MXPA01011313A - Coleopteran-toxic polypeptide compositions and insect-resistant transgenic plants - Google Patents
Coleopteran-toxic polypeptide compositions and insect-resistant transgenic plantsInfo
- Publication number
- MXPA01011313A MXPA01011313A MXPA/A/2001/011313A MXPA01011313A MXPA01011313A MX PA01011313 A MXPA01011313 A MX PA01011313A MX PA01011313 A MXPA01011313 A MX PA01011313A MX PA01011313 A MXPA01011313 A MX PA01011313A
- Authority
- MX
- Mexico
- Prior art keywords
- seq
- nrrl
- plant
- cell
- polypeptide
- Prior art date
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Abstract
Disclosed are novel insecticidal polypeptides, and compositions comprising these polypeptides, peptide fragments thereof, and antibodies specific therefor. Also disclosed are vectors, transformed host cells, and transgenic plants that contain nucleic acid segments that encode the disclosed&dgr;-endotoxin polypeptides. Also disclosed are methods of identifying related polypeptides and polynucleotides, methods of making and using transgenic cells comprising these polynucleotide sequences, as well as methods for controlling an insect population, such as Colorado potato beetle, southern corn rootworm and western corn rootworm, and for conferring to a plant resistance to a target insect species.
Description
COMPOSITIONS OF POLYPEPTIDES TOXICES FOR COLEOPTERS AND TRANSGENIC PLANTS RESISTANT TO INSECTS
This request is based on U.S. Provisional Application No. 60 / 172,240, filed May 4, 1999, the entire contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
The present invention generally relates to the fields of molecular biology. Most particularly, certain embodiments refer to methods and compositions comprising segments of DNA, and proteins derived from bacterial species. Very particularly, it refers to novel genes of Bacillus thuringiensis that encode proteins of toxic crystals to coleoptera. Various methods for making and using these DNA segments, DNA segments encoding synthetically modified d-endotoxin polypeptides, and native and synthetic crystal proteins are described, such as, for example, the use of DNA segments as diagnostic probes and templates for protein production, and the use of proteins, fusion protein carriers and peptides in various immunological and diagnostic applications. Methods for making and using nucleic acid segments in the development of transgenic plant cells containing the polynucleotides described herein are also described.
DESCRIPTION OF THE RELATED TECHNIQUE
Because crops of commercial interest are often the target of insect attack, environmentally sensitive methods to control or eradicate insect infestation are desirable in many cases. This is particularly true for farmers, nursery growers, growers, and commercial and residential areas who seek to control insect populations using environmentally friendly compositions. The most widely used environmentally-sensitive insecticidal formulations developed in recent years have been composed of microbial pesticides derived from the bacterium Bacillus thuringiensis. Bacillus thuringiensis is a gram-positive bacterium that produces crystal proteins or inclusion bodies that are specifically toxic to certain orders and species of insects. It has been shown that many different strains of B. thuringiensis produce insecticidal crystal proteins. Compositions that include strains of B. thuringiensis that produce insecticidal proteins have been commercially available and are used as environmentally acceptable insecticides because they are very toxic to the specific target insect, but are harmless to plants and other non-target organisms.
1. 2.1 d-endotoxins The d-endotoxins are used to control a wide range of caterpillars and beetles that feed on leaves, as well as mosquitoes. These parasporal proteinaceous crystals, also known as insecticidal crystal proteins, crystal proteins, Bt inclusions, crystalline inclusions, inclusion bodies and Bt toinas, are a large collection of insecticidal proteins produced by B. thuringiensis that are toxic when ingested by a susceptible host insect. In the past decade, research on the structure and function of B. thuringiensis toxins has covered all categories of important toxins, and although these toxins differ in specific structure and function, general similarities in structure and function are assumed. Based on the accumulated knowledge of B. thuringiensis toinas, a generalized mode of action of B. thuringiensis toxins has been created and includes: ingestion by the insect, solubilization in the insect's midgut (a combination of stomach and intestine) thin), resistance to digestive enzymes sometimes with partial digestion that actually "activates" the toxin, binding to the cells of the midgut, formation of a pore in the cells of the insect and alteration of cellular homeostasis (English and Slatin, 1992).
One of the features Unique to B. thuringiensis is its production of crystal proteins during sporulation that are specifically toxic to certain orders and insect species. Many different strains of B. thuringiensis have been shown to produce insecticidal crystal proteins. Compositions that include strains of B. thuringiensis that produce proteins that have insecticidal activity against lepidoptera and dipterous insects have been commercially available and have been used as environmentally acceptable insecticides because they are very toxic to the specific target insect., but are innocuous for plants and other non-target organisms. The mechanism of insecticidal activity of the crystal proteins of
B. thuringiensis has been studied extensively in the past decade. It has been shown that crystal proteins are toxic to the insect only after the protein is ingested by the insect. The alkaline pH and proteolytic enzymes in the insect's midgut solubilize the proteins, thus allowing the release of components that are toxic to the insect. These toxic components alter the cells of the midgut, causing the insect to stop feeding and finally leading the insect to death. For this reason, it has been proven that B. thuringiensis is an effective and environmentally safe insecticide to treat several insect pests. As indicated by Hófte et al., (1989), most strains of B. thuringiensis insecticides are active against insects of the order Lepidoptera, that is, caterpillar insects. Other strains of B. thuringiensis are insecticidally active against insects of the order Diptera, that is, flies and mosquitoes, or against other lepidoptera and diptera. In recent years, some strains of B. thuringiensis have been reported to produce crystal proteins that are toxic to insects of the coleoptera order, ie, beetles (Krieg ef al., 1983; Sick et al., 1990; Donovan et al., 1992; Lambert et al., 1992a; 1992b).
1. 2.2 Genes that encode crystal proteins Many of the d-endotoxins are related to several degrees by similarities in their amino acid sequences. Historically, proteins and the genes that encode them were classified based largely on their spectrum of insecticidal activity. The review by Hófte and Whiteley (1989) describes the genes and proteins that were identified in B. thuringiensis before 1990, and exposes the nomenclature and classification scheme that has traditionally been applied to genes and proteins of B. thuringiensis. Cryl genes encode Cryl proteins that are toxic to lepidoptera, and cryll genes encode Cryll proteins that are toxic to both lepidoptera and dipterans. The crylll genes encode Crylll proteins toxic to Coleoptera, while the crylV genes encode CrylV proteins for dipterans. Based on the degree of sequence similarity, the proteins were classified into subfamilies; the most highly related proteins within each family were assigned divisional letters such as CrylA, CrylB, CrylC, etc.
More and more closely related proteins within each division were given names such as CrylCI, CrylC2, etc. Recently, a new nomenclature was developed that systematically classified Cry proteins based on amino acid sequence homology rather than on insect target specificities (Crickmore et al., 1998). The classification scheme for many known toxins, not including allelic variations in individual proteins, is summarized in Section 4.3.
1. 2.3 Glass proteins toxic to coleopteran insects The cloning and expression of the cry3Bb gene has been described (Donovan et al., 1992). This gene encodes a 74-kDa protein that has insecticidal activity against coleoptera such as the Colorado potato beetle (CPB), and the southern corn rootworm (SCRW). A strain of B. thuringiensis, PS201T6, which was reported to have activity against western corn rootworm (WCRW, Diabrotica virgifera virgifera) was described in the U.S. patent. 5,436,002 (specifically incorporated herein by reference in its entirety). This strain also showed activity against Musca domestica, Aedes aegypti and Liriomyza trifoli. The cloning and expression of the cryET29 gene has also been described
(International Patent Application Ser. No. WO 97/17507, 1997). This gene encodes a 25-kDa protein that is active against coleopteran insects, particularly the CPB, SCRW, WCRW, and cat flea Ctenocephalides felis. The cloning and expression of the cryET33 and cryET34 genes have been described (International Patent Application Ser. No. WO 97/17600, 1997). These genes encode SIMBOLO30 and SIMBOL015 kDa proteins, respectively, and are active against coleopteran insects, particularly CPB larvae and the Japanese beetle (Popillia japonica). The viplA gene, which produces a soluble, vegetative insecticidal protein, has been cloned and sequenced (International Patent Application Ser. No. WO 96/10083, 1996). This gene encodes a protein of approximately 80 kDa, which is active against WCRW and the northern corn rootworm (NCRW). Another active endotoxin against coleopteran insects, including WCRW, is Cryl la (International Patent Application Ser. No. WO 90/13651, 1990). The gene encoding this 81-kDa polypeptide has been cloned and sequenced. Additional crystal proteins with toxicity to WCRW have been described (International Patent Application Ser. No. WO 97/40162, 1997). These proteins appear to function as binary toxins and show sequence similarity to mosquitocidal proteins isolated from B. sphaericus. Some strains of B. sphaericus are highly active against mosquito larvae, many of which, under sporulation, produce a crystalline inclusion composed of two protein toxins. The analysis of the genes encoding these proteins has been described by Baumann et al., (1988). Toxins are designated with P51 and P42 based on their predicted molecular masses of 51.4- and 41.9-kDa, respectively, the P42 protein alone is weakly active against mosquito larvae. The P51 protein has no mosquitocida activity by itself. Both P51 and P42 are required for complete insecticidal activity. There are no reports of B. sphaericus crystal proteins having activity on any insects other than mosquitoes (for a recent review see Charles et al., 1996a; 1996b). A second class of mosquitocidal protein toxins are produced by some strains of B. sphaericus. These proteins, known as Mtx toxins, are produced during vegetative growth and do not form a crystalline inclusion. The two Mtx toxins that have been identified, designated Mtx and Mtx2, have molecular masses of 100 and 30.8 kDa, respectively. The cloning and sequencing of the genes of these toxins, designated mtx and mtx2, has been described (Thanabalu et al., 1991, Thanabalu and Porter, 1995). The Mtx and Mtx2 proteins do not share sequence similarity with any other known insecticidal protein, including the crystal proteins of B. sphaericus and B. thuringiensis.
BRIEF DESCRIPTION OF THE INVENTION
The present invention provides novel insecticidal polypeptides and DNA sequences encoding them. For five of these polypeptides, their dissimilarity to known crystal proteins indicates the existence of a new class or subclass of B. thuringiensis crystal proteins, since they share less than 65% amino acid sequence identity with any of the polypeptides. currently known insecticides. The invention also provides novel polypeptides which, when combined, produce insecticidally active crystal proteins. Transformed host cells, transgenic plants, vectors and methods for making and using the novel polypeptides and polynucleotides are also provided. In a first embodiment, the invention provides an isolated CryET69 polypeptide comprising at least 7 contiguous amino acids of SEQ ID NO: 14. Most preferably, the polypeptide comprises at least 9 or at least 11 contiguous amino acids of SEQ ID NO: 14 Most preferably still, the polypeptide comprises at least 13 or at least 15 contiguous amino acids of SEQ ID NO: 14, and most preferably comprises at least 17 or at least 19 contiguous amino acids of SEQ ID NO: 14. In a Illustrative embodiment, the polypeptide comprises the sequence SEQ ID NO: 14. Said polypeptide is preferably encoded by a nucleic acid segment comprising at least one contiguous nucleotide sequence of at least 45 base pairs of SEQ ID NO: 13, and most preferably is encoded by a nucleic acid segment comprising a contiguous sequence of at least 90 base pairs of SEQ ID NO: 13. Most preferably still, said polypeptide is The invention is directed to a nucleic acid segment comprising a contiguous sequence of at least 150 base pairs of SEQ ID NO: 13. Illustrative polynucleotides encoding the insecticidal polypeptide comprise a contiguous nucleotide sequence of at least 300 base pairs of SEQ. ID NO: 13, and in one embodiment comprises the nucleotide sequence of SEQ ID NO: 13. Also described and claimed is an isolated CryET84 polypeptide comprising at least 15 contiguous amino acids of SEQ ID NO: 19. Most preferably, the polypeptide comprises at least 30 to 45 contiguous amino acids of SEQ ID NO: 19. Most preferably still, the polypeptide comprises at least 45 to 90 contiguous amino acids of SEQ ID NO: 19, and most preferably comprises at least 90 to 150 contiguous amino acids of SEQ ID NO: 19. In an exemplary embodiment, the polypeptide comprises the sequence SEQ ID NO. : 19. Said polypeptide is preferably encoded by a nucleic acid segment comprising at least one contiguous nucleotide sequence of at least 45 base pairs of SEQ ID NO: 18, and most preferably is encoded by a nucleic acid segment comprising a contiguous sequence of at least 90 base pairs of SEQ ID NO: 18. Most preferably still, said polypeptide is encoded by a nucleic acid segment which comprises a contiguous sequence of at least 150 base pairs of SEQ ID NO: 18. Illustrative polynucleotides encoding the insecticidal polypeptide comprise a contiguous nucleotide sequence of at least 300 base pairs of SEQ ID NO: 18, and in a embodiment comprises the nucleotide sequence of SEQ ID NO: 18. An isolated CryET75 polypeptide comprising at least 15 contiguous amino acids of SEQ ID NO: 16 is also described and claimed. Most preferably, the polypeptide comprises at least 30 to 45 contiguous amino acids of SEQ ID NO: 16. Most preferably still, the polypeptide comprises at least 45 to 90 contiguous amino acids of SEQ ID NO: 16, and most preferably comprises at least 90 to 150 contiguous amino acids of SEQ ID NO: 16. In an illustrative embodiment, the polypeptide comprises the sequence SEQ ID NO: 16. Said polypeptide is preferably encoded by a nucleic acid segment comprising a nucleotide sequence. contiguous residues of at least 45 base pairs of SEQ ID NO: 15, and most preferably is encoded by a nucleic acid segment comprising at least one contiguous sequence of at least 90 base pairs of SEQ ID NO: 15. Most preferably still, said polypeptide is encoded by a nucleic acid segment that comprises a contiguous sequence of at least 150 base pairs of SEQ ID NO: 15. Illustrative polynucleotides encoding the insecticidal polypeptide comprise a contiguous nucleotide sequence of at least 300 base pairs of SEQ ID NO: 15, and in a embodiment comprises the nucleotide sequence of SEQ ID NO: 15. In another embodiment, the invention describes and claims an isolated CryETdO polypeptide comprising at least 17 contiguous amino acids of SEQ ID NO: 4. Most preferably, the polypeptide comprises at least 20 or at least 23 contiguous amino acids of SEQ ID NO: 4. Most preferably still, the polypeptide comprises at least 26 or at least 29 contiguous amino acids of S EQ ID NO: 4, and most preferably comprises at least 32 or at least 35 contiguous amino acids of SEQ ID NO: 4. In an exemplary embodiment, the polypeptide comprises the sequence SEQ ID NO: 4. Said polypeptide is preferably encoded by a nucleic acid segment comprising at least one contiguous nucleotide sequence of at least 51 base pairs of SEQ ID NO: 3, and most preferably is encoded by a nucleic acid segment comprising a contiguous sequence of at least 60 base pairs of SEQ ID NO: 3. Most preferably still, said polypeptide is encoded by a nucleic acid segment comprising a contiguous sequence of at least 78 base pairs of SEQ ID NO: 3. Illustrative polynucleotides encoding the The insecticidal polypeptide comprises a contiguous nucleotide sequence of at least 96 base pairs of SEQ ID NO: 3, and in one embodiment comprises the nucleotide sequence of SEQ ID NO: 3. In another embodiment, the invention provides an isolated CryET76 polypeptide comprising at least 55 contiguous amino acids of SEQ ID NO: 2. Most preferably, the polypeptide comprises at least 60 or at least 70 contiguous amino acids of SEQ ID NO: 2. Most preferably still, the polypeptide comprises at least 75 or at least 80 contiguous amino acids of SEQ ID NO: 2, and most preferably comprises at least 85 or at least 90 contiguous amino acids of SEQ ID NO: 2. In an illustrative embodiment, the polypeptide comprises the sequence SEQ ID NO: 2. Said polypeptide is preferably encoded by a nucleic acid segment comprising at least one contiguous nucleotide sequence of at least 165 base pairs of SEQ ID NO: 1, and most preferably is encoded by a nucleic acid segment comprising a contiguous sequence of at least 180 base pairs of SEQ ID NO: 1. Most preferably still, said polypeptide is encoded by a nucleic acid segment comprising a contiguous sequence of at least 225 base pairs of SEQ ID NO: 1. Illustrative polynucleotides encoding the insecticidal polypeptide comprise a contiguous nucleotide sequence of at least 270 base pairs of SEQ ID NO: 1, and in one embodiment comprises the nucleotide sequence of SEQ ID NO: 1. In another embodiment, the invention describes and claims an isolated CryET71 polypeptide comprising at least 146 contiguous amino acids of SEQ ID NO: 12. Most preferably, the polypeptide comprises at least 150 or at least 154 contiguous amino acids of SEQ ID NO: 12. Most preferably still, the polypeptide comprises at least 158 or at least 162 contiguous amino acids of SEQ ID NO: 12, and most preferably comprises at least 166 or at least 170 contiguous amino acids of SEQ ID NO: 12. In an exemplary embodiment, the polypeptide comprises the sequence SEQ ID NO: 12. Said preferred polypeptide is encoded by a nucleic acid segment comprising at least one contiguous nucleotide sequence of at least 438 base pairs of SEQ ID NO: 11, and most preferably is encoded by a nucleic acid segment comprising a contiguous sequence of at least 450 base pairs of SEQ ID NO: 11. Most preferably still, said polypeptide is encoded by a nucleic acid segment comprising a sequence contiguous of at least 462 base pairs of SEQ ID NO: 11. Illustrative polynucleotides encoding the insecticidal polypeptide comprise a contiguous nucleotide sequence of at least 510 base pairs of SEQ ID NO: 11, and in one embodiment comprises nucleotide sequence of SEQ ID NO: 11. The invention also provides an isolated CryET74 polypeptide comprising the sequence of SEQ ID NO: 6. Said polypeptide is preferably encoded by a nucleic acid segment comprising a contiguous nucleotide sequence of at least one nucleic acid sequence. minus 45 base pairs of SEQ ID NO: 5; and most preferably is encoded by a nucleic acid segment comprising a contiguous sequence of at least 90 base pairs of SEQ ID NO: 5. Most preferably still, said polypeptide is encoded by a nucleic acid segment comprising an adjoining sequence. of at least 150 base pairs of SEQ ID NO: 5. Illustrative polynucleotides encoding the insecticidal polypeptide comprise a contiguous nucleotide sequence of at least 300 base pairs of SEQ ID NO: 5, and in one embodiment comprises the sequence In addition, the present invention provides an isolated CryET39 polypeptide comprising the sequence of SEQ ID NO: 8. Said polypeptide is preferably encoded by a nucleic acid segment comprising a contiguous nucleotide sequence of polynucleotides of SEQ ID NO: 5. at least 45 base pairs of SEQ ID NO: 7; and most preferably is encoded by a nucleic acid segment comprising a contiguous sequence of at least 90 base pairs of SEQ ID NO: 7. Most preferably still, said polypeptide is encoded by a nucleic acid segment comprising an adjoining sequence. of at least 150 base pairs of SEQ ID NO: 7. Illustrative polynucleotides encoding the insecticidal polypeptide comprise a contiguous nucleotide sequence of at least 300 base pairs of SEQ ID NO: 7, and in one embodiment comprises the sequence of nucleotides of SEQ ID NO: 7. Also, the invention provides an isolated CryET79 polypeptide comprising the sequence of SEQ ID NO: 10. Said polypeptide is preferably encoded by a nucleic acid segment comprising a contiguous nucleotide sequence of at least one nucleic acid sequence. minus 45 base pairs of SEQ ID NO: 9, and most preferably is encoded by a nucleic acid segment comprising a contiguous sequence of p or at least 90 base pairs of SEQ ID NO: 9. Most preferably still, said polypeptide is encoded by a nucleic acid segment comprising a contiguous sequence of at least 150 base pairs of SEQ ID NO: 9. Illustrative polynucleotides which encode the insecticidal polypeptide comprises a contiguous nucleotide sequence of at least 300 base pairs of SEQ ID NO: 9, and in one embodiment comprises the nucleotide sequence of SEQ ID NO: 9. The invention also describes compositions and insecticidal formulations comprising one or more of the polypeptides described herein. Said composition may be a cell extract, cell suspension, cell homogenate, cell lysate, cell supernatant, cell filtrate or cell pellet of a bacterial cell comprising polyucleotides encoding said polypeptides. Exemplary bacterial cells that produce said polypeptides include β. thuringiensis EG4550 (deposited with the NRRL on May 30, 1997 as NRRL B-21784); EG5899 (deposited with the NRRL on May 30, 1997 as NRRL B-21783); EG11529 (deposited with the NRRL on February 12, 1998 as NRRL B-21917); EG4100 (deposited with the NRRL on May 30, 1997 as NRRL B-21786); EG11647 (deposited with the NRRL on May 30, 1997 as NRRL B-21787); EG9444 (deposited with the NRRL on May 30, 1997 as NRRL B-21785); EG11648 (deposited with the NRRL on May 30, 1997 as NRRL B-21788); EG4851 (deposited with the NRRL on February 12, 1998 as NRRL B-21915); and EG11658 (deposited with the NRRL on February 12, 1998 as NRRL B-21916).
The composition as described in detail later in this description can be formulated as a powder, fine powder, tablet, granule, spray, emulsion, colloid, solution or the like, and can be prepared by conventional means such as desiccation, lyophilization, homogenization extraction, filtration, centrifugation, sedimentation or concentration of a cell culture comprising the polypeptide. Preferably, said compositions can be obtained from one or more cultures of the B. thuringiensis cells described herein. In all those compositions containing at least one insecticidal polypeptide, the polypeptide can be present in a concentration of about 1% to about 99% by weight. An illustrative insecticidal polypeptide formulation can be prepared by a method comprising the steps of culturing a suitable B. thuringiensis cell under conditions effective to produce the insecticidal polypeptide (s); and obtaining the insecticidal polypeptide (s) thus produced. For example, the invention describes and claims a method for preparing a d-endotoxin polypeptide having an insecticidal activity against a coleoptera or lepidopteran insect. The method generally involves isolating from an appropriate culture of B. thuringiensis cells that have been cultured under appropriate conditions one or more of the d-endotoxin polypeptides produced by the cells. Such polypeptides can be isolated from the cell culture or supernatant of cells or spore suspensions derived from the cell culture and used in the native form, or otherwise purified or concentrated as appropriate for the particular application. A method of controlling an insect population is also provided with this invention. The method generally involves contacting the population with an insecticidally effective amount of a polypeptide comprising the amino acid sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16 or 19. Such methods can be use to annihilate or reduce the numbers of target insects in a given area, or they can be applied prophylactically to an environmental area to avoid infestation by a susceptible insect. Preferably the insect ingests, or makes contact with, an insecticidally effective amount of the polypeptides. In addition, the invention provides a purified antibody that specifically binds to the insecticidal polypeptides described herein. Methods for preparing said antibody and methods for using the antibody to isolate, identify, characterize and / or purify polypeptides to which said antibody specifically binds are also provided. Immunological equipment and immunodetection methods useful in the identification of said polypeptides and peptide fragments and / or epitopes thereof are provided in detail herein, and also represent important aspects of the present invention. Such antibodies can be used to detect the presence of said polypeptides in a sample, or they can be used as described below in a variety of immunological methods. An illustrative method for detecting a d-endotoxin polypeptide in a biological sample generally involves obtaining a biological sample suspected of containing a d-endotoxin polypeptide; contacting the sample with an antibody that specifically binds to the polypeptide, under conditions effective to allow the formation of complexes; and detect the complexes thus formed. For such methods, the invention also provides immunodetection equipment. Said equipment generally contains, in a suitable container medium, an antibody that binds the d-endotoxin polypeptide, and at least a first immunodetection reagent. Optionally, the kit can provide additional reagents or instructions for using the antibody in the detection of d-endotoxin polypeptides in a sample. The preparation of said antibodies can be achieved using the polypeptides described as an antigen in an animal as described below. Antigenic epitopes, shorter peptides, peptide fusions, vehicle-bound peptide fragments and the like can also be generated from a whole polypeptide sequence or a portion thereof described herein. Another aspect of the invention relates to a biologically pure culture of a B. thuringiensis bacterium as shown in Table 9, deposited in the Agricultural Research Cultivation Deposit, Northern Regional Research Laboratory (NRRL).
A further embodiment of the invention relates to a vector comprising a sequence region encoding a polypeptide comprising one or more of the amino acid sequences described herein, a recombinant host cell transformed with said recombinant vector and biologically pure cultures of recombinant bacteria transformed with a polynucleotide sequence encoding the polypeptide described herein. All strains deposited in the NRRL are submitted to the Patent Crop Deposit under the terms of the Budapest Treaty, and feasibility statements in accordance with the BPM Intemaline Receipt Form is obtained. Described below are examples of vectors, recombinant host cells, transgenic cell lines, pluripotent plant cells and transgenic plants comprising at least a first sequence region encoding a polypeptide comprising one or more of the sequences described herein. In a further embodiment, the invention provides methods for preparing an insecticidal polypeptide composition. In illustrative embodiments, said polypeptides can be formulated for use as an insecticidal agent and can be used to control insect populations in an environment, including agricultural environments and the like. The formulations can be used to kill an insect, either by topical application or by ingestion of the polypeptide composition by the insect. In certain cases it may be convenient to formulate the polypeptides of the present invention to be applied to the soil, or on or near plants, trees, shrubs and the like, plants living in the vicinity, livestock animals, residences, farm equipment, buildings and the like. . The present invention also provides transformed host cells, pluripotent plant cell populations, embryonic plant tissue, plant callus, seedlings and transgenic plants comprising a selected sequence region encoding the insecticidal polypeptide.
Said cells preferably are prokaryotic or eukaryotic cells such as bacteria, fungi or plant cells, illustrative bacterial cells including B. thuringiensis, B. subtilis, B. megaterium, B. cereus, Escherichia,
Salmonella, Agrobacterium or Pseudomonas. The plants and plant host cells are preferably monocotyledonous or dicotyledonous plant cells such as corn, wheat, soybean, oat, cotton, rice, rye, sorghum, sugarcane, tomato, tobacco, kapok, flax, potato, barley, grass cells of peat, grass for pasture, berries, fruits, legumes, vegetables, ornamental plants, shrubs, cactus, succulents and trees. Exemplary transgenic plants of the present invention have preferably incorporated into their genome a selected polynucleotide (or "transgene"), comprising at least a first sequence region encoding one or more of the insecticidal polypeptides described herein.
Likewise, a progeny (descendant, next generations, etc.) of any generation of said transgenic plant also represents an important aspect of the invention. Preferably said progeny comprises the selected transgene and inherits the phenotypic trait of insect resistance demonstrated by the progenitor plant. A seed of any generation of said plants resistant to transgenic insects is also an important aspect of the invention. Preferably, the seed will also comprise the selected frang and will confer on the plants that grow from the seed the phenotypic trait of insect resistance. Insect-resistant, cross-bred transgenic plants comprising one or more transgenes encoding one or more of the polypeptides described herein can be prepared by a method that generally involves obtaining a fertile transgenic plant that contains a chromosomally incorporated transgene which encodes said insecticidal polypeptide; Operably linked to an active promoter in the plant; interlacing of the fertile transgenic plant with a second plant lacking the transgene to obtain a third plant comprising the transgene; and backcrossing from the third plant to obtain a backcrossed transgenic plant. In such cases, the transgene can be inherited through a male parent or through a female parent. The second plant can be endogamically crossed and the third plant can be a hybrid.
