alternatively, the plants may comprise one or DNA polymerase. The probe and sample can then be com more additional transgenes Such as those conferring herbicide bined in a hybridization buffer solution and held at an appro resistance, fungal resistance, bacterial resistance, stress, salt, priate temperature until annealing occurs. Thereafter, the or drought tolerance, improved stalk or root lodging, membrane is washed free of extraneous materials, leaving the increased starch, grain, oil, carbohydrate, amino acid, protein sample and bound probe molecules typically detected and production, and the like. quantified by autoradiography and/or liquid Scintillation counting. 4.10 Isolating Homologous Gene and Gene 0210. Non-radioactive labels include, for example, Fragments ligands such as biotin orthyroxin, as well as enzymes such as 0205 The genes and Ö-endotoxins according to the subject hydrolases or peroxidases, or the various chemiluminescers invention include not only the full length sequences disclosed Such as luciferin, or fluorescent compounds like fluorescein herein but also fragments of these sequences, or fusion pro and its derivatives. The probe may also be labeled at both ends teins, which retain the characteristic insecticidal activity of with different types of labels for ease of separation, as, for the sequences specifically exemplified herein. example, by using an isotopic label at the end mentioned 0206. It should be apparent to a person skill in this art that above and a biotin label at the other end. insecticidal Ö-endotoxins can be identified and obtained 0211 Duplex formation and stability depend on substan through several means. The specific genes, or portions tial complementarity between the two strands of a hybrid, thereof, may be obtained from a culture depository, or con and, as noted above, a certain degree of mismatch can be structed synthetically, for example, by use of a gene machine. tolerated. Therefore, the probes of the subject invention Variations of these genes may be readily constructed using include mutations (both single and multiple), deletions, inser standard techniques for making point mutations. Also, frag tions of the described sequences, and combinations thereof, ments of these genes can be made using commercially avail wherein said mutations, insertions and deletions permit for able exonucleases or endonucleases according to standard mation of stable hybrids with the target polynucleotide of procedures. For example, enzymes Such as Bal31 or site interest. Mutations, insertions, and deletions can be produced directed mutagenesis can be used to systematically cut off in a given polynucleotide sequence in many ways, by meth nucleotides from the ends of these genes. Also, genes which ods currently known to an ordinarily skilled artisan, and per code for active fragments may be obtained using a variety of haps by other methods which may become known in the other restriction enzymes. Proteases may be used to directly future. obtain active fragments of these 6-endotoxins. 0212. The potential variations in the probes listed is due, in 0207 Equivalent 8-endotoxins and/or genes encoding part, to the redundancy of the genetic code. Because of the these equivalent Ö-endotoxins can also be isolated from redundancy of the genetic code, i.e., more than one coding Bacillus strains and/or DNA libraries using the teachings nucleotide triplet (codon) can be used for most of the amino US 2009/0087863 A1 Apr. 2, 2009 27 acids used to make proteins. Therefore different nucleotide of a ribozyme is greater than that of an antisense oligonucle sequences can code for a particular amino acid. Thus, the otide binding the same RNA site. amino acid sequences of the B. thuringiensis 6-endotoxins 0216. The enzymatic nucleic acid molecule may be and peptides can be prepared by equivalent nucleotide formed in a hammerhead, hairpin, a hepatitis 8 virus, group I sequences encoding the same amino acid sequence of the intron or RNaseP RNA (in association with an RNA guide protein or peptide. Accordingly, the Subject invention sequence) or Neurospora VS RNA motif. Examples of ham includes such equivalent nucleotide sequences. Also, inverse merhead motifs are described by Rossi et al. (1992); or complement sequences are an aspect of the Subject inven examples of hairpin motifs are described by Hampel et al tion and can be readily used by a person skilled in this art. In (Eur. Pat. EP 0360257), Hampel and Tritz (1989), Hampelet addition it has been shown that proteins of identified structure al. (1990) and Cech et al. (U.S. Pat. No. 5,631,359; an and function may be constructed by changing the amino acid example of the hepatitis 8 virus motif is described by Perrotta sequence if such changes do not alter the protein secondary and Been (1992); an example of the RNaseP motif is structure (Kaiser and Kezdy, 1984). Thus, the subject inven described by Guerrier-Takada et al. (1983); Neurospora VS tion includes mutants of the amino acid sequence depicted RNA ribozyme motif is described by Collins (Saville and herein which do not alter the protein secondary structure, or if Collins, 1990: Saville and Collins, 1991; Collins and Olive, the structure is altered, the biological activity is substantially 1993); and an example of the Group I intron is described by retained. Further, the invention also includes mutants of Cech et al. (U.S. Pat. No. 4,987,071). All that is important in organisms hosting all or part of a 6-endotoxin encoding a an enzymatic nucleic acid molecule of this invention is that it gene of the invention. Such mutants can be made by tech has a specific Substrate binding site which is complementary niques well known to persons skilled in the art. For example, to one or more of the target gene RNA regions, and that it have UV irradiation can be used to prepare mutants of host organ nucleotide sequences within or Surrounding that Substrate isms. Likewise, such mutants may include asporogenous host binding site which impart an RNA cleaving activity to the cells which also can be prepared by procedures well known in molecule. Thus the ribozyme constructs need not be limited the art. to specific motifs mentioned herein. 0217. The invention provides a method for producing a 4.11 Ribozymes class of enzymatic cleaving agents which exhibit a high 0213 Ribozymes are enzymatic RNA molecules which degree of specificity for the RNA of a desired target. The cleave particular mRNA species. In certain embodiments, the enzymatic nucleic acid molecule is preferably targeted to a inventors contemplate the selection and utilization of highly conserved sequence region of a target mRNA such that ribozymes capable of cleaving the RNA segments of the specific treatment of a disease or condition can be provided present invention, and their use to reduce activity of target with either one or several enzymatic nucleic acids. Such mRNAs in particular cell types or tissues. enzymatic nucleic acid molecules can be delivered exog 0214 Six basic varieties of naturally-occurring enzymatic enously to specific cells as required. Alternatively, the RNAs are known presently. Each can catalyze the hydrolysis ribozymes can be expressed from DNA or RNA vectors that of RNA phosphodiester bonds in trans (and thus can cleave are delivered to specific cells. other RNA molecules) under physiological conditions. In 0218. Small enzymatic nucleic acid motifs (e.g., of the general, enzymatic nucleic acids act by first binding to a target hammerhead or the hairpin structure) may be used for exog RNA. Such binding occurs through the target binding portion enous delivery. The simple structure of these molecules of a enzymatic nucleic acid which is held in close proximity increases the ability of the enzymatic nucleic acid to invade to an enzymatic portion of the molecule that acts to cleave the targeted regions of the mRNA structure. Alternatively, cata target RNA. Thus, the enzymatic nucleic acid first recognizes lytic RNA molecules can be expressed within cells from and then binds a target RNA through complementary base eukaryotic promoters (e.g., Scanlon et al., 1991; Kashani pairing, and once bound to the correct site, acts enzymatically Sabet et al., 1992; Dropulic et al., 1992; Weerasinghe et al., to cut the target RNA. Strategic cleavage of such a target RNA 1991; Ojwang et al., 1992: Chen et al., 1992: Sarver et al., will destroy its ability to direct synthesis of an encoded pro 1990). Those skilled in the art realize that any ribozyme can tein. After an enzymatic nucleic acid has bound and cleaved be expressed in eukaryotic cells from the appropriate DNA its RNA target, it is released from that RNA to search for vector. The activity of such ribozymes can be augmented by another target and can repeatedly bind and cleave new targets. their release from the primary transcript by a second 0215. The enzymatic nature of a ribozyme is advanta ribozyme (Int. Pat. Appl. Publ. No. WO93/23569), and (Int geous over many technologies, such as antisense technology Pat. Appl. Publ. No. WO94/02595), both hereby incorporated (where a nucleic acid molecule simply binds to a nucleic acid in their totality by reference herein; Ohkawa et al., 1992; Taira target to block its translation) since the concentration of et al., 1991; Ventura et al., 1993). ribozyme necessary to affect a therapeutic treatment is lower 0219 Ribozymes may be added directly, or can be com than that of an antisense oligonucleotide. This advantage plexed with cationic lipids, lipid complexes, packaged within reflects the ability of the ribozyme to act enzymatically. Thus, liposomes, or otherwise delivered to target cells. The RNA or a single ribozyme molecule is able to cleave many molecules RNA complexes can be locally administered to relevant tis of target RNA. In addition, the ribozyme is a highly specific Sues ex vivo, or in Vivo through injection, aerosol inhalation, inhibitor, with the specificity of inhibition depending not only infusion pump or stent, with or without their incorporation in on the base pairing mechanism of binding to the target RNA, biopolymers. but also on the mechanism of target RNA cleavage. Single 0220 Ribozymes may be designed as described in (Int. mismatches, or base-substitutions, near the site of cleavage Pat. Appl. Publ. No. WO 93/23569), or (Int. Pat. Appl. Publ. can completely eliminate catalytic activity of a ribozyme. No. WO94/02595) and synthesized to be tested in vitro and in Similar mismatches in antisense molecules do not prevent vivo, as described. Such ribozymes can also be optimized for their action (Woolfetal, 1992). Thus, the specificity of action delivery. While specific examples are provided, those in the US 2009/0087863 A1 Apr. 2, 2009 28 art will recognize that equivalent RNA targets in other species ribozyme-encoding sequences into a DNA expression vector. can be utilized when necessary. Transcription of the ribozyme sequences are driven from a 0221 Hammerhead or hairpin ribozymes may be indi promoter for eukaryotic RNA polymerase I (pol I), RNA vidually analyzed by computer folding (Jaeger et al., 1989), polymerase II pol II), or RNA polymerase III (pol III). Tran to assess whether the ribozyme sequences fold into the appro scripts from pol II or pol III promoters will be expressed at priate secondary structure. Those ribozymes with unfavor high levels in all cells; the levels of a given pol II promoter in able intramolecular interactions between the binding arms a given cell type will depend on the nature of the gene regu and the catalytic core are eliminated from consideration. latory sequences (enhancers, silencers, etc.) present nearby. Varying binding arm lengths can be chosen to optimize activ Prokaryotic RNA polymerase promoters may also be used, ity. Generally, at least 5 bases on each arm are able to bind to, providing that the prokaryotic RNA polymerase enzyme is or otherwise interact with, the target RNA. expressed in the appropriate cells (Elroy-Stein and Moss, 0222 Ribozymes of the hammerhead or hairpin motifmay 1990; Gao and Huang, 1993: Lieber et al., 1993; Zhou et al., be designed to anneal to various sites in the mRNA message, 1990). Ribozymes expressed from such promoters can func and can be chemically synthesized. The method of synthesis tion in mammalian cells (e.g. Kashani-Saber et al., 1992: used follows the procedure for normal RNA synthesis as Owang et al., 1992: Chen et al., 1992: Yu et al., 1993: described in Usman et al. (1992) and in Scaringe et al. (1990) L'Huillier et al., 1992: Lisziewicz et al., 1993). Such tran and makes use of common nucleic acid protecting and cou Scription units can be incorporated into a variety of vectors for pling groups, such as dimethoxytrity1 at the 5'-end, and phos introduction into mammalian cells, including but not phoramidites at the 3'-end. Average stepwise coupling yields restricted to, plasmid DNA vectors, viral DNA vectors (such are typically >98%. Hairpin ribozymes may be synthesized in as adenovirus or adeno-associated vectors), or viral RNA two parts and annealed to reconstruct an active ribozyme vectors (such as retroviral, semliki forest virus, sindbis virus (Chowrira and Burke, 1992). Ribozymes may be modified vectors). extensively to enhance stability by modification with 0226 Ribozymes of this invention may be used as diag nuclease resistant groups, for example, 2-amino. 2'-C-allyl, nostic tools to examine genetic drift and mutations within cell 2'-fluoro. 2'-O-methyl, 2-H (for a review see Usman and lines or cell types. They can also be used to assess levels of the Cedergren, 1992). Ribozymes may be purified by gel electro target RNA molecule. The close relationship between phoresis using general methods or by high pressure liquid ribozyme activity and the structure of the target RNA allows chromatography and resuspended in water. the detection of mutations in any region of the molecule 0223 Ribozyme activity can be optimized by altering the which alters the base-pairing and three-dimensional structure length of the ribozyme binding arms, or chemically synthe of the target RNA. By using multiple ribozymes described in sizing ribozymes with modifications that prevent their degra this invention, one may map nucleotide changes which are dation by serum ribonucleases (see e.g., Int. Pat. Appl. Publ. important to RNA structure and function in vitro, as well as in No. WO92/07065; Perreault et al., 1990; Pieken et al., 1991; cells and tissues. Cleavage of target RNAs with ribozymes Usman and Cedergren, 1992: Int. Pat. Appl. Publ. No. WO may be used to inhibit gene expression and define the role 93/15187: Int. Pat. Appl. Publ. No. WO 91/03162: Eur. Pat. (essentially) of specified gene products in particular cells or Appl. Publ. No. 921 10298.4; U.S. Pat. No. 5,334,711; and cell types. Int. Pat. Appl. Publ. No. WO94/13688, which describe vari ous chemical modifications that can be made to the Sugar 4.12 Recombinant Host Cells moieties of enzymatic RNA molecules), modifications which 0227. The nucleotide sequences of the subject invention enhance their efficacy in cells, and removal of stem II bases to may be introduced into a wide variety of microbial and shorten RNA synthesis times and reduce chemical require eukaryotic hosts. As hosts for recombinant expression of mentS. CryET70 polypeptides, of particular interest will be the 0224 Int. Pat. Appl. Publ. No. WO94/02595 describes the prokaryotes and the lower eukaryotes, such as fungi. Illustra general methods for delivery of enzymatic RNA molecules. tive prokaryotes, both Gram-negative and Gram-positive, Ribozymes may be administered to cells by a variety of include Enterobacteriaceae, such as Escherichia, Erwinia, methods known to those familiar to the art, including, but not Shigella, Salmonella, and Proteus, Bacillaceae, Rhizo restricted to, encapsulation in liposomes, by iontophoresis, or biceae, such as Rhizobium, Spirillaceae, Such as photobac by incorporation into other vehicles, such as hydrogels, terium, Zymomonas, Serratia, Aeromonas, Vibrio, Des cyclodextrins, biodegradable nanocapsules, and bioadhesive ulfo vibrio, Spirillum, Lactobacillaceae, microspheres. For some indications, ribozymes may be Pseudomonadaceae, such as Pseudomonas and Acetobacter, directly delivered ex vivo to cells or tissues with or without Azotobacteraceae, Actinomycetales, and Nitrobacteraceae. the aforementioned vehicles. Alternatively, the RNA/vehicle Among eukaryotes are fungi. Such as Phycomycetes and combination may be locally delivered by direct inhalation, by Ascomycetes, which includes yeast, such as Saccharomyces direct injection or by use of a catheter, infusion pump or stent. and Schizosaccharomyces; and Basidiomycetes yeast, such as Other routes of delivery include, but are not limited to, intra Rhodotorula, Aureobasidium, Sporobolomyces, and the like. vascular, intramuscular, Subcutaneous orjointinjection, aero 0228 Characteristics of particular interest in selecting a Sol inhalation, oral (tablet or pill form), topical, systemic, host cell for purposes of production include ease of introduc ocular, intraperitoneal and/or intrathecal delivery. More ing the genetic constructs of the present invention into the detailed descriptions of ribozyme delivery and administration host cell, availability of expression systems, efficiency of are provided in (Int. Pat. Appl. Publ. No. WO94/02595) and expression, stability of the gene of interest in the host, and the (Int. Pat Appl. Publ. No. WO 93/23569) which have been presence of auxiliary genetic capabilities. incorporated by reference herein. 0229. A large number of microorganisms known to inhabit 0225. Another means of accumulating high concentra the phylloplane (the surface of the plant leaves) and/or the tions of a ribozyme(s) within cells is to incorporate the rhizosphere (the soil Surrounding plant roots) of a wide vari US 2009/0087863 A1 Apr. 2, 2009 29 ety of important crops may also be desirable host cells for result in a screenable or selectable trait and/or which will manipulation, propagation, Storage, delivery and/or impart an improved phenotype to the transformed host cell. mutagenesis of the disclosed genetic constructs. These micro Alternatively, the nucleic acid constructs may contain anti organisms include bacteria, algae, and fungi. Of particular sense constructs, or ribozyme-encoding regions when deliv interest are microorganisms, such as bacteria, e.g., genera ery or introduction of Such nucleic acid constructs are desir Bacillus (including the species and Subspecies B. thuringien able. sis kurstaki HD-1, B. thuringiensis kurstaki HD-73, B. thur ingiensis Sotto, B. thuringiensis berliner; B. thuringiensis thu 4.14 Methods for Preparing Mutagenized ringiensis, B. thuringiensis tolworthi, B. thuringiensis Polynucleotides dendrolimus, B. thuringiensis alesti, B. thuringiensis galle 0233. In certain circumstances, it may be desirable to riae, B. thuringiensis aizawai, B. thuringiensis subtoxicus, B. modify or alter one or more nucleotides in one or more of the thuringiensis entomocidus, B. thuringiensis tenebrionis and polynucleotide sequences disclosed herein for the purpose of B. thuringiensissan diego); Pseudomonas, Erwinia, Serratia, altering or changing the insecticidal activity or insecticidal Klebsiella, Zanthomonas, Streptomyces, Rhizobium, specificity of the encoded polypeptide. In general, the means Rhodopseudomonas, Methylophilius, Agrobacterium, Aceto and methods for mutagenizing a DNA segment are well bacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconos known to those of skill in the art Modifications to such seg toC, and Alcaligenes; fungi, particularly yeast, e.g., genera ments may be made by random, or site-specific mutagenesis Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolo procedures. The polynucleotides may be modified by the myces, Rhodotorula, and Aureobasidium. Of particular inter addition, deletion, or Substitution of one or more nucleotides est are such phytosphere bacterial species as Pseudomonas from the sequence encoding the insecticidally-active syringae, Pseudomonas fluorescens, Serratia marcescens, polypeptide. Acetobacter xylinum, Agrobacterium tumefaciens, Rhodo 0234 Mutagenesis may be performed in accordance with bacter sphaeroides, Xanthomonas Campestris, Rhizobium any of the techniques known in the art such as and not limited melioti, Alcaligenes eutrophus, and Azotobacter vinlandii; to synthesizing an oligonucleotide having one or more muta and phytosphere yeast species such as Rhodotorula rubra, R. tions within the sequence of a particular region. In particular, glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. site-specific mutagenesis is a technique useful in the prepa difluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, ration of mutants, through specific mutagenesis of the under S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyvero lying DNA. The technique flirter provides a ready ability to myces veronae, and Aureobasidium pollulans. prepare and test sequence Variants, for example, incorporat 0230 Characteristics of particular interest in selecting a ing one or more of the foregoing considerations, by introduc host cell for purposes of production include ease of introduc ing one or more nucleotide sequence changes into the DNA. ing a selected genetic construct into the host, availability of Site-specific mutagenesis allows the production of mutants expression systems, efficiency of expression, stability of the through the use of specific oligonucleotide sequences which polynucleotide in the host, and the presence of auxiliary encode the DNA sequence of the desired mutation, as well as genetic capabilities. Other considerations include ease of for a Sufficient number of adjacent nucleotides, to provide a mulation and handling, economics, storage stability, and the primer sequence of sufficient size and sequence complexity to like. form a stable duplex on both sides of the deletion junction 4.13 Polynucleotide Sequences being traversed. Typically, a primer of about 17 to about 75 nucleotides or more in length is preferred, with about 10 to 0231 DNA compositions encoding the insecticidally-ac about 25 or more residues on both sides of the junction of the tive polypeptides of the present invention are particularly sequence being altered. preferred for delivery to recipient plant cells, in the generation 0235. In general, the technique of site-specific mutagen of pluripotent plant cells, and ultimately in the production of esis is well known in the art, as exemplified by various pub insect-resistant transgenic plants. For example, DNA seg lications. As will be appreciated, the technique typically ments in the form of vectors and plasmids, or linear DNA employs a phage vector which exists in both a single stranded fragments, in Some instances containing only the DNA ele and double stranded form. Typical vectors useful in site ment to be expressed in the plant cell, and the like, may be directed mutagenesis include vectors such as the M13 phage. employed. These phage are readily commercially available and their use 0232 Vectors, plasmids, phagemids, cosmids, viral vec is generally well known to those skilled in the art. Double tors, shuttle vectors, baculovirus vectors, BACs (bacterial Stranded plasmids are also routinely employed in site directed artificial chromosomes), YACs (yeast artificial chromo mutagenesis which eliminates the step of transferring the Somes) and DNA segments for use in transforming cells with gene of interest from a plasmid to a phage. a 6-endotoxin-encoding polynucleotide, will, of course, gen 0236. In general, site-directed mutagenesis in accordance erally comprise at least a first gene that encodes the polypep herewith is performed by first obtaining a single-stranded tide in accordance with SEQID NO:2, or a gene that encodes vector or melting apart of two strands of a double stranded a polypeptide that has at least about 80% or 85% or 90% or vector which includes within its sequence a DNA sequence 95% sequence identity to the amino acid sequence disclosed which encodes the desired promoter region or peptide. An in SEQID NO:2. These nucleic acid constructs may comprise oligonucleotide primer bearing the desired mutated sequence one or more genes which one desires to introduce into recipi is prepared, generally synthetically. This primer is then ent cells. These DNA constructs can include structures such annealed with the single-stranded vector, and Subjected to as promoters, enhancers, polylinkers, or regulatory genes as DNA polymerizing enzymes such as E. coli polymerase I desired. The DNA segment or gene chosen for cellular intro Klenow fragment, in order to complete the synthesis of the duction will often encode a polypeptide which will be mutation-bearing Strand. Thus, a heteroduplex is formed expressed in the resultant recombinant cells, such as will wherein one strand encodes the original non-mutated US 2009/0087863 A1 Apr. 2, 2009 30 sequence and the second strand bears the desired mutation. target to form reaction products, excess primers will bind to This heteroduplex vector is then used to transform or transfect the target and to the reaction products and the process is appropriate cells, such as E. coli cells, and clones are selected repeated. Preferably a reverse transcriptase PCRTM amplifi which include recombinant vectors bearing the mutated cation procedure may be performed in order to quantify the sequence arrangement. A genetic selection scheme was amount of mRNA amplified. Polymerase chain reaction devised by Kunkel et al., (1987) to enrich for clones incorpo methodologies are well known in the art. rating the mutagenic oligonucleotide. Alternatively, the use of 0240 Another method for amplification is the ligase chain PCRTM with commercially available thermostable enzymes reaction (referred to as LCR), disclosed in Eur. Pat. Appl. Such as Taq polymerase may be used to incorporate a Publ. No. 320,308, incorporated herein by reference in its mutagenic oligonucleotide primer into an amplified DNA entirety. In LCR, two complementary probe pairs are pre fragment that can then be cloned into an appropriate cloning pared, and in the presence of the target sequence, each pair or expression vector. The PCRTM-mediated mutagenesis pro will bind to opposite complementary Strands of the target cedures of Tomic et al., (1990) and Upender et al., (1995) Such that they abut. In the presence of a ligase, the two probe provide two examples of such protocols. A PCRTM employing pairs will link to form a single unit. By temperature cycling, a thermostable ligase in addition to a thermostable poly as in PCRTM, bound ligated units dissociate from the target merase may also be used to incorporate a phosphorylated and then serve as “target sequences' for ligation of excess mutagenic oligonucleotide into an amplified DNA fragment probe pairs. U.S. Pat. No. 4,883,750, incorporated herein by that may then be cloned into an appropriate cloning or expres reference in its entirety, describes an alternative method of sion vector. The mutagenesis procedure described by Michael amplification similar to LCR for binding probe pairs to a (1994) provides an example of one such protocol. target Sequence. 0237. The preparation of sequence variants of the selected 0241 Qbeta Replicase, described in Intl. Pat. Appl. Publ. Ö-endotoxin-encoding DNA segments using site-directed No. PCT/US87/00880, incorporated herein by reference in its mutagenesis is provided as a means of producing potentially entirety, may also be used as still another amplification useful species and is not meant to be limiting as there are other method in the present invention. In this method, a replicative ways in which sequence variants of DNA sequences may be sequence of RNA which has a region complementary to that obtained. For example, recombinant vectors encoding the of a target is added to a sample in the presence of an RNA desired sequence may be treated with mutagenic agents. Such polymerase. The polymerase will copy the replicative as hydroxylamine, to obtain sequence variants. sequence which can then be detected. 0238. As used herein, the term "oligonucleotide directed 0242 An isothermal amplification method, in which mutagenesis procedure” refers to template-dependent pro restriction endonucleases and ligases are used to achieve the cesses and vector-mediated propagation which result in an amplification of target molecules that contain nucleotide 5'- increase in the concentration of a specific nucleic acid mol C-thiotriphosphates in one Strand of a restriction site ecule relative to its initial concentration, or in an increase in (Walker et al., 1992, incorporated herein by reference in its the concentration of a detectable signal. Such as amplifica entirety), may also be useful in the amplification of nucleic tion. As used herein, the term "oligonucleotide directed acids in the present invention. mutagenesis procedure” also is intended to refer to a process 0243 Strand Displacement Amplification (SDA) is that involves the template-dependent extension of a primer another method of carrying out isothermal amplification of molecule. The term template-dependent process refers to nucleic acids which involves multiple rounds of strand dis nucleic acid synthesis of an RNA or a DNA molecule wherein placement and synthesis, i.e. nick translation. A similar the sequence of the newly synthesized strand of nucleic acid method, called Repair Chain Reaction (RCR) is another is dictated by the well-known rules of complementary base method of amplification which may be useful in the present pairing (Watson, 1987). Typically, vector mediated method invention and is involves annealing several probes throughout ologies involve the introduction of the nucleic acid fragment a region targeted for amplification, followed by a repair reac into a DNA or RNA vector, the clonal amplification of the tion in which only two of the four bases are present. The other vector, and the recovery of the amplified nucleic acid frag two bases can be added as biotinylated derivatives for easy ment. Examples of such methodologies are provided by U.S. detection. A similar approach is used in SDA. Pat. No. 4,237,224, specifically incorporated herein by refer 0244 Still other amplification methods described in Great ence in its entirety. Britain Pat Appl. No. 2 202328, and in Intl. Pat. Appl. Publ. 0239. A number of template dependent processes are No. PCT/US89/01025, each of which is incorporated herein available to amplify the target sequences of interest present in by reference in its entirety, may be used in accordance with a sample. One of the best known amplification methods is the the present invention. In the former application, “modified polymerase chain reaction (PCRTM) which is described in primers are used in a PCRTM like, template and enzyme detail in U.S. Pat. Nos. 4,683,195, 4,683.202 and 4,800,159, dependent synthesis. The primers may be modified by label each of which is incorporated herein by reference in its ing with a capture moiety (e.g., biotin) and/or a detector entirety. Briefly, in PCRTM, two primer sequences are pre moiety (e.g., enzyme). In the latter application, an excess of pared which are complementary to regions on opposite labeled probes are added to a sample. In the presence of the complementary strands of the target sequence. An excess of target sequence, the probe binds and is cleaved catalytically. deoxynucleoside triphosphates are added to a reaction mix After cleavage, the target sequence is released intact to be ture along with a DNA polymerase (e.g., Taq polymerase). If bound by excess probe. Cleavage of the labeled probe signals the target sequence is present in a sample, the primers will the presence of the target sequence. bind to the target and the polymerase will cause the primers to 0245. Other nucleic acid amplification procedures include be extended along the target sequence by adding on nucle transcription-based amplification systems (TAS) (Kwoh et otides. By raising and lowering the temperature of the reac al., 1989; Intl. Pat Appl. PublNo. WO88/10315, incorporated tion mixture, the extended primers will dissociate from the herein by reference in its entirety), including nucleic acid US 2009/0087863 A1 Apr. 2, 2009 sequence based amplification (NASBA) and 3SR. In tive of the amount of protein being produced in the trans NASBA, the nucleic acids can be prepared for amplification formed host cell. This is often due to post-transcriptional by Standard phenol/chloroform extraction, heat denaturation processes, such as splicing, polyadenylation, appropriate of a sample, treatment with lysis buffer and minispin columns translation initiation, and RNA stability, that affect the ability for isolation of DNA and RNA or guanidinium chloride of a transcript to produce protein Such factors may also affect extraction of RNA. These amplification techniques involve the stability and amount of mRNA produced from the given annealing a primer which has crystal protein-specific transgene. As such, it is often desirable to alter the post sequences. Following polymerization, DNA/RNA Hybrids translational events through particular molecular biology are digested with RNase H while double stranded DNA mol ecules are heat denatured again. In either case the single techniques. The inventors contemplate that in certain stranded DNA is made fully double stranded by addition of instances it may be desirable to alter the transcription and/or second crystal protein-specific primer, followed by polymer expression of the polypeptide-encoding nucleic acid con ization. The double stranded DNA molecules are then multi structs of the present invention to increase, decrease, or oth ply transcribed by a polymerase such as T7 or SP6. In an erwise regulate or control these constructs in particular host isothermal cyclic reaction, the RNAs are reverse transcribed cells and/or transgenic plants. into double stranded DNA, and transcribed once against with a polymerase such as T7 or SP6. The resulting products, 4.15.1 Efficient Initiation of Protein Translation whether truncated or complete, indicate crystal protein-spe (0250. The 5'-untranslated leader (5-UTL) sequence of cific sequences. eukaryotic mRNA plays a major role in translational effi 0246 Eur. Pat. Appl. Publ. No. 329,822, incorporated ciency. Many early chimeric transgenes using a viral pro herein by reference in its entirety, disclose a nucleic acid amplification process involving cyclically synthesizing moter used an arbitrary length of viral sequence after the single-stranded RNA (“ssRNA), ssDNA, and double transcription initiation site and fused this to the AUG of the stranded DNA (dsDNA), which may be used in accordance coding region. More recently studies have shown that the with the present invention. The ssRNA is a first template for 5'-UTL sequence and the sequences directly surrounding the a first primer oligonucleotide, which is elongated by reverse AUG can have a large effect in translational efficiency in host transcriptase (RNA-dependent DNA polymerase). The RNA cells and particularly certain plant species and that this effect is then removed from resulting DNA:RNA duplex by the can be different depending on the particular cells or tissues in action of ribonuclease H(RNase H, an RNase specific for which the message is expressed. RNA in a duplex with either DNA or RNA). The resultant 0251. In most eukaryotic mRNAs, the point of transla ssDNA is a second template for a second primer, which also tional initiation occurs at the AUG codon closest to the 5' cap includes the sequences of an RNA polymerase promoter (ex of the transcript. Comparison of plant mRNA sequences and emplified by T7 RNA polymerase) 5' to its homology to its site directed mutagenesis experiments have demonstrated the template. This primer is then extended by DNA polymerase existence of a consensus sequence Surrounding the initiation (exemplified by the large “Klenow' fragment of E. coli DNA codon in plants, 5'-UAAACAAUGGCU-3' (SEQ ID NO:4) polymerase I), resulting as a double-stranded DNA (“ds (Joshi, 1987; Lutcke et al., 1987). However, consensus DNA) molecule, having a sequence identical to that of the sequences will be apparent amongst individual plant species. original RNA between the primers and having additionally, at For example, a compilation of sequences Surrounding the one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA initiation codon from 85 maize genes yields a consensus of copies of the DNA. These copies can then re-enter the cycle 5'-(C/G)AUGGCG-3 (Luehrsenet al., 1994). In tobacco pro leading to very swift amplification. With proper choice of toplasts, transgenes encoding B-glucuronidase (GUS) and enzymes, this amplification can be done isothermally without bacterial chitinase showed a 4-fold and an 8-fold increase in addition of enzymes at each cycle. Because of the cyclical expression, respectively, when the native sequences of these nature of this process, the starting sequence can be chosen to genes were changed to encode 5'-ACCAUGG-3 (Gallie et al. be in the form of either DNA or RNA. 1987b; Jones et al., 1988). 0247 PCT Intl. Pat. Appl. Publ. No. WO 89/06700, incor 0252. When producing chimeric transgenes (i.e. trans porated herein by reference in its entirety, disclose a nucleic genes comprising DNA segments from different sources acid sequence amplification Scheme based on the hybridiza operably linked together), often the 5'-UTL of plant viruses tion of a promoter/primer sequence to a target single-stranded are used. The alfalfa mosaic virus (AMV) coat protein and DNA (“ssDNA) followed by transcription of many RNA brome mosaic virus (BMV) coat protein 5'-UTLs have been copies of the sequence. This scheme is not cyclic; i.e. new shown to enhance mRNA translation 8-fold in electroporated templates are not produced from the resultant RNA tran tobacco protoplasts (Gallie et al., 1987a; 1987b). A 67-nucle scripts. Other amplification methods include “RACE (Fro otide derivative (S2) of the 5'-UTL of tobacco mosaic virus hman, 1990), and “one-sided PCRTM” (Ohara, et al. 1989) RNA (TMV) fused to the chloramphenicol acetyltransferase which are well-known to those of skill in the art. (CAT) gene and GUS gene has been shown to enhance trans 0248 Methods based on ligation of two (or more) oligo nucleotides in the presence of nucleic acid having the lation of reporter genes in vitro (Gallie et al., 1987a; 1987b; sequence of the resulting “di-oligonucleotide', thereby Sleat et al., 1987; Sleat et al., 1988). Electroporation of amplifying the di-oligonucleotide (Wu and Dean, 1996, tobacco mesophyll protoplasts with transcripts containing the incorporated herein by reference in its entirety), may also be TMV leader fused to reporter genes CAT, GUS, and LUC used in the amplification of polynucleotide sequences of the produced a 33-, 21-, and 36-fold level of enhancement, present invention. respectively (Gallie et al., 1987a; 1987b; Gallie et al., 1991). Also in tobacco, an 83-nt 5'-UTL of potato virus X RNA was 4.15 Post-Transcriptional Events Affecting shown to enhance expression of the neomycin phosphotrans Expression of Transgenes in Plants ferese II (NptII) 4-fold (Poogin and Skryabin, 1992). 0249. In many instances, the level of transcription of a (0253) The effect of a 5"-UTL may be different depending particular transgene in a given host cell is not always indica on the plant, particularly between dicots and monocots. The US 2009/0087863 A1 Apr. 2, 2009 32