Also, an insect-resistant hybrid transgenic plant can be prepared by a method that generally involves crossing to a first and a second endogamically crossed plant, wherein one or both of the first or second endogamically cross plants comprises a chromosomally incorporated transgene that encodes the selected polypeptide operably linked to a promoter expressible in the plant expressing the transgene. In illustrative embodiments, the first and second endogamically crossed plants may be monocotyledonous plants selected from the group consisting of: maize, wheat, rice, barley, oats, rye, sorghum, peat grass and sugar cane. In a related embodiment, the invention also provides a method for preparing an insect resistant plant. The method generally involves contacting a recipient plant cell with a DNA composition comprising at least a first transgene encoding an insecticidal polypeptide under conditions that allow assimilation of the DNA composition; selecting a recipient cell comprising a chromosomally incorporated transgene encoding the polypeptide; regenerate a plant from the selected cell; and identifying a fertile transgenic plant having increased insect resistance in relation to the corresponding non-transformed plant. A method for producing transgenic seed generally involves obtaining a fertile transgenic plant comprising a chromosomally incorporated transgene encoding a polypeptide comprising one or more of the amino acid sequences described herein, operably linked to a promoter that expresses the transgene in a plant; and cultivate the plant under appropriate conditions to produce the transgenic seed. A method for producing progeny of any generation of a fertile transgenic plant increased in resistance to insects is also provided with this invention. The method generally involves harvesting transgenic seed from a transgenic plant comprising a chromosomally integrated transgene encoding said polypeptide, operably linked to a promoter that expresses the transgene in the plant; sow the collected transgenic seed; and cultivate the transgenic plants of seed progeny. These methods for creating transgenic plants, progeny and seed may involve contacting the plant cell with the DNA composition using one of the well-known methods for plant cell transformation such as microprojectile bombardment, electroporation or Agrobacterium-mediated transformation. These and other embodiments of the present invention will be apparent to those skilled in the art from the following examples and claims, which have benefit from the teachings of the present specification.
2. 1 Polynucleotide Segments The present invention provides nucleic acid segments that can be isolated from almost any source, which are free of total genomic DNA and which code for the novel insecticidal polypeptides and peptide fragments thereof which are described herein. The polynucleotides encoding these peptides and polypeptides can encode active insecticidal proteins or fragments of peptides, polypeptide subunits, functional domains or the like of one or more of CryET84, CryET80, CryET76, CryET71, CryET69, CryET75, CryET39, CryET79, CryET74 and proteins. of crystal related as - or the polypeptides described herein. In addition, the invention comprises nucleic acid segments that can be fully synthesized in vitro using methods that are well known to those skilled in the art encoding the novel polypeptides, peptides, peptide fragments, subunits or functional domains described herein. As used herein, the term "nucleic acid segments" or
"polynucleotide" refers to a nucleic acid molecule that has been isolated from total genomic DNA of particular species. Thus, a segment of nucleic acid or polynucleotide that encodes an endotoxin polypeptide refers to a nucleic acid molecule comprising at least one
The first sequence encoding a crystal protein but which is isolated from, or purified from, the genomic DNA of the species from which the nucleic acid segment is obtained, which in the present case is the genome of the bacterial gram-positive genus, Bacillus. , and in particular, the Bacillus species known as S. thuringiensis. Included within the term "nucleic acid segment" are the polynucleotide segments and smaller fragments of said segments, and also recombinant vectors including for example plasmids, phagemid cosmids, phages, virions, baculoviruses, artificial chromosomes, viruses and the like. Accordingly, polynucleotide sequences having between about 70% and about 80%, or most preferably between about 81% to about 90%, or most preferably still between about 91% to about 99% sequence identity of nucleic acids or functional equivalence to the polynucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO : 13, SEQ ID NO: 15 or SEQ ID NO: 18 will be the sequences that are "essentially as set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 18". Highly preferred sequences are those which are about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% adrenic or functionally equivalent to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 18. Other preferred sequences encoding the related polypeptide sequences are those which are approximately 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% identical or functionally equivalent to the polynucleotide sequence set forth in one or more of these sequence identifiers . Likewise, sequences that are approximately 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80% identical or functionally equivalent to the polynucleotide sequence exposed in one or more of these sequence identifiers is also contemplated as being useful in the practice of the present invention. Similarly, a polynucleotide comprising a gene or region of sequence isolated, purified or selected for a polynucleotide which may include in addition to peptide coding sequences, some other elements such as regulatory sequences, substantially isolated in isolation from other naturally occurring genes or protein coding sequences. In this regard, the term "gene" is used for purposes of simplicity to refer to a functional protein or polypeptide coding unit. As will be understood by those skilled in the art, this functional term includes genomic sequences, operator sequences and segments of smaller genetically engineered genes that express, or can be adapted to express proteins, polypeptides or peptides. In some embodiments, a nucleic acid segment will comprise at least a first gene encoding one or more of the polypeptides described herein. To allow expression of the gene and translation of the mRNA into mature polypeptide, the nucleic acid segment preferably also comprises at least a first promoter operably linked to the gene to express the gene product in a host cell transformed with the nucleic acid segment . The promoter may be an endogenous promoter or alternatively a heterologous promoter selected for its ability to promote expression of the gene in one or more particular cell types. For example, in the creation of transgenic plants and pluripotent plant cells comprising a selected gene, the heterologous promoter of choice is one that is expressible by the plant, and in many cases, preferably it can be a promoter expressible by the plant that is specific to tissue cycle or cell cycle. The selection of promoters expressible by the plant is well known to those skilled in the plant transformation art, and suitable illustrative promoters are described herein. In some embodiments, the promoter expressible by the plant can be selected from the group consisting of corn sucrose synthetase 1, corn alcohol dehydrogenase 1, corn light harvest complex, corn heat shock protein, RuBP carboxylase subunit small pea, synthase plasmid Ti, nopalin synthase of plasmid Ti, chalcone isomerase petunia, protein 1 rich in bean glycine, potato patatin, lectin, CaMV 35S and RuBp carboxylase promoter small subunit S-E9. "Sequentially isolated from other coding sequences" means that the gene of interest, in this case, a gene encoding a bacterial crystal protein, forms the significant part of the coding region of the DNA segment, and the DNA segment does not contains large portions of naturally occurring coding DNA, such as large chromosomal fragments or other functional genes or operon coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes, recombinant genes, synthetic linkers or coding regions added to the last segment by human hand. In particular embodiments, the invention relates to isolated polynucleotides (such as DNA, RNA, antisense DNA, antisense RNA, ribozomes and APN) and recombinant vectors comprising polynucleotide sequences that encode one or more of the polypeptides described herein. The term "a sequence essentially as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 19"means that the sequence corresponds substantially to a portion of the sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 , SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 19 and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of any of these sequences. The term "biologically functional equivalent" is well understood in the art and is defined in detail here (for example, see Illustrative Modalities). Accordingly, sequences having between about 70% and about 80%, or most preferably between about 81% and about 90% or even most preferably between about 91% to about 99% amino acid sequence identity or equivalence functional to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, 5 SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 19 will be the sequences that are "essentially as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 19". Highly preferred sequences are those which preferably are about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or functionally equivalent to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 19. Other
Preferred sequences are those that are approximately 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% identical or functionally equivalent to the amino acid sequence of SEQ ID. NO: 2, SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 19. Similarly, sequences that are approximately 71%, 72%, 73%, 74%, 75 %, 76%, 77%, 78%, 79% or 80% $
identical or functionally equivalent to the polypeptide sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 19 are also contemplated to be useful in practice of the present invention.
It will also be understood that the amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal or 5 'or 3' amino acid sequences and yet be essentially as set forth in one of the sequences herein. describes, as long as the sequence meets the criteria set forth above, including the maintenance of biological activity of the protein in which the expression of the protein is involved. The addition of terminal sequences is particularly applicable to nucleic acid sequences which may, for example, include several non-coding sequences that flank either the 5 'or 3' portion of the coding region or may include several internal sequences, i.e., introns, that are known to occur within genes. The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other encoding segments and the like, in such a way that their overall length can vary considerably. Therefore, it is contemplated that a nucleic acid fragment of almost any length can be employed with full length being preferably limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. For example, nucleic acid fragments can be prepared that include a contiguous and short extension encoding the peptide sequence described in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 19, or which are identical or complementary to the nucleic acid sequences encoding the peptides described in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 19, and particularly those nucleic acid segments described in SEQ ID NOS: 1, 3, 13, 15 or 18. e.g., nucleic acid sequences such as about 23 nucleotides, and which are up to about 10,000, about 5,000, about 3,000 , approximately 2,000, approximately 1,000, approximately 500, approximately 200, approximately 100, approximately 50 and approximately 23 or base pairs in length. tud (including all intermediary lengths) comprising a contiguous nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO : 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 18 or those encoding a contiguous amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 19 are considered to be particularly useful. It will be readily understood that "intermediate lengths" in the context of polynucleotide sequences, or nucleic acid segments, or primers or probes specific for the described gene, means any length between the aforementioned ranges, such as about 24, 25, 26 , 27, 28, 29, efe; 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, efe; 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, efe; 100, 101, 102, 103, 104, efe; 110, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 180, 190, efe; including all integers in the ranges of approximately 200-500; 500-1, 000; 1, 000-2,000; 2,000-3,000; 3,000-5,000; and up to and including sequences of about 10,000 or 12,000 or nucleotides and the like. Likewise, it will be understood that "intermediate lengths", in the context of polypeptides or peptides, means any length between the said ranges of contiguous amino acids. For example, when considering the disclosed insecticidal polypeptides, all lengths between about 7 and about 300 contiguous amino acid sequences are contemplated to be useful in particular embodiments described herein. For example, peptides comprising contiguous amino acid sequences having about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, approximately 20, approximately 21, approximately 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 , 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, efe, 70, 75, etc., 80, 85, efe, 90, 95, etc., and even those peptides comprising at least about 96, 97, 98, 99, 100, 101, 102, 103, and 104 or more contiguous amino acids of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 19 is considered explicitly which fall within the scope of the present invention. In addition, also one skilled in the art will easily understand that
"intermediate lengths", in the context of larger insecticidally active polypeptides, means any length between the said ranges of contiguous amino acids comprising said polypeptide. For example, when considering the polypeptides of the present invention, all sequence lengths of about 100 and about 1000 contiguous amino acids are contemplated to be useful in particular embodiments described herein. For example, polypeptides comprising a contiguous amino acid sequence having at least about 100, about 101, about 102, 103, 104, 105, 106, 107, 108, 109, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, efe, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 220, 230, 240, 250, 260, 270, 280, 290, efe, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, efe, 410, 430, 450, 470, 490, etc., 500, 525, 550, 575, 600, 650, 700, efe, 750, efe, and even those polypeptides comprising at least about 775 or more amino acids are explicitly considered to fall within the scope of this invention. Particularly in the case of fusion proteins comprising a complete amino acid sequence or a portion thereof of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 19 Longer polypeptide sequences may be preferred, including sequences comprising approximately 760, 770, 780, 790, or even approximately 800 or 900 or more amino acids in length. It will also be understood that this invention is not limited to the particular nucleic acid sequences encoding peptides of the present invention, or encoding the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 19 including the DNA sequence that is particularly described in SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 18. Recombinant vectors or isolated DNA segments can thus variously include the coding regions of polypeptides themselves, coding regions having selected alterations or modifications in the basic coding region, or they can encode larger polypeptides which nevertheless include those coding regions of peptides or they can encode biologically equivalent proteins or peptides s that have variable amino acid sequences. The DNA segments of the present invention comprise biologically functional equivalent peptides, said sequences can originate as a sequence of codon degeneracy and functional equivalence that is known to occur naturally within the nucleic acid sequences and the proteins thus encoded. Alternatively, proteins or functionally equivalent peptides can be created by the application of recombinant DNA technology, in which changes in the structure of the protein can be manipulated, based on considerations of the properties of the amino acids that are being exchanged. Man-made changes can be introduced through the application of self-directed mutagenesis techniques, for example, to introduce improvements to the antigenicity of the protein or to test mutants in order to examine activity at the molecular level. Alternatively, native polynucleotides and / or polypeptides, still unknown or not yet structurally identified and / or functionally related to the sequences described herein that fall within the scope of the present invention, can also be identified. Said polynucleotides are those polynucleotides that encode a polypeptide structurally and / or functionally similar or identical to the polypeptide characterized herein as a polynucleotide that encodes crystal protein. Since the designations "CryET39", "CryET69", "CryET71", "CryET74", "CryET76", "CryET79", "CryET80", "CryET84" and "CryET75" are arbitrary names chosen to easily identify polypeptides comprising the amino acid sequences described herein. , it is likely that many other polypeptides can be identified that are highly homologous to (or even identical to) this sequence, but that may have been isolated from different organisms or sources, or alternatively, even that may have been fully synthesized, or partially de novo. As such, all polypeptide sequences, either naturally occurring or artificially created, which are structurally homologous to the primary amino acid sequences as described herein, and which have similar insecticidal activity against the target insects described herein, are considered that fall within the scope of this description. Likewise, all polynucleotide sequences, either naturally occurring or artificially created, which are structurally homologous to the nucleotide sequences described herein, or which encode a polypeptide that is homologous, and biologically-functionally equivalent to the sequence of amino acids described herein is also considered to fall within the scope of this description. If desired, proteins and fusion peptides can also be prepared, for example, wherein the peptide coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification purposes or immunodetection (e.g., proteins that can be purified by affinity chromatography and coding regions of enzyme labels, respectively). Recombinant vectors form additional aspects of the present invention. Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether it encodes full-length insecticidal protein or a more complete peptide, is located under the control of a promoter. The promoter can be in the form of a promoter that is naturally associated with the gene encoding peptides of the present invention, since they can be obtained by isolating the 5 'non-coding sequences towards the 5' end of the coding or exon segment, for example, using recombinant cloning technology and / or PCR ™, in connection with the compositions described herein. In many cases, the promoter may be a native promoter, or alternatively, a heterologous promoter, such as those of bacterial origin (including promoters of other crystal proteins), of fungal, viral, phage or phagemid origin (including promoters such as CaMV35, and its derivatives, promoters T3, T7 SYMBOL PAGE 23 AND SYMBOL and the like), or of plant origin, including constitutive, inducible and / or tissue-specific promoters and the like).
2. 1.1 Characteristics of the polypeptides CryET76. CryETdO and
CrvET84 ISOLATED EG4851 The present invention provides a novel peptide that defines all or a portion of a crystal protein CryET76, CryET84 or CryETdO of B. thuringiensis. In a preferred embodiment, the invention describes and claims an isolated and purified CryET76 protein. The CryET76 protein isolated from EG4851 comprises a sequence of 3d7 amino acids and has a calculated molecular mass of approximately 43, d00 Da. CryET76 has a calculated isoelectric constant (pl) equal to 5.39. In a preferred embodiment, the invention describes and claims an isolated and purified CryETdO protein. The CryETdO protein isolated from EG4851 comprises a sequence of 132 amino acids and has a calculated molecular mass of approximately 14,800 Da. CryET80 has a calculated isoelectric constant (pl) equal to 6.03. In a preferred embodiment, the invention describes and claims an isolated and purified CryET84 protein. The CryETd4 protein isolated from EG4851 comprises a sequence of 341 amino acids and has a calculated molecular mass of approximately 37,884 Da. CryET84 has a calculated isoelectric constant (pl) equal to 5.5. In strain EG4851, the cryETQO and cryET76 genes are preferably located in a single DNA segment and are separated by approximately 95 nucleotides. The gene for CryET76 extends from nucleotide 514 to nucleotide 1674 of SEQ ID NO: 5, and the gene encoding
CryETdO extends from nucleotide 23 to nucleotide 41 d of SEQ ID
NO: 5. In the present invention, the cryET80 and cryET76 genes can preferably be located in a single DNA segment. In strain EG4d51, the cryET84 gene is located immediately
? for the cryET80 and cryET76 genes. The nucleotide sequence of the gene
CryET84 is shown in SEQ ID NO: 18 and the deduced amino acid sequence of the CryET84 protein is shown in SEQ ID NO: 19. In the present invention, the CryET80, CryET84 and CryET76 genes can be preferably located in a single DNA segment. (ie, SEQ ID NO: 17).
2.2 Nucleic Acid Segments as Probes and Hybridization Initiators In addition to their use in the direction of crystal peptide or protein expression of the present invention, the nucleic acid sequences described herein also have a variety of other uses. For example, they have utility as probes or primers in nucleic acid hybridization modalities. The invention provides a method for detecting a nucleic acid sequence encoding a d-endotoxin polypeptide. The method generally involves obtaining nucleic acids from the sample that are suspected of encoding a d-endotoxin polypeptide; contacting the nucleic acids of sample 15 with an isolated nucleic acid segment comprising one of the sequences described herein, under conditions effective to allow hybridization of substantially complementary nucleic acids; and detecting the hybridized complementary nucleic acids thus formed. Nucleic acid detection equipment is also provided
comprising, in a suitable container means, at least a first segment of nucleic acid comprising at least 23 contiguous nucleotides of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 18, and at least one first detection reagent. The ability of said nucleic acid shortages to hybridize specifically to the crystal protein coding sequences will allow them to be useful for detecting the presence of complementary sequences in a given sample. However, other uses are contemplated, including the use of sequence information for the preparation of primers of mutant species or primers for use in the preparation of other genetic constructs. Nucleic acid molecules that have regions of sequences that consist of contiguous nucleotide spreads of about 23 to about 50, or even up to and including sequences of about 100-200 nucleotides or so, identical or complementary to the DNA sequences of the present, are particularly contemplated as hybridization probes to be used for example in Southern and Northern blotting. Fragments of intermediate size are also generally used in hybridization modalities wherein the length of the contiguous complementary region can be varied, such as between about 25-30, or between about 30 and about 40 etc., nucleotides, but can be used larger contiguous complementary extensions, such as those from about 200 to about 300, or from about 300 to about 400 or 500 etc., nucleotides in length, according to the length of the complementary sequences to be detected. It is even possible that regions of longer contiguous sequences can be included including those sequences comprising at least about 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 or more contiguous nucleotides from one of the sequences that auqí are described. Of course, fragments can also be obtained by other techniques such as, for example, by mechanical cutting or by digestion with restriction enzymes. Small nucleic acid segments or fragments can be easily prepared, for example, by direct synthesis of the fragment by chemical means, as is common practice using an automated oligonucleotide synthesizer. Also, fragments can be obtained by the application of nucleic acid reproduction technology, such as PCR ™ technology of the U.S. Patents. 4,683,195 and 4,683,202 (each incorporated herein by reference), introducing selected sequences into recombinant vectors for recombinant production and by other recombinant DNA techniques generally known to those skilled in the art of molecular biology. Accordingly, the nucleotide sequences of the invention can be used for their ability to selectively form duplex molecules with complementary extensions of DNA fragments. Depending on the contemplated application, it will be desired to employ variable hybridization conditions to achieve varying degrees of probe selectivity towards the target sequence. For applications requiring high selectivity, it will typically be desirable to employ relatively stringent conditions to form the hybrids. "Highly stringent" hybridization conditions, for example, typically employ conditions of relatively low salt content and / or high temperature, such as are provided by NaCl of about 0.02 M to about 0.15 M at temperatures of about 50 ° C. at approximately 70 ° C. Such selective conditions tolerate little, if any, mismatch between the probe and the target template or strand, and would be particularly suitable for isolating DNA segments encoding crystal protein. The detection of DNA segments by hybridization is well known to those skilled in the art and the teachings of the U.S. Patents. 4,965.1 dd and 5,176,995 (each incorporated herein by reference) are illustrative of the methods of analysis by hybridization. The teachings such as those found in the texts of Maloy et al., 1990; Maloy 1994; Segal, 1976; Prokop and Bajpai, 1991; and Kuby, 1994, are particularly important. Of course, some applications, for example, where it is desired to prepare mutants using a mutant initiator strand hybridized to an underlying template or where it is sought to isolate crystal protein coding sequences from related species, functional equivalents or the like, Less stringent hybridization will typically be necessary in order to form the heteroduplex. In these circumstances, it is desired to employ "low stringency" or "low stringency" hybridization conditions such as those employing salt of about 0.15 M to about 0.9 M, at temperatures ranging from about 20 ° C to about 55 °. C. Cross-hybridizing species therefore can easily be identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be made more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same way as it increases the temperature. Therefore, the hybridization conditions can be easily manipulated and therefore will generally be a method of choice depending on the desired results. Regardless of which particular combination of salts (such as NaCl or Na citrate and the like), organic pH regulators (including, for example, formamide and the like), and incubation or washing temperatures are employed, one skilled in the art will be able to easily employ Hybridization conditions that are of "high", "medium" or "low" stringency, and may interpret the results of the hybridization assays using those conditions to determine the relative homology of said target nucleic acid sequence with that of the sequence of particular novel polynucleotide probe employed from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 , SEQ ID NO: 15 or SEQ ID NO: 13. In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with appropriate means such as a label, to determine hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin / biotin, which are capable of giving a detectable signal. In preferred embodiments, it will probably be desirable to employ a fluorescent label or an enzyme label, such as urease, alkaline phosphatase or peroxidase, in place of radioactive reagents or other environmentally undesirable reagents. In the case of enzyme labels, it is known that calorimetric indicator substrates can be used to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with samples containing nucleic acid, complementary. In general, it is contemplated that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in modalities employing a solid phase. In embodiments employing a solid phase, the test DNA (or RNA) is adsorbed or otherwise fixed to a selected matrix or surface. The fixed single stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the content of G + G, type of target nucleic acid, nucleic acid source, size of hybridization probe, etc.). After washing the hybridized surface to remove non-specifically bound probe molecules, specific hybridization is detected or even quantified by means of the label.
2. 3 Vectors and methods for recombinant expression of Cry-related polypeptides In other embodiments, it is contemplated that certain advantages will be obtained by placing the coding DNA segment under the control of a recombinant, or heterologous promoter. As used herein, a heterologous recombinant or promoter refers to a promoter that is not normally associated with a segment of DNA encoding crystal peptide or protein in its natural environment. Such promoters may include promoters normally associated with other genes and / or promoters isolated from any bacteria, viruses, eukaryotic or plant cells. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell, organism or even animal type, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those skilled in the art of molecular biology, for example, see Sambrook et al., (19d9). The promoters employed may be constitutive or inducible and may be used under appropriate conditions to direct the high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of peptides or recombinant proteins. Suitable promoter systems contemplated for use in high level expression include but are not limited to the Pichia expression vector system (Pharmacia LKB Biotechnology). In relation to the expression modalities for preparing peptides and recombinant proteins, it is contemplated that longer DNA segments will be used more often are segments of DNA encoding whole peptide sequences being most preferred. However, it will be appreciated that the use of shorter DNA segments to direct the expression of crystal peptides or epitope center regions such as those that can be used to generate crystal anti-protein antibodies also fall within the scope of the invention. DNA segments encoding peptide antigens of about 50 amino acids in length, or most preferably from about 8 to about 30 amino acids in length, or most preferably still about 20 amino acids in length are contemplated to be particularly tools. Said peptide epitopes may be amino acid sequences comprising a contiguous amino acid sequence as described herein.
2. 4 Transgenic Plants Expressing CrvET Polypeptides In yet another aspect, the present invention provides methods for producing a transgenic plant expressing a select nucleic acid segment comprising a sequence region encoding the novel endotoxin polypeptides of the present invention. The process for producing transgenic plants is well known in the art. In general, the method consists of transforming a suitable plant host cell with a DNA segment containing a promoter operably linked to a coding region that encodes one or more of the described polypeptides. Said coding region is generally operably linked to at least a first transcription termination region, whereby the promoter is capable of activating the transcription of the coding region in the cell, and thus providing the cell with the ability to produce the polypeptide in vivo. Alternatively, in cases where it is convenient to control, regulate or reduce the amount of a particular recombinant crystal protein expressed in a particular transgenic cell, the invention also provides for the expression of crystal protein antisense mRNA. The use of antisense mRNA as a means to control or reduce the amount of a given protein of interest in a cell is well known in the art. Another aspect of the invention comprises transgenic plants that express a gene, gene segment or sequence region encoding at least one or more of the novel polypeptide compositions described herein. How it is used here, the term "transgenic plant" refers to a plant that has incorporated DNA sequences including but not limited to genes that are perhaps not normally present, DNA sequences that do not normally transcribe to RNA or are translated into protein ("expressed" ) or any other genes or DNA sequences that are desired to be introduced into the non-transformed plant, such as genes that are normally present in the non-transformed plant but which are desired to be genetically engineered or have altered expression. It is contemplated that in some cases the genome of a transgenic plant of the present invention has been increased by the stable introduction of one or more transgenes, either native, synthetically modified or mutated, that encodes an insecticidal polypeptide that is identical or highly homologous to the polypeptide that Here is described. In some cases, more than one transgene will be incorporated into the genome of the cell of the transformed host plant. Such is the case when more than one segment of DNA encoding crystal protein is incorporated into the genome of said plant. In certain situations, it may be desirable to have one, two, three, four or even more B. thuringiensis crystal proteins (either native or recombinantly engineered) incorporated and stably expressed in the transformed transgenic plant. Alternatively, a second transgene can be introduced into the plant cell to confer additional genotypic traits to the plant. Such transgenes can confer resistance to one or more insects, bacteria, fungi, viruses, nematodes or other pathogens. A preferred gene that can be introduced includes, for example, a DNA sequence encoding crystal protein of bacterial origin, and particularly one or more of those described herein that are obtained from Bacillus spp. Highly preferred nucleic acid sequences are those obtained from B. thuringiensis or any of those sequences that have been genetically engineered to reduce or increase the insecticidal activity of the crystal protein in said transformed host cell. Means for transforming a plant cell and the preparation of pluripotent plant cells, and regeneration of a transgenic cell line from a transformed cell, cell culture, embryo or callus tissue are well known in the art and are discussed herein. Vectors (including plasmids, cosmic, phage, phagemids, baculovirus, viruses, virions, BACs [bacterial artificial chromosomes], YACs [yeast artificial chromosomes]) comprising a first segment of nucleic acid encoding an insecticidal polypeptide for use in transformation of said cells will of course comprehend in general either the operons, genes or sequences derived from genes of the present invention, either native, or synthetically derived, and particularly those which encode the crystal proteins described. These nucleic acid constructs may further include structures such as promoters, enhancers, polylinkers, introns, terminators or even sequences of genes having positive or negatively regulatory activity on the cloned d-endotoxin gene as desired. The DNA or gene segment can encode either a native or modified crystal protein, which will be expressed in the resulting recombinant cells, and / or confer to a transgenic plant comprising said segment, an improved phenotype (in this case, the resistance). increased to attack, infestation or colonization of insects). The preparation of a transgenic plant comprising at least one polynucleotide sequence encoding an insecticidal polypeptide for the purpose of increasing or improving the resistance of said plant to attack by a target insect represents an important aspect of the invention. In particular, the inventors describe here the preparation of monocotyledonous or dicotyledonous plants resistant to insects, incorporating into said plant a segment of transgenic DNA that encodes one or more insecticidal polypeptides that are toxic to a coleoptera or lepidopteran insect. In a related aspect, the present invention also comprises a seed produced by the transformed plant, a progeny of said seed and a seed produced by the progeny of the original transgenic plant, produced according to the above procedure. Said progeny and seeds will have a crystal protein coding transgene stably incorporated in their genome, and said plants of the progeny will inherit the traits acquired by introducing a stable transgene in a Mendelian manner. All those transgenic plants that have transgenic DNA segments encoding one or more crystal proteins or polypeptides incorporated into their genome are aspects of this invention. As is well known to those skilled in the art, it is understood that a progeny of a plant means any descendant or next generation of said plant.
2. 5 Crystal Protein Screening and Selection Equipment The present invention contemplates methods and equipment for selecting samples suspected of containing crystal protein polypeptides or crystal protein-related polypeptides, or cells producing said polypeptides. A kit may contain one or more antibodies specific for the amino acid sequences described, or one or more antibodies specific for a peptide derived from one of the described sequences and may also contain reagents to detect an interaction between a sample and an antibody of the present invention. invention. The reagents provided can be radiolabeled, fluorescently labeled or enzymatically labeled. The kit may contain a known radiolabelled agent capable of binding or interacting with a nucleic acid or antibody of the present invention. The equipment reagents can be supplied as a liquid solution, fixed to a solid support or as a dry powder. Preferably, when the reagents are supplied in a liquid solution, the liquid solution is in aqueous solution. Preferably, when the provided reagents are fixed to a solid support, the solid support can be a chromatography medium, a test plate having a plurality of wells or a microscope slide. When the reagents provided are a dry powder, the powder can be reconstituted by the addition of a suitable solvent that can be supplied. In additional embodiments, the present invention relates to immunodetection methods and associated equipment. It is proposed that the crystal proteins or peptides of the present invention can be used to detect antibodies having reactivity thereto, or alternatively antibodies prepared according to the present invention, can be used to detect proteins or peptides containing epitope related to crystal protein In general, these methods will first include obtaining a sample suspected of containing said protein, peptides or antibody, contacting the sample with an antibody or peptide according to the present invention, as the case may be, under conditions effective to allow the formation of an immunocomplex, and then detect the presence of the immune complex. In general, the detection of immunocomplex formation is well known in the art and can be achieved by the application of numerous approaches. For example, the present invention contemplates the application of ELISA, RIA, immunoblot (spot spot), indirect immunofluorescence techniques and the like. Generally, immunocomplex formation will be detected by the use of a label, such as a radiolabel or an enzyme tag (such as alkaline phosphatase, horseradish peroxidase or the like). Of course, additional advantages can be found by the use of a secondary binding ligand such as a second antibody or a biotin / avidin ligand binding arrangement, as is known in the art. For testing purposes, it is proposed that almost any sample that is suspected to comprise either a crystal protein or peptide or a peptide related to crystal protein or antibody is to be detected, as the case may be, can be employed. It is contemplated that said modalities may have application in the titration of antigen or antibody samples, in the selection of hybridomas and the like. In related embodiments, the present invention contemplates the preparation of equipment that can be used to detect the presence of crystal proteins or related peptides and / or antibodies in a sample. Samples may include cells, cell supernatants, cell suspensions, cell extracts, enzyme fractions, protein extracts or other cell-free compositions that are suspected to contain crystal proteins or peptides. Generally speaking, the equipment according to the present invention will include a suitable crystal protein, peptide or an antibody directed against said protein or peptide, together with an immunodetection reagent and means for containing the antibody or antigen and the reagent. The immunodetection reagent will typically comprise a label associated with the antibody or antigen or associated with a secondary binding ligand. Illustrative ligands may include a secondary antibody directed against the first antibody or antigen or a biotin or avidin ligand (or streptavidin) having an associated label. Of course, as indicated above, many example markers are known in the art and all such markers can be employed in conjunction with the present invention. The container will generally include a vial in which the antibody, antigen or detection reagent can be placed, and preferably will be in suitable aliquots. The kits of the present invention will also typically include a means for containing the containers of antibody, antigen and reagent in close confinement for commercial scale. Such containers may include injection or blow molded plastic containers in which the desired bottles are retained.
2. 6 Insecticidal compositions and methods of use The inventors contemplate that the polypeptide compositions described herein will find particular utility as insecticides for topical and / or systemic application to field crops, grasslands, fruits and vegetables, lawns, trees and / or plants of ornament. Alternatively, the polypeptides described herein can be formulated as a spray, powder, fine powder, other aqueous, atomized or aerosolized forms to kill an insect, or to control a population of insects. The polypeptide compositions described herein can be prophylactically or alternatively administered to an environment once the target insects, such as WCRW, have been identified in the particular environment to be treated. The polypeptide compositions may comprise a single Cry polypeptide or may contain various combinations of the polypeptides described herein. Regardless of the method of application, the amount of the active polypeptide component is applied in an insecticidally effective amount, which will vary depending on factors such as, for example, the specific target insects to be controlled, the specific environment, location, plant, culture. or agricultural site to be treated, the environmental conditions and the method, rate, concentration, stability and amount of application of the insecticidally active polypeptide composition. The formulations may also vary with respect to weather conditions, environmental considerations and / or frequency of application and / or severity of insect infestation. The disclosed insecticidal compositions can be made by formulating either the bacterial cell, crystal suspension and / or spores, or components of isolated protein with the desired agriculturally acceptable vehicle. The compositions may be formulated prior to administration in an appropriate medium such as freeze-dried, freeze dried, dried or in an aqueous vehicle, medium or suitable diluent, such as saline or other pH regulator. The formulated compositions may be in the form of a fine powder or granular material or an oil suspension (vegetable or mineral), or water or oil / water emulsions or as a wettable powder, or in combination with another suitable vehicle material for agricultural application. Such agricultural vehicles can be solid or liquid and are well known in the art. The term "agriculturally acceptable vehicle" covers all adjuvants, inert components, dispets, surfactants, adhesives, binders, etc., which are ordinarily used in insecticide formulation technology; these are well known to experts in the formulation of insecticides. The formulations can be mixed with one or more solid or liquid adjuvants and prepared by various methods, for example, homogeneously by mixing, combining and / or milling the insecticidal composition with suitable adjuvants using conventional formulation techniques.
2. 6.1 Flowable suspensions in oil In a preferred embodiment, a bioinsecticide composition comprises a flowable suspension in oil of bacterial cells expressing the novel crystal protein described herein. Exemplary bacterial species include those such as B. thuringiensis, B. cereus, E. coli, Salmonella spp. Agrobacterium spp. or Pseudomonas spp.
2. 6.2 Water Dispersible Granules In another important embodiment, the bioinsecticide composition comprises a water dispersible granule. This granule comprises bacterial cells expressing a novel crystal protein described herein.
Preferred bacterial cells include bacteria such as B. megaterium, β. subtilis, B. cereus, E. coli, Salmonella spp. Agrobacterium spp. or Pseudomonas spp. Cells transformed with DNA segment described herein and expressing the crystal protein are also contemplated as useful.
2. 6.3 Formulations in powders, fine powders and spores In a third important embodiment, the bioinsecticide composition comprises a wettable powder, fine powder, formulation of spore crystals, cell tablets or colloidal concentrate. This powder comprises bacterial cells expressing a novel crystal protein described herein. Preferred bacterial cells include B. thuringiensis, or cells from strains of bacteria such as B. megaterium, B. subtilis, B. cereus, E. coli, Salmonella spp. Agrobacterium spp. or Pseudomonas spp. and the like, can also be transformed with one or more nucleic acid segments as described herein. Said dried forms of the insecticidal compositions can be formulated to dissolve immediately upon wetting, or alternatively, they can be dissolved in a controlled release manner, sustained release or otherwise time dependent manner. Such compositions can be applied to the target insect, or can be ingested by it, and as such can be used to control many insects, or control the spread of said insects in a given environment.
2. 6.4 Aqueous suspensions and filtrates or lysates of bacterial cells In a fourth important embodiment, the bioinsecticide composition comprises an aqueous suspension of bacterial cells or an aqueous solution of paraespore crystals, or an aqueous suspension of lysates or filtrates of bacterial cells, etc., such as those described above that express the crystal protein. Said aqueous suspensions may be provided as a concentrated supply solution which is diluted before application or alternatively as a diluted solution ready to be applied. For these methods involving the application of bacterial cells, the cell host containing the crystal protein gene or genes can be cultured in any convenient nutrient medium, where DNA construction provides a selective advantage, providing a selective means of so that substantially all or all cells retain the B. thuringiensis gene. These cells can be harvested according to conventional means. Alternatively, the cells can be treated before harvesting. When the insecticidal compositions comprise according to the invention intact B. thuringiensis cells expressing the protein of interest, said bacteria can be formulated in a variety of ways. They can be used as wettable powders, granules or fine powders, mixing it with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates and the like) or botanical materials (powdered corncobs, rice s, walnut shells) and similar). The formulations may include spreader-adhesive adjuvants, stabilizing agents, other pesticidal additives or surfactants. The liquid formulations may be aqueous or non-aqueous based and may be employed as foams, suspensions, emulsifiable concentrates or the like. The ingredients can include rheological agents, surfactants, emulsifiers, dispersants or polymers. Alternatively, novel insecticidal polypeptides can be prepared by means of native or recombinant bacterial expression systems in vitro and can be isolated for subsequent application in the field. Said proteins can be in lysates, suspensions or crude colloids of cells, etc., or alternatively they can be purified, refined, regulated in their pH and / or subsequently processed, before formulating them in an active biocidal formulation. Also, under certain circumstances, it may be convenient to isolate crystals and / or spores of bacterial cultures expressing the crystal protein and to apply solutions, suspensions or colloidal preparations of said crystals and / or spores as the active bioinsecticide composition.
2. 6.5 Multifunctional Formulations In certain modalities, when it is desired to control multiple insect species, the insecticidal formulations described herein may also comprise one or more chemical pesticides, (also as chemical pesticides, mematicides, fungicides, viricides, microbicides, amebocides, insecticides, etc.), and / or one or more d-endotoxin polypeptides having the same or different insecticidal or insecticidal activities or specificities, than the insecticidal polypeptide identified herein. The insecticide polypeptides may also be used in conjunction with other treatments such as fertilizers, herbicides, cryoprotectants, surface active agents, detergents, insecticidal soaps, dormant oils, polymers, and / or release vehicle formulations over time or biodegradable which allow for a metered dosage. long term after a single application of the formulation. Also, the formulations can be prepared in ingestible "baits" or they can be placed in "traps" for insects to allow feeding or ingestion by a target insect of the insecticidal formulation. The insecticidal compositions of the invention can also be used in consecutive or simultaneous application to an environmental site or individually or in combination with one or more insecticides, pesticides, chemical compounds, fertilizers or other additional compounds.
2. 6.6 Application methods and effective cups The insecticidal compositions of the invention are applied to the environment of the target insect, typically on the foliage of the plant or crop to be protected, by conventional methods, preferably by spraying. The resistance and duration of insecticide application will be set with respect to the specific conditions of the pest, particular crop to be treated as well as the particular environmental conditions. The proportional ratio of active ingredient to vehicle will normally depend on the chemical nature, safety and stability of the insecticidal composition, as well as the particular formulation contemplated. Other application techniques, including dusting, spraying, soil soaking, soil injection, seed coating, seedling coating, foliar spraying, aeration, spraying, atomization, spraying, aerosolization and the like are also feasible and may be required under certain circumstances. such as, for example, insects that produce infestation on the root or stem, or for application to delicate vegetation or delicate ornamental plants. These application methods are also well known to those skilled in the art. The insecticidal compositions of the present invention can also be formulated for preventive or prophylactic application for an area and in certain circumstances can be applied to pets, livestock animals, animal sheds or around farm equipment, stables, houses or agricultural facilities or industrial and similar. The concentration of insecticidal composition that is used for environmental, systemic, topical or foliar application will vary widely depending on the nature of the particular formulation, means of application, environmental conditions and degree of biocidal activity. Typically, the bioinsecticide composition will be present in the formulation applied at a concentration of at least 1% by weight and can be up to and including about 99% by weight. The dry formulations of the polypeptide compositions can be from about 1% to about 99% or more by weight of the protein composition, while the liquid formulations can generally comprise from about 1% to about 99% or more of the ingredient. active in weight. As such, a variety of formulations can be prepared, including those formulations comprising from about 5% to about 95% or more by weight of the insecticidal polypeptide, including those formulations comprising from about 10% to about 90% or more in weight in insecticidal polypeptide. Naturally, compositions comprising from about 15% to about d5% or more by weight of the insecticidal polypeptide and formulations comprising from about 20% to about 10% or more by weight of the insecticidal polypeptide will also be considered within the scope of the present invention. description. In the case of compositions in which intact bacterial cells containing the insecticidal polypeptide are included, the preparations will generally contain from about 10 4 to about 10 8 cells / mg, although in some embodiments it may be convenient to use formulations comprising about about 104 cells / mg, or when more concentrated formulations were desired, compositions comprising from about 108 to about 1010 or
1011 cells / mg can also be formulated. Alternatively, cell pastes, spore concentrates or crystal protein suspension concentrations containing the equivalent of about 100% can be prepared.
1012 to 1013 cells / mg of the active polypeptide, and said concentrates can be diluted before application. The above-described insecticidal formulation can be administered to a particular plant or to a target area in one or more applications as needed, with a typical field application rate per hectare ranging from the order of about 50 g / hectare to about 500 g / hectare of active ingredient or alternatively from about 500 g / hectare to about 1000 g / hectare can be used. In certain cases, it may be convenient to apply the insecticidal formulation to a target area at an application rate of about 1000 g / hectare to about 5000 g / hectare or more of active ingredient. In fact, all application rates in the range of about 50 g of active polypeptide per hectare of approximately 10,000 g / ha are contemplated to be useful in the management, control and annihilation of target insect pests, using such insecticidal formulations. As such, the cups are approximately 100 g / hectares, approximately 200 g / hectares, approximately 300 g / hectares, approximately 400 g / hectares, approximately 500 g / hectares, approximately 600 g / hectares, approximately 700 g / hectares, approximately dOO. g / hectares, approximately 900 g / hectares, approximately 1 kg / hectares, approximately 1.1 kg / hectares, approximately 1.2 kg / hectares, approximately 1.3 kg / hectares, approximately 1.4 kg / hectares, approximately 1.5 kg / hectares, approximately 1.6 kg / hectares, approximately 1.7 kg / hectares, approximately 1.8 kg / hectares, approximately 1.9 kg / hectares, approximately 2.0 kg / hectares, approximately 2.5 kg / hectares, approximately 3.0 kg / hectares, approximately 3.5 kg / hectares, approximately 4.0 kg / hectares, approximately 4.5 kg / hectares, approximately 6.0 kg / hectares, approximately 7.0 kg / hectares, approximately 8.0 kg / hectares, approx. per pound 8.5 kg / ha, approximately 9.0 kg / ha, and up to and including approximately 10.0 kg / ha or greater of active polypeptide may be used in certain agricultural, industrial and domestic applications of the pesticide formulations described above.
2. 7 Sequences of epitopic centers The present invention is also directed to protein or peptide compositions, free of total cells and other peptides, comprising a purified peptide that incorporates an epitope that is immunologically reactive cross-linked with one or more antibodies that are specific for the polypeptide sequences described. In particular, the invention relates to sequences of epitope centers derived from one or more of the polypeptides described herein. As used herein, the term "incorporating an epitope (s) that is immunologically reactive cross-linked to one or more antibodies that are specific for the polypeptide sequence described" refers to a peptide or protein antigen that includes a primary structure, secondary or tertiary similar to an epitope located within the described polypeptide. The level of similarity will generally be such that the monoclonal or polyclonal antibodies directed against the crystal protein or crystal polypeptide will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein antigen. Various methods of immunoassays can be employed with such antibodies, such as, for example, Western blotting, ELISA, RIA, and the like, all of which are known to those skilled in the art. The identification of immunodominant epitopes and / or their functional equivalents, suitable for use in vaccines is a relatively straightforward matter. For example, the Hopp methods can be employed, as taught in the U.S. patent. 4,554,101, incorporated herein by reference, which teaches the identification of epitopes of amino acid sequences on the basis of hydrophilic character. The methods described in other documents and software programs based thereon can also be used to identify sequences of epitopic centers (see, for example, Jameson and Wolf, 198d; Wolf et al., 19dd; U.S. Patent 4,554,101). The amino acid sequence of these "epitope center sequences" can then be easily incorporated into peptides, either through the application of peptide synthesis or recombinant technology. The peptides used according to the present invention will generally be from about d to about 20 amino acids in length, and most preferably from about d to about 15 amino acids in length. It is proposed that peptides derived from antigenic crystal protein provide advantages in certain circumstances, for example, in the preparation of immunological detection tests. Illustrative advantages-include the ease of preparation and purification, the relatively low cost and the improved reproduction capacity of production and advantageous biodistribution. It is proposed that particular advantages of the present invention can be achieved through the preparation of synthetic peptides including modified and / or extended epitope / immunogenic site sequences resulting in a "universal" epitope peptide targeting crystal proteins and related sequences. These sequences of epitopic centers are identified here in particular aspects as hydrophilic regions of the particular polypeptide antigen. It is proposed that these regions represent those that most likely promote the stimulation of T cells or B cells and therefore induce the production of specific antibodies.
A sequence of epitope centers, as used herein, is a relatively short stretch of amino acids that is "complementary" to, and will therefore bind to, antigen binding sites on the crystal protein-directed antibodies described herein. In addition, alternatively, an epitope center sequence is one that will induce antibodies that are reactive cross-linked with antibodies directed against the peptide compositions of the present invention. It will be understood that in the context of the present description, the term "complementary" refers to amino acids or peptides that have an attractive force towards each other. Therefore, certain epitope sequences of the present invention can be operationally defined in terms of their ability to compete with or perhaps displace the binding of the desired protein antigen with the corresponding protein directed antisera. In general, the size of the polypeptide antigen is not believed to be particularly crucial, as long as it is at least large enough to cut the identified sequence or center sequences. The smallest useful center sequence unforeseen by the present disclosure will generally be of the order of about d amino acids in length, with sequences of the order of 10 to 20 being most preferred. Therefore, this size will generally correspond to the peptide antigens. smaller prepared according to the invention. However, the size of the antigen may be larger where desired, as long as it contains a basic epitope center sequence.
The identification of epitope center sequences is known to those skilled in the art, for example, as described in the U.S. patent. 4,554, 101, incorporated herein by reference, which teaches the identification and preparation of epitopes of amino acid sequences on the basis of hydrophilic character. In addition, numerous computer programs are available for use in the prediction of antigenic portions of proteins (see, for example, Jameson and Wolf, 193d; Wolf et al., 19dd). Computerized peptide sequence analysis programs (eg, DNAStar®, DNAStar, Inc., Madison, Wl) may also be useful for designing synthetic peptides according to the present disclosure. The synthesis of epitope sequences, or peptides that include an antigenic epitope within their sequence, is easily achieved using conventional synthetic techniques such as the solid phase method (e.g., by the use of commercially available peptide synthesizer such as Applied Biosystems Model 430A Peptide Sinthesizer). The peptide antigens synthesized in this manner can be included in aliquots in predetermined amounts and stored in conventional forms, such as aqueous solutions or very preferably still, a powder or lyophilized state depending on the use. In general, due to the relative stability of the peptides, they can be easily stored in aqueous solutions for very long periods if desired, for example, up to 16 months or more, in almost any aqueous solution without appreciable degradation or loss of antigenic activity. However, where extended aqueous storage is contemplated it will generally be desirable to include agents containing pH regulators such as Tris or phosphate pH regulators to maintain a pH of about 7.0 to about 7.5. In addition, it may be convenient to include agents that inhibit microbial growth, such as sodium acid or Merthilate. For prolonged storage in an aqueous state, it will be convenient to store the solutions at about 4 ° C, or most preferably, in frozen form. Of course, where the peptides are stored in a lyophilized or powdered state, they can be stored almost indefinitely, for example, in measured aliquots that can be rehydrated with a predetermined amount of water (preferably distilled) or pH regulator before being used .
2. d Definitions The following words and phrases have the meanings set out below.
Expression: The combination of intracellular processes, including transcription and translation undergone by a coding DNA molecule such as a structural gene to produce a polypeptide. Pluripotential: A term used to describe developmental plasticity. A pluripotential cell is able to differentiate into a number of different cell types and lineages. For example, a stem cell in the bone marrow can give rise to many different lineages of circulating blood cells. This contrasts with a differentiated cell, which is generally aimed at a particular developmental pathway. Promoter: A recognition site on a DNA sequence or group of DNA sequences that provides an expression control element for a structural gene and for which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene . Regeneration: The process of growing a plant from a plant cell (for example, protoplasm or plant explant). Structural gene: A gene that is expressed to produce a polypeptide. Transformation: A process of introducing an exogenous DNA sequence (eg, a vector, a recombinant DNA molecule) into a cell or protoplast in which that exogenous DNA is incorporated into a chromosome or is capable of autonomous replication. Transformed cell: A cell whose DNA has been altered by introducing an exogenous DNA molecule into that cell. Transgenic cell: Any cell derived from or generated from a cell transformed or derived from a transgenic cell. Exemplary transgenic cells include plant keys derived from a transformed plant cell and particular cells such as leaf cells, radicals, from the stem, for example, somatic cells, or reproductive (germinative) cells obtained from a transgenic plant. Transgenic plant: A plant or progeny of any generation of the plant that was derived from a cell or protoplast of transformed plant, wherein the plant nucleic acids contain an exogenous selected nucleic acid sequence region that was not originally present in a non-transgenic native plant of the same strain. The term "transgenic plant" and "transformed plant" have sometimes been used in the art as synonyms to define a plant whose DNA contains an exogenous DNA molecule. However, it is thought that it is more scientifically correct to refer to a regenerated plant or key obtained from a cell or protoplast of transformed plant or cells of transformed pluripotent plants as a transgenic plant. Preferably, the transgenic plants of the present invention include those plants that comprise at least one first selected polynucleotide that encodes an insecticidal polypeptide. This selected polynucleotide is preferably a d-endotoxin (or gene) coding region operably linked to at least a first promoter that expresses the coding region for producing the insecticidal polypeptide in the transgenic plant. Preferably, the transgenic plants of the present invention that produce the encoded polypeptide demonstrate a phenotype of improved resistance to target insect pests. Said transgenic plants, their progeny, descendants and seeds of any of those generations are preferably insect resistant plants. Vector: A nucleic acid molecule capable of replicating in a host cell and / or to which another nucleic acid segment can be operably linked to carry out the replication of the attached segment. Plasmids, phages, phagemids and cosmids are all examples of vectors. In many embodiments, the vectors are used as a vehicle to introduce one or more selected polynucleotides into a host cell, thereby generating a "transformed" or "recombinant" host cell.
BRIEF DESCRIPTION OF THE DRAWINGS
The drawings are part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention can be better understood with reference to or more of these drawings in combination with the detailed description of specific embodiments presented herein. Figure 1 is a restriction map of pEG1337. Figure 2 is a restriction map of pEG1921. Figure 3 is an SDS-PAGE analysis of spore-crystal suspensions of C2 cultures of EG1165d, EG12156, and EG1215d. Twenty-five microliters (μl) of the suspensions were diluted with 75 μl of sterilized water and prepared for electrophoresis as described in example 11. Ten μl were loaded per line into the 15% acrylamide gel. A serial dilution of bovine serum albumin (BSA) was diluted as a standard. Lines 1-3, EG11658; lines 4-6, EG12156; lines 7-d, EG12158. M = molecular weight standards (Sigma M-0671) in kilodaltons. The bands corresponding to CryET76, CryETdO, and CryETd4 are indicated by arrows.
DESCRIPTION OF ILLUSTRATIVE MODALITIES
4. 1 Some advantages of the invention The present invention provides novel d-endotoxins which are highly toxic to insects such as WCRW, SCRW, and CPB. These proteins have amino acid sequences that are only distantly related to those of other d-endotoxins that are toxic to Diptera or Coleoptera insects. Based on the guidelines established for the B. thuringiensis crystal protein nomenclature (Crickmore et al., 199d), two of these polypeptides, designated CryET76 and CryETdO, represent a new subclass of insecticidal active crystal proteins of Coleoptera.
4. 2 Insect plaque Almost all field crops, plants and commercial agricultural areas are susceptible to attack by or more insect pests.
Particularly problematic are the pests of lepidoptera and coleoptera identified in table 1. For example, crops of vegetables and cabbages such as artichokes, rutabagas, arugula, leeks, asparagus, lentils, beans, lettuce (for example, orejona, Roman, Italian) , beets, bok choy, malanga, broccoli, melons (for example, Chinese melon, watermelon, crinkly peel melon, h drop melon, white melon, Brussels brussels, cabbage, cardoni, carrot, napa, cauliflower, okra, onions , celery, parsley, chickpeas, parsnips, chicory, peas, Chinese cabbage, peppers, kale, potato, cucumber, pumpkins, cucurbits, radishes, dry bulb onions, rutabaga, eggplant, salsify, escaroles, escalonas, endivia, soy, garlic, spinach, green onions, yellow squash, edible leafy vegetables, sugar beets, sweet potatoes, turnip, Swiss chard, horseradish, tomatoes, cabbage, turnips and a variety of species are sensitive in gestation by an om More of the following insect pests: alfalfa measuring caterpillar, soldier worm, soldier beetworm, artichoke vilano moth, cabbage gall worm, cabbage measuring worm, cabbage worm, worm of corn cob, celery leaf devourer, cross vein cabbage worm, European corn borer, diamond back moth, clover green worm, imported cabbage worm, melon worm, omnivorous leafroller, worm cucumber, cascara worm, saline caterpillar, soy measuring caterpillar, tobacco gillworm, tomato fruit worm, tomato horned worm, thin tomato worm, velvety bean caterpillar and veined worm yellow
Also, pasture and hay crops, such as alfalfa, grassland and encilage are often attacked by pests such as soldier worm, cattle soldier worm, alfalfa caterpillar, European jumper, a variety of caterpillars and weaver worms. , as well as yellow vein welded worms. Fruit crops and vineyards such as apples, apricots, cherries, tangerines, peaches, pears, plums, prunes, almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, citrus fruits, blackberries, blueberries, big blackberries, blueberries, currants, American raspberries, raspberries, grapes, avocados , bananas, kiwi, plaque, pomegranate, pineapple, tropical fruits are often susceptible to attack and defoliation by sphinx moth, amorbia, soldier worm, citrus cutter worm, banana borer, red head worm, blueberry leafroller blue, cancer worm, cherry fruit worm, cherry cutter worm, blueberry narrowing worm, eastern caterpillar, autumn weaver worm, filler filler of hazelnut, hazelnut weaver worm, fruit tree leafroller, grape fence moth, grape leafroller, grape leaf skeletonizer, green fruit worm, gummy-Batrachedra commosae, gypsy moth, worm of the American walnut bark, horned worms, measuring caterpillars, umbilicated orange worm, oblique band leafroller, omnivorous leafroller, omnivorous measuring caterpillar, orange tortrix, orange worm, oriental fruit moth, pandemic leafroller , peach tree borer, walnut borer, red band leafroller, red back caterpillar, rough skin cutworm, saline caterpillar, worm gauge, lizard, basilids key-key, gallworm Tobacco, tortrious moth, apple moth plume moth, variegated leafroller, walnut caterpillar, western lizard and yellow vein welded worm. Fields of cultivation such as canola / colsa seeds, donkey grass, prairie foam, corn (field, rosette), cotton, hops, jojoba, peanut, rice, safflower, small grains (barley, oats, rye, wheat, etc.), sorghum, soy, sunflower and tobacco are often targets for insect ingestion including armyworms, Asian corn borers and other corn boragers, veined sunflower moth, soldier beetworm, worm of the cotton capsule, cabbage measuring caterpillar, corn rootworm (including southern and western varieties), cotton leaf borer, diamondback moth, European corn borer, green cloverworm, cottonworm cabezuela, cabezuela worm, worm imported from cabbage, caterpillar measuring (including Anacamptodes spp.), oblique band leafroller, omnivore leaf attacker, podworm, caterpillar of the salina, southwestern borer corn, soybean measuring caterpillar, spotted cutworm, sunflower moth, tobacco weaver worm, horned tobacco worm, velvety bean caterpillar.