TMV 5'-UTL has been shown to be more effective in tobacco derived form the gene. Significant experience in using Syn protoplasts (Gallie et al., 1989) than in maize protoplasts thetic genes to increase expression of a desired protein has (Gallie and Young, 1994). Also, the 5'-UTLs from TMV-2 been achieved in the expression of Bacillus thuringiensis in (Gallie et al., 1988), AMV-coat (Gehrke et al., 1983; Jobling plants. Native B. thuringiensis genes are often expressed only and Gehrke, 1987), TMV-coat (Goelet et al., 1982), and at low levels in dicots and sometimes not at all in many BMV-coat (French et al., 1986) worked poorly in maize and species of monocots (Koziel et al., 1996). Codon usage in the inhibited expression of a luciferase gene in maize relative to native genes is considerably different from that found in its native leader (Koziel et al., 1996). However, the 5'-UTLs typical plant genes, which have a higher G+C content. Strat from the cauliflower mosaic virus (CaMV)35S transcript and egies to increase expression of these genes in plants generally the maize genes glutelin (Boronat et al., 1986), PEP-carboxy alter the overall G+C content of the genes. For example, lase (Hudspeth and Grula, 1989) and ribulose biphosphate synthetic B. thuringiensis crystal-protein encoding genes carboxylase showed a considerable increase in expression of have resulted in significant improvements in expression of the luciferase gene in maize relative to its native leader (Ko these endotoxins in various crops including cotton (Perlaket Ziel et al., 1996). al., 1990: Wilson et al., 1992), tomato (Perlak et al., 1991), 0254 These 5'-UTLs had different effects in tobacco. In potato (Perlak et al., 1993), rice (Cheng et al., 1998), and contrast to maize, the TMV S.25'-UTL and the AMV coat maize Koziel et al., 1993). protein 5'-UTL enhanced expression in tobacco, whereas the 0258. In a similar fashion the inventors contemplate that glutelin, maize PEP-carboxylase and maize ribulose-1,5-bis the genetic constructs of the present invention, because they phosphate carboxylase 5'-UTLs did not show enhancement contain one or more genes of bacterial origin, may in certain relative to the native luciferase 5'-UTL (Koziel et al., 1996). circumstances be altered to increase the expression of these Only the CaMV 35S 5'-UTL enhanced luciferase expression prokaryotic-derived genes in particular eukaryotic host cells in both maize and tobacco (Koziel et al., 1996). Furthermore, and/or transgenic plants which comprise Such constructs. the TMV and BMV coat protein 5'-UTLs were inhibitory in Using molecular biology techniques which are well-knownto both maize and tobacco protoplasts (Koziel et al., 1996). those of skill in the art, one may alter the coding or noncoding sequences of the particular CryET70-encoding gene 4.15.2 Use of Introns to Increase Expression sequences to optimize or facilitate its expression in trans formed plant cells at levels Suitable for preventing or reducing 0255 Including one or more introns in the transcribed insect infestation or attack in Such transgenic plants. portion of a gene has been found to increase heterologous gene expression in a variety of plant systems (Callis et al. 4.15.4 Chloroplast Sequestering and Targeting 1987: Maas et al., 1991; Mascerenhas et al., 1990; McElroy et al., 1990; Vasil et al., 1989), although not all introns produce 0259 Another approach for increasing expression of A+T a stimulatory effect and the degree of stimulation varies. The rich genes in plants has been demonstrated in tobacco chlo enhancing effect of introns appears to be more apparent in roplast transformation. High levels of expression of an monocots than indicots. Tanaka et al., (1990) has shown that unmodified Bacillus thuringiensis crystal protein-encoding use of the catalase intron 1 isolated from castor beans genes in tobacco has been reported by McBride et al. (1995). increases gene expression in rice. Likewise, the first intron of 0260 Additionally, methods of targeting proteins to the the alcohol dehydrogenase 1 (Adh1) has been shown to chloroplast have been developed. This technique, utilizing increase expression of a genomic clone of Adh1 comprising the pea chloroplast transit peptide, has been used to target the the endogenous promoter in transformed maize cells (Callis enzymes of the polyhydroxybutyrate synthesis pathway to et al., 1987: Dennis et al., 1984). Other introns that are also the chloroplast Nawrath et al., 1994). Also, this technique able to increase expression of transgenes which contain them negated the necessity of modification of the coding region include the introns 2 and 6 of Adh1 (Luehrsen and Walbot, other than to add an appropriate targeting sequence. 1991), the catalase intron (Tanaka et al., 1990), intron 1 of the 0261 U.S. Pat. No. 5,576,198 (specifically incorporated maize bronze 1 gene (Callis et al., 1987), the maize sucrose herein by reference) discloses compositions and methods synthase intron 1 (Vasil et al., 1989), intron 3 of the rice actin useful for genetic engineering of plant cells to provide a gene (Luehrsen and Walbot, 1991), rice actin intron 1 (McEl method of controlling the timing or tissue pattern of expres roy et al., 1990), and the maize ubiquitin exon 1 (Christensen sion of foreign DNA sequences inserted into the plant plastid et al., 1992). genome. Constructs include those for nuclear transformation 0256 Generally, to achieve optimal expression, the which provide for expression of a viral single subunit RNA selected intron(s) should be present in the 5' transcriptional polymerase in plant tissues, and targeting of the expressed unit in the correct orientation with respect to the splice junc polymerase protein into plant cell plastids. Also included are tion sequences (Callis et al., 1987; Maas et al., 1991; Mas plastid expression constructs comprising a viral gene pro cerenhas et al., 1990; Oard et al., 1989: Tanaka et al., 1990; moter region which is specific to the RNA polymerase Vasil et al., 1989). Intron 9 of Adh1 has been shown to expressed from the nuclear expression constructs described increase expression of a heterologous gene when placed 3' (or above and a heterologous gene of interest to be expressed in downstream of) the gene of interest (Callis et al., 1987). the transformed plastid cells. 4.15.3 Use of Synthetic Genes to Increase Expression of 4.15.5 Effects of 3' Regions on Transgene Expression Heterologous Genes in Plants 0262 The 3'-end regions of transgenes have been found to 0257. When introducing a prokaryotic gene into a eukary have a large effect on transgene expression in plants (Ingel otic host, or when expressing a eukaryotic gene in a non brecht et al., 1989). In this study, different 3' ends were oper native host, the sequence of the gene must often be altered or ably linked to the neomycin phosphotsferase II (NptII) modified to allow efficient translation of the transcript(s) reporter gene and expressed in transgenic tobacco. The dif US 2009/0087863 A1 Apr. 2, 2009