Plants for straw bed, flower plants, ornamental plants, vegetables and farm are frequently attacked by many insect pests such as soldier worms, azalea moth, soldier beetworm, diamondback moth, worm horned, Florida fern caterpillar, moth and / or, measuring caterpillar, oleander moth, omnivorous leafroller, omnivorous measuring caterpillar and tobacco weaverworm. Forest trees, ornamental fruits and nuts, as well as shrubs and other nursery trees are often susceptible to attack by various insects such as bag worm, black-headed gillworm, brown tail moth, oak worm from California, douglas fir lizard, elm measuring worm, autumn weaver worm, fruit tree leafroller, maple green streak worm, gypsy moth, pine gillworm, mimosa weaver worm, pine butterfly , caterpillar of red back, caterpillar of back in the form of saddle, caterpillar with prominence in the form of saddle, worm of the cancer of the spring and autumn, worm of the spruce of the spruce, lagarta, tortrícidos and western lagarga moth. Likewise, peat grasses are often attacked by pests such as welded worms, turf weaver worm, and tropical turf weaver worm.
TABLE 1
Taxonomy of pests of coleopteran pests in the Archostemata and Polyphaga suborders
Infraorder SuperFamily Subfamily Tribe Genus Species family Cupedidae (beetles Priacma P. serrata reticulados) Bostrichí- Dermestidae (beetles of Attagenus A. pellio formia piel and from the store Chrysomeli- Cerambycidae (beetles of Agopanthia Agopanthia sp. Formia long horns) Lepturinaé Leptura Leptura sp (flower-horned beetle) Rhagium Rhagium sp. to Megacyllene M. robiniae Prioninae Derobrachus D. geminatus Tetraopes T. tetrophtal-mus Chrysomelidae (Chlamisinae beetles Exema E. neglecta leaves) Chrysomelinae Chrysomelini Chrysomela C. tremula, Chrysomela sp.
Infraorder SuperFamily Subfamily Tribe Genus Species family Oreina O. cacaliae Doryphorini Chrysoline Chrysolina sp. Leptinotarse L. decemlineata (beetle of the potato of olorado) Gonioctenini Gonioctena G. fornicata, G. Holdausi, G. Intermedia, G.interpostia, G. Kamikawai, G. Linneana, G. Nigroplagiata, G. Occidentalis, G. Olivaceae, G. Paluda, G. Quinquepunctata, G. Rubripennis, G. Rufipes, G. Tredecim-maculata, G. Variabilis, G. viminalis 8 Trimarchini Timarcha Timarcha sp. Criocerinae Oulema Oulema sp. Galerucinae Galerucini Monoxia M. inornata, Monoxia sp. Orphaella O. arctica, O. artemisiae, O. bilneata, O. commune, O. conferia, O. cribata, O. notata, O. notulata, O. nuda, O. pilosa, O.sexvittata, O. slobodkini Cerotoma C trifurcata
Infraorder SuperFamily Subfamily Tribe Gender Species family Diabrotica D. barberi (northern root worm), D. Undecimpunctata (southern root worm), D. Virgifera (western root worm) Not classified Lachnaia Lachnaia sp. Chrysomelidae Epitrix E. cucumeris (Harris) (potato flea beetle), E. Fuscala (flea beetle of eggplant) Curculionidae Curculioninae Authonomus A. grandis (weevil of the (weevil) capsule) w Entiminae Naupactini Aramigus A. conirostris, A Globoculuc, A. Intermedius, A. Planioculuc, A. tesselatus Otiorhynchus Otiorhynchus sp. Diaprepes D. abbreviata Phillobiini Phyllobius Phyllobius sp. Galapaganus G. galapagoensis
Infraorder Superfamily Subfamily Tribe Genus Species family Hyperinae Hypera H. brunneipennis (Egyptian weevil of alfalfa), H. Popstica (alfalfa weevil, H. punctata (clover leaf weevil) Molytinae Pissodes P. affinis, P. Nemorensis, P. schwarzm P. strobi, O. terninalis Rhynchophorinae Sitophilini Sitophilus S. granarius (barn weevil), S. zeamais (corn weevil) Nemonychidae Lebanorhinus L. succinus Scolytidae Ips I. acuminatuas, I. amitinus, I. cembrae, I duplicatus, I. mannsfeldi, I. sexdentatus, I. fS typographus Orthotomicus O. erosus Tomicus T. minor Cucujiformia Coccinellidae Epilachna E. borealis (ladybird of the (ladybugs) yellow squash), E. varivstis (beetle of the Mexican bean) Cucujidae ' Cryptolestes C. ferrugineus (flat bark beetle)
Infraorder Superfamily Subfamily Tribe Genus Species family Oryzaephilus O. surinamensis (beetle (sawtooth beetles of the grgano) grain) Lagriidae (large Lagria beetles Lagria sp.) Meloidae (blister beetles Epicauta £ funebris) Meloe M. proscarabaeus Rhipiphoridae Rhipiphorus R. fasciatus Tenebrionidae Alphitobius A. diaperinusK (minor worm of the 83 (dark tenebrous beetles) of the soil) Hegeter H. amaroid, H. brevicollis, H. costipennis, H. fernandezi, H. glaber, H. gomerensis, H. grancanariensis , H. impressus, H. intercedens, H. lateralis, H. plicifrons, H. politus, H. subrotundatus, H. tenuipunctatus, H. transversu, H. webbians Misolampus M. goudoti
Infraorder Superfamily Subfamily Tribe Gender Species family Palorus P. ficicola, P. Ratzeburgi (small leaf beetle of flour), P Subdepressus (depressed flour beetle) Pimelia P. baetica, P. Canariensis, P. Criba, P. Elevata , P. Estevezi, P. Fernan-dezlopezi, P. Grandis, P. Granulicollis, P. Integra, P. Lutaria, P. Interjecta, P. Laevigata, P. Lutaria, P. Radula, P. Sparsa, P. variolosa Tenebrio T. molitor (yellow flour worm) 7"Obscurus (dark flour worm) Tentyria T.brevicornis, T. castaneum (red flour beetle g), T. confusum (confused flour beetle), T. Freemani, T. madens Tribolium Z. atratus Zophobas Z. rugipes
Elateriformia Elateroidea Octinodes Octinodes so.
Infraorder Superfamily Subfamily Tribe Genus Species family Scarabaeiformia Pyrophorus P plagio-phtalamus Lucanidae (deer Dorcus D. parallelo-pipedus flounders) Lucanus L. cervus Scarabeidae Allomyrna A. dichotoma (lamelicomian beetles) Cetoniinae (Pachnoda beetle P. marginata of flowers) 8R Dynaastinae Xyloryctes X. faunus Geotrupinae Geotrupes G. stercorosus (soil-boring dung beetles) Melonlonthinae Costelytra C. zealandica (bumblebees) Holotrichia H. diomphalia Melolontha M. melolontha (bumblebee) Odontria O. striata, 0. variegata
Infraorder SuperFamily Subfamily Tribe Gender Species family Prodontria P. bicolorata, P. capito, P. lewisi, P. tarsis, P. modesta, P. pinguis, P. praelatella, P. truncatta, Prodontria sp. Scythrodes S. squalidus Rutelinae (Popillia bumblebees P. japonica (Japanese beetle) bright leaves Scarabaeinae Copris C. lunarias (black dung beetle Scarabaeus Scarabaeus sp. (Beetles) Staphyliniformia Hydrophilidae Cercyon Cercyon sp. SR Silphidae Nicrophorus N. americanus, N. Marginatus, N. Orbicollis, N. tomentosus Staphylinidae Carpelimus Carpelimus sp. (Wandering beetles) Quedius Q. mesomelinus Tachyporus Tachyporus sp. Xantholinus Xantholinus sp.
4. 3 Nomenclature of B. thuringiensis d-endotoxins
Table 2 contains a list of the traditional nomenclature and
currently recognized for the known. Access numbers to the gene bank for the polypeptides and sequenced polynucleotides are also shown.
TABLE 2
Nomenclature of b. Thuringiensis or known d-endotoxins
No. of access to the bank New Viejo of genes Cry1Aa1 CrylA (a) M11250
CryiAa2 CrylA (a) M10917
Cry1Aa3 Cry1A (a) DOO34d Cry1Aa4 Cry1A (a) X13535
Cry1Aa5 CrylA (a) D175162
Cry1Aa6 CrylA (a) U43605
Cry1Aa7 AFO81790
CrylAad 126149
Cry1Aa9 ABO26261
Cry1Ab1 CrylA (b) M13896
Cry1Ab2 CrylA (b) M12661
Cry1Ab3 CrylA (b) M15271
Cry1Ab4 CrylA (b) DOOI17 Bank access number
New Old Genes
Cry1Ab6 CrylA (b) M37263
CrylAb7 CrylA (b) X13233
Cry 1 Abd CrylA (b) M 16463
Cry1Ab9 CrylA (b) X54939
Cry1Ab10 Cry? A (b) A29125
Cry1Ab11 112419
Cry1Ab12 AFO57670
CrylAd CrylA (c) M11O6d
Cry1Ac2 CrylA (c) M35524
Cry1Ac3 CrylA (c) X54159
Cry1Ac4 CrylA (c) M73249
Cry1Ac5 CrylA (c) M73246
Cry1Ac6 CrylA (c) U43606
Cry1Ac7 CrylA (c) U67793
CrylAcd CrylA (c) U87397
Cry1Ac9 CrylA (c) U89872
Cry 1Ac10 CrylA (c) AJ002514
Cry1Ac11 AJI30970
CrylAc12 112418 Bank access number
New Old Gene Cry1Ad2 A27531 CrylAel CrylA (e) M65252 CrylA l U32003 CrylAgl AFO3124d
CrylBal CrylB XO6711
CrylBa2 X95704 CrylBbl ET5 L32020 CrylBcl Crylb (c) Z46442 Cry1Bd1 CryE1 U 70726 CrylCal CrylC XO751d Cry1Ca2 CrylC X13620 Cry1Ca3 CrylC M73251 CrylCa4 CrylC A27642 CrylCad CrylC X966d2 CrylCad CrylC X966d3 Cry1Ca7 CrylC X966d4 CrylCbl CrylC (b) M97d80 Cryl Dai CrylD X54160 Cry1Da2 176415 CrylDbl prtb Z22511 Bank access number
New Old Genes
Cry1Ea2 CrylE X56144
Cry1Ea3 CrylE M73252
Cryl Ea4 U94323
Cry1 Ea5 A15535
Cry1 Eb1 CrylE (b) M73253
Cry1Fa1 CrylF M63397
Cry1Fa2 CrylF M63397
Cry1Fb1 PrtD Z22512
CrylFb2 Z22512
Cry1Fb3 AFO62350
Cry1Fb4 173695
CrylGa 1 PrtA Z2251
Cry1Ga2 CrylM Y09326
CrylGbl CryH2 U 70725
CrylHal PrtC Z22513
CrylHbl U35730
Cry11al CryV X62621
Cry1 la2 CryV M9d544
Crylla3 CryV L36333
Crylla4 CryV L49391
Cry1 la5 CryV YOd920 Bank access number
New Old Gene Cry11bl CryV UO7642 Cryllcl AFO56933
CrylJal ET4 L32019 CrylJbl ETI U31527 CrylJcl AFO56933
CrylKal U2dd01 Cry2Aal CryllA M31738 Cry2Aa2 CryllA M23723 Cry2Aa3 D860d4 Cry2Aa4 AFO4703d
Cry2Aa5 AJ 132464
Cry2Aa6 AJ 1324635
Cry2Aa7 AJ 132463
Cry2Ab1 CryllB M23724 Cry2Ab2 CryllB X55416 Cry2Ac1 CryllC X57252 Cry3Aa1 Cryll I A M22472 Cry3Aa2 CrylllA JO2976 Cry3Aa3 CrylllA YOO420
Cry3Aa4 CrylllA M30503 Cry3Aa5 CrylllA M37207 Bank access number
New Old Gene Cry3Aa7 AJ237900
Cry3Ba1 X17123 Cry3Ba2 Cryll IB IB Cryl I AO7234 Cry3Bb1 Cryl 11 B2 M69794 Cry3Bb2 CrylllC (b) U31633 Cry3Ca1 Cryll ID Cry3Bb3 115475 X59797 Cry4Aa2 Cry4Aal CrylV A YO0423 CrylV A DO0248 Cry4Ba1 X07423 Cry4Ba2 CrylVB X07082 Cry4Ba3 CrylVB M20242 Cry4Ba4 CrylVB CrylVB DO0247
CrydAal CryV A (a) L07025 CrydAbl CryVA (b) L07026 CrydAcI 134543 CrydBal PS66Q3 U 19725 CrydAal CryVIA L07022 Cry6Bai Cry VI B L07024 Cry7Aal CrylllC M64476
Cry7Abl CrylllCb U04367 Bank access number
New Old CrydAal Cryl gene CrydBal ME U04364 U04365 U04366 CrydCal CrydBal CrylllG U04365 U04366 CrydCal CrylllF Cry9Aal CrylG Cry9Aa2 CrylG X58534 X53120 X75019 Cry9Bal CrylX Cry9Cal CrylH Z37527 Cry? Dal N14I Dd5560 Cry9Da2 AF042733 Cry9Eal
CrylOAal CrylVC M 12662 Cry10Aa2 EOO614
Cry11Aa1 CrylVD M31737
CryllAa2 CrylVD M22d60
Cry11 Bal JegdO Xd6902 CrylIBbl AF017416
Cry12Aa1 CryVB LO7027
Cryl3Aal CryVC LO7023
Cry14Aal CryVD U 13955
Cryl5Aa1 34kDa M76442 Bank access number
New Old Gene Cryl7Aa1 cbm71 X99478 CryldAal CryBPI X99049 Cry19Aal Jeg65 YOd920 Cry20Aal Ud251d Cry21Aal 132932 Cry22Aa1 134547 CrY23Aa1 AFO304d
Cry24Aal Uddldd Cry25Aa1 LJdd1d9
Cry26Aa1 - AF122697
Cry27Aal AB023293
Cry28Aa1 AF 132926
CytlAal CytA X03132 Cyt1Aa2 CytA XO4333 CytlAa3 CytA YO0135
Cyt1Aa4 CytA M35968
CytlAbl CytM X98793 CytBai U37196 Cyt2Aal CytB Z14147 Cyt2Bal Cyt2Bal CytBa U52043 Cyt2Ba2 CytBa AFO20739 Bank access number New Old Cyt2Ba4 gene "CytB" AF022dd5 Cyt2Ba5"CytB" AF022dd6 Cyt2Ba6 AFO34926 Cyt2Bbl U82519 Cyt2Bbl U82519
a Adapted from: http://epunix.bioIs.susx.ac.uk Home / Neil_Crickmore / Bt / toxins.htmI (April 27, 1999).
4. 4 Probes and primers In another aspect, the DNA information provided by the invention allows the preparation of relatively short DNA (or RNA) sequences that have the ability to specifically hybridize to the gene sequences of the selected polynucleotides described herein. In these aspects, nucleic acid probes of an appropriate length are prepared based on a consideration of a gene sequence encoding crystal protein, for example, a sequence such as the one described herein. The ability of such nucleic acid DNA probes to specifically hybridize to a gene sequence encoding a crystal protein gives them particular utility in a variety of modalities. More important, the probes can be used in a variety of tests to detect the presence of complementary sequences in a given sample. In some embodiments, it is advantageous to use oligonucleotide primers. The sequence of said primers is designed using a polynucleotide of the present invention to be used in the detection, amplification or mutation of a defined segment of a crystal protein gene from β. thuringiensis using PCR ™ technology. Related crystal protein segments from other species can also be amplified by PCR ™ using such primers. To provide some of the advantages according to the present invention, a preferred nucleic acid sequence employed for hybridization or hybridization assays includes sequences that are complementary to at least about 23 to about 40, etc., the extension of nucleotides long of a sequence encoding crystal protein, such as the one shown here. A size of at least 14 or 15 nucleotides in length helps ensure that the fragment is of sufficient length to form a duplex molecule that is both stable and selective. Molecules having complementary sequences in extensions greater than about 23 bases in length are generally preferred although to increase the stability and selectivity of the hybrid, and therefore to improve the amount and degree of specific hybrid molecules obtained. It will be preferred to design nucleic acid molecules having complementary extensions of genes of about 14 to about 20 nucleotides or even longer as desired. Such fragments can be easily prepared, for example, by direct synthesis of the fragment by chemical means by the application of nucleic acid reproduction technology such as PCR ™ technology of the U.S. Patents. 4.6d3,195, and 4,663,202, specifically incorporated herein by reference or by cutting DNA fragments selected from recombinant plasmids having appropriate inserts and appropriate restriction sites.
4. Expression Vectors The present invention contemplates a polynucleotide of the present invention that is comprised within one or more expression vectors. Thus, in one embodiment, an expression vector comprises a nucleic acid segment that contains a gene encoding crystal protein operably linked to a promoter that expresses the gene. In addition, the coding region can also be operably linked to a transcription termination region, whereby the promoter activates the transcription of the coding region and the transcription termination region stops transcription at a 3 'site in the coding region. As used herein in the term "operably linked" it means that a promoter is connected to a coding region in such a way that the transcription of that coding region is controlled and regulated by that promoter. Techniques for operatively linking a promoter to a coding region are known in the art. In a preferred embodiment, the recombinant DNA expression encoding the crystal proteins of the present invention is preferable in a Bacillus host cell. Preferred host cells include B. thuringiensis, B. megaterium, B. subtilis, and related bacilli, with host cells of B. thuringiensis being highly preferred. Promoters that function in bacteria are well known in the art. An illustrative and preferred promoter for the crystal proteins derived from bacilli include any of the crystal protein gene promoters, including. the cry gene promoters themselves. Alternatively, mutagenized or recombinant promoters can be genetically engineered by man and used for promoter expression of the novel gene segments described herein. In an alternative embodiment, the recombinant expression of the DNAs encoding the crystal proteins of the present invention is performed using a transformed Gram negative bacterium such as an E. coli host cell or Pseudomonas spp. Promoters that function in high level expression of target polypeptides in E. coli and other Gram negative host cells are also well known in the art. Where a vector of the present invention is to be used to transform a plant, a promoter having the ability to activate expression in plants is selected. Promoters that function in plants are also well known in the art. Useful in the expression of the polypeptide in plants are promoters that are inducible, viral, synthetic, constitutive as described (Poszkowski et al., 19d9; Odell et al., 19d5) and temporarily regulated, spatially regulated and temporally regulated space (Chau et al. al., 19d9). A promoter is also selected for its ability to direct the transcription activity of the cells of the transformed plant or of the transgenic plant to the coding region. Structural genes can be activated by a variety of promoters in plant tissues. The promoters can be almost constitutive, such as the CaMV 35S promoter, or tissue-specific or developmental specific promoters to the dicotyledonous or monocotyledonous. Where the promoter is almost a constitutive promoter such as CaMV 35S, increases in the expression of polypeptides are found in a variety of tissues of transformed plants (e.g., callus, leaf, seed and root). Alternatively, transformation effects can be directed to specific plant tissues using plant integration vectors containing a tissue-specific promoter. An illustrative tissue-specific promoter is the lectin promoter, which is specific for seed tissue. The lectin protein in soybean seeds is encoded by a single gene (Lei) that is expressed only during seed maturation and represents from about 2 to about 5% of the total mRNA of the seed. The gene of lectin and the seed-specific promoter have been fully characterized and used to direct the specific expression of the seed in transgenic tobacco plants (Vodkin et al., 19d3; Lindstrom et al., 1990). An expression vector comprising a coding region encoding a polypeptide of interest is genetically engineered to control the lectin promoter and that vector is introduced into plants using, for example, a protoplast transformation method. (Dhir et al., 1991a). The expression of the polypeptide is directed specifically to the seed of the transgenic plant. A transgenic plant of the present invention produced from a plant cell transformed with a tissue-specific promoter can be crossed with a second transgenic plant developed from a plant cell transformed with a different specific promoter to produce a hybrid transgenic plant that shows the effects of the transformation on more than one specific tissue. Illustrative tissue-specific promoters are sucrose synthetase 1 (Yang et al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1939), corn light harvest complex (Simpson, 19d6), corn heat shock protein (Odell et al., 19d5), small subunit RuBP carboxylase of pea (Poulsen et al., 19d6; Cashmore et al., 1963), mannopine synthase of Ti plasmid (Langridge et al., 1969) , nopalina synthase of plasmid Ti (Langridge et al., 19d9), calcone isomerase of petunia (Van Tunen et al., 19dd), protein 1 rich in bean glycine (Keller et al., 19d9), transcript of CaMV 35S ( Odell ef al., 19d5) and potato patatina (Wenzler ef al., 19d9). The promoters preferred are the cauliflower mosaic virus (CaMV 35S) and the RuBP carboxylase promoter of small subunit S-E9. The choice of which expression vector and finally to which promoter a polypeptide coding region is operably linked depends directly on the desired functional properties, for example, the location and time of protein expression, and the host cell to be transformed . These are well-known limitations inherent in the construction technique of recombinant DNA molecules. However, a vector useful in the practice of the present invention is capable of directing the expression of the polypeptide coding region to which it is operably linked. Typical vectors useful for the expression of genes in higher plants are well known in the art and include vectors derived from the tumor inducing plasmid (Ti) of Agrobacterium tumefaciens described (Rogers et al., 1937). However, it is known that other plant integrator vector systems function in plants including the pCaMVCN transfer control vector (Fromm et al., 1965). PCaMVCN (available from Pharmacia, Piscataway, NJ) includes the CaMV 35S promoter of the cauliflower mosaic virus. In preferred embodiments, the vector used to express the polypeptide includes a selection marker that is effective in a plant cell, preferably a drug resistance selection marker. A preferred drug resistance selection marker is the gene whose expression results in resistance to kanamycin, ie, the chimeric gene comprising the nopaline synthase promoter, Tn5 phosphotransferase II neomycin (nptll) and the untranslated region of nopaline synthase 3 '(Rogers et al., 1988). RNA polymerase transcribes a coding DNA sequence through a site where polyadenylation occurs. Typically, DNA sequences located a few hundred base pairs toward the 3 'end to the polyadenylation site serve to terminate transcription. Those DNA sequences are known as transcription termination regions. Those regions are required for efficient polyadenylation of described messenger RNA (mRNA). Means for preparing expression vectors are well known in the art. The expression (transformation vectors) used to transform plants and methods for making those vectors are described in the U.S. Patents. 4, 971, 908, 4,940,635, 4,769,061 and 4,757,011, the descriptions of which are specifically incorporated herein by reference in their entirety. Those vectors can be modified to include a coding sequence according to the present invention. A variety of methods have been developed to operatively insert a segment of DNA into a vector through shaved ends or complementary cohesive terminations. For example, complementary homopolymer tracts can be added to the DNA segment to be inserted and to the vector DNA. The vector and the DNA segment are then linked by hydrogen bonding between the homopolymeric tails complementary to the recombinant DNA molecules. A coding region that encodes a polypeptide having the ability to confer insecticidal activity to a cell is preferably a gene encoding for B. thuringiensis crystal protein in preferred embodiments said polypeptide having the amino acid residue sequence of one of the sequences herein are described or a functional equivalent thereof. According to said embodiments, a coding region comprising the DNA sequence of said sequence is also preferred.
4. 6 Nomenclature of novel proteins The inventors have arbitrarily assigned allocations to the novel proteins of the invention. Also, arbitrary gene designations have been assigned to the novel nucleic acid sequence encoding these polypeptides. The formal assignment of gene and protein designations based on the revised nomenclature of crystal protein endotoxins will be assigned by a committee on the nomenclature of B. thuringiensis, formed to systematically classify crystal proteins B. thuringiensis. The inventors contemplate that the arbitrarily assigned designations of the present invention will be superseded by the official nomenclature assigned to those sequences.
4. 9 Transformed host cells and transgenic plants Methods and compositions for transforming a bacterium, a yeast cell, a plant cell or another whole plant with one or more expression vectors comprising a crystal protein coding gene segment are additional aspects of this description. A transgenic bacterium, yeast cell, plant cell or plant derived from said transformation process or the progeny and seeds of said transgenic plant are also embodiments of the invention. Means for transforming bacteria and yeast cells are well known in the art. Typically, transformation media are similar to those well known means used to transform other bacteria or yeasts such as £. coli or Saccharomyces cerevisiae. Methods for transforming DNA from plant cells include the transformation of Agrobacterium-mediated plants, protoplast transformation, gene transfer into pollen, injection into reproductive organs, injection into immature embryos and bombardment of particles. Each of these methods has different advantages and disadvantages. Therefore, a particular method of introducing genes into a particular silver strain may not necessarily be the most effective for another plant strain, but it is well known that methods are useful for a particular plant strain. There are many methods for introducing transforming DNA segments into cells, but not all are suitable for delivering DNA to plant cells. Suitable methods are believed to include almost any method by which DNA can be introduced into a cell, such as Agrobacterium infection, direct DNA delivery such as, for example, PEG-mediated protoplast transformation (Omirulleh et al., 1993), by DNA absorption mediated by desiccation / inhibition, by electroporation, by agitation with silicon carbide fibers, by acceleration of DNA coated particles, etc. In certain embodiments, acceleration methods are preferred and include, bombardment of microprojectiles and the like. The technology for the introduction of DNA into cells is well known to those skilled in the art. Four general methods for delivering a gene in cells have been described: 1 chemical methods (Graham and van der Eb, 1973; Zatloukal et al., 1992); 2 methods such as microinjection
(Capecchi, 1930), electroporation (Wong and Neumann, 19d2; Fromm et al., 19d5; patent of U.S. 5,334,253) and the gene gun (Johnston and Tang, 1994;
Fynan et al., 1993); 3 viral vectors (Clapp, 1993; Lu ef al., 1993; Eglitis and Anderson, 19dda; Eglitis et al., 19dd); and 4 mechanisms mediated by receiver
(Curiel et al., 1991; 1992; Wagner et al., 1992).
4. 9.1 Electroporation The application of brief high-voltage electrical pulses to a variety of animal and plant cells leads to the formation of nanometer-sized pores in the plasma membrane. The DNA is taken directly into the cytoplasm of the cell either through these pores or as a consequence of the redistribution of membrane components that accompany the closing of the pores. Electroporation can be extremely efficient and can be used for transient gene expression of clones and for the establishment of cell lines that cut integrated copies of the gene of interest. Electroporation, unlike calcium phosphate-mediated transfection and protoplast fusion, it often gives rise to cell lines that carry one, or at most a few, integrated copies of the foreign DNA. The introduction of DNA by means of electroporation is well known to those skilled in the art. In this method, some enzymes, cell wall degraders such as pectin degrading enzymes, are employed to make target receptor cells more susceptible to transformation by electroporation than untreated cells. Alternatively, the recipient cells become more susceptible to transformation by mechanical injury. To effect the transformation by electroporation, either friable tissues such as a cell culture in suspension, or embryogenic callus, or alternatively, immature embryos or other directly organized tissues can be transformed. The cell walls of the chosen cells are partially degraded by exposing them to the pectin degrading enzymes (pectoliases) or by mechanical injury in a controlled manner. Said cells would be receptors for DNA transfer by electroporation, which can be carried out in this step, and the transformed cells are then identified by a suitable screening or sieving protocol depending on the nature of the newly incorporated DNA.
4. 9.2 Microprojectile bombardment A further advantageous method for delivering transformant DNA segments to plant cells is the bombardment of microprojectiles. In this method, the particles can be coated with nucleic acids and delivered to cells by force of proportion. Illustrative particles include those composed of tungsten, gold, platinum and the like. An advantage of microprojectile bombardment, besides being an effective means of transforming monocots in reproducibly stable form, is that protoplast isolation or susceptibility to infection by Agrobacterium is not required. An illustrative embodiment of a method for delivering DNA into corn cells by acceleration is a biolistic particle delivery system, which can be used to propel DNA coated particles, or cells through a sieve, such as a steel screen. stainless steel or Nytex, on a filter surface covered with corn cells grown in suspension. The sieve disperses the particles so that they are no longer delivered to the recipient cells in large aggregates. It is believed that a sieve that intervenes in the projectile apparatus and the cells to be bombarded reduces the size of projectile aggregates and can contribute to a higher frequency of transformation by reducing the damage produced to the recipient cells by projectiles that are too large. For bombardment, cells in suspension are preferably concentrated in filters or in a solid culture medium. Alternatively, immature embryos or other target cells can be placed in solid culture medium. The cells that are going to be bombed; they are placed at an appropriate distance below the macroprojectile stop plate. If desired, one or more sieves are also placed between the acceleration device and the cells to be bombarded. By using the techniques discussed here, up to one thousand or more foci of cells transiently expressing a marker gene can be obtained. The number of cells in a focus expressing the exogenous gene product 4d hours after bombardment often varies from 1 to 10 and on average from 1 to 3. In the bombardment transformation, the pre-bombardment culture conditions can be optimized and the bombing parameters to obtain the maximum numbers of stable transformants. Both physical and biological parameters for bombardment are important in this technology. The physical factors are those that involve manipulation of the DNA / microprojectile precipitate or those that affect the flight and velocity of either macro or microprojectiles. Biological factors include all steps involved in the manipulation of cells before and immediately after bombardment, the osmotic adjustment of the cells or target to help alleviate the trauma associated with the bombardment and also the nature of the transforming DNA, such as a DNA linearized or intact superhelical plasmids. It is believed that pre-bomb manipulations are really important for the successful transformation of immature embryos. Accordingly, it is contemplated that it is desired to adjust several of the bombardment parameters in small scale studies to fully optimize the conditions. It may be particularly desirable to adjust the physical parameters such as gap distance, flight distance, tissue distance and helium pressure. Trauma reduction factors (TRFs) can also be reduced to a minimum by modifying the conditions that influence the physiological state of the recipient cells and that therefore influence transformation and integration efficiencies, for example; The osmotic state, tissue hydration and subculture stage or cell cycle of the recipient cells can be adjusted for optimal transformation. The execution of other routine adjustments will be known to a person skilled in the art in light of the present description.
4 > 9.3 Agrobacterium-mediated transfer Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the
DNA can be introduced into whole plant tissues, thus avoiding the need for regeneration of an intact plant from a protoplast. The use of plant-integrating vectors mediated by Agrobacterium to introduce
DNA in plant cells is well known in the art. See, for example, the described methods (Rogers et al; 19d7). In addition, the integration of Ti-DNA is a relatively accurate procedure that results in few rearrangements. The region of DNA to be transferred is defined by the contiguous sequences, and the intervening DNA is generally inserted into the genome of the plant as described (Spielmann et al; Jorgensen et al;
1987). Agrobacterium transformation vectors are capable of replicating in E. Coli as well as Agrobacterium allowing convenient manipulations as described (Klee et al, 1985). In addition, recent technological advantages in gene transfer vectors mediated by Agrobacterium have improved gene ordering and restriction sites in vectors
'to facilitate the construction of vectors capable of expressing several genes encoding polypeptide. The vectors described (Rogers et al., 1987), have multi-lane regions flanked by a promoter and a polyadenylation site for the direct expression of inserted polypeptide-encoding genes and are suitable for the following purposes. In addition, Agrobacteium containing unarmed Ti genes can be used for transformations. In those strains of plants in which the transformation mediated by Agrobacterium is efficient, this is the method of choice due to the easy and defined nature of gene transfer. Agrobacterium-mediated transformation of leaf discs and other tissues such as cotyledons and hypocotyls appear to be limited to plants that are naturally infected by Agrobacterium. The transformation mediated by Agrobacterium is more efficient in dicotyledonous plants. Some monocots appear to be the natural hosts for Agrobacterium, although asparagus transgenic plants have been produced using Agrobacterium vectors as described (Bytebier et al., 1987). Therefore, commercially important cereal grains such as rice, corn and wheat should generally be processed using alternative methods. However, as mentioned before, the transformation of asparagus using Agrobacterium can also be achieved (see for example, Bytebier et al., 1987). A transgenic plant formed by transformation methods by Agrobacterium typically contains a single gene on a chromosome. Said transgenic plants can be referred to as heterosigotics for the added gene. However, while the use of the word "heterozygous" generally implies the presence of a complementary gene in the same locus of the second chromosome of a pair of chromosomes, and there is no such gene in a plant that contains an added gene like here, it is believed that a more precise name for said plant is a dependent segregator because the exogenous gene added segregates independently during mitosis and meiosis. A transgenic plant that is homozygous for the added structural gene, i.e., a transgenic plant that contains two added genes, a gene at the same locus on each chromosome of a pair of chromosomes is very preferred. A homozygous transgenic plant can be obtained by sexually matching (self-matching) an independent segregating transgenic plant containing a single added gene, germinating some of the seeds produced and analyzing, the resulting plants produced for increased carboxylase activity in relation to a control (native non-transgenic) or an independent segregating transgenic plant. It should be understood that two different transgenic plants can also be matched to produce offspring containing two independently added exogenous segregating genes. The appropriate progeny self matching can produce plants that are homozygous for added exogenous genes that encode a polypeptide of interest. Backcrossing to a parent plant is also contemplated and crossed with a non-transgenic plant.
4. 9.4 Other transformation methods The transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation and combination of these treatments (see e.g. Potrykus ef al., 1985; Lorz et al. al., 1985, Fromm ef al., 1986; Uchymiya ef al., 1987;
Callis et al., 1985., 1987; Marcotte et al., 1988). The application of these systems to different strains of plants depends on the ability to regenerate that particular plant strain from protoplasts. Illustrative methods are described for the regeneration of cereals from protoplasts (Fujimura et al., 1985; Toriyama et al., 1986;
Yamada et al., Abdullha ef a /., 1986). To transform strains of plants that can not be successfully regenerated from protoplasts, other forms can be used to introduce DNA into intact cells. For example, the regeneration of cereals from immature embryos or explants can be carried out as described (Vasil, 1988). In addition, the "particle gun" or high-speed microprojectile technology can be used (Vasil et al., 1992). Using the latest technology, DNA is carried through the cell wall and into the cytoplasm on the surface of small metal particles (Klein et al., 1987, Klein et al., 198d, McCabe et al., 198d). The metallic particles penetrate through several layers of cells and thus allow the transformation of cells into tissue explants.
4. 9.5 Expression of genes in plants Although a breakthrough has been obtained in recent years with respect to the preparation in transgenic plants expressing bacterial proteins such as B. thuringiensis crystal proteins, the results of expressing native bacterial genes in plants are a often disappointing. Unlike microbial genetics, the early plant geneticists knew little about the factors that affect the heterologous expression of foreign genes in plants. In recent years, however, several potential factors have been implicated as being responsible in varying degrees for the level of protein expression from a particular coding sequence. For example, scientists now know that maintaining an important level of a particular mRNA in the cell is indeed a crucial factor. Unfortunately, the causes for low steady state levels of mRNA that encode foreign proteins are many. First, the synthesis of full-length RNA may not occur at a high frequency. This, for example, could be caused by the premature termination of RNA during transcription or due to unexpected mRNA processing during transcription. Second, full length RNA can be produced in the plant cell, but can then be processed (splice, poly A addition) in the nucleus in a manner that results in a non-functional mRNA. If RNA is not properly synthesized, terminated and polyadenylated, it can not move into the cytoplasm for translation. Similarly, in the cytoplasm, if the mRNA has reduced half-lives (which are determined by its primary or secondary sequence) an insufficient protein product will be produced. In addition there is an effect, whose magnitude is uncertain, of translation efficiency over the half-life of the mRNA. In addition, each RNA molecule is bent into a particular structure, or perhaps a family of structures, which is determined by its sequence. The particular structure of any RNA could lead to more or less stability in the cytoplasm. The structure itself is also probably a determinant of a processing of mRNA in the nucleus. Unfortunately, it is possible to predict, and almost impossible to determine, the structure of any RNA (except tRNA) in vitro or in vivo. However, it is likely that the drastic change in the sequence of an RNA will have a large effect on its bent structure. It is likely that the structure by itself or particular structural characteristics, also have a role in the determination of RNA stability. To overcome these limitations in foreign gene expression, the researchers have identified particular sequences and signals in RNA that have the potential to have a specific effect on RNA stability. In some embodiments of the invention, therefore, it is desired to optimize the expression of the nucleic acid segments described in plant. A particular method to do this is by altering the bacterial gene to remove sequences or motifs that reduce the expression in a transformed plant cell. The processing of genetic manipulation of a coding sequence for optimal expression in plant is often referred to as "planting" a DNA sequence. Particularly problematic sequences are those that are rich in A + T. Unfortunately, since B. thuringiensis has a genome rich in A + T, the sequences of native crystal protein genes must often be modified for optimal expression. The sequence motif ATTTA (or AUUUA as it appears in RNA) has been implicated as a destabilizing sequence in mammalian mRNA (Shaw and Kamen, 1986). Many short-lived mRNAs have 3 'untranslated regions rich in A + T, and those regions often have the sequence ATTTA, sometimes present in multiple copies or as multimers (for example ATTTATTTA ...). Shaw and Kamen showed that the transfer of the 3 'end of an unstable mRNA to a stable RNA (globin or VA1) decreased the stable RNA half-life dramatically. They also showed that an ATTTA pentamer had a profound destabilizing effect on a stable message, and that this signal could exert its effect whether it was located at the 3 'end or within the coding sequence. However, the number of ATTTA sequences and / or the sequence context in which they occur could also be important in determining whether they function as destabilizing sequences. Shaw and Kamen demonstrated that an ATTTA trimer had a much smaller effect than a pentamer on mRNA stability and a dimer or monomer had no effect on stability (Shaw and Kamen, 1987). Note that ATTTA multimers such as a pentamer automatically create a region rich in A + T. This showed that it was a nuclear cytoplasmic effect. In other unstable RNAs, the ATTTA sequence may be present in only one copy, but is often contained in a region rich in A + T. From the animal cell data collected to date, it seems that ATTTA is at least in some contexts important in stability, but it is not yet possible to predict which occurrences of ATTTA are destabilizing elements or if any of these effects are likely to Look at the plants. Some studies on mRNA degradation in animal cells also indicate that RNA degradation may begin in some cases with nucleolytic attack in regions rich in A + T. It is not clear if these cuts occur in ATTTA sequences. There are also examples of mRNAs that have differential stability dependent on the cell type in which they are expressed or of the stage within the cell cycle to which they are expressed. For example, histone mRNAs are stable during DNA synthesis but unstable if DNA synthesis is altered. The 3 'end of certain histone mRNAs seems to be responsible for this effect (Pandey and Marzluff, 1987). It does not seem to be mediated by ATTTA nor is it clear that it controls the differential stability of this mRNA. Another example is a differential stability of lm &mRNA; G of B lymphocytes during the maturation of B cells (Genovese and Milcarek, 198d). These examples all provide evidence that the stability of the mRNA can be mediated by the cell type or specific factors of the cell cycle. In addition, this type of instability is not yet associated with specific sequences. Given these uncertainties, it is not possible to predict which RNAs are likely to be unstable in a given cell. In addition, even the ATTTA motif can act differentially according to the nature of the cell in RNA. Shaw and Kamen (1987) have reported that activation of protein kinase C can block ATTTA-mediated degradation. The addition of a polyadenylate chain to the 3 'end is common for most eukaryotic mRNAs, both plants and animals. The currently accepted view of addition of poIyA is that the nascent transcript extends beyond the mature 3 'terminal. Contained within this transcript are the signals for polyadenylation and the appropriate 3 'end formation. This processing at the 3 'end involves cutting the mRNA and adding the poIyA to the mature 3' end. Searching for consensus sequences near the tract of the poIyA in both plant and animal mRNA, it has been possible to identify consensus sequences that are apparently involved in the addition of poIyA and end cutting. The same consensus sequences seem to be important for these two processes. Those signals are typically a variation on the AATAAA sequence. In animal cells, some variants of that sequence that are functional have been identified; in plant cells there seems to be an extended range of functional sequences (Wickens and Stephenson, 1984, Dean et al., 1986). Because all these consensus sequences are variations on AATAAA, they are all sequences rich in A + T.
This sequence is typically found 15 to 20 bp before the poIyA tract in the mRNA. Studies in animal cells indicate that this sequence is involved in the addition of the poIyA and 3 'maturation. Mutations directed to the site in this sequence can alter these functions (Conway and Wickens, 1988, Wickens et al., 1987). However, it has also been observed that sequences up to 50 to 100 bp 3 'to the putative poIyA signal are also required; that is, a gene that has a normal AATAAA but that has been replaced or altered towards the 3 'end is not appropriately polyadenylated (Gil and Proundfoot, 1984; Sadofsky and Alwine, 1984; McDevitt et al., 1984). That is, the poiyA signal itself is not sufficient to complete and for proper processing. It is not yet known which sequences towards the specific 3 'end are required in addition to the poiyA signal, or if there is a specific sequence having this function. Therefore, the sequence analysis can identify only potential poiyA signals. In naturally occurring mRNAs, which are normally polyadenylated, it has been observed that alteration of this process, either by altering the poiyA signal or other sequences in the mRNA, can obtain profound effects in the functional mRNA. This has been observed in several mRNAs that occur naturally, with the result that they are so far gene-specific. It has been shown that in the appropriate polyadenylation of natural mRNAs it is important in the accumulation of mRNA, and that the alteration of this process can significantly affect mRNA levels. However, there is insufficient knowledge to predict the effect of changes in a normal gene. In a heterologous gene, it has been even harder to predict the consequences. However, it is possible that the putative sites identified are dysfunctional. That is, these sites may not act as appropriate polyA sites, but instead, they may function as aberrant sites that give rise to unstable mRNA. In systems of animal origins, AATAAA is for most of the common signals identified in mRNA towards the 5 'end of the poiyA, but at least four variants have also been found (Wickens and Stephenson, 1984). In the plants, an analysis that approaches this has not been done, but it is clear that multiple sequences similar to AATAAA can be used. The sites of the plants in table 3 called major or minor refer only to the study by Dean et al., (1936) who analyzed only 3 types of plant gene. The designation of polyadenylation sites as major or minor refers only to the frequency of their occurrence as functional sites in naturally occurring genes that have been analyzed. In the case of plants, this is a very limited database. It is difficult to predict with certainty that a site designated as major or minor is more or less likely to function partially or completely when it is in a heterologous gene such as those encoding the crystal proteins of the present invention.
TABLE 3 Polyadenylation Sites in Plant Genes
PA ATA Greater consensus site
P1A AATAAT Larger site in the plant
P2A AACCAA Minor site in the plant
P3A ATATAA Minor site on the ground
P4A AATCAA Minor site on the ground
P5A ATACTA Minor site on the ground
P6A ATA Minor site on the ground
P7A ATGAAA Minor site on the ground
P3A AAGCAT Minor site in the plant
P9A ATTAAT Minor site on the ground
P10A ATACAT Minor site in the plant
P11A ATA Minor site on the ground
P12A ATA Petite site in animal
P13A ATA Petite site in animal
P14A ATACA Minor site in animal
P15A CATAAA Petite site in animal
The present invention provides a method for preparing genes from synthetic plants that express their protein product at significantly higher levels than the wild-type genes commonly used in plant transformation up to now. In another aspect, the present invention also provides novel synthetic plant genes that encode non-plant proteins. As described above, the expression of native plant genes is often problematic. The nature of the coding sequences of B. thuringiensis genes distinguishes them from plant genes as well as many other expressed heteorological genes used in plants. In particular, the genes of ß. thuringiensis are very rich in adenine (-62%) and thymine (A) while the genes of plants and most other bacterial genes that have been expressed in plants are of the order of 45-55% A + T. Due to the degeneracy of the genetic code and the limited number of codons for any amino acid, the majority of the A + T in "excess" of the structural coding structures of some Bacillus species are in the third position in the codons. That is, the genes of some Bacillus species have A or T as the third nuleotide in many codons. Therefore, the content of A + T can partly determine the derivation of use of the codon. In addition, it is clear that genes evolve for maximum function in the organism in which they evolve. This means that particular nucleotide sequences found in a gene of an organism, e they can play a role except coding for a particular extension of amino acids, have a potential to be recognized as gene control elements in another organism (such as promoters or transcription terminators, Syrian addition of poiyA, splice sites of introns, or signals of mRNA degradation). It is perhaps surprising that such erroneously read signals are not a more common characteristic of the heteorologist gene, but this can be explained in part by the relatively homogeneous A + T content (-50%) of many organisms. This content of A + T plus the nature of the genetic code places clear restrictions on the probability of occurrence of any particular oligonucleotide sequence. Therefore, an E. coli gene with an A + T content of 50% is much less likely to contain any segment rich in A + T than a gene from B. thuringiensis. Typically, to obtain high level expression of the d-endotoxin genes in plants the existing structural coding sequence ("structural gene") coding for d-endotoxin is modified by removal of ATTTA sequences and putative polyadenylation signals by mutagenesis directed to the DNA site comprising the structural gene. It is more preferred that substantially all of the polyadenylation signals and ATTTA sequences be removed although increased expression levels are observed only with partial removal of any of the sequences previously identified. Alternatively, if a synthetic gene encoding the expression of the following protein is prepared, codons are selected to avoid the ATTTA sequence and putative polyadenylation signals. For purposes of the present invention, putative polyadenylation signals include, but are not necessarily limited to AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATAAA, AATACA and CATAAA. By replacing the ATTTA sequences and polyadenylation signals, codons are preferably used that avoid the codons that are rarely found in plant genomes. The selected DNA sequence is scanned to identify "with more than four consecutive adenine (A) and thymine (T) nucleotides." A + T regions are scanned for potential polyadenylation signals in plants, although the absence of 5 or more nucleotides of consecutive A or T eliminates most of the polyadenylation signals of plants, if there is more than one of the minor polyadenylation signals identified within ten nucleotides of each other, then the nucleotide sequence of this region is preferably altered to remove these signals while maintaining the original encoded amino acid sequence The second step is to consider the approximately 15 to approximately 30 nucleotide residues that surround the A + T rich region identified in step 1. If the A + T content of the surrounding region is less than 30%, the region should be examined for signs of polladenylation. The alteration of the region based on polyadenylation signals depends on (1) the number of polyadenylation signals present and (2) the presence of a greater polyadenylation signal in plants. The extended region is examined for the presence of plant polyadenylation signals. The polyadenylation signals are removed by site-directed mutagenesis of the DNA sequence. The extended region is also examined for multiple copies of the ATTTA sequence that are also removed by mutagenesis. It is also preferred that regions comprising many consecutive A + T bases or G + C bases be altered since in those regions it is predicted that they are more likely to form a pin structure due to self-complementarity. Therefore, the insertion of heterogeneous base pairs would reduce the likelihood of self-complementary secondary structure information that is known to inhibit transcription and / or translation in some organisms. In most cases, adverse effects can be minimized by using sequences that contain no more than five consecutive A + T or G + C.
4. 9.6 Synthetic oligonucleotides for mutagenesis When oligonucleotides are used in mutagenesis, it is convenient to maintain the appropriate amino acid sequence and the appropriate reading frame, without introducing restriction sites such as BglU, HindlW, Sacl,
Kpnl, EcoR \, Ncol, Psti and Salí in the modified gene. These restriction sites are found at polylinker insertion sites of many cloning vectors. Of course, the introduction of new polyadenylation signals, ATTTA sequences or consecutive extensions of more than 5 A + T or G + C should also be avoided. The preferred size for the oligonucleotides is from about 40 to about 50 bases, but fragments ranging from about 1d to about 100 bases have been used. In most cases, a minimum of about 5 to about d base pairs of template DNA homology at both ends of the synthesized fragment is maintained to ensure proper hybridization of the primer to the template. Oligonucleotides should avoid sequences greater than 5 base pairs A + T or G + C. The codons used in the replacement of wild-type codons should preferably avoid the doublet of TA or GC whenever possible. The codons are selected from a preferred codon picture of plants (such as Table 4 below) to avoid codons that are seldom found in plant genomes, and efforts must be made to select codons to preferably adjust the G content. + C to approximately 50%.
TABLE 4
Use of preferred codons in plants
Percentage of use in
Amino acid system plants ARG CGA 7 CGC 11
CGG 5
CGU 25
AGA 29
AGG 23
LEU CUA * 8 CUC 20
CUG 10
CUU 2d
UUA 5
UUG 30
BE UCA 14
UCC 26
UCG 3
UCU 21
AGC 21
AGU 15 Percent of use in
Amino acid fodder plants
THR ACA 21
ACC 41
ACG 7
ACU 31
PRO CCA 45
CCC 19
CCG 9 CCU 26
ALA GCA 23
GCC 32
GCG 3
GCU 41
GLY GGA 32
GGC 20
GGG 11
GGU 37
1LE AUA 12
AUC 45
AUU 43 Percent of use in
Amino acid? Odón plants
VAL GUA 9 GUC 20 GUG 28 GUU 43 LYS AAA 36 AAG 64 ASN AAC 72 AAU 28 GLN CAA 64 CAG 36 HIS CAC 65 CAU 35
GLU GAA 48 GAG 52
ASP GAC 48
GAU 52
ASP GAC 48
GAU 52 - Percent of use in Amino Acid? Odón plants TYR UAC 68 UAU 32
CYS UGC 78 UGU 22
PHE UUC 56 UUU 44
MET AUG 100
TRP UGG 100
Regions with many A + T bases or consecutive G + C bases are predicted to have a higher probability of forming pin structures due to self-complementarity. Altering these regions by inserting heterogeneous base pairs is preferred and should reduce the likelihood of the formation of self-complementary secondary structures such as pins that are known to inhibit transcription (transcription terminators) and translation (attenuators) in some organisms . Alternatively, a completely synthetic gene for a given amino acid sequence can be prepared with regions of 5 or more nucleotides of A + T or G + C being avoided. The codons are selected by avoiding doublets of TA and GC in codons whenever possible. The use of codons can be normalized against a preferred codon usage frame in plants (such as Table 4) and the G + C content can preferably be adjusted to approximately 50%. The resulting sequence should be examined to ensure that there are polyadenylation signals from minimal putative plants and minimal ATTTA sequences. Restriction sites found in commonly used cloning vectors are also preferred to avoid. However, the placement of several unique restriction sites throughout the gene is useful for the analysis of gene expression or construction of gene variants.
4. Methods for producing insect resistant transgenic plants Transforming a suitable host cell, such as a plant cell, with a segment containing recombinant cry gene, the expression of the encoded crystal protein (i.e., a bacterial crystal protein or polypeptide having insecticidal activity against coloptera) can result in the formation of insect resistant plants. By way of example, an expression vector containing a coding region for the crystal protein B. thuringiensis and a suitable selectable marker can be used to transform a suspension of embryonic plant cells, such as wheat or corn cells to deliver the DNA coated on microprojectiles in the recipient cells. The transgenic plants are then regenerated from transformed embryonic calli expressing the insecticidal proteins. The formation of transgenic plants can also be achieved using other cell transformation methods that are known in the art such as Agrobacterium-mediated DNA transfer (Fraley et al., 1983). Alternatively, DNA can be introduced into plants by direct DNA transfer into pollen (Zhou et al., 1983; Hess, 1987; Lou ef al., 19d8), by injecting DNA into reproductive organs of a plant (Pena et al., 1987), or by direct injection of DNA into the cells * of immature embryos followed by the rehydration of dried embryos (Neuhaus et al. to the.,
Benbropk ef a /., 1986). The regeneration, development and cultivation of plants from protoplast transformants of individual plants or from several transformed explants is well known in the art (Weissbach and Weissbach, 19dd). This process of regeneration and growth typically includes the steps of selecting transformed cells, cultivating those individualized cells through the usual stages of embryonic development through the rooted seedling stage. Embryos and transgenic seeds are regenerated in a similar way. The resulting transgenic rooted shoots are then planted in an appropriate plant growth medium such as soil.
The development or regeneration of plants containing the foreign exogenous gene encoding a polypeptide of interest produced by Agrobacterium from leaf explants can be achieved by methods well known in the art such as those described (Horsch et a .., 1985 ). In this procedure, the transformants are grown in the presence of a selection agent and in a medium that induces shoot regeneration in the strain of the plant that is being transformed as described (Fraley et al., 1983). This procedure typically produces shoots within two to four months and those shoots are then transferred to an appropriate root inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. The shoots that take root in the presence of the selective agent to form seedlings are then transplanted to the soil or other means to allow the production of roots. Those procedures vary depending on the particular plant strain employed, such variations being well known in the art. Preferably, the regenerated plants are self-pollinated, to provide homozygous transgenic plants as described above. Otherwise, the pollen obtained from the regenerated plants is crossed with plants that grow from seed of preferably agriculturally important inbred lines. A transgenic plant of the present invention containing a desired polypeptide is cultured using methods well known to one skilled in the art.
A transgenic plant of this invention, therefore, has an increased amount of a coding region (e.g., a gene) that encodes a polypeptide as described herein. A preferred transgenic plant is an independent segregant and can transmit that gene and its activity to its progeny. A more preferred transgenic plant is homozygous for that gene, and transmits that gene to all its descendants by sexual reproduction. The seed of a transgenic plant can be grown in the field or greenhouse and the sexually mature transgenic plants are self-pollinated to regenerate true inbred plants. The progeny of these plants are converted into true inbred lines which are evaluated, by way of example, for insecticidal capacity increased against coleopteran insects, preferably in the field, under a range of environmental conditions. The inventors contemplate that the present invention will find particular utility in the creation of transgenic plants of commercial interest including various pastures of peat and pasture, rye, wheat, maiz, kapok, flax, rice, oats, sugar cane, cotton tomato, potato, soybeans and other legumes, tobacco, sorghum, as well as a variety of ornamental plants including cactus and succulents, fruits, berries, vegetables, and also a number of trees and plants containing nuts and fruits. Transgenic plants comprising one or more transgenes encoding a polypeptide as described herein, preferably will exhibit an improved or uninfested insect resistance phenotype for the target Coleoptera and Lepidoptera insects as described herein. These plants will preferably provide transgenic seeds, which will be used to create lineages of transgenic plants (ie, progeny or advanced generations of the original transgenic plant) that can be used to produce seed, or can be used as food for animals or humans, or to produce fibers, oil, fruits, grains or other plant products of commercial importance or components derived from plants. In such cases, the progeny and seed obtained from any generation of the transformed plants will contain the selected duly integrated transgene encoding the d-endotoxin of the present invention. The transgenic plants of the present invention can be crossed to produce hybrids or inbred lines with one or more plants having desirable properties. In certain circumstances, it may also be desirable to create transgenic plants, seeds and progeny that contain one or more additional transgenes incorporated therein than to their genome, in addition to the transgene encoding the polypeptide of the invention. For example, the transgenic plants may contain a second gene encoding the same or different insect resistance polypeptide, or alternatively, the plants may comprise one or more additional transgenes such as those conferring herbicide resistance, fungal resistance, resistance to bacteria, tolerance to adverse conditions, salt or drought, fixation of the stem or root improved, production of starch, grain, oil, carbohydrate, amino acid, protein, increased and similar.