ferent 3' ends used were obtained from the octopine synthase gene, the 2S seed protein from Arabidopsis, the Small Subunit TABLE 7-continued of rbcS from Arabidopsis, extension form carrot, and chal cone synthase from Antirrhinum. In stable tobacco transfor TISSUE SPECIFICPLANT PROMOTERS mants, there was about a 60-fold difference between the best Tissue Specifi expressing construct (small subunit rbcS 3' end) and the c Promoter Tissue(s) Reference lowest expressing construct (shalcone synthase 3' end). Sh phloem tissue Graham et al., 1997 CMd endosperm Grosset et al., 1997 Bnm1 pollen Treacy et al., 1997 TABLE 6 rice tungro phloem Yin et al., 1997a: 1997b bacilliform virus PLANT PROMOTERS S2-RNase pollen Ficker et al., 1998 LeB4 Seeds Baumlein et al., 1991 Promoter Reference? gf-2.8 Seeds; seedlings Berna and Bernier, 1997 Viral Each reference is specifically incorporated herein by reference in its entirety. Figwort Mosaic Virus (FMV) U.S. Pat. No. 5,378,619 Cauliflower Mosaic Virus (CaMV) U.S. Pat. No. 5,530,196 0263. The ability to express genes in a tissue specific man U.S. Pat. No. 5,097,025 U.S. Pat. No. 5,110,732 ner in plants has led to the production of male and female Plant sterile plants. Generally, the production of male sterile plants involves the use of anther-specific promoters operably linked Elongation Factor U.S. Pat. No. 5,177,011 Tomato Polygalacturonase U.S. Pat. No. 5,442,052 to heterologous genes that disrupt pollen formation (U.S. Pat. Arabidopsis Histone H4 U.S. Pat. No. 5,491,288 Nos. 5,689,051; 5.689,049; 5,659,124, each specifically Phaseolin U.S. Pat. No. 5,504,200 incorporated herein by reference). U.S. Pat. No. 5,633,441 Group 2 U.S. Pat. No. 5,608,144 (specifically incorporated herein by reference) discloses a Ubiquitin U.S. Pat. No. 5,614,399 method of producing plants with female genetic sterility. The P119 U.S. Pat. No. 5,633,440 C-amylase U.S. Pat. No. 5,712,112 method comprises the use of style-cell, Stigma-cell, or style Viral enhancer/Plant promoter and Stigma-cell specific promoters that express polypeptides that, when produced in the cells of the plant, kills or signifi CaMV 35Senhancertmannopine U.S. Pat. No. 5,106,739 cantly disturbs the metabolism, functioning or development synthase promoter of the cells. Each reference is specifically incorporated herein by reference in its entirety. TABLE 8

INDUCIBLE PLANT PROMOTERS TABLE 7 Promoter Reference TISSUE SPECIFICPLANT PROMOTERS heat shock promoter U.S. Pat. No. 5,447,858 Em U.S. Pat. No. 5,139,954 Tissue Specifi Adh1 Kyozoka et al., 1991 c Promoter Tissue(s) Reference? HMG2 U.S. Pat. No. 5,689,056 Blec epidermis U.S. Pat. No. 5,646,333 cinnamyl alcohol dehydrogenase U.S. Pat. No. 5,633,439 malate synthase seeds; seedlings U.S. Pat. No. 5,689,040 asparagine synthase U.S. Pat. No. 5,595,896 isocitrate lyase Seeds; seedlings U.S. Pat. No. 5,689,040 GST-II-27 U.S. Pat. No. 5,589,614 patatin tuber U.S. Pat. No. 5,436,393 ZRP2 root U.S. Pat. No. 5,633,363 Each reference is specifically incorporated herein by reference in its ZRP2(2.0) root U.S. Pat. No. 5,633,363 entirety. ZRP2(1.0) root U.S. Pat. No. 5,633,363 RB7 root U.S. Pat. No. 5,459.252 root U.S. Pat. No. 5,401,836 4.16 Peptide Nucleic Acid Compositions ruit U.S. Pat. No. 4,943,674 meristem U.S. Pat. No. 5,589,583 0264. In certain embodiments, the inventors contemplate guard cell U.S. Pat. No. 5,538,879 the use of peptide nucleic acids (PNAs) in the practice of the Stannen U.S. Pat. No. 5,589,610 methods of the invention. PNA is a DNA mimic in which the SodA1 pollen; middle layer; Van Camp et al., 1996 stomium of anthers nucleobases are attached to a pseudopeptide backbone (Good SodA2 vasular bundles; stomata; Van Camp et al., 1996 and Nielsen, 1997). PNAS are able to be utilized in a number axillary buds; pericycle; methods that traditionally have used RNAS or DNAs. Often stomium; pollen PNA sequences perform better in techniques than the corre CHS15 flowers; root tips Faktor et al., 1996 Psam-1 phloem tissue; cortex; Vander et al., 1996 sponding RNA or DNA sequences and have utilities that are root tips not inherent to RNA or DNA. An excellent review of PNA ACT11 elongating tissues and Huang et al., 1997 including methods of making, characteristics of, and methods organs; pollen; ovules of using, is provided by Corey (1997) and is incorporated ZnOBS pollen; endosperm Russell and Fromm, 1997 Zn227 endosperm Russell and Fromm, 1997 herein by reference. OSAGP endosperm Russell and Fromm, 1997 OSGT1 endosperm Russell and Fromm, 1997 4.16.1 Methods of Making PNAS RolC phloem tissue; bundle Graham et al., 1997 sheath; vascular 0265 According to Corey, PNAS have 2-aminoethyl-gly parenchyma. cine linkages replacing the normal phosphodiester backbone of DNA (Nielsen et al., 1991; Hanvey et al. 1992: Hyrup and US 2009/0087863 A1 Apr. 2, 2009 34