4. 11 Gene isolation and homologous gene fragments The genes and d-endotoxin according to the present invention include not only the full-length sequences described herein but also fragments of this sequence or fusion proteins, which retain the characteristic insecticidal activity. of the sequences that are specifically illustrated here. It will be apparent to one skilled in the art that d-endotoxins can be identified and obtained by various means. The specific genes, or portions thereof, can be obtained from a culture deposit or they can be synthetically constructed, for example, by the use of a gene machine. Variations of these genes can be easily constructed using standard techniques to make point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to nuclear procedures. For example, enzymes such as ßa / 31 or site-directed mutagenesis can be used to systematically cut nucleotides from the ends of these genes. Also, genes encoding active fragments can be obtained using a variety of other other restriction enzymes. Proteases can be used to directly obtain active fragments of these dendotoxins. The equivalent d-endotoxins and / or genes encoding these equivalent d-endotoxins can also be isolated from Bacillus strains and / or DNA libraries using the teachings provided herein. For example, antibodies to the d-endotoxins described and claimed herein may be used to identify and isolate other d-endotoxins from a mixture of proteins. Specifically, antibodies can be produced for the portions of the d-endotoxins that are more constant and more distinct from other Thuringiensis d-endotoxins. These antibodies can then be used to specifically identify equivalent d-endotoxins with the characteristic insecticidal activity by immunoprecipitation, enzyme-linked immunoassay (ELISA) Western blotting. Another method to identify the d-endotoxins and genes of the present invention is by the use of oligonucleotide probes. These probes are nucleotide sequences that have a detectable label. As is known in the art, if the probe molecule and the nucleic acid sample hybridize to form a strong bond between the two molecules, it can reasonably be assumed that the probe and the sample are essentially identical. The detectable probe label provides a means to determine in a known manner whether hybridization has occurred. Said probe analysis provides a rapid method for identifying d-endotoxin-formicidal genes of the present invention. The nucleotide segments that are used as probes according to the invention can be synthesized by the use of DNA synthesizers using standard procedures. During the use of the nucleotide segments as probes, the particular probe is labeled with any suitable marker known to those skilled in the art, including radioactive and non-radioactive labels. Typical radioactive labels include 32 P, 125 I, 35 S, or the like. A probe labeled with a radioactive isotope can be constructed from a nucleotide sequence complementary to the DNA sample by conventional nick translation reaction, using DNAse and DNA pilomerase. The probe and the sample can then be combined in a hybridization buffer and maintained at an appropriate temperature until quenching occurs. Subsequently, the membrane is washed free of foreign materials, leaving the sample and bound probe molecules typically detected and quantified by autoradiography and / or liquid scintillation counting. Non-radioactive labels include, for example, ligands such as biotin or thyroxine, as well as enzymes such as hydrolases or peroxidases., the various chemiluminescers such as luciferin, or fluoridating compounds, such as fluoricin and its derivatives. The zone can also be marked at both ends with different types of markers for ease of separation, for example by the use of an isotopic label at the end mentioned above and a biotin label at the other end. Duplex formation and stability depend on the substantial complementarity of the two strains of a hybrid and, as noted above, a certain degree of mismatch can be tolerated. Therefore, the probes of the present invention include mutations (both single and multiple), deletions, insertions of the described sequences and combinations thereof, wherein said mutations, insertions and deletions allow the formation of stable hybrids with the target polynucleotide. of interest. Mutations, insertions and deletions can occur in a polynucleotide sequence given in many ways, by methods currently known to one skilled in the art, and perhaps by other methods that may be known in the future. The potential variations in the probes listed are partly due to the redundancy of the genetic code. Due to the redundancy of the genetic code, ie more than one nucleotide triplet of encoder (codon) can be used for most of the amino acids used to make proteins. Therefore, different nucleotide sequences can encode an amino acid. Therefore, the amino acid sequences of the d-endotoxins and B.Thuringiensis peptides can be prepared by equivalent nucleotide sequences encoding the same amino acid sequence of the protein or peptide. Accordingly, the present invention includes said equivalent nucleotide sequences. Also reverse or complement sequences are an aspect of the present invention and can be easily used by one skilled in the art. It has also been shown that proteins of structure and function identified can be constructed by changing the amino acid sequence if said changes do not alter the secondary structure of the protein (Kaiser and Kezdy, 1934). Therefore, the present invention includes mutants of the amino acid sequence illustrated herein that does not alter the secondary structure of the protein, or if the structure is altered, the biological activity is substantially retained. In addition, the invention also includes mutants of organisms that contain all or part of a gene encoding d-endotoxin of the invention. Such mutants can be made by techniques well known to those skilled in the art. For example, UV irradiation can be used to prepare mutants of host organisms. Likewise, said mutants may include asporogenic host cells which may also be prepared by methods well known in the art.
4. 13 Recombinant Host Cells The nucleotide sequences of the present invention can be introduced into a wide variety of microbial and eukaryotic hosts. As hosts for the recombinant expression of Cry polypeptides, of particular interest, will be the prokaryotes and the lower eukaryotes, such as fungi. Illustrative prokaryotes, both gram-negative and gram-positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spiríllaceae, such as Photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrip, Spirillum; Lactobacillaceae; Pseudomonadeceae, such as Pseudomonas and Acetobacter; Azotobacteraceae, Actinomycetales, and Nitrobacteraceae. Among the eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which include yeasts, such as Saccharomyces and Schizosaccharomyces; and the yeast Basidiomycetes, such as Rhodotorula, Aureobasidium, Sporobolomyces and the like. Characteristics of particular interest in the selection of a host cell for production purposes include the ease of introduction of the genetic constructs in the present invention into the host cell, availability of expression systems, expression efficiency, stability of the gene of interest in the host cell. the host and the presence of auxiliary genetic capabilities. A large number of microorganisms that are known to inhabit the phylloplane (the surface of the leaves of plants) and / or the rozósfera (the soil that surrounds the roots of plants) of a wide variety of important crops can also be host cells desirable for manipulation, propagation, storage, supply and / or mutagenesis of the described genetic constructs. These microorganisms include bacteria, algae and fungi. Of particular interest are microorganisms such as bacteria, for example, from the genera Bacillus (including the species and subspecies ß. Thuringiensis kurstaki HD-1, ß.thuringiensis HD-73, ß.thuringiensis sotto, B. thuringiensis berliner, B. thuringiensis thuringiensis, B. thuringiensis tolworthi, B. thuringiensis dendrolimus, B. thuringiensis alesti, B. thuringiensis galleriae, B. thuringiensis aizawai, B. thuringiensis subtoxicus, B. thuringiensis entomocidus, B. thuringiensis tenebrionis and B. thuringiensis san diego); Pseudomonas, Erwinia, Serratia, Klebsiella, Zanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, in particular yeasts, for example the genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula and Aureobasidium. Of particular interest are those bacterial species of the phytosphere such as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodobacter sphaeroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes eutrophus and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R. glutiis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S.pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. Odorus, Kluyveromyces veronae and Aureobasidium pollulans. Characteristics of particular interest in the selection of a host cell for production purposes include the ease of introduction of a selected genetic construct in the host, availability of expression systems, expression efficiency, stability of the polynucleotide in the host and the presence of auxiliary genetic capabilities. Other considerations include ease of formulation and handling, economy, storage stability and the like.
4. 14 Polynucleotide sequences The DNA compositions encoding the insecticidally active polypeptides of the present invention are particularly preferred for delivery to cells of recipient plants, in the generation of pluripotent plant cells and finally in the production of transgenic plants resistant to insects. For example, DNA segments in the form of vectors and plasmids, or linear DNA fragments, in some cases containing only the DNA element to be expressed in the plant cell, and the like, may be employed. Vectors, plasmids, phagemids, cosmids, viral vectors, shuttle vectors, baculovirus vectors, BAC (bacterial articular chromosomes), YAC (yeast artificial chromosomes), and DNA segments to be used in the transformation of cells with a polynucleotide encoding d-endotoxin, of course will generally comprise at least a first gene encoding the polypeptide according to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 19, or a gene encoding a polypeptide having at least about 80% or 85% or 90% or 95% of sequence identity with respect to the amino acid sequence described in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 19. These nucleic acid constructs may comprise one or more genes that are desired to be introduced into recipient cells. These DNA constructs can include structures such as promoters, enhancers, polylinkers or regulatory genes as desired. The DNA segment or gene chosen for cellular introduction will often encode a polypeptide that will be expressed in the resulting recombinant cells, such that a screened or screened trait will result and / or impart an improved phenotype to the transformed host cell. Alternatively, nucleic acid constructs may contain antisense constructions or ribosomal coding regions when supplies or introduction of such nucleic acid constructs are desirable.
4. Methods for preparing mutagenized polynucleotides In certain circumstances it may be inconvenient to encode or alter one or more nucleotides in one or more of the polynucleotide sequences described herein for the purpose of altering or changing the insecticidal activity or insecticidal specificity of the purified polypeptide. The mutant sequence is subsequently amplified. Methods for mutagenizing and amplifying a DNA segment are well known to those skilled in the art. Mutagenesis of the DNA segments can be done by random mutagenesis or site-specific mutagenesis procedures. The polynucleotides can be modified by the addition, deletion or substitution of one or more nucleotides of the sequence encoding the insecticidally active polypeptide.
The particular methods of multagenesis and amplification that can be used in the practice of the present invention are described in Tomic et al., Michael, et al., Upender et al., Kwoh et al., Frohman, et al., Ohara et al. al., Wu, ef al., Walker et al; US patents No. 4,683,195, 4,683,202, 4,800,159, 4,8d3J50 Y; EP320,308 EP329,822; GB2202323; PCT / US87 / 00680; PCT / US89 / 01025; WO dd / 10315, WO 69/06700, each of which is incorporated herein by reference in its entirety.
4.16 Post-transcription events that affect the expression of transgenes in plants In many cases, the level of transcription of a particular transgene in a given host cell is not always indicative of the amount of protein that is being produced in the host cell transformed. This is often due to post-transcription procedures, such as splicing, polyadenylation, appropriate translation initiation, and stability of RANs, which affect the ability of a transcript to produce protein. Said factors may also affect the stability and amount of mRNA produced from the given transgene. As such, it is often convenient to alter post-translational events by particular molecular biology techniques. The inventors contemplate that in certain cases it may be convenient to alter the transcription and / or expression of the nucleic acid constructs encoding polypeptide of the present invention to increase, reduce or otherwise regulate or control those constructs in particular host cells and / or particular transgenic plants.
4. 16.1 Efficient initiation of protein translation The leader sequence untranslated to 5 '(5'-UTL) eukaryotic mRNAs play an important role in translation efficiency. Many early chimeric transgenes that use a viral promoter that used an arbitrary length of viral sequence after the transcription initiation site and functioned to the AUG of the coding region. More recent studies have shown that the sequence of 5'-UTL and the sequences that directly surround AUG may have a large defect in translation deficiency in host cells and particularly in certain plant species and that this effect may be different depending on the particular cells or tissues in which the message is expressed. In most eukaryotic mRNAs, the translation initiation point occurs at the AUG codon closest to the 5 'end of the transcript. Comparison of plant mRNA sequences and site-directed mutagenesis experiments have demonstrated the existence of consensus sequence surrounding the initiation codon in plants, 5'-U AAACAAU GGCU-3 '(Joshi, 1967; Lutcke et al. al., 19d7). However, consensus sequences will be evident between individual plant species. For example, a co-sequence of sequences surrounding the initiation codon of d5 maize genes gives a consensus of 5 '- (C / G) AUGGCG-5' (Luehrsen et al., 1994). In tobacco protoplasts the transgenes encoding delta-glucuronidase (GUS) and bacterial chitinase showed a 4-fold increase and an 8-fold increase in expression, respectively, when the native sequences of these genes changed to encode 5'-ACCAjJGG-3 '(Galli et al., 1987bJones et al., 19dd). When chimeric transgenes (ie, transgenes comprising segments of DNA from different sources operably linked to each other) are produced, 5'-UTL of plant viruses is often used. The (AMV) of the alfalfa mosaic virus (BMV) cover protein and the cover of the bromeliaceous mosaic virus 5'-UTL have been shown to increase the translation of mRNA d times in electroporated tobacco protoplast (Gallie et al. ., 1987a; 1987b). A 67-nucleotide (O) derivative of the 5'-UTL of mosaic virus RNA (TMV) fused to the chloramphenicol acetyltransferase (CAT) gene and gene (GUS) have been shown to increase the translation of reporter genes in vitro (Gallei et al., 1987a; 1987b; Sleat et al., 1987; Sleat et al., 198d). The electroporation of tobacco mesophilic protoplasts with transcripts containing TMV leader fused to CAT, GUS, and LUC reporter genes produced a level of 33, 21, and 36 fold increase, respectively (Gallie et al., 1987a; 1987b; Gallie et al., 1991). Also in tobacco, a 5'-UTL of 83 RNA nucleotides of potato X virus was shown to increase the expression of neomycin phosphotransferase II (Nptll) 4 times (Poogin and Skryabin, 1992).
The effect of a 5'-UTL may be different depending on the plant, particularly between dicots and monocots, The 5'-UTL of TMV has been shown to be more effective in tobacco protoplasts (Gallie et al., 1989) than in corn protoplasts (Gallie and Young 1994). Also, the 5'-UTL of TMV-O (Gallie et al., 1988), covered by AMV (Gehrke et al., 1983; Jobling and Gehrke, 1987), covered by TMV (Goelet et al., 1982), and BMV cover (French et al., 1986) functioned poorly on corn and inhibited the expression of a luciferase gene in corn in relation to its native leader (Koziel et al., 1996). However, the 5'-UTL of the 35S transcrof the cauliflower mosaic virus (CaMV) and the glutein of maize genes (Boronat et al., 1986), PEP-carboxylase (Hudspeth and Gruía, 1989) and ribulose bisphosphate carboxylase showed a considerable increase in the expression of the luciferase gene in corn in relation to its native leader (Koziel et al., 1996). These 5'-UTL had different effects on tobacco. In contrast to corn, the 5'-UTL of TMV O and the 5'-UTL of the AMV coat protein increased expression in tobacco, while the 5'-UTL of glutein, PEP-carboxylase of corn and corn ribulose-1, 5-bisphosphate carboxylase showed an increase in relation to the 5'-UTL of the native luciferase (Koziel et al., 1996). Only 35S 5'-UTL of CaMV increased the expression of luciferase in both corn and tobacco (Koziel et al., 1996). In addition, the 5'-UTL of the TMV and BMV coat protein were inhibitors in the protoplasts of both maize and tobacco (Koziel et al., 1996).
4. 16.2 Use of introns to increase expression The inclusion of one or more introns in the transcribed portion of a gene has been found to increase the expression of the heterologous gene in a variety of plant systems (Callis et al., 1987; Maas et al. ., 1991, Mascerenhas ef al., 1990, McEroy et al., 1990, Vasil et al., 1989), although not all introns produce a stimulating effect and the degree of stimulation varies. The effect of increasing introns seems to be more evident in monocots than in dicots. Tanaka et al., (1990) have shown that the use of the 1 -nucleon of the isolated catalase of beans increases the. expression of the gene in rice. Also, the first intron of alcohol dehydrogenase 1 (Adhl) has been shown to increase the expression of an Adhl genomic clone comprising the endogenous promoter in transformed maize cells (Callis et al., 1987; Dennis et al., 1984). Other introns that can also increase the expression of the transgenes that contain them include Adhl's introns 2 and 6 (Luehrsen and Walbot, 1991), in catalase intron (Tanaka et al., 1990), intron 1 of the bronze gene 1 of corn (Callis et al., 1987), intron 1 of corn sucrose synthetase (Vasil et al., 1989), intron 3 of the rice actin gene (Luehrsen and Walbot, 1991), intron 1 of rice actin (McEIroy et al., 1990), and exon 1 of corn ubiquitin (Christensen et al., 1992). In general, to achieve optimal expression, the selected introns must be present in the transcron unit 5 'in the correct orientation with respect to the splice junction sequences (Callis et al., 1987; Maas et al., 1991; Mascerenhas ef al., 1990, Oard ef al., 1989, Tanaka ef al., 1990, Vasil ef a /., 1989). Adhl intron 9 has been shown to increase the expression of a heterologous gene when placed towards the 3 'end of the gene of interest (Callis et al., 1987).
4. 16.3 Use of synthetic genes to increase the expression of heterologous genes in plants When a prokaryotic gene is introduced into a prokaryotic host, or when a prokaryotic gene is expressed in a non-reactive host, the sequence of the gene often must be altered or modified to allow the efficient translation of transcr derived from the gene. Significant experience in the use of synthetic genes to increase the expression of a desired protein has been achieved in the expression of B. thuringiensis. The genes of ß. native thuringiensis are often expressed only at low dicotyledonous levels and sometimes not all in many monocot species (Koziel et al., nineteen ninety six). The use of codons in the native genes is considerably different from that found in genes of typical plants, which have a higher G + C content. Strategies to increase the expression of these genes in plants generally alter the overall G + C content of the genes. For example, genes encoding synthetic crystal protein from B. thuringiensis have resulted in significant improvements in expression of these endotoxins in several crops including cotton (Perlak et al., 1990; Wilson et al., 1992), tomato (Perlak et al., 1991), potato (Perlak et al., 1993), rice (Cheng ef al., 1998) and maize (Koziel et al., 1993). Similarly, the inventors contemplate that the genetic constructs of the present invention, because they contain one or more genes of bacterial origin, in some circumstances can be altered to increase the expression of these prokaryotic-derived genes, in particular eukaryotic host cells and / or transgenic plants comprising said constructions. Using molecular biology techniques that are well known to those skilled in the art, the coding or non-coding sequences of the sequences of particular Cry-encoding genes can be altered to optimize or facilitate their expression in cells of transformed plants at suitable levels to prevent or reduce the infestation or attack of insects in said transgenic plants.
4. 16.4 Sequestration and direction of chloroplasts Another approach to increase the expression of genes rich in A + T in plants has been demonstrated in the transformation of chloroplast in tobacco. High levels of gene expression encoding crystal protein of ß. Thuringiensis unmodified in tobacco has been reported in McBride et al., (1995). In addition, methods of targeting chloroplast proteins have been developed. This technique, which uses the pea chloroplast transient peptide, has been used to direct the oaks of the synthesis pathway of polyhydroxygutirate to the chloroplast (Nawrath et al., 1994). Also, that technique negates the need for modification of the coding region other than the addition of an appropriate target sequence. Patent of E.U.A. 5,576,198 (specifically incorporated herein by reference) discloses compositions and methods useful for genetically manipulating plant cells to provide a time control method or pattern of expression of foreign DNA sequences from the tissue inserted into the plant plastid genome. The constructions include those for nuclear transformation that provide expression of a RNA polymerase of a single viral safety in plant tissues and direct the poimerase protein expressed in plastids of plant cells. Also included are plastid expression constructs comprising a promoter region that is specific for the RNA polymerase expressed from nuclear expression constructs described above and a heterologous gene of interest to be expressed in the transformed plastid cells. Alternatively, the gene can be transformed / localized to the chloroplast / plastid genome and expressed thereafter using promoters well known in the art (see Maliga, et al).
4. 16.5 Effects of the 3 'regions on the expression of transgenes The 3' regions of transgenes have been found to have a large effect on transgene expression in plants (Ingelbrecht et al., 1989).
In this study, different 3 'ends were operably linked to the reporter gene of neomycin phosphotransferase II (Nptll) and expressed in transgenic tobacco. The different 3 'ends used were obtained from the octopine synthase gene, the 2S seed protein of Arabidopsis, the small subunit of rbcS from Arabidopsis, carrot extension form, and chalcone synthase from Antirrhinum. In stable tobacco transformants, there was approximately a 60-fold difference between the best expression construct (rbcS 3 'end of small subunit) and the lowest expression construct (3' end of chalcone synthase).
TABLE 5 Plant promoters Promoters Reference8
Viral Virus of the scrofularia mosaic (FMV) Patent of E.U.A. No. 5,378,619 Cauliflower mosaic virus (CaMV) Patent of E.U.A. No. 5,530, 196 Patent of E.U.A. No. 5,097,025 Patent of E.U.A. No. 5, 110, 732
Plant Lengthening factor Patent of E.U.A. No. 5,177,011 Polygalacturonase from tomato Patent of E.U.A. No. 5, 442, 052 Histone of Arabidopsis H4 Patent of E.U.A. No. 5,491,288 Faseolin Patent of E.U.A. No. 5,504,200
Group 2 Patent of E.U.A. No. 5,608,144 Ubiquitin Patent of E.U.A. No. 5, 614,399
P119 Patent of E.U.A. No. 5,633,440 a-amylase Patent of E.U A No. 5,712,112
Promoters Reference *
Viral enhancer / plant promoter
Incrementor of 35S CaMV Patent of E.U.A. No. 5,106,739
Synthesis promoter
9Each reference is specifically incorporated here by reference
In its whole.
TABLE 6 Specific plant tissue promoters
Tissue specific promoter Fabric (s) Reference Blec epidermis Patent of E.U.A. No. 5,646,333
malate synthase seeds; Seedlings Patent of E.U.A. No. 5,689,040
isocitrate lyase seeds; Seedlings Patent of E.U.A. No. 5,689,040
patatina tubercle Patent of E.U.A. No. 5,436,393
ZRP2 Root Patent of E.U.A. No. 6,633,363
ZRP2 (2.0) root Patent of E.U.A. No. 5,633,363
ZRP2 (1.0) root Patent of E.U.A. No. 5,633,363
RB7 Root Patent of E.U.A. No. 5,459,252
Patent root of E.U.A. No. 5,401, 836
fruito Patent of E.U.A. No. 4,943,674
meristem Patent of E.U.A. No. 5,589,583 • Protective cell Patent of E.U.A. No. 5,538,879 Specific tissue promoter Fabric (s) Reference stamen Patent of E.U.A. No. 5,589,610
SodA1 pollen; middle layer; Van Camp ef al, 1986 stoma of the anthers SodA2 vascular bundles; Van Camp et al., 1996 stomas; axillary buds; Stomile pericycle pollen CHS15 flowers; tip of the root Faktor ef al., 1996 Psam-1 phloem tissue; Cortex; Vander et al., 1996 root tip ACT11 tissues and organs of Huang et al., 1997 lengthening; pollen; ovules zmGBS pollen; endosperm Russell and Fromm, 1997 zmZ27 endosperm Russell and Fromm, 1997 osAGP endosperm Russell and Fromm, 1997 osGTI endosperm Russell and Fromm, 1997 RolC phloem tissue; Graham pod et al., 1997 beams; vascular parenchyma Tissue specific promoter Tissue (s) Reference Sh phloem tissue Graham et al., 1997 CMd endosperm Grosset ef a /., 1997 Bnm 1 pollen Treacy ef a /., 1997 Bacilliform rice virus floem Yin ef a /., 1997a; 1997b S2-Rnasa pollen Ficket ef a /., 1998 LeB4 seeds Baumlein et al., 1991 gf-2.8 seeds; Berna and Bernier seedlings, 1997 aEvery reference is incorporated here specifically for its reference in its entirety. The ability to express genes in a specific way from plant tissue has led to the production of male and female sterile plants. In general, the production of male sterile plants involves the use of anther-specific promoters linked to heterologous generators that alter pollen formation (U.S. Patent Nos. 5,689,051; 5,689,049; 5,659,124, each specifically incorporated herein by reference). The Patent of E.U.A. No. 5,633,441 (specifically incorporated herein by reference) describes a method for producing plants with female genetic sterility. The method involves the use of style cell promoters, stigma cells or cell style-specific promoters and stigma cells that express polypeptides that, when produced in plant cells, significantly annihilate or alter metabolism, functioning or development of cells.
TABLE 7 Inducible plant promoters
Promoters Reference "Heat shock promoter Patent of U.S. No. 5,447,858
Em Patent of E.U.A. No. 5,139,954
Adh l Kyozoka ef a /., 1991 HMG2 Patent of E.U.A. No. 5,689,056 alcohol cinnamic dehydrogenase Patent of US Pat. No. 5,633,439 asparagine sistasa Patent of E.U.A. No. 5,595,896
GST-ll-27 Patent of E.U.A. No. 5,589,614 to each reference is incorporated herein by reference in its entirety.
4. 18 Antibody compositions and methods for making them In particular embodiments, the inventors contemplate the use of monoclonal or polyclonal antibodies that bind to one or more of the polypeptides described herein. The means for preparing and characterizing antibodies are well known in the art (see, for example Harlow and Lane, 1988, incorporated herein by reference). The methods for generating monoclonal antibodies (mAbs) are generally along the lines that those to prepare polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition according to the present invention and collecting antiserum from that immunized animal. A wide range of animal species can be used for the production of antiserum. Typically the animal used for the production of the antiserum is the rabbit, a mouse, a rat, a hamster, a coballo or a goat. Due to the relatively large blood volume of rabbits, a rabbit is a preferred choice for the production of polyclonal antibodies. mAbs can be easily prepared using well-known techniques, such as those illustrated in U.S. Patent No. 4,196,265 incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, for example, a purified or partially purified protein, polypeptide or peptide. The immunizing composition is administered in an effective manner to stimulate the antibody producing cells. Rodents such as mice and rats are preferred animals, however the use of rabbit, sheep or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB / c mouse being the most preferred since this is the one that is used more routinely and generally gives a higher percentage of stable mergers.
4. ELISA and Immunoprecipitation ELISA can be used in conjunction with the invention. The production and use of Elisa or equipment employing said ELISAs are well known to those skilled in the art.
4. Western Blots The compositions of the present invention will find an important use in the analysis of Immunoblot or Westem blot. Anti-peptide antibodies can be used as high affinity primary reagents for the identification of immobilized proteins on a solid support matrix, such as nitrocellulose, nylon or combinations thereof. Together with immunoprecipitation, followed by gel electrophoresis, these can be used as a single passage reagent for use in the detection of antigens against which the secondary reagents used in the detection of the antigen produce an adverse effect. This is especially useful when the antigens studied are immunoglobulins (precluding the use of immunoglobulins that bind to bacterial cell wall components), the antigens studied react cross-reactive with the detection agent, or migrate to the same relative molecular weight as a signal of cross reaction. The immunologically based detection methods for use in conjunction with Western blotting include secondary antibodies enzymatically, radiolabelled or fluorescently labeled against the toxin portion are considered to be of particular use in this regard.