Nielsen, 1996: Neilsen, 1996). This chemistry has three in PNA-DNA or PNA-RNA duplexes increases the melting important consequences: firstly, in contrast to DNA or phos temperature (T) and reduces the dependence of T on the phorothioate oligonucleotides, PNAS are neutral molecules: concentration of mono- or divalent cations (Nielsen et al., secondly, PNAS are achiral, which avoids the need to develop 1991). The enhanced rate and affinity of hybridization are a stereoselective synthesis; and thirdly, PNA synthesis uses significant because they are responsible for the Surprising standard Boo (Dueholm et al., 1994) or Fmoc (Thomson et al. ability of PNAS to perform strand invasion of complementary 1995) protocols for solid-phase peptide synthesis, although sequences within relaxed double-stranded DNA. In addition, other methods, including a modified Merrifield method, have the efficient hybridization at inverted repeats Suggests that been used (Christensen et al., 1995). PNAs can recognize secondary structure effectively within 0266 PNA monomers or ready-made oligomers are com double-stranded DNA. Enhanced recognition also occurs mercially available from PerSeptive Biosystems with PNAS immobilized on surfaces, and Wang et al., have (Framigham, Mass., USA). PNA syntheses by either Boc or shown that support-bound PNAs can be used to detect hybrid Fmoc protocols are straightforward using manual or auto ization events (Wang et al., 1996). mated protocols (Norton et al., 1995). The manual protocol 0271. One might expect that tight binding of PNAS to lends itself to the production of chemically modified PNAS or complementary sequences would also increase binding to the simultaneous synthesis of families of closely related similar (but not identical) sequences, reducing the sequence PNAS. specificity of PNA recognition. As with DNA hybridization, 0267 As with peptide synthesis, the success of a particular however, selective recognition can be achieved by balancing PNA synthesis will depend on the properties of the chosen oligomer length and incubation temperature. Moreover, sequence. For example, while in theory PNAs can incorporate selective hybridization of PNAS is encouraged by PNA-DNA any combination of nucleotide bases, the presence of adjacent hybridization being less tolerant of base mismatches than purines can lead to deletions of one or more residues in the DNA-DNA hybridization. For example, a single mismatch product. In expectation of this difficulty, it is Suggested that, within a 16 bp PNA-DNA duplex can reduce the T by up to in producing PNAS with adjacent purines, one should repeat 15°C. (Egholm et al., 1993). This high level of discrimination the coupling of residues likely to be added inefficiently. This has allowed the development of several PNA-based strategies should be followed by the purification of PNAS by reverse for the analysis of point mutations (Wang et al., 1996; Carls phase high-pressure liquid chromatography (Norton et al., son et al., 1996: Thiede et al., 1996; Webb and Hurskainen, 1995) providing yields ad purity of product similar to those 1996: Perry-O'Keefe et al., 1996). observed during the synthesis of peptides. 0272 High-affinity binding provides clear advantages for 0268. Further discussed by Corey are desired modifica molecular recognition and the development of new applica tions of PNAs for given applications. Modifications can be tions for PNAS. For example, 11-13 nucleotide PNAS inhibit accomplished by coupling amino acids during Solid-phase the activity of telomerase, a ribonucleo-protein that extends synthesis or by attaching compounds that contain a carboxy telomere ends using an essential RNA template, while the lic acid group to the exposed N-terminal amine. Alternatively, analogous DNA oligomers do not (Norton et al., 1996). PNAs can be modified after synthesis by coupling to an 0273 Neutral PNAS are more hydrophobic than analo introduced lysine or cysteine. The ease with which PNAS can gous DNA oligomers, and this can lead to difficulty solubi be modified facilitates optimization for better solubility or for lizing them at neutral pH, especially if the PNAs have a high specific functional requirements. Once synthesized, the iden purine content or if they have the potential to form secondary tity of PNAS and their derivatives can be confirmed by mass structures. Their solubility can be enhanced by attaching one spectrometry. Several studies have made and utilized modi or more positive charges to the PNA termini (Nielsen et al., fications of PNAs (Norton et al., 1995; Haaima et al., 1996: 1991). Stetsenko et al., 1996: Petersen et al., 1995: Ulmann et al., 1996: Koch et al., 1995: Orum et al., 1995; Footer et al., 1996; 4.16.3 Applications of PNAS Griffith et al., 1995; Kremsky et al., 1996: Pardridge et al., 0274 Findings by Alfrey and colleagues suggest that 1995; Boffa et al., 1995; Landsdorp et al., 1996: Gambacorti Strand invasion will occur spontaneously at sequences within Passerini et al., 1996; Armitage et al., 1997: Seeger et al., chromosomal DNA (Boffa et al., 1995; Boffa et al., 1996). 1997: Rusckowski et al., 1997). U.S. Pat. No. 5,700,922 These studies targeted PNAS to triplet repeats of the nucle discusses PNA-DNA-PNA chimeric molecules and their uses otides CAG and used this recognition to purity transcription in diagnostics, modulating protein in organisms, and treat ally active DNA (Boffa et al., 1995) and to inhibit transcrip ment of conditions susceptible to therapeutics. tion (Boffa et al., 1996). This result suggests that if PNAS can be delivered within cells then they will have the potential to be 4.16.2 Physical Properties of PNAS general sequence-specific regulators of gene expression. 0269. In contrast to DNA and RNA, which contain nega Studies and reviews concerning the use of PNAS as antisense tively charged linkages, the PNA backbone is neutral. In spite and anti-gene agents include Nielsen et al., (1993b), Hanvey of this dramatic alteration, PNAS recognize complementary et al. (1992), and Good and Nielsen (1997). Koppelhus et al. DNA and RNA by Watson-Crick paring (Egholm et al., (1997) have used PNAS to inhibit HIV-1 inverse transcription, 1993), validating the initial modeling by Nielsen et al., showing that PNAS may be used for antiviral therapies. (1991). PNAS lack 3' to 5' polarity and can bind in either 0275 Methods of characterizing the antisense binding parallel or antiparallel fashion, with the antiparallel mode properties of PNAS are discussed in Rose (1993) and Jensen being preferred (Egholm et al., 1993). et al., (1997). Rose uses capillary gel electrophoresis to deter (0270 Hybridization of DNA oligonucleotides to DNA mine binding of PNAS to their complementary oligonucle and RNA is destabilized by electrostatic repulsion between otide, measuring the relative binding kinetics and Stoichiom the negatively charged phosphate backbones of the comple etry. Similar types of measurements were made by Jensen et mentary Strands. By contrast, the absence of charge repulsion al, using BIAcoreTM technology. 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(0276. Other applications of PNAS include use in DNA 0281 mAbs may be readily prepared through use of well strand invasion (Nielsen et al., 1991), antisense inhibition known techniques, such as those exemplified in U.S. Pat. No. (Hanvey et al., 1992), mutational analysis (Orum et al., 1993), 4,196,265, incorporated herein by reference. Typically, this enhancers of transcription (Mollegaard et al., 1994), nucleic technique involves immunizing a suitable animal with a acid purification (Orum et al., 1995), isolation of transcrip selected immunogen composition, e.g., a purified or partially tionally active genes (Boffa et al., 1995), blocking of tran purified crystal protein, polypeptide or peptide. The immu scription factor binding (Vickers et al., 1995), genome cleav nizing composition is administered in a manner effective to age (Veselkov et al., 1996), biosensors (Wang et al., 1996), in stimulate antibody producing cells. Rodents such as mice and situ hybridization (Thisted et al., 1996), and in a alternative: to rats are preferred animals, however, the use of rabbit, sheep, Southern blotting (Perry-O'Keefe, 1996). or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are 4.17 Antibody Compositions and Methods of preferred, with the BALB/c mouse being most preferred as Making this is most routinely used and generally gives a higher per centage of stable fusions. 0277. In particular embodiments, the inventors contem 0282 Following immunization, somatic cells with the plate the use of antibodies, either monoclonal or polyclonal potential for producing antibodies, specifically B lympho which bind to one or more of the polypeptides disclosed cytes (B cells), are selected for use in the mAb generating herein. Means for preparing and characterizing antibodies are protocol. These cells may be obtained from biopsied spleens, well known in the art (See, e.g., Harlow and Lane, 1988: tonsils or lymph nodes, or from a peripheral blood sample. incorporated herein by reference). The methods for generat Spleen cells and peripheral blood cells are preferred, the ing monoclonal antibodies (mAbs) generally begin along the former because they are a rich Source of antibody-producing same lines as those for preparing polyclonal antibodies. cells that are in the dividing plasmablast stage, and the latter Briefly, a polyclonal antibody is prepared by immunizing an because peripheral blood is easily accessible. Often, a panel animal with an immunogenic composition in accordance with of animals will have been immunized and the spleen of ani the present invention and collecting antisera from that immu mal with the highest antibody titer will be removed and the nized animal. A wide range of animal species can be used for spleen lymphocytes obtained by homogenizing the spleen the production of antisera. Typically the animal used for with a syringe. Typically, a spleen from an immunized mouse production of anti-antisera is a rabbit, a mouse, a rat, a ham contains approximately 5x107 to 2x10 lymphocytes. ster, a guinea pig or a goat. Because of the relatively large (0283. The antibody-producing B lymphocytes from the blood volume of rabbits, a rabbit is a preferred choice for immunized animal are then fused with cells of an immortal production of polyclonal antibodies. myeloma cell, generally one of the same species as the animal 0278. As is well known in the art, a given composition may that was immunized. Myeloma cell lines suited for use in vary in its immunogenicity. It is often necessary therefore to hybridoma-producing fusion procedures preferably are non boost the host immune system, as may be achieved by cou antibody-producing, have high fusion efficiency, and enzyme pling a peptide or polypeptide immunogen to a carrier. Exem deficiencies that render them incapable of growing in certain plary and preferred carriers are keyhole limpet hemocyanin selective media which support the growth of only the desired (KLH) and bovine serum albumin (BSA). Other albumins fused cells (hybridomas). Such as ovalbumin, mouse serum albumin or rabbit serum 0284 Any one of a number of myeloma cells may be used, albumin can also be used as carriers. Means for conjugating a as are known to those of skill in the art (Goding, pp. 65-66, polypeptide to a carrier protein are well known in the art and 1986; Campbell, pp. 75-83, 1984). For example, where the include glutaraldehyde, m-maleimidobencoyl-N-hydrox immunized animal is a mouse, one may use P3-X63/Ag8, y succinimide ester, carbodiimide and bis-biazotized benzi X63-Ag8.653, NS1/1. Ag 4 1, Sp210-Ag14, FO, NSO/U, dine. MPC-11, MPC11-X45-GTG 1.7 and S194/5XXO Bul; for 0279. As is also well known in the art, the immunogenicity rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and of a particular immunogen composition can be enhanced by 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and the use of non-specific stimulators of the immune response, UC729-6 are all useful in connection with humancell fusions. known as adjuvants. Exemplary and preferred adjuvants (0285. One preferred murine myeloma cell is the NS-1 include complete Freund's adjuvant (a non-specific stimula myeloma cell line (also termed P3-NS-1-Ag4-1), which is tor of the immune response containing killed Mycobacterium readily available from the NIGMS Human Genetic Mutant tuberculosis), incomplete Freund's adjuvants and aluminum Cell Repository by requesting cell line repository number hydroxide adjuvant. GM3573. Another mouse myeloma cell line that may be used 0280. The amount of immunogen composition used in the is the 8-azguanine-resistant mouse murine myeloma SP2/0 production of polyclonal antibodies varies upon the nature of non-producer cell line. the immunogen as well as the animal used for immunization. 0286 Methods for generating hybrids of antibody-pro A variety of routes can be used to administer the immunogen ducing spleen or lymph node cells and myeloma cells usually (Subcutaneous, intramuscular, intradermal, intravenous and comprise mixing Somatic cells with myeloma cells in a 2:1 intraperitoneal). The production of polyclonal antibodies ratio, though the ratio may vary from about 20:1 to about 1:1, may be monitored by sampling blood of the immunized ani respectively, in the presence of an agent or agents (chemical mal at various points following immunization. A second, or electrical) that promote the fusion of cell membranes. booster, injection may also be given. The process of boosting Fusion methods using Sendai virus have been described and tittering is repeated until a suitable titer is achieved. When (Kohler and Milstein, 1975; 1976), and those using polyeth a desired level of immunogenicity is obtained, the immunized ylene glycol (PEG), such as 37% (vol.?vol.) PEG, (Gefter et animal can be bled and the serum isolated and stored, and/or al., 1977). The use of electrically induced fusion methods is the animal can be used to generate mabs. also appropriate (Goding, 1986, pp. 71-74). US 2009/0087863 A1 Apr. 2, 2009 36

0287 Fusion procedures usually produce viable hybrids at blocking of nonspecific adsorption sites on the immobilizing low frequencies, about 1x10 to 1x10. However, this does Surface and thus reduces the background caused by nonspe not pose a problem, as the viable, fused hybrids are differen cific binding of antisera onto the Surface. tiated from the parental, unfused cells particularly the 0292. After binding of antigenic material to the well, coat unfused myeloma cells that would normally continue to ing with a non-reactive material to reduce background, and divide indefinitely) by culturing in a selective medium. The washing to remove unbound material, the immobilizing Sur selective medium is generally one that contains an agent that face is contacted with the antisera or clinical or biological blocks the de novo synthesis of nucleotides in the tissue extract to be tested in a manner conducive to immune com culture media. Exemplary and preferred agents are aminop plex (antigen/antibody) formation. Such conditions prefer terin, methotrexate, and azaserine. Aminopterin and methotr ably include diluting the antisera with diluents such as BSA, exate block de novo synthesis of both purines and pyrim bovine gamma globulin (BGG) and phosphate buffered saline idines, whereas azaserine blocks only purine synthesis. (PBS)/Tween(R). These added agents also tend to assist in the Where aminopterin or methotrexate is used, the media is reduction of nonspecific background. The layered antisera is Supplemented with hypoxanthine and thymidine as a source then allowed to incubate for from about 2 to about 4 hr, at of nucleotides (HAT medium). Where azaserine is used, the temperatures preferably on the order of about 25°C. to about media is Supplemented with hypoxanthine. 27°C. Following incubation, the antisera-contacted surface is 0288 The preferred selection medium is HAT. Only cells washed so as to remove non-immunocomplexed material. A capable of operating nucleotide Salvage pathways are able to preferred washing procedure includes washing with a solu survive in HAT medium. The myeloma cells are defective in tion such as PBS/Tween(R), or borate buffer. key enzymes of the Salvage pathway, e.g., hypoxanthine 0293 Following formation of specific immunocomplexes phosphoribosyltransferase (HPRT), and they cannot survive. between the test sample and the bound antigen, and Subse The B-cells can operate this pathway, but they have a limited quent washing, the occurrence and even amount of immuno life span in culture and generally die within about two weeks. complex formation may be determined by Subjecting same to Therefore, the only cells that can survive in the selective a second antibody having specificity for the first. To provide media are those hybrids formed from myeloma and B-cells. a detecting means, the second antibody will preferably have 0289. This culturing provides a population of hybridomas an associated enzyme that will generate a color development from which specific hybridomas are selected. Typically, upon incubating with an appropriate chromogenic Substrate. selection of hybridomas is performed by culturing the cells by Thus, for example, one will desire to contact and incubate the single-clone dilution in microtiter plates, followed by testing antisera-bound Surface with a urease or peroxidase-conju the individual clonal supernatants (after about two to three gated anti-human IgG for a period of time and under condi weeks) for the desired reactivity. The assay should be sensi tions which favor the development of immunocomplex for tive, simple and rapid, such as radioimmunoassays, enzyme mation (e.g., incubation for 2 hr at room temperature in a immunoassays, cytotoxicity assays, plaque assays, dot PBS-containing solution such as PBS Tween(R). immunobinding assays, and the like. 0294. After incubation with the second enzyme-tagged 0290. The selected hybridomas would then be serially antibody, and Subsequent to washing to remove unbound diluted and cloned into individual antibody-producing cell material, the amount of label is quantified by incubation with lines, which clones can then be propagated indefinitely to a chromogenic Substrate Such as urea and bromocresol purple provide mabs. The cell lines may be exploited for mAb or 2,2'-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid production in two basic ways. A sample of the hybridoma can (ABTS) and H.O., in the case of peroxidase as the enzyme be injected (often into the peritoneal cavity) into a histocom label. Quantitation is then achieved by measuring the degree patible animal of the type that was used to provide the somatic of color generation, e.g., using a visible spectra spectropho and myeloma cells for the original fusion. The injected ani tOmeter. mal develops tumors secreting the specific monoclonal anti 0295 The anti-crystal protein antibodies of the present body produced by the fused cell hybrid. The body fluids of the invention are particularly useful for the isolation of other animal. Such as serum or ascites fluid, can then be tapped to crystal protein antigens by immunoprecipitation. Immuno provide mabs in high concentration. The individual cell lines precipitation involves the separation of the target antigen could also be cultured in vitro, where the mabs are naturally component from a complex mixture, and is used to discrimi secreted into the culture medium from which they can be nate or isolate minute amounts of protein. For the isolation of readily obtained in high concentrations. mAbs produced by membrane proteins cells must be solubilized into detergent either means may be further purified, if desired, using filtra micelles. Nonionic salts are preferred, since other agents such tion, centrifugation and various chromatographic methods as bile salts, precipitate at acid pH or in the presence of such as HPLC or affinity chromatography. bivalent cations. 0296. In an alternative embodiment the antibodies of the 4.18 Elisas and Immunoprecipitation present invention are useful for the close juxtaposition of two antigens. This is particularly useful for increasing the local 0291 ELISAs may be used in conjunction with the inven tion. In an ELISA assay, proteins or peptides incorporating ized concentration of antigens, e.g. enzyme-substrate pairs. crystal protein antigen sequences are immobilized onto a selected Surface, preferably a surface exhibiting a protein 4.19 Western Blots affinity such as the wells of a polystyrene microtiter plate. 0297. The compositions of the present invention will find Afterwashing to remove incompletely adsorbed material, it is great use in immunoblot or western blot analysis. The anti desirable to bind or coat the assay plate wells with a nonspe peptide antibodies may be used as high-affinity primary cific protein that is known to be antigenically neutral with reagents for the identification of proteins immobilized onto a regard to the test antisera Such as bovine serum albumin Solid Support matrix. Such as nitrocellulose, nylon or combi (BSA), casein or solutions of milk powder. This allows for nations thereof. In conjunction with immunoprecipitation, US 2009/0087863 A1 Apr. 2, 2009 37 followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which sec TABLE 9- continued ondary reagents used in the detection of the antigen cause an adverse background. This is especially useful when the anti Amino Acids Codons gens studied are immunoglobulins (precluding the use of Tryptophan Trp W. UGG immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with the detecting agent, or Tyrosine Tyr Y UAC UAU they migrate at the same relative molecular weight as a cross reacting signal. 0298 Immunologically-based detection methods for use 0300 For example, certainamino acids may be substituted in conjunction with Western blotting include enzymatically for other amino acids in a protein structure without appre radiolabel-, or fluorescently-tagged secondary antibodies ciable loss of interactive binding capacity with structures against the toxin moiety are considered to be of particular use Such as, for example, antigen-binding regions of antibodies or in this regard. binding sites on Substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's 4.20 biological functional equivalents biological functional activity, certain amino acid sequence Substitutions can be made in a protein sequence, and, of 0299 Modification and changes may be made in the struc course, its underlying DNA coding sequence, and neverthe ture of the peptides of the present invention and DNA seg less obtain a protein with like properties. It is thus contem ments which encode them and still obtain a functional mol plated by the inventors that various changes may be made in ecule that encodes a protein or peptide with desirable the peptide sequences of the disclosed compositions, or cor characteristics. The following is a discussion based upon responding DNA sequences which encode said peptides changing the amino acids of a protein to create an equivalent, without appreciable loss of their biological utility or activity. or even an improved, second-generation molecule. In particu 0301 In making such changes, the hydropathic index of lar embodiments of the invention, mutated crystal proteins are amino acids may be considered. The importance of the hydro contemplated to be useful for increasing the insecticidal pathic amino acid index in conferring interactive biologic activity of the protein, and consequently increasing the insec function on a protein is generally understood in the art (Kyte ticidal activity and/or expression of the recomibinant trans and Doolittle, 1982, incorporate herein by reference). It is gene in a plant cell. The amino acid changes may be achieved accepted that the relative hydropathic character of the amino by changing the codons of the DNA sequence, according to acid contributes to the secondary structure of the resultant the codons given in Table 9. protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, Substrates, TABLE 9 receptors, DNA, antibodies, antigens, and the like. Amino Acids Codons 0302 Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge char Alanine Ala A GCA GCC GCG GCU acteristics byte and Doolittle, 1982), these are: isoleucine Cysteine Cys C UGC UGU (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); Aspartic acid Asp D GAC GAU glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2): Glutamic acid Glu B GAA GAG glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); aspar Phenylalanine Phe F UUC UUU agine (-3.5); lysine (-3.9); and arginine (-4.5). 0303. It is known in the art that certain amino acids may be Glycine Gly G. GGA. GGC GGG GGU Substituted by otheramino acids having a similar hydropathic Histidine His H CAC CAU index or score and still result in a protein with similar bio logical activity, i.e., still obtain a biological functionally Isoleucine Ile I AUA AUC AUU equivalent protein. In making Such changes, the Substitution Lysine Lys K AAA AAG of amino acids whose hydropathic indices are within t2 is preferred, those which are within +1 are particularly pre Leucine Lell L UUA UUG CUA CUC CUG. CUU ferred, and those within +0.5 are even more particularly pre Methionine Met M AUG ferred. 0304. It is also understood in the art that the substitution of Asparagine Asn N AAC AAU like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein Proline Pro P CCA CCC CCG CCU by reference, states that the greatest local average hydrophi Glutamine Glin Q CAA CAG licity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of Arginine Arg R AGA AGG CGA CGC CGG CGU the protein. Serine Ser S AGC AGU UCA, UCC UCG UCU 0305 As detailed in U.S. Pat. No. 4,554,101, the follow ing hydrophilicity values have been assigned to amino acid Threonine Thir T ACA ACC ACG ACU residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1): Waline Wall W GUA GUC GUG GUU glutamate (+3.0+1); serine (+0.3); asparagine (+0.2): glutamine (+0.2); glycine (O); threonine (-0.4); proline (-0. 5+1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); US 2009/0087863 A1 Apr. 2, 2009

methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine of the coleopteran insect Diabrotica virgifera virgifera (West (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3. ern corn rootworm, WCRW). Because WCRW larvae are 4). delicate, bioassays with this insect are time-consuming and 0306. It is understood that an amino acid can be substi frequently result in high control mortalities. To limit the num tuted for another having a similar hydrophilicity value and ber of WCRW bioassays that needed to be performed, still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In Such changes, the coleopteran-toxic B. thuringiensis strains were first identified substitution of amino acids whose hydrophilicity values are by performing bioassay screens against larvae of the Colo within +2 is preferred, those which are within +1 are particu rado potato beetle (Leptinotarsa decemlineata, CPB), an larly preferred, and those within +0.5 are even more particu insect that shows greater sensitivity than WCRW to larly preferred. coleopteran-toxic B. thuringiensis Cry proteins. Strains with 0307 As outlined above, amino acid substitutions are gen CPB-toxicity were further prioritized by performing bioassay erally therefore based on the relative similarity of the amino screens against larvae of the coleopteran insect Diabrotica acid side-chain Substituents, for example, their hydrophobic undecempunctat howardii (Southern corn rootworm, ity, hydrophilicity, charge, size, and the like. Exemplary Sub SCRW), a closely related species of rootworm that is hardier stitutions which take various of the foregoing characteristics and easier to bioassay than the WCRW. B. thuringiensis into consideration are well known to those of skill in the art strains exhibiting toxicity towards SCRW and/or CPB larvae and include: arginine and lysine; glutamate and aspartate; were evaluated in bioassays against the WCRW. In this man serine and threonine; glutamine and asparagine; and valine, ner, several hundred B. thuringiensis Strains were screened in leucine and isoleucine. bioassay and strains exhibiting WCRW-toxicity were identi fied. Strain EG4096, a wild-type B. thuringiensis stain that 5.0 EXAMPLES produces a coleopteran-toxic protein, CryET29, was used as 0308 The following examples are included to demon a positive-control in these bioassays. strate preferred embodiments of the invention. It should be 0311 B. thuringiensis strains were grown in C2 medium appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques (Donovan et al., 1988) at 25°C. for four days at which time discovered by the inventor to function well in the practice of sporulation and lysis had occurred. The sporulated cultures the invention, and thus can be considered to constitute pre were used directly in screens against CPB. For SCRW and ferred modes for its practice. However, those of skill in the art WCRW bioassays, the cultures were harvested by centrifu should, in light of the present disclosure, appreciate that many gation, washed in approximately 2.5 times the original Vol changes can be made in the specific embodiments which are ume with water, and resuspended in 0.005% TritonX-100R) at disclosed and still obtain a like or similar result without one-tenth the original culture Volume. The spore-crystal Sus departing from the spirit and scope of the invention pensions were used directly in bioassay. 0312 Insecticidal activity against CPB and SCRW larvae 5.1 Example 1 was determined via a Surface contamination assay on an Isolation of B. thuringiensis EG4140 artificial diet in a plastic feeding cup (175 mm surface) as described by Rupar et al. (1991). All bioassays were per 0309 Crop dust samples were obtained from various Sources throughout the U.S. and abroad, typically grain stor formed using 128-well trays containing approximately 1 ml age facilities. The crop dust samples were treated and spread of diet per well with perforated mylar sheet covers (C-D on agar plates to isolate individual Bacillus-type colonies as International Inc., Pitman, N.J.). A first instar larva was described (Donovan et al., 1992). EG4140 is a wild-type B. placed in each cup and scored for mortality after 3-5 days thuringiensis stain isolated from a crop dust sample from (CPB) or 7 days (SCRW). Thirty-two larvae were tested per Pennsylvania. Phase contrast microscopy was used to visu bioassay screen at 50ul of a spore-crystal Suspension per well ally examine the morphology of parasporal crystals produced by the bacterial colonies from this crop dust. The colony of diet. designated EG4140 contained endospores and crystalline 0313 Bioassay screens against WCRW larvae were per inclusions of a unique morphology resembling spindles. The formed via a Surface contamination assay using an artificial complement of native plasmids contained within isolated B. diet (20 gagar, 50g wheat germ, 39 g Sucrose, 32 g casein, 14 thuringiensis EG4140 was determined by modified Eckhardt gfiber, 9 g Wesson salts mix, 1 g methyl paraben, 0.5g Sorbic agarose gel electrophoresis as described by Gonzalez et al. acid, 0.06 g cholesterol, 9 g Vaderzant's vitamin mix, 0.5 ml (1982). The pattern of native plasmids did not correspond to linseed oil, 2-5 ml phosphoric/propionic acid per liter). A first patterns of typical known serovars (Gonzalez and Carlton, instar larva was placed in each diet cup and scored for mor 1984). The plasmid sizes are approximately 110 MDa, 80 tality after 7 days. MDa and 33 MDa. 0314. A sporulated and lysed culture of strain EG4140 exhibited toxicity towards CPB larvae in a bioassay screen. 5.2 Example 2 Subsequently, the strain was screened in bioassay against Bioassay Evaluation of B. thuringiensis Strains for SCRW larvae and exhibited little, if any, toxicity. Neverthe Toxicity Towards Coleopteran Insects less, the strain was screened in bioassay against WCRW larvae and was found to exhibit significant insecticidal activ 0310. A three-tiered bioassay scheme was adopted to ity (Table 10). EG4140 also showed toxicity towards larvae of identify B. thuringiensis strains with toxicity towards larvae the lepidopteran pest, Plutella xylostella. US 2009/0087863 A1 Apr. 2, 2009 39

0319. The FASTA algorithm (Lipman and Pearson, 1985) TABLE 10 in the PCGene program (Intelligenetics, Mountain View, Calif.) was used to compare the N-terminal sequence of the INSECTICIDAL ACTIVITY OF STRAIN EG414OTO CryET70 polypeptide with amino acid sequences of all B. WCRW LARVAE thuringiensis crystal proteins of which the inventors were Sample % Mortality aware of at the time, including the sequences of all B. thur ingiensis crystal proteins which had been published in the EG4140 100% EG4096 100% Scientific literature, international patent applications or Control 31% issued patents. The N-terminal sequence of the CryET70 polypeptide was not found to be similar to sequences of the Dose: known B. thuringiensis crystal proteins. EG4096 - 31 g well EG4140-38 Lig?well 5.4 Example 4 5.3 Example 3 Cloning of the CryET70 Gene 0320 Total DNA was extracted from EG4140 by the fol Characterization of the CryET70 Polypeptide lowing procedure. Vegetative cells were resuspended in a 0315 EG4140 was grown in C2 (Donovan et al., 1988) lysis buffer containing 50 mM glucose, 25 mM Tris-HCl medium for four days at 25°C. during which time the culture (pH8.0), 10 mM EDTA, and 4 mg/ml lysozyme. The suspen grew to stationary phase, sporulated and lysed, thus releasing sion was incubated at 37°C. for one hr. Following incubation, the protein inclusions into the medium. Two hundred micro SDS was added to 1%. The suspension was then extracted liters of the culture were added to one milliliter of 1M NaCl, with an equal Volume of phenol:chloroform:isoamyl alcohol 1 mM EDTA, pH 8 and centrifuged to pellet the spores and (50:48:2). DNA was precipitated from the aqueous phase by crystals. The pellet was resuspended in water at one-half the the addition of one-tenth volume 3M sodium acetate, and two original Volume. volumes of 100% ethanol. The precipitated DNA was col 0316 Crystal protein was solubilized by incubating the lected with a glass rod, washed with 70% ethanol, and resus spore-crystal suspension in a SDS solubilization buffer at pended in dH.O. 100° C. for 5 min and size fractionated by SDS-polyacryla 0321. The DNA was partially digested with the restriction mide gel electrophoresis (SDS-PAGE) (Laemmli, 1970). endonuclease MboI and size fractionated on a 0.6% agarose After size fractionation, the proteins were visualized by Coo gel in 1xTAE overnight at 2 V/cm. The gel was stained with massie Brilliant Blue R-250 staining. This analysis showed ethidium bromide so that the digested DNA could be visual that the major crystal protein present in the sporulated culture ized when exposed to long-wave UV light. Gel slices con of EG4140 is approximately 86 kDa in size. This novel pro taining DNA fragments between about 9 and about 12 kb in tein was designated CryET70. Additional proteins of 39 kDa, size were excised from the gel with a razor blade. The DNA 34 kDa, and 32 kDa are also produced by this strain. was extracted from the gel using the GENECLEAN IIR) kit 0317. To further characterize CryET70, the NH-terminal (BIO 101 Inc., La Jolla, Calif.) and eluted in H.O. amino acid sequence of the protein was determined. A sporu 0322 To create a library in E. coli, the 9- to 12-kb MboI lated culture of EG4140 was washed in water and resus restriction fragments of DNA from EG4140 were ligated into pended at the original Volume in water. The proteins were size the E. coli/B. thuringiensis shuttle vector pHT315 (Arantes fractionated by SDS-PAGE following the protocol described and Lereclus, 1991). The vector pH1315 can replicate in E. by Brussock and Currier (1990). The procedure was modified coli and carries the gene for resistance to the antibiotic ampi to eliminate the neutralization step with 3M HEPES. After cillin, which may be used as a selectable marker. The vector electrophoresis the proteins were transferred to a Immo pH1315 can also replicate at a high copy number in B. thur bilon-P membrane (Millipore, Bedford, Mass.) following ingiensis, and carries the gene for resistance to the antibiotic standard Western blotting procedures (Towbinet al., 1979). erythromycin, which may be used as a selectable marker in After transfer, the membrane was rinsed four times in dEIO this organism. The fragments from EG4140 were ligated with and washed in 0.025% Coomassie Blue R-250, 40% metha pHT315 which had been digested with BamHI and treated nol. The filter was destained in 50% methanol and rinsed 4x with calf intestine alkaline phosphatase (Boehringer Man in dHO. The portion of the filter containing the approxi nheim, Indianapolis, Ind.) to remove the terminal 5' phos mately 86-kDa protein band was excised with a razor blade. phates from the digested plasmid, thereby preventing re-liga 0318. The determination of the NH2-terminal amino acid tion of the vector to itself. T4 ligase and a ligation buffer sequence of the purified CryET70 polypeptide immobilized (Promega Corporation, Madison, Wis.) were added to the on the membrane was performed in the Department of Physi reaction containing the digested pHT315 and the Mbol frag ology at the Tufts Medical School, Boston, Mass. using stan ments. These were incubated at room temperature for several dard automated Edman degradation procedures. The NH hours to allow the insertion and ligation of the Mbol frag terminal sequence was determined to be: ments into the pHT315 vector DNA. 0323. The ligation mixture described above was used to transform electroporation-competent SURETM cells (Strat (SEQ ID NO:3) agene, La Jolla, Calif.) to amplicillin resistance following 1. 2 3 4. 5 6 7 8 9 1O 11 12 procedures described by the manufacturer. Aliquots of the Ala Ser Asp Tyr Ile Asp Ile Arg Ser Ile Phe Glin transformed cells were frozen at -70° C. in LB (Maniatis et 13 al., 1982) containing 15% glycerol. The transformed E. coli Thir cells were later thawed and plated on LB agar plates contain ing 50 ug/ml amplicillin and incubated overnight at 37°C. The US 2009/0087863 A1 Apr. 2, 2009 40 growth of amplicillin-resistant colonies indicated the presence 0330 Several colonies hybridized to AM24. These colo of a recombinant plasmid in the cells of each colony. nies were identified by lining up the signals on the autorad 0324. To isolate the colonies harboring the cloned frag iogram with the colonies on the original transformation ments that contain the cryET70 gene sequences, the trans plates. The isolated colonies were then grown in LB-amplicil formed E. coli colonies were first transferred to nitrocellulose lin liquid medium from which the cells could be harvested filters. This was accomplished by placing a circular filter and recombinant plasmid prepared by the standard alkaline (Millipore HATF 085 25, Millipore Corp., Bedford, Mass.) lysis miniprep procedure (described in Maniatis et al., 1982). directly on top of the LB-amplicillinagarplates containing the The isolated plasmids were digested with the restriction transformed colonies. When the filter is slowly peeled off of endonuclease EcoRI. The DNA from these plasmid digests the plate the colonies stick to the filter giving an exact replica were resolved by agarose gel electrophoresis and transferred of the pattern of colonies from the original plate. Enough cells to an Immobilon-NC nitrocellulose filter (Millipore Corp., from each colony are left on the plate that five to six hr of Bedford, Mass.) according to the method of Southern (1975). growth at 37°C. will restore the colonies. The plates are then DNA was fixed to the filter by baking at 80°C. in a vacuum stored at 4°C. until needed. The nitrocellulose filters with the OVC. transferred colonies were then placed, colony-side up, on 0331. Subsequently, the blot was hybridized with the oli gonucleotide, AM24, that had been radioactively labeled at fresh LB-ampicillinagar plates and allowed to grow at 37°C. the 5' end with Y-P using T4 polynucleotide kinase. EcoRI until they reached a size of approximately 1 mm in diameter. fragments from five of the 16 plasmids hybridized to the 0325 To release the DNA from the recombinant E. coli probe, confirming the presence of a cryET70 gene in those cells onto the nitrocellulose filter, the filters were placed, clones. One of the plasmids containing a cryET70 gene was colony-side up, on Whatman 3 mM Chromatography paper designated pEG1648 (FIG. 1). The E. coli strain containing (Whatman International LTD., Maidstone, England) soaked pEG1648 was designated EG1 1841. with 0.5 N NaOH, 1.5 M NaCl for 15 min. The filters were then neutralized by placing the filters, colony-side up, on Whatman papersoaked with 1 MNH-acetate, 0.02 MNaOH 5.5 Example 5 for 10 min. The filters were then rinsed in 3xSSC, 0.1% SDS, Sequencing of The CryET70 Gene and Determina air dried, and baked for one hr at 80°C. in a vacuum oven to tion of the Encoded Amino Acid Sequence prepare them for hybridization. 0326 In order to identify the gene encoding the CryET70 0332 A partial DNA sequence of the gene cloned on polypeptide, an oligonucleotide probe specific for the NH pEG1648 was determined following established dideoxy terminal amino acid sequence of the protein was designed chain-termination DNA sequencing procedures (Sanger et using codons: typically found in B. thuringiensis toxin genes. al., 1977). Preparation of the double stranded plasmid tem The following oligonucleotide, designated AM24, was Syn plate DNA was accomplished using a Wizard RSV Miniprep thesized by Integrated DNA Technologies, Inc. (Coralville, Kit (Promega, Madison, Wis.) following the manufacturer's Iowa): procedures or a Qiagen Plasmid Kit (Qiagen Inc., Chat sworth, Calif.) following the manufacturer's procedures, fol lowed by a phenol: chloroform: isoamyl alcohol (50:48:2) (SEQ ID NO : 4) extraction and then a chlorform:isoamyl alcohol (24:1) s' - GCITCIGATTATATTGATATTAGATCAATTTTTCAAAC-3' extraction. The sequencing reactions were performed using the SequenaseTM Version 2.0 DNA Sequencing Kit (United 0327 where I-inosine. States Biochemical/Amersham Life Science Inc., Cleveland, 0328. The NH-terminal oligonucleotide specific for the Ohio) following the manufacturer's procedures and using cryET70 gene, AM24, was labeled at the 5' end with Y-'Pand S-dATP as the labeling isotope (obtained from DuPont used as a hybridization probe. To radioactively label the NENR Research Products, Boston, Mass.). Denaturing gel probe, 40 pmoles AM24 were added to a reaction (20 ul total electrophoresis of the reactions was performed on a 6% (wt./ volume) containing Y--PATP (3 ul of 3,000 Ci/mmole at 10 Vol.) acrylamide, 42% (wt.?vol.) urea sequencing gel or on a mCi/ml), 2 ul of a 10x reaction buffer (700 mM Tris-HCl, CastAwayTM Precast 6% acrylamide sequencing gel (Strat pH7.8, 100 mM MgCl, 50 mM DTI), and 10 units T4 poly agene, La Jolla, Calif.). The dried gel was exposed to Kodak nucleotide kinase (Promega Corporation, Madison, Wis.). X-OMAT ARX-ray film (Eastman Kodak Company, Roch The reaction was incubated for 20 min at 37° C. to allow the ester, N.Y.) overnight at room temperature. transfer of the radioactive phosphate to the 5' end of the 0333. The NH2-terminal specific oligonucleotide AM24 oligonucleotide, thus making it useful as a hybridization was used as the initial sequencing primer. A partial DNA probe. sequence for the cryET70 gene on pEG1648 was obtained by 0329. The labeled probe was added to the filters in 3xSSC, using the procedures described above. Successive oligo 0.1% SDS, 10xDenhardt's reagent (0.2% bovine serum albu nucleotides to be used for priming sequencing reactions were min(BSA), 0.2%polyvinylpyrrolidone, 0.2% FicollR), 0.2% designed from the sequencing data of the previous set of mg/ml heparin and incubated overnight at 37°C. These con reactions. An approximately 5.8 kb-EcoRI-HindIII fragment ditions were chosen to allow hybridization of the labeled containing the entire cryET70 gene was subcloned from oligonucleotide to related sequences present on the nitrocel pEG1648 into puC18. This plasmid, designated pG 1657 lulose blots of the transformed E. coli colonies. Following the (FIG. 1) was used to complete the cryET70 sequence using incubation, the filters were washed in three changes of automated sequencing. Five hundred nanograms of purified 3xSSC, 0.1% SDS at 40°C. The filters were blotted dry and plasmid was added to 100 ng of primer in a total volume of 20 exposed to Kodak X-OMAT ARX-ray film (Eastman Kodak ul. All the primers were designed using the OLIGO primer Company, Rochester, N.Y.) overnight at -70° C. with an analysis software (National Biosciences, Inc., MN). DNA intensifying screen (Fisher Biotech, Pittsburgh, Pa.). samples were sequenced using the ABI PRISM Dye Deoxy US 2009/0087863 A1 Apr. 2, 2009

sequencing chemistry kit (Applied Biosystems, CA) accord ing to the manufacturer's suggested protocol. The completed - Continued reactions were run on as ABI377 automated DNA sequencer. AAGCAGAAAAAAGTATCAAAGTCCTTGTTCTAGGAGAACCAAGCATTGAA Overlapping DNA sequence fragments were analyzed and GCAAATAATGTTGAGATTGAAAAAGACGAAAGGTTCGATCCATTAACAGA assembled using Sequencher v2.1 DNA analysis software (Gene Codes Corporation, Mich.). The DNA sequence of TTCAAGAGTAGGTCTCCGTGCAAAAGACTCATTAGGCAAAGATATTACGA cryET70 is shown in Sections 5.5.1 (SEQID NO:1). The ATGATGTGAAAGTAAAATCAAGTAATGTGGATActroAAAAccAggAGAA protein coding region begins at nucleotide 92. The deduced amino acid sequence of the CryET70 polypeptide is shown in TATGAAGTTGTATTTGAAGTGACTGATCGTTTTGGTAAATATGTAAAGAA Section 5.5.1 (SEQID NO:2). ATTGATTGTAGTTATAGTACCAGTAATTGATGATGAATGGGAAGATGGAA