4. 21 Biological Functional Equivalents Modifications and changes can be made in the structure of the peptides of the present invention and DNA segments that encode them and yet obtain a functional molecule that encodes a protein or peptide with desirable characteristics. The following is a discussion based on changing the amino acids of a protein to create an equivalent, or even an improved second generation molecule. In particular embodiments of the invention, mutated crystal proteins are contemplated which are useful for increasing the insecticidal activity of the proteins, and consequently increasing the insecticidal activity and / or expression of the recombinant transgene in a plant cell. The amino acid changes can be achieved by changing the codons of the DNA sequence, according to the codons given in Table 8.
TABLE 8
Amino Acids Codons
Alanina Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU Aspartic Acid Asp D GAC GAU Glutamic Acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly GGA GGC GGG GGU
Amino Acids Codons Histidine His H CAC CAU Isoleucine He 1 AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gin Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serina Ser S AGC AGU UCA UCC UCG UCU
Treonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tiptofan Trp w UGG Tyrosine Tyr and UAC UAU
For example, some amino acids can substitute for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structure such as, for example, binding regions of antibody antigens as binding sites on substrate molecules. Since it is the interactive ability and nature of a protein that defines that functional biological activity of the protein, some substitutions of amino acid sequences can be made in a protein sequence and of course the underlying DNA coding sequence, and yet obtain a protein with similar properties. In this way the inventors contemplate that various changes can be made in the peptide sequences of the compositions described, or corresponding DNA sequences encoding said peptides without appreciable loss of their usefulness or biological activity. By making those changes, you can consider the hydropathic amino acid index. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens and the like. A hydropathic index has been assigned to each amino acid on the basis of its hydrophobic character and loading characteristics (Kyte and Doolittle, 1982), these are: isoieucine (+4.5); valina (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); Alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
It is known in the art that certain amino acids can be substituted by other amino acids having a similar hydropathic index or hydropathic score and yet results in a protein with similar biological activity, i.e., a functionally equivalent biological protein is obtained. In making such changes, substitution of amino acids whose hydropathic indices are within ± 2 is preferred, those within ± 1 are particularly preferred and those within ± 0.5 are still more preferred. It is also understood in the art that substitution of similar amino acids can be done effectively on the basis of hydrophilicity. The patent of E.U.A. 4,554,101, incorporated herein by reference, states that the greater total average hydrophilic character of a protein, as regulated by the hydrophilic character of its adjacent amino acids, correlates with a biological property of the protein. As detailed in the patent of E.U.A. 4,554,101, the following hydrophilic character values have been assigned to amino acid residues: arginine (+3.0); licina (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); Alanine (09.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); Soleucine (-1.8); tirosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can replace another having a similar hydrophilic character value and yet obtain a biologically equivalent protein and in particular an immunologically equivalent protein. In such changes, substitution of amino acids whose hydrophilic character values are within ± 2 is preferred, those that are within ± 1 are particularly preferred of those that are within ± 5 are even more preferred. As indicated above, amino acid substitutions are generally based on the relative similarity of the amino acid side chain substituents, for example, their hydrophobicity, hydrophilicity, charge size and the like. Illustrative substitutions which consider several of the above characteristics are known to those skilled in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
EXAMPLES
The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art should appreciate that the techniques described in the following examples represent techniques discovered by the inventor to function well in the practice of the invention and therefore can be considered to constitute preferred modes for their practice. However, those skilled in the art, in light of the present disclosure, should appreciate that changes can be made in the specific embodiments described and yet obtain a similar or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1 Isolation of strains EG4550 and EG5899 from Bacillus thuringiensis
Culture dust samples were obtained from various sources in the United States and abroad, typically from grain storage facilities. The culture powder samples were treated and dispersed on agar plates to isolate individual colonies of the Bacillus type, for example B. thuringiensis, as described in the patent of E.U.A. 5,187,091, specifically incorporated herein by reference in its entirety. Phase contrast microscopy was used to visually identify cells with crystalline inclusions in the colonies that grew after this treatment. The crystal producing strains were characterized by electrophoresis in modified Eckhardt agarose gel as described in González ef al., (1982). This procedure allows visualization of the arrangement of native plasmids in a strain of β. thuringiensis. The plasmid arrangements can be compared with those of servo known varieties of ß. thuringiensis to facilitate the identification of wild type strains (Carlton and González, 1985). Strain EG4550 is a strain of crystal producing B. thuringiensis isolated from a culture powder sample from New York. The crystallized inclusions of sporulated EG4550 have a different morphology and resemble thin canes. The EG4550 plasmid arrangement does not resemble the arrangement of any of the servovaries known varieties of B. thuringiensis. Strain EG5899 is a strain of ß. glass producing thuringiensis isolated from a California crop dust sample. Crystal inclusions of sporulated EG5899 are uncommon as they appear to be multiple bound crystals with an irregular morphology. The EG5899 plasmid arrangement does not resemble the arrangement of any of the known servo varieties of B. thuringiensis. The insect bioassay of strains EG4550 and EG5899 of B. thuringiensis indicated that these strains are toxic to larvae of coleopteran insects, including SCRW, suggesting that the crystals in these strains contained novel insecticidal proteins. EG4550 and EG5899 were deposited in the ARS patent culture deposit and the numbers of NRRL B-21784 and B-21783, respectively, have been designated. These strains and other strains of the present invention are listed in Table 9.
TABLE 9
Bacterial strains of wild type and recombinant9 No of Date of Gen (s) cry Marker of
Strain access deposit of Organism Plasmid insert cloned Vector present antibiotic of
NRRL NRRLb plasmid
EG4550 B-21784 30-5-97 B. thuringiensis ci? ET39, 74, 75 EG5899 B-21783 30-5-97 B. thuringiensis - - - cryET39, 74, 75 - EG11582 - - pEG1337 8.4- b Mpdlll pUC18 cryET39 , 74, 75 Amp
EG11525 - - pEG1321 8.4-kb HintiW pEG597 cryET39, 74, 75 Amp
EG11529 B-21917 12-2-98 B. thuringiensis pEG1321 8.4-kb HináWl pEG597 cryET39, 74, 75 Cm
EG11521 - - pEG1319 8.4-kb Hin? Ti pBluescript cryET39, 74, 75 Amp
EG11934 - - B. thuringiensis pEG1918 4.5-kb Mndll pHT315 cryET75 Eryth
EG 11935 - - B. thuringiensis pEG1919 3.2-kb H / ndll- £ coR1 pHT315 cryET74 Eryth
EG11936 - - B. thuringiensis pEG1920 3.7-kb H¡ná \ l \ pHT315 cryET39, 74 Eryth
EG11937 - - B. thuringiensis pEG1921 1.4-kb H / ndlll pEG1915 cryET39 Eryth
EG4100 B-21786 30-5-97 B. thuringiensis - - cryET69 - EG11647 B-21787 30-5-97 B. thuringiensis pEG1820 Mbo partial pHT315 cryET69 Eryth
EG9444 B-21785 30-5-97 B, thuringiensis - - - cryET71, 79 - EG11648 B-21788 30-5-97 B, thuringiensis pEG1821 Mbo partial pHT315 cryET71, 79 Eryth
EG4851 B-21915 12-2-98 B, thuringiensis - - - cryET76, 80, 84 - EG11658 B-21916 12-2-98 B, thuringiensis pEG1823 Mbo partial pHT315 cryEpß, 80, 84 Eryth
present invention will be irrevocably removed upon granting a patent that describes them. The crops shown in Table 3 will be deposited in the permanent deposit of the culture deposit of the Agricultural Research Service, Northern Regional Research Laboratory (NRRL), domiciled in 1815
N. University Street, Peoria, IL 61604, under the terms of the Budapest treaty.
EXAMPLE 2 Evaluation of the crystal proteins of EG4550 and EG5899
Strains EG4550 and EG5899 were cultured in a C2 depopulation medium (Donovan, et al., J. Biol. Chem., 263: 561-567, 1988) for 3 days at 30 ° C during which the cultures grew to the stationary phase, sporulated and were lysed, released the protein inclusions in the medium. The cultures were centrifuged to harvest tablets of cells containing the spores and crystals. The tablets were washed with suspension in a Triton X-100® solution at 0.005% and centrifuged. The washed tablets were resuspended to one tenth of the original volume in Triton X-100® at 0.005%. The crystal proteins were solubilized from the spore-crystal suspensions incubating in solubilizing pH regulator [0.14 M Tris-HCl 8.0, 2% (weight / vol.) Of sodium dodecyl sulfate to the details of the construction of the previously listed strains are included in the following examples. bThe present crops have been deposited under conditions that ensure that access to the crops is available while this patent application is pending to a determined by the Director of Patents and Trademarks who is entitled to it under 37 C..F.R. § 1.14 and 35 U.S.C. § 122. Deposits are available as required by foreign patent laws in countries where counterparts of the present application for their progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license for the practice of the present invention in derogation of patent rights granted by governmental action. The culture reservoirs of the present invention will be stored and made available to the public in accordance with the provisions of the Budapest Treaty for the deposit of microorganisms, that is, they will be stored with all necessary care to keep them viable and decontaminated for a period of time. at least five years after the most recent request for the termination of a deposit sample, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the effective life of any patent that can be issued that describes the crops. The depositor recognizes the obligation to replace the deposits if the depositary is unable to obtain a sample when required, due to the condition of the deposits. All restrictions on the availability to the public of the culture deposits of the present invention will be irrevocably removed upon granting a patent describing them. The crops shown in Table 3 will be deposited in the permanent deposit of the Agricultural Research Service's culture deposit, Northern Regional Research Laboratory (NRRL), located at 1815 N. University Street, Peoria, IL 61604, under the terms of the treaty. of Budapest.
EXAMPLE 2 Evaluation of the crystal proteins of EG4550 and EG5899
Strains EG4550 and EG5899 were cultured in a C2 depopulation medium (Donovan, et al., J. Biol. Chem., 263: 561-567, 1988) for 3 days at 30 ° C during which the cultures grew to the stationary phase, sporulated and were lysed, released the protein inclusions in the medium. The cultures were centrifuged to harvest tablets of cells containing the spores and crystals. The tablets were washed with suspension in a Triton X-100® solution at 0.005% and centrifuged. The washed tablets were resuspended to one tenth of the original volume in Triton X-100® at 0.005%. The crystal proteins were solubilized from the spore-crystal suspensions by incubating in solubilizing pH buffer [0.14 M Tris-HCl 8.0, 2% (weight / vol.) Of sodium dodecyl sulfate (SDS), 2-mercaptoethanol 5% (vol./vol.), 10% glycerol (vol./vol.) And 1% bromophenol blue] at 100 ° C for 5 minutes. The solubilized crystal proteins were fractionated in size by SDS-PAGE using a gel with an acrylamide concentration of 12.5%. After fractionation in size, the proteins were visulized by tinsion with Coomassie brilliant blue R-250. Strain EG4550 displayed proteins at molecular weights of approximately 45 and 15 Kda. Strain EG5899 displayed proteins of molecular weights of approximately 160 Da, 45 kDa, 35 kDa, and 15 kDa.
EXAMPLE 3 Characterization of the crystal protein CryET39 of EG4550
The NH2-terminal sequence of the approximately 45-kDa protein of EG4550, designated as CryET39, was determined. A sporulated culture of EG4550 was washed and resuspended. The crystal proteins in the suspension were solubilized and run on a 10% acrylamide gel following the SDS-PAGE analysis procedures. After electrophoresis the proteins were transferred to a BioRad PVDF membrane using standard western blotting procedures. After the transfer the membrane was rinsed 3 times in distilled H20 and stained for one minute using Amido Black 1013 (Sigma Chemical Co., St. Louis, MO). The filter was destained for 1 minute in 5% acetic acid and rinsed in 3 changes of distilled H20. The portion of the filter containing the 45-kDa CryET39 band of approximately 45 kDa was cut with a shaving blade. This procedure resulted in a pure form of CryET39 which was obtained as a spotted protein band on a PVDF membrane (BioRad, Hercules, CA). The determination of the NH2-terminal amino acid sequence of the purified CryET39 protein immobilized on the membrane was performed at the Department of Phisiology at Tufts Medical School, Boston, MA using standard Edman degradation procedures. The NH2-terminal sequence was determined to be: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Met Leu Asp Thr Asn Lys Val Tyr Glu lie As As His Ala Asn (SEQ ID NO: 20) computer algorithms (Kom and Queen, 1984) to compare the NH2-terminal sequence of the CryET39 protein with the amino acid sequences of all the B. thuringiensis crystal proteins of which the inventors were aware of including the sequences of all the B. thuringiensis crystal proteins that had been published in the scientific literature, international patent applications or patents issued.
A list of the crystal proteins whose sequences have been published and to which a gene / protein designation has been assigned is shown in Table 2.
EXAMPLE 4 Isolation of a DNA fragment comprising the CryET39 gene
In order to identify the gene encoding CryET39, an oligonucleotide probe specific for the NH2-terminal amino acid sequence of the protein was designated. Using codons typically found in β-toxin genes. thuringiensis, an oligonucleotide of 41 nucleotides was synthesized by Integrated DNA Technologies, Inc. (Coralville, IA) and was designated as wd271. The sequence of wd27 is: 5? TGITTAGATACAAATAAAGTATATGAAATTTCAAATCATGC-3 '(SEQ ID NO: 21) wd271 relatively labeled was used as a probe in Southern hybridization experiments, as described below, to identify a restriction fragment containing the cryET39 gene . Total DNA was extracted from strains EG4550 and EG5899 by the following procedure. Vegetative cells were suspended in a lysis pH regulator containing 50 mM glucose, 25 mM tris-HCl (pH 8.0), 10 mM EDTA and 4 mg / ml lysozyme. The suspension was incubated at 37 ° C for 1 hour. After incubation, the suspension was extracted once with a total volume of phenol, then once with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1), and once with an equal volume of croformo: Isoamyl alcohol (24: 1). The DNA was precipitated from the aqueous phase by the addition of one tenth of the volume of 3 M sodium acetate, then two volumes of 100% ethanol. The precipitated DNA was collected by centrifugation, washed with 70% ethanol and resuspended in dH20. The extracted DNA was then digested in separate reactions, with various restriction endonucleases, including EcoRI and HindIII, using conditions recommended by the manufacturer (Promega Corp..Madison, Wl). The digested DNA was fractionated in size by electrophoresis through a 0.8% agarose gel in IX TBE (0.089 M Tris-borate, 0.089 M boric acid, 0.002 M EDTA) overnight at volts / cm in length gel. The fractionated DNA fragments were then transferred to a Millipore Immobilon-NC® nictocellulose filter (Millipore Corp., Bedford, MA) according to the method of Southern (1975). The DNA fragments were fixed to the nitrocellulose by heating the filter at 80 ° C according to the vacuum oven. Identification of DNA fragment (s) containing the NH2-terminal-encoding sequence of the CryET39 protein (see Example 3) was achieved using the oligonucleotide wd271 as a hybridization probe. To reactive label the probe, 1 to 5 pmoles of wd271 were added to the reaction containing [y-32P] ATP (3 μl of 3,000 Ci / mmoles at 10 mCi / ml in a reaction volume of 20 μl), a 10X reaction pH buffer (700 mM Tris-HCl (pH 7.8), 100 mM MgCl2, 50 mM DTT, and 10 units of 4 kinase polynucleotide (Promega Corp) The reaction was incubated for 20 minutes at 37 ° C to allow the transfer of the radioactive phosphate to the 5 'end of the polynucleotide, thus marking it useful as a hybridization probe.The labeled area was then incubated with the nitrocellulose filter overnight at 45 ° C in 3X SSC, 0.1% SDS , 10X Denhardt's reagent (0.2% BSA, 0.2% polyvinylpyrrolidone, 0.2% Ficoll®), 0.2 mg / ml heparin After the incubation, the filter was washed in several changes of 3X SSC, 0.1% SDS a 45 ° C. The filter was dried by block and exposed to an X-OMAT AR X- X-ray film from Kodak (Eastman Kodak Company, Rochester, NY ) • overnight at -70 ° C with a DuPont Cronex Lightning Plus screen to obtain an radioiodine. The autoradiogram examination identified a hybridized EcoRI gragmeter dw of 272 of 2.5-kb in DNA of an EG4550 strain. Strain EG4899 had a Hindlll restriction fragment of approximately 8.4 kb that hybridized to wd271. This result indicated that EG4550 and EG5899 contained related or identical copies of the cryET39 gene.
EXAMPLE 5 Cloning of the cryET39 gene
The first cloning study included the isolation of the EcoRI fragment of 2.5-kb EG4550 to express and characterize the CryET39 protein of EG4550. When this fragment was cloned and expressed in a recombinant B.thuringiensis strain, however, only the 15-kDa protein was produced, indicating that the 2.5-kb EcoRI fragment did not contain a complete and functional cryET39 gene. This result also indicated that the genes for the 15-kDa crital protein and CryET39 were nevertheless in close proximity. The recombinant B.thuringiensis strain expressing the 15-kDa protein, designated EG11467, was not toxic to SCRW larvae. The approximately 8.4 kb Hindill restriction fragment containing the cryET39 gene of EG5899 was isolated from the genomic DNA as described in section 5.4. The DNA was digested with Hindlll and electrophoresed through cm gel length. The gel was stained with ethidium bromide so that the digested DNA could be visualized when exposed to long-wave ultraviolet light. Slices containing DNA fragments of approximately 8.0-9.0 kb were cut from the gel with a shaving blade. The DNA fragments were purified and then from the gel slice using the Geneclean® method (Bio 101, Vista, CA).
The isolated DNA fragments were ligated to the phagemid pBluescript® II SK + (Stratagene, LaJolla, CA) a library in E. coli of the Hindlll restriction fragment selected in size. The pBluescript (R) II SK + phagemid vector could be replicated to a high copy number in E. coli and carried on the gene for resistance to antibiotic ampicillin, which can be used as a selected marker. The fragments were mixed with Hindlll digested by pBluescript® II SK + which have been treated with bacterial alkaline phosphatase (GibcoBRL, Gaithersburg, MD) to remove the 5 'phosphates from the targeted plasmid to avoid religation of the vector itself. T4 ligase and a binding pH regulator (Promega Corp.) were added to the reaction containing the digested phagemid and the Hindlll fragments selected in size. These were incubated at room temperature for 1 hour to allow the insertion and ligation of the Hindlll fragments into the pBluescript® II SK + SK + vector. The ligation mixture was introduced into transformation competent E. coli DH5a ™ cells (GibcoBRL) following procedures described by the manufacturer. Transformed E.coli cells were plated on LB agar plates containing 50μg / ml ampicillin and incubated overnight at 37 ° C. The growth of several hundred resistant colonies of ampicillin in each plate indicated the presence of the recombinant plasmid in the cells of each of those colonies.
To isolate the colonies that cut the cloned 8.4-kb Hindlll fragment containing the cryET39 gel, the colonies were first transferred to nitrocellulose filters. This was achieved by placing a circular filter (Millipore HALTF 085 25, Millipore Corp., Bedfor, MA) directed on the upper part of the LB-ampicillin agar plates containing the transformed colonies. When the filter slowly detached from the plate the colonies were stuck to the filter giving an exact replica of the colonies pattern of the original plate. Sufficient cells from each colony were left on the plate so that 5 to 6 hours of growth at 37 ° C restored the colonies. Plates were stored at 4 ° C as needed. The nitrocellulose filters with the transferred colonies were then placed, with the colonies upwards, on fresh LB-ampicillin agar plates and allowed to grow at 37 ° C until the colonies reached a diameter of approximately 1 mm. To release the DNA from the recombinant E. coli cells the nitrocellulose filters were placed with the colonies up on two sheets of Whatman 3MM chromatographic paper (Whatman International Ltd., Maidstone, England) soaked with 0.5 N NaOH, 1.5 M NaCl for 15 minutes. This treatment smooth the cells and denatured the released DNA allowing it to stick to the nitrocellulose filter. The filters were then neutralized by placing the filters with the colony up on two sheets of Whatman paper soaked with 1M ammonium acetate, 0.02M NaOH for 10 minutes. The filters were then rinsed in 3X SSC, air-dried and baked for 1 hour at 80 ° C in a vacuum oven to prepare them for hybridization. The NH-III oligonucleotide specific for the cryET39 gene, wd271, was labeled at the 5 'end at y-32 and the T4 polynucleotide kinase as described above. The labeled probe was added to the filters in 3X SSC, 0.1% SDS, 10X Denhardt's reagent (0.2% BSA, 0.2% polyvinylpyrrolidone, 0.2% Ficoll®), 0.2 mg / ml heparin and incubated during the night at 40 ° C. These conditions were chosen to allow hybridization of the labeled oligonucleotide for related sequences present in the nitrocellulose stains of the transformed E. coli colonies. After incubation, the filters were washed in several changes of 3X SSC, 0.1% SDS at 45 ° C. The filters were dried by spotting and exposed to a Kodak X-OMAT AR (Eastman Kodak) X-ray film overnight at -70 ° C with a DuPont Cronex Lightning Plus screen. Several colonies of this transport hybridized to wd271. These colonies were identified by aligning the autoradiogram signals with the colonies on the original transformation plates. The isolated colonies were then grown in a liquid medium of LB-ampicillin from which the cells could be harvested and prepared with recombinant plastid by the lysis minipreparation procedure with standard alkaline material (Maniatis et al., 1982). The isolated plasmids were digested with the restriction enzyme HindIII which indicated that the cloned DNA fragments EG5899 were of the expected size, ie 8.4-kb. The Hindill-digested DNA plasmid from six of the hybridizing colonies was electrophoresed through an agarose gel and transcribed to nitrocellulose as described above. The blot was then hybridized with oligonucleotide wd271 which had been labeled relatively at the 5 'end with y32 and T4 polynucleotide kinase. Insect fragments of approximately 8.4-kb of all six digested plasmids hybridized with wd271 confirming the presence of the cryET39 gene. One of the plasmids with the 8.4 kb insert containing the cryET39 gene was designated with pEG1319. The E.coli strain containing pEG1319 had been designated EG11521. AX PLO 6 Expression of recombinant proteins from EG11529
To characterize the properties of the CryET39 protein it was necessary to express the cryET39 gene cloned in β cells. thuringiensis that did not produce any crystal protein (Cry). To accomplish this, the cloned 8.4-kb Hindlll fragment from pEG1319 was inserted into a plasmid capable of replicating in B. thuringiensis, thus allowing expression of the cryET39 gene and production of the encoded protein. pEG1319 was digested with Hindlll to cut the cloned 8.4-kb fragment. The digested plasmid was dissolved in an agarose gel and a slice of gel containing 8.4-kb fragment was cut. The 8.4-kb Hind lll fragment was purified from silica gel using the GeneClean method (Bio 101). The fragment was ligated into a shuttle vector B. thuringiensis / E. coli that had been digested with Hindlll and treated with bacterial alkaline phosphatase. This shuttle vector designated by pEG597 was described by Baum et al. (1990). pEG597 is able to replicate in E. coli and β. thuringiensis, conferring resistance to ampicillin E. coli and resistance to chloramphenicol to B. thuringiensis. This ligation mixture was introduced into DH5a ™ cells of E. coli using the transformation procedure described by the manufacturer (GibcoBRL). The plasmid DNA was prepared from Amp R transformants and the restriction enzyme analysis was performed to confirm the proper construction. A plasmid containing 8.4-kb Hindlll fragment inserted into the vector pEG597 was designated pEG1321. The E. coli strain pEG1321 was designated by EG11525. pEG1321 was introduced into a strain of Cry B. thuringiensis, EG10368, by electroporation (Macaluso and Metrus 1991). Transformed cells for chloramphenicol resistance were selected by incubation overnight on LB agar plate containing 3μg / ml chloramphenicol. The plasmid DNA was isolated from several of the transformants of B. thuringiensis. The isolated plasmid was digested with HindIII and subjected to electrophoresis through an agarose gel. All transformants had restriction fragments corresponding to 8.4 kb cryET39 fragment and pEG597 vector. To verify the construction of the correct plasmid, the restriction fragments were subjected to block to a nitrocellulose filter which was then hybridized with oligo wd271, specific for cryET39, as described above. The wd271 probe hybridized to such cloned 8.4 kb Hindlll fragments confirming that pEG 1321 contains the cryET39 gene and that it has been successfully introduced into β. thuringiensis. The recombinant strain B. thuringiensis containing pEG1321 was designated EG11529. EG11529 was deposited with the NRRL and given accession number B-21917. EG11529 was cultured in a glucose + DSM sporulation medium containing 5μg / ml chloramphenicol [0.8% (w / v.) Difco nutrient broth, 0.5% (w / v) glucose, 10mM K2HP04, 10mM KH2PO4. , Ca (NO3) 2 1nM, 0.5mM MgSO4, 10μM MnCl2, 10μM FeS04] for 3 days at 30 ° C during which the culture grew to stationary phase, sporulated and smooth, thus releasing the protein inclusions in the medium . The cultures were harvested by centrifugation. Tablets consisting of spores and protein crystals were washed in a solution of TritonX-100®, 0.005%, 2 mM EDTA and centrifuged. The tablet was washed, suspended in one tenth of the original volume in Triton X-100®, at 0.005% 2 mM EDTA. The crystal proteins were solubilized from the spore suspension and a crystal incubated the suspension in pH buffer for solubilization [Tris-HCl 0.14 M pH 8.0, 2% sodium dodecyl sulfate (SDS), 2-mercaptoethanol 5% (vol./vol.), 10% glycerol (vol./vol./) and bromophenol blue 0.1%] at 0 ° C for 5 minutes. The solubilized crystal proteins were fractionated in size by SDS-PAGE. After fractionation to size the proteins were visualized by staining with Coomassie brilliant blue R-250. This analysis showed that three distinct crystal proteins were produced in strain EG115299. In addition to the CryET39 44-kDa toxin, polypeptides of approximately 15 and 35 kDa were also produced. The crystal protein of kDa expressed in EG11529 of ß. thuringiensis could be separated from 44-kDa (CryET39) and 15-kDa proteins with centrifugation through a gradient of sucrose cases (steps: 55%, 68%, 72%, 79%) as described in section 5.12. The determination of the NH2-terminal amino acid sequence of the isolated 35 kDa protein was accompanied using procedures written in section 5.3. The NH2-terminal amino acid sequence of the 35 kDa protein was shown to be: SILNLQDLSQKYMTAALNKI (SEQ ID NO: 22) The NH2-terminal comparison of the 35 kDa protein with the amino acid sequence deduced from CryET39 confirmed that it was not an processed form of the protein. The approximately 35 kDa protein was designated as CryET75 and the gene encoding it (residing in the 8.4-kb fragment from EG5899) was designated as cryET75. The sucrose fraction gradient containing CryET39 also contained the approximately 15 kDa protein, designated CryET74, the NH2-terminal amino acid sequence CryET74 was determined as described for CryET39 in Section 5.3. The NH2-terminal amino acid sequence of the isolated CryET74 protein was determined to be:
SARQVHIQINNKTRH (SEQ ID NO: 23) Comparison of this sequence with that of CryET39 and CryET175 showed that CryET74 was a unique protein encoded by a third gene, designated as cryET74, which was contained in the 8.4-kb Hindlll fragment cloned from of EG5899.