ATGTGAATGGATGGAAATTCTATGCGGGGCAAGACATCACACTGTTGAAA 5. 5.1 DNA SEQUENCE OF THE CRYET7 O GENE (SEQ ID NO: 1) GATCCTGAAAAAGCATATAAAGGAGAATATGTATTCTATGATTCTAGGCA GTAATAGTAGTTATTTAGCAGGAATAAAAGGGAGGGTATCGAATACTTTC TGCTGCTATTTCTAAAACAATCCCAGTAACAGATTTACAAGTGGGAGGGA AAATGAAGACTGAAAATTTACAAATAGAAGGAGAGAAAAGTATGAAAGAT ATTATGAAATTACAGTATATGTTAAAGCAGAAAGCGGTGATCATCACCTA TCAATTTCAAAGGGATATGATGAAATAACAGTGCAGGCAAGTGATTATAT AAAGTGACGTACAAGAAAGACCCGAAAGGTCCGGAGGAACCACCAGTTTT TGATATTCGCTCAATTTTTCAAACGAATGGATCTGCAACATTTAATTCAA CAATAGACTTATTAGTACAGGGAAATTGGTGGAAAAAGACTATAGAGAAT CCACTAATATTACAACTTTAACGCAAGCTACAAATAGTCAAGCGGGAGCA TAAAAGGAACATTCCGTGTAACGGAATTAAACCAAGCACCATTGATAATC ATTGCAGGGAAGACAGCTTTAGATATGAGACATGATTTTACTTTTAGAGC GTAGAGAATTTTGGTGCTGGATATATAGGTGGAATTAGAATTGTGAAAAT TGATATTTTTCTTGGAACTAAAAGTAATGGAGCAGATGGTATTGCGATAG ATCGTAATAAAAACAGATAAAATAGAGAGAGGAACGCTGGATAGCGTTCC CATTTCATAGAGGATCAATTGGTTTTGTTGGGGAGAAGGGTGGAGGACTT TCTTTTCAGTTTAGTGTACCATCTCCAGGTCCAGTTCATTTTTT GGGATTTTAGGCGCCCTAAAAGGTATAGGATTTGAATTAGACACATATGC 5. 5.2 AMINO ACID SEQUENCE OF THE CRYET7 O POLY GAATGCTCCTCAAGATGAACAAGGAGATTCTTTTGGACATGGAGCAATGA PEPTIDE MKDSISKGYDEITVOASDYIDIRSIFOTNGSATFNSTTNITTLTOATNSO GAGGCCTATTCCCTGGTTTCCCAAATGGATATCCACATGCTGGTTTTGTA AGAIAGKTALDMRHDFTFRADIFLGTKSNGADGIAIAFHRGSIGFWGEKG AGTACGGATAAAAATAGAGGTTGGTTATCTGCCTTAGCTCAGATGCAGCG GGLGILGALKGIGFELDTYANAPODEQGDSFGHGAMRGLFPGFPNGYPHA AATAGCTGCTCCAAATGGGCGTTGGAGACGTCTGGCGATTCATTGGGATG GFWSTDKNRGWLSALAOMORIAAPNGRWRRLAIHWDARNKKLTANLEDLT CTCGCAATAAAAAATTAACTGCAAACCTTGAGGATTTAACTTTTAATGAT FNDSTVLVKPRTPRYARWELSNPAFELDOKYTFWIGSATGASNNLHOIGI TCAACGGTATTAGTGAAACCACGTACTCCAAGATATGCAAGATGGGAGTT IEFDAYFTKPTIEANNWSWPWGATFNPKTYPGINLRATDEIDGDLTSEII ATCAAATCCTGCATTTGAACTTGATCAAAAGTATACTTTTGTTATTGGTT WTDNNVNTSKSGWYNWTYYWKNSYGESDEKTIEWTVSNPTIIASDWEE CAGCGACGGGTGCATCTAATAACCTACATCAGATTGGTATTATAGAATTT KGESFNPLTDSRVRLSAODSLGNDITSKVKVKSSNVDTSKPGEYDVWFEW GATGCATACTTTACTAAACCGACAATAGAGGCGAATAATGTAAGTGTTCC TDNFGGKAEKEIKVTVLGOPSIEANDWELEIGDLFNPLTDSOVGLRAKDS GGTGGGAGCAACATTTAATCCGAAAACATATCCAGGAATAAATTTAAGAG LGKDITNDWKWKSSNWDTSKPGEYEWWFEWTDRFGKKAEKSIKWLWLGEP CAACTGATGAAATAGATGGTGATTTGACATCTGAAATTATTGTGACAGAT SIEANNWEIEKDERFDPLTDSRWGLRAKDSLGKDITNDWKWKSSNWDTSK AATAATGTTAATACGTCGAAATCTGGTGTGTATAATGTGACGTATTATGT PGEYEVWFEVTDRFGKYWKKLIVVIVPVIDDEWEDGNVNGWKFYAGODIT AAAGAATAGCTATGGGGAAAGTGATGAAAAAACAATCGAAGTAACTGTGT LLKDPEKAYKGEYWFYDSRHAAISKTIPVTDLOWGGNYEITVYVKAESGD TTTCAAACCCTACAATTATTGCAAGTGATGTTGAAATTGAAAAAGGTGAA HHLKVTYKKDPKGPEEPPVFNRLISTGKLVEKDYRELKGTFRVTELNOAP TCGTTTAATCCATTAACAGACTCAAGAGTGAGGCTGTCTGCACAAGATTC LIIWENFGAGYIGGIRIWKIS ATTGGGTAATGATATTACTTCAAAAGTAAAGGTGAAATCAAGTAATGTGG ATACTTCGAAACCAGGTGAATATGATGTTGTGTTTGAAGTGACCGATAAT 5.6 Example 6 TTTGGTGGGAAAGCAGAAAAAGAAATCAAGGTTACAGTTTTAGGGCAGCC Analysis of Sequence Homologies to CryET70/ AAGTATTGAAGCGAATGATGTTGAATTAGAAATAGGTGATTTATTTAATC CryET70 Sequences CGTTAACAGATTCACAAGTAGGCCTTCGTGCAAAAGACTCATTAGGCAAA 0334 Database searches were conducted to determine if the amino acid sequence of the CryET70 polypeptide shares GATATTACGAATGATGTGAAAGTAAAGTCAAGTAATGTGGATACTTCAAA identity with the sequences of other characterized proteins, ACCAGGAGAATATGAAGTTGTATTTGAAGTGACCGATCGTTTTGGAAAAA especially other insecticidal proteins. Database searches using on-line servers were performed using the amino acid sequence of CryET70 with the BLASTP program (Altschulet US 2009/0087863 A1 Apr. 2, 2009 42 al, 1990) provided by the National Center for Biotechnology toxic protein Cry3Bb (Donovan et al., 1992) was grown and Information (Bethesda, Md.). The BLASTP searches were harvested in the same manner. Toxin proteins from the run with the BLOSUM62 matrix. The searched database con samples were quantified by SDS-PAGE as described by Brus sisted of non-redundant GenBank CDS translations plus the sock and Currier (1990). The procedure was modified to PDB, SwissProt, SPupdate, and PIR databases. eliminate the neutralization step with 3M HEPES. 0335. Only one protein in these databases was identified with any significant similarity to CryET70. Cry22, a 0342 WCRW larvae were bioassayed via surface con hymenopteran toxic protein from B. thuringiensis PS211B2 tamination of an artificial diet (20g agar, 50g wheatgerm, 39 identified as SEQID NO:51 of U.S. Pat. No. 5,596,071 (spe g Sucrose, 32 g casein, 14 g fiber, 9 g Wesson salts mix, 1 g cifically incorporated herein by reference), showed 88.9% methyl paraben, 0-5 g sorbic acid, 0.06 g cholesterol, 9 g sequence identity to CryET70. Vanderzant's vitamin mix, 0.5 ml linseed oil, 2.5 ml phos 0336 A B. thuringiensis toxins database (which includes phoric/propionic acid per 1 liter). Each bioassay of EG1 1839 all toxin genes and proteins published to date) was searched (CryET70) and EG11204 (Cry3Bb) consisted of eight serial for proteins with homology to CryET70 using the FASTA aqueous dilutions with aliquots applied to the Surface of the algorithm (Lipman and Pearson, 1985) in the PCGene pro diet. After the diluent (an aqueous 0.005% Triton X-100R gram (Intelligenetics, Mountain View, Calif.). Again, only solution) had dried, first instar larvae were placed on the diet Cry22 showed significant sequence identity to CryET70. and incubated at 28°C. Thirty-two larvae were tested per dose. Mortality was scored after seven days. Data from rep 5.7 Example 7 licated bioassays were pooled for probit analysis (Daum, 1970) with mortality being corrected for control death, the Expression of Recombinant CryET70 Polypeptide control being diluent only (Abbott, 1925). Results are 0337 To characterize the properties of the CryET70 reported as the amount of crystal protein perwell (175 mm of polypeptide, it was necessary to express the cloned cryET70 diet surface) resulting in an LCso, the concentration killing gene in a B. thuringiensis strain that does not produce other 50% of the test insects. 95% confidence intervals are also crystal proteins (i.e. a Cry strain). The plasmid containing reported for the LCs values (Table 11). the cloned cryET70 gene, pFG 1648, contains a B. thuring iensis origin of replication as well as an origin that directs TABLE 11 replication in E. coli, as described above. The plasmid, pEG1648, was used to transform the Cry B. thuringiensis INSECTICIDAL ACTIVITY OF THE CRYETTO PROTEIN strain EG10650 to erythromycin resistance (Em") by elec TO WCRW LARVAE troporation (Macaluso and Mettus, 1991). Cells transformed Crystal LCso LC95 to Em' were selected by incubation overnight on LB agar Sample Protein (g protein well) 95% C.I. (g protein well) plates containing 25 ||g/ml erythromycin. One Em' colony EG11839 CryET70 8.4 6-11 69 from each transformation was selected for further analysis. EG11204 Cry3 Bb 11.4 3-23 437 One isolate was designated EG1 1839. 0338 EG1 1839 was grown in C2 medium containing 25 Control mortality 12.5% ug/ml erythromycin for four days at 25°C., at which point 0343. The results shown in Table 11 demonstrate that the sporulation and cell lysis had occurred. Microscopic exami CryET70 polypeptide has significant activity on larvae of the nation of the sporulated cultures demonstrated that the western corn rootworm. CryET70 is at least as active as recombinant strain was producing large, dark spindle-shaped Cry3Bb. crystalline inclusions. These crystals resemble the crystals produced by the wild-type strain EG4140, indicating that the 0344) A spore-crystal suspension of EG1 1839, prepared cryET70 gene in the recombinant strain EG1 1839 is a func as described in Example 2, was screened for activity against tional gene capable of directing the expression of the lepidopteran insects. EG2001, the Cry 1-containing strain CryET70 polypeptide HD1, was assayed as a control. EG2001 was grown in C2 0339. The sporulated culture of EG1 1839 was harvested medium as described in Example 2, washed and resuspended by centrifuigation, washed, and resuspended at one-tenth the in an original volume of 0.005% Triton X-100R). EG11839 original Volume in H2O. The crystal protein in the Suspension exhibited no significant activity against Agrotis ipsilon, Heliothis virescens, Helicoverpa zea, Ostrinia nubialis, was characterized by SDS-PAGE analysis which revealed the Spodoptera exigua, Spodoptera frugiperda, Diabroica production of an approximately 86-kDa protein. undecimpunctata howardi, and Leptinotarsa decemlineata. EG1 1839 exhibited some activity against Plutella xylostella 5.8 Example 8 and Trichoplusia ni (Table 12). Toxicity OF CryET70 to Insects (0340. The toxicity of CryET70 polypeptide towards TABLE 12 WCRW larvae (Diabrotica virgifera virgifera) was deter INSECTICIDAL ACTIVITY OF EG11839 AGAINST mined. PXYLOSTELLA AND TNT (0341 EG1 1839 was grown in C2 medium at 25° C. for Sample Pxylostella (% mortality) T. ni (% mortality) four days until sporulation and cell lysis had occurred. The culture was harvested by centrifugation, washed in approxi EG11839* 31 40 mately 2.5 times the original volume with H2O, and resus EG2001.** 25 81 pended in 0.005% Triton X-100R at one-tenth the original *Dose - 10,000 inl/well volume. For comparison with EG1 1839, a recombinant B. **Dose - 10 n? well thuringiensis Strain, EG 1 1204, producing the coleopteran US 2009/0087863 A1 Apr. 2, 2009

(0345 Thus, CryET70 exhibits toxicity towards larvae of tified by colony blot hybridization experiments using a both coleopteran and lepidopteran species. cryET70-specific hybridization probe. Wild-type strains (0346) CryET70 is 88.90% homologous to Cry22. The were patched onto LB plates and incubated at 30° C. for four Eschericia coil strain NRRL B-21150, containing the plas hr. A Nytran?R Maximum-Strength Plus (Schleicher and mid pMYC2371 that carries the cry22 gene, was obtained Schuell, Keene, N.H.) circular (82 mm) membrane filter was from the USDA-ARS, Patent Culture Collection (Peoria, Ill.). then placed on the plates and the plates and filters were The plasmid was isolated using the Wizard R. SV Miniprep incubated at 25°C. overnight. The filters, which contained an Kit (Promega, Madison, Wis.), and the plasmid DNA was exact replica of the patches, were then placed on fresh LB used to transform BTEG10650 to Em. The resulting strain plates, and the filters and the original plates were incubated at is EG11860. EG11860 and EG11839 were grown as 30° C. for 4 hr to allow for growth of the colonies. After four described in Example 2, and screened against WCRW as hr, filters were denatured, neutralized, washed, and baked as described above. The results are shown in Table 13. described in Example 4. TABLE 13 0350 Primers were designed based on the cryET70 Sequence. INSECTICIDAL ACTIVITY OF CRYET7OAND CRY22 AGAINSTWCRW AM34: 20 Mortality s' - GACATGATTTTACTTTTAGAGC-3' (SEO ID NO. 5)

Sample 20 ug? well 200 lug? well AM43: EG11839 (CryET70) 81.5 1OO s' - CATCACTTTCCCCATAGC-3' (SEQ ID NO : 6) EG11860 (Cry22) O 18.5 0351 A PCRTM with primers AM34 and AM 43 was used to amplify a cryET70 fragment from pEG1648 DANA. This (0347 CryET70 exhibits significant toxicity towards the WCRW, while Cry22 does not. The differences in sequence PCRTM product was labeled with O'PdATP using the between CryET70 and Cry22 impart WCRW activity on Prime-a-Gene(R) kit (Promega Corporation, Madison, Wis.) to CryET70. generate a cryET70-specific probe. Hybridizations were per 0348 An alignment of the CryET70 and Cry22Aa amino formed as described in Example 4 with the exception that the acid sequences revealed that the amino acid sequence differ hybridization temperature was 63°C. Filters were washed in ences are not localized to a particular region, but are scattered 1xSSC, 0.1% SDS at 63° C. Hybridizing colonies were throughout the length of the two proteins. Nevertheless, sev detected by autoradiography using Kodak X-OMAT AR eral regions of dissimilarity are notable. For instance, the X-ray film. The stains identified by colony blot hybridization amino terminal sequences of the two proteins from positions are listed in Table 14. 3-8 are completely different. In addition, Cry22Aa contains an extra alanine residue at position 17. The CryET70 5.10 Example 10 sequence from residues 506-516 shows only 45% sequence identity with the homologous sequence 507-517 in Cry22Aa Production of Antibody to CryET70 Using the ANTIGEN program based on the method of Hopp and Woods (1981) in the PC/GENE sequence analysis pack 0352 CryET70 polypeptide was prepared for antibody age, the CryET70 sequence from 508-514 was determined to production so that CryET70 antibodies could be used to iden be significantly more hydrophilic than the homologous tify CryET70-related proteins. EG1 1839 was grown in C2 Cry22A sequence. Since hydrophilic regions tend to be local medium for four days at 25°C. The culture was washed in ized at the Surface of globular proteins and, consequently, 2.5.x volume H2O and resuspended at /20 the original volume may be involved in interactions with other molecules, this in 0.005% Triton X-100R). The spore-crystal suspension was hydrophilic region may be important in determining the dif then loaded on a Sucrose step gradient consisting of 79%, ferences in insecticidal activity noted between CryET70 and 72% and 55% sucrose. The gradient was spun overnight in a Cry22Aa Using the SOAP program based on the method of Beckman SW28 at 18,000 rpm. CryET70 crystals banded Kyle and Doolittle (1982), in the PC/GENE sequence analy between the 79% and the 72% sucrose layers. The CryET70 sis package, the hydropathy indices for CryET70 and crystals were washed several times in H2O and resuspended Cry22Aa could be compared. The two proteins showed in 0.005% Triton X-100R). The purified crystals were then apparent differences in hydrophobicity at the amino terminus, solubilized in 50 mM sodium carbonate (pH 10), 5 mM DTT, near residue Q124 in CryET70, near residues 506-516 in and any contaminating vegetative cells or spores were ET70, near residues 569-573 in CryET70, and near residue removed by centrifugation. The Supernatant was neutralized K662 in CryET70. These regions represent targets for future with boric acid to pH8.4, and the solubilized crystals were investigations. For instance, each unique CryET70 region sent to Rockland Laboratories (Gilbertsville, Pa.) for anti could be systematically introduced into the cry22Aa gene by body production in rabbits according to standard procedures. in vitro mutagenesis to identity those regions that can confer The rabbits received two intradermal injections on days zero WCRW toxicity on Cry22Aa and seven with 50% CryET70 polypeptide in sterile phos phate buffered saline, 50% complete Freund's adjuvant. Two 5.9 Example 9 additional boosts were given Subcutaneously on days 14 and Additional Strains with Sequences Related to 28 before a test bleed on day 38. Two hundred fifty ug of CryET70 CryET70 were used per rabbit for the initial injection, and 0349 To date, 26 wild-type strains have been identified 125 ug of CryET70 were used per rabbit for the subsequent with sequences related to cryET70. These strains were iden boosts. On day 56 the rabbits were boosted again, as before, US 2009/0087863 A1 Apr. 2, 2009 44 prior to a production bleed on day 71. The final boost was with 160 ug CryET70 on day 80, followed by a termination bleed TABLE 14-continued on day 90. STRAINS CONTAINING CRYET70-RELATED GENES

5.11 Example 11 Strains Southern blot Western blot % Control WCRW

Analysis of Strains Containing CryET70-Related EG4448 -- 100 Sequences EG4SO3 -- 56 EG4S41 -- 72 0353 Stains identified in Example 9 as containing EG458O -- -- 33 EG4640 95 sequences related to cryET70 were screened in bioassay EG4737 72 against WCRW as described in Example 2. In addition, the EG4741 -- 73 strains were examined further by Southern and Western blot EG5233 52 analyses. EGS366 -- 69 0354 Total DNA was prepared from the strains as EG5370 16 described in Example 4. Total DNA was digested with EcoRI EGS422 8 and separated on a 0.8% agarose gel in TAE buffer (40 mM Tris-acetate, 2 mM NaEDTA, pH 8). The DNA was blotted 0357 The above data indicate that there is a family of onto an Immobilon-NC nitrocellulose filter (Millipore Corp., cryET70-related genes, some of which encode CryET70 Bedford, Mass.) according to the method of Southern (1975). related proteins that have activity against WCRW. Related DNA was fixed to the filter by baking at 80°C. in a vacuum proteins may be found with increased activity against WCRW OVC. or with an expanded spectrum of insecticidal activity. 0355 The blot was then hybridized with the cryET70 specific probe described in Example 9. Hybridizations were 5.12 Example 12 performed as in Example 4 with the exception that the hybrid Isolation of Transgenic Plants ization and wash temperature was 60° C. Strains containing hybridizing DNA fragments are listed in Table 14. 5.12.1 Plant Gene Construction 0356. For the Western blot analysis, proteins from 10-fold 0358. The expression of a plant gene which exists in concentrated cultures of the strains were run on a 10% SDS double-stranded DNA form involves transcription of messen polyacrylamide gel (Owl Separation Systems, Woburn, ger RNA (mRNA) from one strand of the DNA by RNA Mass.). Twenty ul of culture was added to 10 ul of 3x Laem polymerase enzyme, and the Subsequent processing of the mli buffer and heated at 100° C. for five minutes. Fifteen oil mRNA primary transcript inside the nucleus. This processing were loaded per lane. Following electrophoresis, the gel was involves a 3' non-slated region which adds polyadenylate blotted to nitrocellulose following standard Western blotting nucleotides to the 3' end of the RNA. Transcription of DNA procedures (Towbinet al., 1979). The filter was blocked with into mRNA is regulated by a region of DNA usually referred TBSN (10 mM Tris, pH 7.8, 0.9% NaCl, 0.1% globulin-free to as the “promoter'. The promoter region contains a BSA, 0.03% NaNs)+2% BSA. The filter was then washed sequence of bases that signals RNA polymerase to associate with TBSN twice and then incubated with a 1/1,000 dilution with the DNA and to initiate the transcription of mRNA using of anti-CryET70 in TBSN. The filter was washed in TBSN one of the DNA strands as a template to make a corresponding after incubation with antibody and then incubated with the Strand of RNA. sheep anti-rabbit IgG conjugated with alkaline phosphatase 0359 A number of promoters which are active in plant (1/1,000 dilution in TBSN). After washing in TBSN, the cells have been described in the literature. Such promoters proteins antigenically related to CryET70 were detected with may be obtained from plants or plant viruses and include, but ImmunoPureR NBT/BCIP Substrate Kit (Pierce, Rockford, are not limited to, the nopaline synthase (NOS) and octopine Ill.). The results of the Southern blot, Western blot, and bio synthase (OCS) promoters (which are carried on tumor-in assay analyses are shown in Table 14. ducing plasmids of Agrobacterium tumefaciens), the cauli flower mosaic virus (CaMV) 19S and 35S promoters, the TABLE 1.4 light-inducible promoter from the small subunit of ribulose 1.5-bisphosphate carboxylase (ssRUBISCO, a very abundant STRAINS CONTAINING CRYET70-RELATED GENES plant polypeptide), and the Figwort Mosaic Virus (FMV)35S Strains Southern blot Western blot % Control WCRW promoter. All of these promoters have been used to create various types of DNA constructs which have been expressed EG2929 -- -- 26 in plants (see e.g., U.S. Pat. No. 5,463,175, specifically incor EG3218 -- 30 EG3221 -- 63 porated herein by reference). EG3303 -- 15 0360. The particular promoter selected should be capable EG3304 -- O of causing Sufficient expression of the enzyme coding EG3707 -- 45 sequence to result in the production of an effective amount of EG38O3 O EG3953 -- 100 protein. One set of preferred promoters are constitutive pro EG3966 -- 7 moters such as the CaMV35S or FMV35S promoters that EG4113 40 yield high levels of expression in most plant organs (U.S. Pat. EG413S -- -- 45 No. 5,378,619, specifically incorporated herein by refer EG41SO 64 EG4268 -- 46 ence). Another set of preferred promoters are root enhanced EG4375 100 or specific promoters such as the CaMV derived 4 as-1 pro EG4447 -- O moter or the wheat POX1 promoter (U.S. Pat. No. 5,023,179, specifically incorporated herein by reference: Hertig et al., US 2009/0087863 A1 Apr. 2, 2009

1991). The root enhanced or specific promoters would be region encoding the insecticidal polypeptide must have a particularly preferred for the control of corn rootworm (Di suitable sequence composition (Diehn et al., 1996). abroticus spp.) in transgenic corn plants. 0367 To place a cry gene in a vector suitable for expres 0361. The promoters used in the DNA constructs of the sion in monocotyledonous plants (i.e. under control of the present invention may be modified, if desired, to affect their enhanced Cauliflower Mosaic Virus 35S promoter and link to control characteristics. For example, the CaMV35S promoter the hsp70 intron followed by a nopaline synthase polyadeny may be ligated to the portion of the ssRUBISCO gene that represses the expression of ssRUBISCO in the absence of lation site as in U.S. Pat. No. 5,424,412, specifically incorpo light, to create a promoter which is active in leaves but not in rated herein by reference), the vector is digested with appro roots. The resulting chimeric promoter may be used as priate enzymes such as NcoI and EcoRI. The larger vector described herein. For purposes of this description, the phrase band of approximately 4.6 kb is then electrophoresed, puri “CaMV35S' promoter thus includes variations of CaMV35S fied, and ligated with T4 DNA ligase to the appropriate promoter, e.g., promoters derived by means of ligation with restriction fragment containing the plantized cry gene. The operator regions, random or controlled mutagenesis, etc. Fur ligation mix is then transformed into E. coli, carbenicillin thermore, the promoters may be altered to contain multiple resistant colonies recovered and plasmid DNA recovered by "enhancer sequences' to assist in elevating gene expression. DNA miniprep procedures. The DNA may then be subjected 0362. The RNA produced by a DNA construct of the to restriction endonuclease analysis with enzymes such as present invention also contains a 5' non-translated leader NcoI and EcoRI (together), NotI, and PstI to identify clones sequence. This sequence can be derived from the promoter containing the cry gene coding sequence fused to the hsp70 selected to express the gene, and can be specifically modified intron under control of the enhanced CaMV35S promoter). so as to increase translation of the mRNA. The 5' non-trans 0368 To place the gene in a vector suitable for recovery of lated regions can also be obtained from viral RNAs, from stably transformed and insect resistant plants, the restriction Suitable eucaryotic genes, or from a synthetic gene sequence. fragment from pMON33708 containing the lysine oxidase The present invention is not limited to constructs wherein the coding sequencefused to the hsp70 intron under control of the non-translated region is derived from the 5' non-translated sequence that accompanies the promoter sequence. enhanced CaMV35S promoter may be isolated by gel elec 0363 For optimized expression in monocotyledenous trophoresis and purification. This fragment can then be plants such as maize, an intron should also be included in the ligated with a vector such as pMON30460 treated with NotI DNA expression construct. This intron would typically be and calf intestinal alkaline phosphatase (pMON30460 con placed near the 5' end of the mRNA in untranslated sequence. tains the neomycin phosphotransferase coding sequence This intron could be obtained from, but not limited to, a set of under control of the CaMV35S promoter). Kanamycin resis introns consisting of the maize hsp70 intron (U.S. Pat. No. tant colonies may then be obtained by transformation of this 5,424,412; specifically incorporated herein by reference) or ligation mix into E. coli and colonies containing the resulting the rice Act 1 intron (McElroy et al., 1990). As shown below, plasmid can be identified by restriction endonuclease diges the maize hsp70 intron is useful in the present invention. tion of plasmid miniprep DNAS. Restriction enzymes such as 0364. As noted above, the 3' non-translated region of the NotI, EcoRV, HindIII, NcoI, EcoRI, and BglII can be used to chimeric plant genes of the present invention contains a poly identify the appropriate clones containing the restriction frag adenylation signal which functions in plants to cause the ment properly inserted in the corresponding site of addition of adenylate nucleotides to the 3' end of the RNA. pMON30460, in the orientation such that both genes are in Examples of preferred 3' regions are (1) the 3' transcribed, tandem (i.e. the 3' end of the cry gene expression cassette is non-translated regions containing the polyadenylate signal of linked to the 5' end of the nptII expression cassette). Expres Agrobacterium tumor-inducing (Ti) plasmid genes, such as sion of the CryET70 polypeptide by the resulting vector is the nopaline synthase (NOS) gene and (2) plant genes such as then confirmed in plant protoplasts by electroporation of the the pea ssRUBISCO E9 gene (Fischhoffet al., 1987). vector into protoplasts followed by protein blot and ELISA analysis. This vector can be introduced into the genomic 5.12.2 Plant Transformation and Expression DNA of plant embryos such as maize by particle gun bom 0365 A transgene containing a structural coding sequence bardment followed by paromomycin selection to obtain corn of the present invention can be inserted into the genome of a plants expressing the cry gene essentially as described in U.S. plant by any suitable method such as those detailed herein. Pat. No. 5,424,412, specifically incorporated herein by refer Suitable plant transformation vectors include those derived ence. In this example, the vector was introduced via cobom from a Tiplasmid of Agrobacterium tumefaciens, as well as bardment with a hygromycin resistance conferring plasmid those disclosed, e.g., by Herrera-Estrella (1983), Bevan into immature embryo scutella (IES) of maize, followed by (1983), Klee (1985) and Eur. Pat Appl. Publ. No. EP0120516. hygromycin selection, and regeneration. Transgenic corn In addition to plant transformation vectors derived from the Ti lines expressing the CryET70 polypeptide are then identified or root-inducing (R1) plasmids of Agrobacterium, alternative by ELISA analysis. Progeny seed from these events are then methods can be used to insert the DNA constructs of this Subsequently tested for protection from Susceptible insect invention into plant cells. Such methods may involve, for feeding. example, the use of liposomes, electroporation, chemicals that increase free DNA uptake, free DNA delivery via micro 5.13 Example 13 projectile bombardment, and transformation using viruses or pollen (Fromm et al., 1986; Armstrong et al., 1990: Fromm et Modification of CryET70 Genes for Expression in al., 1990). Plants 5.12.3 Construction of Plant Expression Vectors for CryET70 0369. Many wild-type genes encoding bacterial crystal Transgenes proteins are known to be expressed poorly in plants as a 0366 For efficient expression of the polynucleotides dis full-length gene or as a truncated gene. Typically, the G+C closed herein in transgenic plants, the selected sequence content of a cry gene is low (37%) and often contains many US 2009/0087863 A1 Apr. 2, 2009 46

A+Trich regions, potential polyadenylation sites and numer vector and compared for expression of the crystal protein by ous ATTTA sequences. Table 15 shows a list of potential Western blot or ELISA immunoassay in leaf and floral tissue. polyadenylation sequences which should be avoided when The FMV promoter has been used to produce relatively high preparing the “plantized' gene construct. levels of crystal protein infloral tissue compared to the CaMV promoter. TABL E 15 5.14 Example 14 LIST OF SEQUENCES OF POTENTIAL POLYADENYLATION SIGNALS Expression of Synthetic Cry Genes with AATAAA* AAGCAT ssRUBISCO Promoters and Chloroplast Transit Pep tides AATAATk ATTAAT 0373 The genes in plants encoding the small subunit of AACCAA ATACAT RUBISCO (SSU) are often highly expressed, light regulated and sometimes show tissue specificity. These expression ATATAA AAAATA properties are largely due to the promoter sequences of these AATCAA ATTAAAikik genes. It has been possible to use SSU promoters to express heterologous genes in transformed plants. Typically a plant ATACTA AATTAAikik will contain multiple SSU genes, and the expression levels and tissue specificity of different SSU genes will be different. ATAAAA AATACAkk The SSU proteins are encoded in the nucleus and synthesized ATGAAA CATAAAkk in the cytoplasm as precursors that contain an N-terminal * indicates a potential major plant polyadenylation extension known as the chloroplast transit peptide (CTP). The site. CTP directs the precursor to the chloroplast and promotes the * * indicates a potential minor animal polyadenyla uptake of the SSU protein into the chloroplast. In this process, tion site. the CTP is cleaved from the SSU protein. These CTP All others are potential minor plant polyadenyla sequences have been used to direct heterologous proteins into tion sites. chloroplasts of transformed plants. 0370. The regions for mutagenesis may be selected in the 0374. The SSU promoters might have several advantages following manner. All regions of the DNA sequence of the cry for expression of heterologous genes in plants. Some SSU gene are identified which contained five or more consecutive promoters are very highly expressed and could give rise to base pairs which were A or T. These were ranked in terms of expression levels as high or higher than those observed with length and highest percentage of A+T in the Surrounding the CaMV35S promoter. The tissue distribution of expression sequence over a 20-30 base pair region. The DNA is analysed from SSU promoters is different from that of the CaMV35S for regions which might contain polyadenylation sites or promoter, so for control of some insect pests, it may be ATTTA sequences. Oligonucleotides are then designed advantageous to direct the expression of crystal proteins to which maximize the elimination of A+T consecutive regions those cells in which SSU is most highly expressed. For which contained one or more polyadenylation sites or ATTTA example, although relatively constitutive, in the leaf the sequences. Two potential plant polyadenylation sites have CaMV35S promoter is more highly expressed in vascular been shown to be more critical based on published reports. tissue than in some other parts of the leaf, while most SSU Codons are selected which increase G+C content, but do not promoters are most highly expressed in the mesophyll cells of generate restriction sites for enzymes useful for cloning and the leaf. Some SSU promoters also are more highly tissue assembly of the modified gene (egg, BamHI, BglII, SacI. specific, so it could be possible to utilize a specific SSU NcoI, EcoRV, etc.). Likewise condons are avoided which promoter to express the protein of the present invention in contain the doublets TA or GC which have been reported to be only a Subset of plant tissues, if for example expression of infrequently-found codons in plants. such a protein in certain cells was found to be deleterious to 0371 Although the CaMV35S promoter is generally a those cells. For example, for control of Colorado potato beetle high level constitutive promoter in most plant tissues, the in potato, it may be advantageous to use SSU promoters to expression level of genes driven the CaMV35S promoter is direct crystal protein expression to the leaves but not to the low in floral tissue relative to the levels seen in leaf tissue. edible tubers. Because the economically important targets damaged by 0375 Utilizing SSU CTP sequences to localize crystal some insects are the floral parts or derived from floral parts proteins to the chloroplast might also be advantageous. (e.g., cotton Squares and boils, tobacco buds, tomato buds and Localization of the B. thuringiensis crystal proteins to the fruit), it is often advantageous to increase the expression of chloroplast could protect these from proteases found in the crystal proteins in these tissues over that obtained with the cytoplasm. This could stabilize the proteins and lead to higher CaMV35S promoter. levels of accumulation of active toxin. cry genes containing 0372. The 35S promoter of Figwort Mosaic Virus (FMV) the CTP may be used in combination with the SSU promoter is analogous to the CaMV35S promoter. This promoter has or with other promoters such as CaMV35S. been isolated and engineered into a plant transformation vec tor. Relative to the CaMV promoter, the FMV 35S promoter 5.15 Example 15 is highly expressed in the floral tissue, while still providing Targeting of CryET70 Polypeptides to the Extracel similar high levels of gene expression in other tissues such as lular Space or Vacuole Using Signal Peptides leaf. A plant transformation vector, may be constructed in which the full length synthetic cry gene is driven by the FMV 0376. The B. thuringiensis polypeptides described here 35S promoter. Tobacco plants may be transformed with the are primarily localized to the cytoplasm of the plant cell, and US 2009/0087863 A1 Apr. 2, 2009 47 this cytoplasmic localization results in plants that are insec like compartment found in seeds. A signal peptide DNA ticidally effective. However, in certain embodiments, it may sequence for the B-subunit of the 7S storage protein of com be advantageous to direct the B. thuringiensis polypeptide to mon bean (Phaseolus vulgaris), PvuB has been described other compartments of the plant cell. Localizing B. thuring (Doyle et al., 1986). Based on the published these published iensis proteins incompartments other than the cytoplasm may sequences, genes may be synthesized chemically using oli result in less exposure of the B. thuringiensis proteins to gonucleotides that encode the sisal peptides for PR1b and cytoplasmic proteases leading to greater accumulation of the PvuB. In some cases to achieve secretion or compartmental protein yielding enhanced insecticidal activity. Extracellular ization of heterologous proteins, it may be necessary to localization could lead to more efficient exposure of certain include some amino acid sequence beyond the normal cleav insects to the B. thuringiensis proteins leading to greater age site of the signal peptide. This may be necessary to insure efficacy. If a B. thuringiensis protein were found to be delete proper cleavage of the signal peptide. rious to plant cell function, then localization to a noncyto plasmic compartment could protect these cells from the pro 6.O REFERENCES tein. 0380. The following references, to the extent that they 0377. In plants as well as other eukaryotes, proteins that provide exemplary procedural or other details Supplementary are destined to be localized either extracellularly or in several to those set forth herein, are specifically incorporated herein specific compartments are typically synthesized with an by reference: N-terminal amino acid extension known as the signal peptide. (0381 U.S. Pat. No. 4,196,265, issued Apr. 1, 1980. This signal peptide directs the protein to enter the compart 0382 U.S. Pat. No. 4,237,224, issued Dec. 2, 1980 mentalization pathway, and it is typically cleaved from the 0383 U.S. Pat. No. 4,554,101, issued Nov. 19, 1985. mature protein as an early step in compartmentalization. For 0384 U.S. Pat. No. 4,683,195, issued Jul. 28, 1987. an extracellular protein, the secretory pathway typically 0385 U.S. Pat. No. 4,683.202, issued Jul. 28, 1987. involves cotranslational insertion into the endoplasmic 0386 U.S. Pat. No. 4,757,011, issued Jul 12, 1988. reticulum with cleavage of the signal peptide occurring at this (0387 U.S. Pat. No. 4,769,061, issued Sep. 6, 1988. stage. The mature protein then passes through the Golgi body 0388 U.S. Pat. No. 4,800,159, issued Jan. 24, 1989. into vesicles that fuse with the plasma membrane thus releas 0389 U.S. Pat. No. 4,883,750, issued Nov. 28, 1989. ing the protein into the extracellular space. Proteins destined 0390 U.S. Pat. No. 4,940,835, issued Feb. 23, 1990. for other compartments follow a similar pathway. For 0391 U.S. Pat. No. 4,943,674, issued Jul. 24, 1990. example, proteins that are destined for the endoplasmic 0392 U.S. Pat. No. 4,965,188, issued Oct. 23, 1990. reticulum or the Golgi body follow this scheme, but they are 0393 U.S. Pat. No. 4,971,908, issued Nov. 20, 1990. specifically retained in the appropriate compartment. In 0394 U.S. Pat. No. 4,987,071, issued Jan. 22, 1991 plants, some proteins are also targeted to the vacuole, another 0395 U.S. Pat. No. 5,097,025, issued Mar. 17, 1992. membrane bound compartment in the cytoplasm of many 0396 U.S. Pat. No. 5,106,739, issued Apr. 21, 1992. plant cells. Vacuole targeted proteins diverge from the above 0397 U.S. Pat. No. 5,110.732, issued May 5, 1992. pathway at the Golgi body where they enter vesicles that fuse 0398 U.S. Pat. No. 5,139,954, issued Aug. 19, 1992. with the vacuole. 0399 U.S. Pat. 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SEQUENCE LISTING

<16 Oc NUMBER OF SEO ID NOS: 6

<210 SEQ ID NO 1 <211 LENGTH: 2344 &212> TYPE: DNA <213> ORGANISM: Baccillus thuringiensis &220s FEATURE: <221 NAME/KEY: CDS <222> LOCATION: (92) ... (2254)

<4 OO SEQUENCE: 1 gtaat agtag tt atttagca ggaataaaag ggagggitatic gaat actitt C aaatgalagac 60 tgaaaattta caaatagaag gagagaaaag. t atgaaa gat tca att toa aag 112 Met Lys Asp Ser Ile Ser Lys 1. 5

gga t at gat gala at a aca gtg cag gca agt gat tat att gat att CC 16 O Gly Tyr Asp Glu Ile Thr Val Glin Ala Ser Asp Tyr Ile Asp Ile Arg 10 15 2O

tda att titt caa acg aat gga t cit gca acattt aat tca acc act aat 2O8 Ser Ile Phe Glin Thr Asn Gly Ser Ala Thr Phe Asin Ser Thr Thr Asn 25 3 O 35

att aca act tta acg caa got aca aat agt caa goggga gca att gca 256 Ile Thr Thr Lieu. Thr Glin Ala Thr Asn Ser Glin Ala Gly Ala Ile Ala 4 O 45 SO 55

US 2009/0087863 A1 Apr. 2, 2009 58

- Continued ccg gag gaa cca cca gtt ttcaat aga citt att agt aca ggg aaa ttg 2128 Pro Glu Glu Pro Pro Val Phe Asn Arg Lieu. Ile Ser Thr Gly Lys Lieu. 665 670 675 gtg gala aaa gac tat aga gaa tta aaa gga aca ttic ct gta acg gala 2176 Val Glu Lys Asp Tyr Arg Glu Lieu Lys Gly Thr Phe Arg Val Thr Glu 68O 685 690 695 tta aac caa goa cca ttgata atc gta gag aat titt ggit gct gga tat 2224 Lieu. Asn Glin Ala Pro Lieu. Ile Ile Val Glu Asn. Phe Gly Ala Gly Tyr 7 OO 7Os 71O ata ggit gga att aga att gtg aaa at a tog taataaaaac agataaaata 2274 Ile Gly Gly Ile Arg Ile Val Lys Ile Ser 71s 72 O gaga.gaggaa cqctggatag cqttic ct citt ttcagtt tag titac Catct C Caggit ccag 2334 tt catttitt t 2344

<210 SEQ ID NO 2 <211 LENGTH: 721 &212> TYPE: PRT <213> ORGANISM: Baccillus thuringiensis

<4 OO SEQUENCE: 2 Met Lys Asp Ser Ile Ser Lys Gly Tyr Asp Glu Ile Thr Val Glin Ala 1. 5 1O 15 Ser Asp Tyr Ile Asp Ile Arg Ser Ile Phe Gln Thr ASn Gly Ser Ala 2O 25 3O

Thir Phe Asn. Ser Thir Thir Asn. Ile Thir Thir Lieu. Thir Glin Ala Thir Asn 35 4 O 45 Ser Glin Ala Gly Ala Ile Ala Gly Lys Thir Ala Lieu. Asp Met Arg His SO 55 6 O Asp Phe Thir Phe Arg Ala Asp Ile Phe Lieu. Gly. Thir Lys Ser Asn Gly 65 70 7s 8O Ala Asp Gly Ile Ala Ile Ala Phe His Arg Gly Ser Ile Gly Phe Val 85 90 95 Gly Glu Lys Gly Gly Gly Lieu. Gly Ile Lieu. Gly Ala Lieu Lys Gly Ile 1OO 105 11 O Gly Phe Glu Lieu. Asp Thr Tyr Ala Asn Ala Pro Glin Asp Glu Glin Gly 115 12 O 125 Asp Ser Phe Gly His Gly Ala Met Arg Gly Leu Phe Pro Gly Phe Pro 13 O 135 14 O Asn Gly Tyr Pro His Ala Gly Phe Val Ser Thr Asp Lys Asn Arg Gly 145 150 155 160 Trp Lieu. Ser Ala Lieu Ala Gln Met Glin Arg Ile Ala Ala Pro Asn Gly 1.65 17O 17s Arg Trp Arg Arg Lieu Ala Ile His Trp Asp Ala Arg Asn Llys Llys Lieu 18O 185 19 O Thir Ala Asn Lieu. Glu Asp Lieu. Thir Phe Asn Asp Ser Thr Val Lieu Val 195 2OO 2O5 Llys Pro Arg Thr Pro Arg Tyr Ala Arg Trp Glu Lieu. Ser Asn Pro Ala 21 O 215 22O Phe Glu Lieu. Asp Gln Lys Tyr Thr Phe Val Ile Gly Ser Ala Thr Gly 225 23 O 235 24 O Ala Ser Asn. Asn Lieu. His Glin Ile Gly Ile Ile Glu Phe Asp Ala Tyr 245 250 255 US 2009/0087863 A1 Apr. 2, 2009 59

- Continued

Phe Thr Llys Pro Thr Ile Glu Ala Asn Asn Val Ser Val Pro Val Gly 26 O 265 27 O Ala Thr Phe Asin Pro Llys Thr Tyr Pro Gly Ile Asn Lieu. Arg Ala Thr 27s 28O 285 Asp Glu Ile Asp Gly Asp Lieu. Thir Ser Glu Ile Ile Val Thr Asp Asn 29 O 295 3 OO Asn Val Asn Thr Ser Lys Ser Gly Val Tyr Asn Val Thr Tyr Tyr Val 3. OS 310 315 32O Lys Asn Ser Tyr Gly Glu Ser Asp Glu Lys Thr Ile Glu Val Thr Val 3.25 330 335 Phe Ser Asn Pro Thr Ile Ile Ala Ser Asp Val Glu Ile Glu Lys Gly 34 O 345 35. O Glu Ser Phe Asin Pro Lieu. Thir Asp Ser Arg Val Arg Lieu. Ser Ala Glin 355 360 365 Asp Ser Lieu. Gly Asn Asp Ile Thir Ser Llys Val Llys Wall Lys Ser Ser 37 O 375 38O Asn Val Asp Thr Ser Lys Pro Gly Glu Tyr Asp Val Val Phe Glu Val 385 390 395 4 OO Thir Asp Asn. Phe Gly Gly Lys Ala Glu Lys Glu Ile Llys Val Thr Val 4 OS 41O 415 Lieu. Gly Glin Pro Ser Ile Glu Ala Asn Asp Val Glu Lieu. Glu Ile Gly 42O 425 43 O Asp Lieu. Phe Asn Pro Lieu. Thir Asp Ser Glin Val Gly Lieu. Arg Ala Lys 435 44 O 445 Asp Ser Lieu. Gly Lys Asp Ile Thr Asn Asp Val Llys Wall Lys Ser Ser 450 45.5 460 Asn Val Asp Thr Ser Lys Pro Gly Glu Tyr Glu Val Val Phe Glu Val 465 470 47s 48O Thir Asp Arg Phe Gly Llys Lys Ala Glu Lys Ser Ile Llys Val Lieu Val 485 490 495 Lieu. Gly Glu Pro Ser Ile Glu Ala Asn. Asn Val Glu Ile Glu Lys Asp SOO 505 51O Glu Arg Phe Asp Pro Lieu. Thir Asp Ser Arg Val Gly Lieu. Arg Ala Lys 515 52O 525 Asp Ser Lieu. Gly Lys Asp Ile Thr Asn Asp Val Llys Wall Lys Ser Ser 53 O 535 54 O Asn Val Asp Thr Ser Lys Pro Gly Glu Tyr Glu Val Val Phe Glu Val 5.45 550 555 560 Thir Asp Arg Phe Gly Llys Tyr Val Lys Llys Lieu. Ile Val Val Ile Val 565 st O sts Pro Val Ile Asp Asp Glu Trp Glu Asp Gly Asn Val Asn Gly Trp Llys 58O 585 59 O Phe Tyr Ala Gly Glin Asp Ile Thr Lieu. Lieu Lys Asp Pro Glu Lys Ala 595 6OO 605 Tyr Lys Gly Glu Tyr Val Phe Tyr Asp Ser Arg His Ala Ala Ile Ser 610 615 62O Lys. Thir Ile Pro Val Thr Asp Leu Glin Val Gly Gly Asn Tyr Glu Ile 625 630 635 64 O Thr Val Tyr Val Lys Ala Glu Ser Gly Asp His His Lieu Lys Val Thr 645 650 655 US 2009/0087863 A1 Apr. 2, 2009 60

- Continued Tyr Lys Lys Asp Pro Llys Gly Pro Glu Glu Pro Pro Val Phe Asn Arg 660 665 67 O

Lieu. Ile Ser Thr Gly Llys Lieu Val Glu Lys Asp Tyr Arg Glu Lieu Lys 675 68O 685

Gly Thr Phe Arg Val Thr Glu Lieu. Asn Glin Ala Pro Leu. Ile Ile Val 69 O. 695 7 OO

Glu Asn. Phe Gly Ala Gly Tyr Ile Gly Gly Ile Arg Ile Wall Lys Ile 7 Os 71O 71s 72O

Ser

<210 SEQ ID NO 3 <211 LENGTH: 13 &212> TYPE: PRT <213> ORGANISM: Bacillus thuringiensis

<4 OO SEQUENCE: 3 Ala Ser Asp Tyr Ile Asp Ile Arg Ser Ile Phe Glin Thr 1. 5 1O

<210 SEQ ID NO 4 <211 LENGTH: 38 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &22OY FEATURE: <223> OTHER INFORMATION: Cry ET7 O Oligonucleotide probe &220s FEATURE: <221 NAMEAKEY: modified base <222> LOCATION: (3) . . (6) &223> OTHER INFORMATION: N = Inosine &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: (3) . . (6) &223> OTHER INFORMATION: N = I

<4 OO SEQUENCE: 4 gcnt cngatt at attgat at tagat caatt tttcaaac 38

<210 SEQ ID NO 5 <211 LENGTH: 22 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Oligonucleotide probe to Cry ET70

<4 OO SEQUENCE: 5 gacatgattt tacttittaga gc 22

<210 SEQ ID NO 6 <211 LENGTH: 18 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Oligonucleotide probe to Cry ET70

<4 OO SEQUENCE: 6 catcacttitc cccatago 18 US 2009/0087863 A1 Apr. 2, 2009

1-22. (canceled) 25. An immunodetection kit comprising, in Suitable con 23. A purified antibody that specifically binds to a polypep tainer means, the antibody of claim 23 and an immunodetec tide comprising an amino acid sequence that is at least 99% tion reagent. identical to SEQID NO:2. 24. A method for detecting a 6-endotoxin polypeptide in a 26-101. (canceled) biological sample, comprising the steps of 102. The antibody of claim 23, wherein said antibody is a) obtaining a biological sample Suspected of containing a generated by using said polypeptide as an immunogen. Ö-endotoxin polypeptide; 103. The antibody of claim 23, wherein said antibody is b) contacting said sample with the antibody of claim 23, produced by a hybridoma, said hybridoma being generated under conditions effective to allow the formation of using said polypeptide. complexes; and c) detecting the complexes so formed. c c c c c