EXAMPLE 7 Sequencing of the CRY genes and determination of the amino acid sequence of the purified polypeptides
To facilitate the cryET39, cryET74, and cryET75 gene sequences, the 8.4-kb Hindlll fragment of pEG1319 was subcloned into HindIII digested by pUC18 (Yanisch-Perron et al., 1985). The plasmid was designated pEG1337 and is shown in Figure 1. The preparation of a double stranded plasmid template DNA was achieved using either a standard alkaline lysis procedure or a Kit Qiagen plasmid kit (Qiagen Inc., Chatworth, CA) following the manufacturer's procedures. Sequencing reactions were performed using the DNA sequencing kit version 2.0 of Sequensase ™ (United States Biochemical / Amersham Life Science Inc., Cleveland, OH) following the manufacturer's procedures and using 35S- [dATP] as the labeling isotope (DuPont NEN Research Products, Boston, MA). Denaturing gel electrophoresis of the reactions was performed on 6% (w / v) acrylamide, 42% (w / v) urea sequencing gel. The dried gel was exposed to Kodak X-OMAT AR X-ray film. (Eastman Kodak) overnight at room temperature. The NH2-terminal specific wd271 oligonucleotide was used as the sequencing primer. The complete sequence for the cryET39 gene was determined using the methods described above. The successive oligonucleotides to be used for initiator sequencing reactions were designated from the sequencing data of the above reaction set. In this way, DNA sequencing progressed along the upper and inner chain of the cryET39 gene in a two-step manner. An oligonucleotide primer in the NH2-terminal amino acid sequence of the CryET175 protein was designated for use in the sequencing of the cryET75 gene. The oligonucleotide was designated MR51 and had the sequence: 5'-TCACAAAAATATATGAGAAGC-3 '(SEQ ID NO: 24) Using the DNA sequencing methods described above, a partial nucleotide sequence of the cryET75 gene was determined with the complement of the sequence being achieved using automated sequencing. DNA samples were sequenced using ABI PRISM® DyeDeoxy sequencing chemistry in accordance with (Applied Biosystems, Inc., CA) the manufacturer's protocol. The completed reactions were run on an automated ABL 377 DNA sequencer. The DNA sequence data was analyzed using Sequencher v3.0 DNA analysis software (Gene Codes Corporation, Ann Arbor, Ml). The amino acid sequence of the CryET75 protein was then derived by translating the open reading frame of cryET75. The NH2-terminal sequence of CryET75 was identical to the NH2-terminal amino acid sequence from the nucleotide sequence. Studies in which the Hindlll 8.4-kb fragment from EG11529 was subsequently digested and the subcloned fragments to express the crystal protein genes individually, or in combination are described in Section 5.11. The expression of the CryET39 protein was dependent on the cloning of the cryET74 gene on the same restriction fragment. This suggested that the cryET74 gene be located towards the 5 'end of the cryET39 gene and that the promoter for cryET74 also directs the expression of cryET39. Oligonucleotides specific for the 5 'DNA sequence to start the cryET39 gene were designated to be used as primers for automated sequencing. Successive initiators were designated in the data derived from each of the sequencing reactions. In this way, the region towards the 51 end of cryET39 was sequenced in some way by steps. A translation of the DNA sequence revealed an open reading frame that encodes the CyET74 protein. Examination of the derived amino acid sequence found a region identical to the determined NH2-terminal amino acid sequence of CryET74, identified by the open reading frame as the cryET74 gene.
. 7.1 CrvET39 The DNA sequence of the CryET39 gene is represented by SEQ ID NO: 7, and encodes the amino acid sequence of the CryET39 polypeptide, represented by SEQ ID NO: 8.
. 7.13 Characteristics of the CryET39 polypeptide isolated from EG5899 The CryET39 polypeptide comprises a sequence of 385 amino acids, has a calculated molecular mass of 44.245 Da, and has a calculated isoelectric constant (pl) equal to 5.47. The amino acid composition of the CryET39 polypeptide is given in Table 11.
TABLE 11
Amino acid composition of CrvET39
No. of% of% of% of
Amino Acid Total Waste Amino Acid Total Waste
Wing 6 1.5 Leu 33 8.5 Arg 3 OJ Lys 39 10.1
Asn 31 8.0 Met 8 2.0
Asp 23 5.9 Phe 6 1.5
Cys 2 0.5 Pro 16 4.1
Gin 17 4.4 Ser 30 1.7 Glu 27 7.0 Thr 36 9.3
Gly 19 4.9 Trp 7 1.8
His 8 2.0 Tyr 24 6.2
lie 32 8.3 Val 18 4.6
Acid (Asp + Glu) 50
Basic (Arg + Lys) 42
Aromatic (Phe + Trp + Tyr) 37
Hydrophobic (Aromatic + lie + Leu + Met + Val) 126
. 7.2 CrvET74 The DNA sequence of the CryET74 gene is represented by SEQ
ID NO: 5, and encodes the amino acid sequence of the CryET74 polypeptide,
represented by SEQ ID NO: 6 5.7.2.3 Characteristics of the CryET74 polypeptide
The CryET74 polypeptide comprises a sequence of 119 amino acids, has a calculated molecular mass of 13.221 Da, and has a calculated pl equal to 6.21. The amino acid composition of the CryET74 polypeptide
it is given in table 12.
TABLE 12 Amino acid composition of CryET74
No. of% of% of% of
Amino Acid Total Waste Amino Acid Total Waste
Wing 4 3.3 Leu 6 5.0
Arg 6 5.0 Lys 7 5.8
Asn 6 5.0 Met 2 1.6
Asp 7 5.8 Phe 4 3.3
Cys 1 0.8 Pro 3 2.5 Gin 3 2.5 Ser 13 10.9
Glu 8 6.7 Thr 12 10.0
Gly 10 8.4 Trp 1 0.8 His 5 4.2 Tyr 4 3.3 He 9 7.5 Val 8 6.7
Acid (Asp + Glu) 15 Basic (Arg + Lys) 13 Aromatic (Phe + Trp + Tyr) 9 Hydrophobic (Aromatic + lie + Leu + Met + Val) 34 5.7.3 CrvET75
The DNA sequence of the CryET75 gel is represented by SEQ
ID NO: 15, and verify the amino acid sequence of the CryET75 polypeptide,
represented by SEQ ID NO: 16.
. 7.3.3 Characteristics of the CryET75 polypeptide
The CryET75 polypeptide comprises a sequence of 310
amino acids, has a calculated molecular mass of 34.259 Da, and has a calculated pl equal to 5.67. The amino acid compositions of the polypeptide
CryET75 is given in table 13.
TABLE 13
Amino acid composition of CryET75
AminoNo.de% Total Amino-Acid No.% Acid Residues Total Waste
Wing 15 4.8 Leu 24 7.7
Arg 5 1.6 Lys 29 9.3
Asn 15 4.8 Met 7 2.2
Asp 17 5.4 Phe 11 3.5
Cys 2 0.6 Pro 9 2.9
Gin 11 3.5 Ser 34 10.9
Glu 22 7.0 Thr 33 10.6
Giy 17 5.4 Trp 1 0.3
His 6 1.9 Tyr 11 3.5 lie 22 7.0 Val 19 6.1 AminoNo.de% Total Amino-acid No. of% acid Waste Total Waste
Acid (Asp + Glu) 39 Basic (Arg + Lys) 34 Aromatic (Phe + Trp + Tyr) 23 Hydrophobic (Aromatic + lie + Leu + Met + Val) 95
EXAMPLE
Homology analysis for CryET39
The amino acid sequence of the sequence deduced from the
CryET39 protein was used to challenge sequence databases
electronics for related protein homologies. The database of
SWISS-PROT ALL (small) was questioned using FASTA version 3.15 (Pearson
and Lipman, 1988) on the FASTA European Bioinformatics Institute server (http://ww.ebi.ac.uk) under the following parameters (matrix = pam 150, ktup = 2,
gapcost = -12, gapxcost = -2). The results of the search of the database
showed that CryET39 presented an amino acid sequence identity of
-25% in a 322 amino acid region of the 42 kDa mosquitocidal crystal protein of CryET39 also showed a sequence entity -20% in one
343 amino acid region of the 51 kDa crystal protein of β. sphaericus
No other protein sequence in the database showed similarity of
significant sequence with the sequence of CryET39. The amino acid sequence of CryET39 was also used to challenge the non-redundant database (nr) of National Center Biotechnology Information (NCBI) using version 2.0 of BLASTP (Altschul et al., 1997) using the following parameters: matrix = blosum62, alignment with space, other parameters = fixes by default. The nr database comprises sequence input of PDB, SWISS-PROT, PIR, and GenBank CDS translations. The results of this search were in accordance with those obtained using FASTA search.
EXAMPLE 9 Search of database for protein related to CryET74
The amino acid sequence deduced for CryET74 was also used to challenge the SWISS-PROT ALL databases and nr using FASTA and BLASTP as described in section 5.8. No proteins were found that showed any significant sequence similarity by CryET74.
EXAMPLE 1Q Search of database for protein related to CryET75
The deduced amino acid sequence of CryET75 was also used to challenge the SWISS-PROT ALL and nr databases using FASTA and BLASTP as described in Section 5.8. The search for FASTA revealed that CryETJd showed an identity of 28.1% with Cry 15 Aa (Genbank accession number M76442) in a region of 121 amino acids. The BLASTP analysis revealed 23% sequence activity in a region of 231 amino acids.
EXAMPLE 11 Subcloning and expression of the cryET39 and cryET74 genes
The sucrose gradient fraction of paraspore crystals obtained from lysed cultures of strain EG11529 contained the polypeptides
CryET39 and CryET74. The bioassay evaluation of the preparation of CryET39 and
CryET74 demonstrated that this preparation was toxic to WCRW larvae as the total crystal protein prepared from EG11529. To determine the insecticidal activity of the CryET39 protein alone it was necessary to clone the cryET39 gene towards the 3 'end from another promoter. As described below, this was accomplished using PCR ™, to amplify the promoter region for the crystal protein of B. thuringiensis, Cry2Ac (Wu et al., 19991) and placing it towards the 5 'end of the cryET39 PCR ™ gene in a bead vector, thus allowing the expression of only the CryET protein 39 in a recombinant B. thuringiensis strain. The oligonucleotides were designed to be used as primers in the application of PCR ™ and subsequent cloning of the regulatory region of the cry2Ac gene; including the open reading frames ORFl and ORF2, the ribosome binding site, and the start codon for Cry2Ac. Oligo mr47 includes the 1124 base pairs of the EcoRI restriction site towards the 5 'end from the start codon of the cry2Ac coding region. The sequence of mr47 is: 5'-ATATCTATASMrrGGCAATTCGTCCATGTG-3 '(SEQ ID
NO: 25) EcoRI The complementary oligonucleotide primer, mr43, consists of the inverted complementary sequence for the ribosome binding cyto and the start codon (Met) for the cry2Ac gene, a site in Hindlll has been incorporated between the
RBS and the Met codon to allow an insertion in the frame of the cryET39 gene sequence. The sequence of mr43 is: 5'-CAGTATTCATATAAGCTTCCTCCTTTAATA-3 '(SEQ ID NO: 26) Met Hind lll RBS The PCR ™ reaction to amplify the cryET39 gene consisted of the following: four deoxynucleoside triphosphates-dATP, dTTP, dCTP, dGTP- at a final concentration of 200μM; 5μl 10X Taq Extender ™ (Stratagene Cloning systems) for a final IX concentration; 10 ng pEG1273, which consisted of pUC18 in which the cry2Ac gene has been cloned; Oligonucleotide primers mr47 and mr43 at a final concentration of 2.5 μM each; 2.5 units Taq Extender ™ (Stratagene Cloning Systems): 2.5 units Taq
Polymerase (Promega Corp.) and dH20 at a final reaction volume of 50μl. The reaction was performed on a PowerBIock ™ EasyCycler ™ temperature cycler (Ericomp, Inc. San Diego, CA). The cyclization conditions consisted of a denaturation step of 2 minutes at 94 ° C, followed by 30 cycles of 94 ° C for 1 minute, annealing at 50 ° C for 1 minute and extension at 72 ° C for 2 minutes. After cyclization, 5μl of the reaction was subjected to electrophoresis through a 0.8% agarose gel to verify that a band of approximately 2-kb product was produced by PCR ™. The rest of the reaction product was purified using a QIAquick ™ spin column following the manufacturer's instructions (QIAGEN, Inc.). The cry2Ac promoter amplified by PCR ™ was then cloned into the vector pHT315 of the broiler E. coli / ß.thuringiensis (Arantes and Lereclus, 1991), this was accomplished by digesting both pHT315 and the PCR ™ product, in separate reactions, with restriction enzymes EcoRI and Hindlll These enzymes cut within the multiple cloning of pHt315 and near the ends of the PCR ™ product, within the sequences specified by the nucleotides mr47 and mr43.The digested PCR ™ product was isolated running the reaction through an agarose gel followed by purification of the approximately 2-kb fragment using the Geneclean® procedure (Bio 101), pHT315 digested was purified in a similar manner, the fragment was ligated after pHT315 digested in a reaction that it contained T4 DNA ligase and a binding pH regulator (Promega Corp.).
The ligation mixture was introduced into transformation-competent E. coli DH5a ™ cells (GibcoBRL) following the procedures described by the manufacturer. The E. coli cell was placed on LB agar plates containing 505 μg / ml ampicillin and incubated overnight at 37 ° C. pH% 315 contains a gene that confers resistance to ampicillin to recombinant cells in which it has been successfully introduced. Plasmid DNA was prepared from several clones resistant to ampicillin and digested with EcoRI and HindIII to confirm the presence of 2-kb insert. One of these plasmids, designated pEG1915, was used for cloning and expressing the cryET39 gene. PCR ™ was used to amplify cryET39 from the 8.4-kb Hindlll fragment in pEG1337. Oligonucleotide primers were designed to facilitate the insertion of cryET39 into pEG1915 so that the gene could be expressed from the cry2Ac promoter. The specific oligonucleotide cryET39, 15 mr44, includes the start codon (Met) for Hindlll for the Hindlll site genetically manipulated 5 'to the start codon. The sequence of mr44 is: 5, -AAGGTGAASCIITTAIfiTTAGATACTAATAAAGTTTATG-3, (SEQ ID NO: 27) Hlnd lll Met A second primer designated mr45, was designed to be complementary to a sequence of 212 base pairs 3 'towards the end of the cryET39 coding region. A Hindlll site was incorporated into the sequence of mr45.
'-CCGGAATAGAAG_ £ IITGCATATGG-3 '(SEQ ID NO: 28) Hind lll The generated cryET39 PCR ™ product using mr44 and mr45 as primers was cut with Hindlll and inserted into the Hindlll mr43 site in plasmid pEG1915. This places the Met codon of the cryET39 gene 7 base pairs towards the 3 'end of the ribosome binding site of the cloned cry2Ac promoter. Said configuration was expected to allow efficient expression of the protein of
Recombinant CryET39. The ligation reaction that was performed to insert the cryET39 gene into pEG1915 was used to transform E. coli DH5a ™ for ampicillin resistance. The plasmid DNA was prepared and subjected to restriction enzyme analysis to identify a clone in which the cryET39 gene had been inserted into pEG1915 in the proper orientation. It was necessary for the sense strand of cryET39 to be oriented in the same direction as that of the cry2Ac regulatory region for efficient transitions to occur. Restriction digestions using the enzymes shown in Figure 2 identified a plasmid containing cryET39 in the proper orientation. This plasmid was designated pEG1921. A strain of Cry of B. thuringiensis was transformed for resistance to erythromycin by the introduction of pEG1921. The recombinant strain designated EG11937 was cultured in a C2 sporulation medium until sporulation and crystal formation had occurred. Phase contrast microscopy clearly identified crystalline inclusions in the form of elongated rectangles, or needles, in the culture. Non-attached spores, crystals and spores were harvested by centrifugation. The material in the tablet was washed twice in a solution of Triton X-100®, 0.005%, 10 mM Tris-HCl, pH7.5 and suspended at one half of the original volume in the wash solution. SDS-PAGE was used to visualize the protein in the crystal. 25μl of
0. 5 NaOH N was added to 100 μl of the sample to inhibit the proteolytic activity that can destroy the protein as the crystal is solubilized. After 2.5 minutes at room temperature, 65 μl of 3X Laemmli sample pH regulator (30% glycerol, 15% 2-mercaptoethanol, 3% SDS, 0.01% Tris 1875 M, 0.01% Tris) was added to the samples. 0.01% bromophenol). The sample was heated at 100 ° C for 5 minutes, centrifuged briefly to remove insoluble material and loaded with an acrylamide gel. Protein bands were visualized by staining with Coomassie bright blue R-250. This analysis demonstrated that EG11937 expressed the 44-kDa CryET39 protein and not the 13-kDa (CryET74) or 34-kDa (CryET75) proteins produced by the recombinant strain EG11529. The copy generated by cryET39 of the PCR ™ gene in pEG1921 was sequenced to confirm that it was identical to the wild-type copy of pEG1337. Strain EG11937 was cultured and prepared by bioassays in WCRW larvae. Unexpectedly the EG11937 crystal protein had no activity on the insects. This result suggested that the CryET39 protein required the presence of CryET74 that was toxic, or that CryET74 is the active toxin protein.
pEG1337 was digested in the restriction enzymes Hindlll and EcoRI to release an approximately 3.2-kb fragment containing the cryET74 gene and only a small piece of the cryET39 gene. This fragment was isolated on an agarose gel was purified and cloned in the roaster vector pHT315, 5 was digested with Hindlll and EcoRI, using previously described procedures. This plasmid, designated pEG1919, was introduced into the Cry strain of B. thuringiensis, EG10650, by electroporation, transforming the recombinant cells for erythromycin resistance. A transformant, designated as EG11935, was cultured in a C2 sporulation medium to determine whether the cloned cryET74 e gene could direct the expression of the crystal protein. The culture was harvested and the crystal protein was analyzed with SDS-PAGE as described above. EG11935 produced only CryEt74 and had no activity on the WCRW larvae. The observations that CryET39 and CryET74, individually, do not have activity on WCRW larvae indicates that the two proteins interact to form a toxic protein composition. PCR ™ was used to generate a DNA fragment containing the genes for CryET39 and CryET74, but not the gene for CryET75 also present in the 8.4-kb fragment of pEG1337 (see map of pEG1337). M13 / pUC to the sequencing primer 20 (GibcoBRL) and mr45 were used to amplify an approximately 3.7-kb product containing CryET39 and CryET74. PCR ™ was performed using conditions described above using pEG1337 as the template. The PCR ™ product was gel purified, digested with HindlW and cloned in pHT315 which had been cut with Hindi and treated with bacterial alkaline phosphatase. The resulting plasmid, designated pEG1920, was used to transform the B. thuringiensis strain, EG10650, for resistance to erythromycin. A recombinant, designated EG11936, was cultured to evaluate crystal protein production. EG11936 produced both the 44-kDa CryET39 polypeptide and the CryET74 polypeptide of approximately 13-kDa.
EXAMPLE 12 Toxicity of crystal proteins to insects
. 12.1 Toxicity of crystal proteins EG11529 to SCRW larvae The larval toxicity of SCRW (Diabrotica undecimpunctata howardí) was determined for the recombinant strain EG11529, which expressed CryET39, CryET74 and CryET75 polypeptides. EG11529 was cultured in a C2 medium at 30 ° C for 3 days until sporulation and lysis had occurred. The cultures were harvested by centrifugation, washed twice in an original volume 1X of Triton X-100 ^ at 0.005% and suspended in 1/10 of the original culture volume of Triton X-100R at 0.005%. For comparison, EG11535, a strain of recombinant B. thuringiensis expressing the toxic protein for Coleoptera Cry3B2 (Donovan et al., 1992) was cultured and harvested in the same manner. SDS-PAGE was used to visualize the proteins. Proteins were quantified by comparison with a standard loading of a known amount of bovine serum albumin (Sigma Chemical Co., St. Louis, MO) using a computational densitometer 5, Model 300A (Molecular Dynamics, Sunnyvale, CA), following the manufacturer's procedures. The SCRW larvae were bioassayed by surface contamination of an artificial diet similar to Marrone et al., (1992), but without formalin. Each bioassay consisted of eight aqueous serial dilutions with aliquots applied to the surface of the diet. After the diluent (Triton X-100% aqueous 0.005% solution) had dried, the first instar larvae were placed in the diet and incubated at 28 ° C. Thirty-two larvae were tested per dose. Mortality was evaluated after 7 days. The data from the replicated bioassays were saved for sample analysis
(Daum, 1970), mortality being corrected to control death, control being diluted only (Abbot, 1925). The results were reported as the amount of crystal protein per well (175 mm2 of diet surface) resulting in an LC50, the concentration killing 50% of the test insects, 95% confidence intervals were also reported.
TABLE 14
Insecticidal activity of EG11529 proteins of SCRW larvae
Sample LC50 (μg protein / well) 95% LC. EG11529 34.1 28-41 EG11535 (cry3B2) 49.5 33-83
The results shown in the previous table showed that the
crystal proteins of EG11529, had a significant activity on the
larvae of SCRW. The LC50 value for EG11529 was lower than that given for the Cry3B2 control protein, although 95% confidence intervals
they overlapped, indicating that the difference had not been significant.
. 12.2 Toxicity of CrvET39 v CrvET74 to WCRW larvae
The toxicity of WCRW larvae (Diabrotica virgifera virgifera) is
determined for EG11529, as well as the recombinant strains constructed for
produce the individual crystal proteins of EG11529. The strains
recombinants and the crystal proteins they produced are shown in the
Table 15
TABLE 15
Recombinant strain Bt crystal protein expressed PM (kDa) * EG11529 CryET39 44 kDa CryET74 13 kDa CryET75 34 kDa
EG11934 CryET75 34 kDa EG11935 CryET74 13 kDa EG11936 CryET39 + CryET74 44 kDa + 13 kDa EG11937 CryET39 44 kDa * Molecular weights are estimated by migration of the protein on an SDS-PAGE gel and by comparison with molecular weight standards
= known ig.
A series of bioassays was performed to determine the activity of
the crystal proteins essentially as described for the tests of
SCRW, with the exception that neonatal larvae were used in place of
First stage larvae. The purified crystal proteins were prepared for 15 tests using step gradients of sucrose. EG11529 was cultivated during
three days at 30 ° C in a sporulation medium of C2. Spurulated cultures and lysates were harvested by centrifugation and washed twice in volumes
equal pH regulator Iavdo (Tris-HCI-10 mM, pH 7.5, Triton X-100R at 0.005%), and suspended to 1/10 of the original volume in the wash solution.
Step gradients of sucrose were prepared by stratification of solutions of increasing concentrations of sucrose, in the solution of
washing, in 25 x 89 mm Ultra-Clear centrifuge tubes (Beckman Instruments, Inc., Palo Alto, CA). The steps consisted of 7.5 ml each of the following concentrations of sucrose (from bottom to top): 79% -72% -68% -55%. 5 ml of spore / crystal suspension were stratified on top of the gradient. The gradients were centrifuged at 18,000 rpm at 4 ° C in an ultracentrifuge of L8-70M (Beckman Instruments) overnight. The crystal proteins of EG11529 were separated into two distinct bands. One band, at the 60% -72% interface, contained only the CryET75 protein. The second band, at the 72% -79% interface, contained CryET39 and CryET74. The bands were detached with a pipette and washed twice in the pH regulator of the wash. The protein sample was then run on a second gradient to ensure complete separation of CryET75 from CryET39 and CryET74. The protein samples were run on an SDS-PAGE gel to verify the integrity of the sample. The samples were then quantified using a standard protein test (Bio-Rad Laboratories, Hercules, CA), following the
manufacturer's procedures. A test was performed comparing the toxicity to WCRW larvae of CryET39 + CryET74 and the purified crystal protein samples CryET75 with the toxicity of EG11529. EG11529 was prepared as a spore / crystal suspension and the amount of protein was determined by SDS-PAGE and
densitomefría. The test data were stored for test analysis (Daum, 1970) with mortality corrected to control death, control being only diluent (Abbot, 1925). The results were reported as the amount of crystal proteins per well (175 mm ^ of diet surface) giving
resulting in an LC50, the concentration killing 50% of the insects
proof. 95% confidence intervals are also reported in table 16.
TABLE 16
Insecticidal protein activity EG11529 on WCRW larvae
Sample LC50 (μg / well) 95% of I.C. CryET75 No activity * EG11529 8.6 6.6-10.6 CryET39 + CryET74 9.7 7.2-12.7 * 6% mortality at a dose of 45 μg / well.
This test clearly demonstrated that the CryET75 protein
purified was not toxic towards the WCRW larvae. The sample containing the mixture of CryET39 and CryET74 had activity similar to that of EG11529, indicating
that CryET75 did not play a synergistic role in the toxicity of EG11529 at
larvae of WCRW.
To determine if CryET74 is a toxic component of the strain
EG11529, a spore / crystal suspension of EG11935, which produces only CryET74, was compared in bioassay with spore / glass suspensions of
EG11529 and EG11936, which produce CryET39 and CryET74. The data from
replicated bioassays were stored for test analysis (Daum, 1970)
being corrected mortality control death, being the control only
diluent (Abbot, 1925). The results were reported as the amount of crystal proteins per well (175 mnr 2 of diet surface) resulting in an LC50, concentrating by killing 50% of the test insects. 95% confidence intervals are also reported in the following table 17. TABLE 17 Insecticidal activity of B. thuringiensis proteins on WCRW larvae
Sample LC5o (μg / well) 95% of I.C. EG11935 No activity at 80 μg / well EG11529 9.78 6.9-12.5 EG11936 14.5 9.7-19.5
The CryET74 protein produced by EG11935 had no activity on WCRW larvae, suggesting that the CryET39 protein, either alone or in combination with CryET74, was responsible for the insecticidal activity seen in EG11529 and EG11936. A test was performed that compared the spore / crystal suspension of EG11937, which produces only the CryET39 crystal proteins, are suspensions of EG11936 and EG11937. Also included in this test are mixtures of 50:50 of EG11935 + EG11937 to see if a mixture of CryET39 and CryET74 had activity similar to that of EG11936. The data (Table 18) are expressed as control percent, which is the mortality at a given corrected dose to control the mortality in the diluent control. Two samples
identical of EG11937 were prepared for repetition purposes.
TABLE 18
Dosage Percent Sample (μg / well) of control EG11935 80 0 EG11935 760 0 EG11936 80 100 EG11936 160 100 EG11937 (1) 80 10.5
EG11937 (1) 160 6.7 EG11937 (2) 80 13.3 EG11937 (2) 160 0 EG11935 + EG11937 (1) 80 100 EG11935 + EG11937 (1) 160 100 EG11935 + EG11937 (2) 80 100 EG11935 + EG11937 (2) 160 93.3
The results of this test clearly demonstrated that the
CryET39 protein alone, as expressed in EG11937, does not explain the observed activity
in EG11936 or EG11529. The addition of CryET74 to the CryET39 protein, however, resulted in a toxic composition for WCRW larvae. These data suggest that CryET39 and CryET74 interact to form the toxic component of EG11529 and EG11936.
. 12.3 Toxicity of EG11529 crystal proteins for larvae and CPB A sporulated culture of EG11529 was harvested, washed and suspended as described above, to determine whether the crystal proteins produced by EG11529 were toxic to larvae of the beetle of the pap. of the Colorado (CPB). The CPB larvae test was performed using techniques similar to those of the SCRW test, except for the substitution of the BioServe's # 9380 insect diet (with added potato flakes) for the artificial diet. Mortality was assessed after three days instead of seven days. For this test, 16 insects were used at a single dose of 140 μg / well. At this dose, 100% of the larvae were killed by demonstrating that EG11529 is toxic to CPB larvae.
EXAMPLE 13 Identification of genes encoding related d-endotoxin polypeptides
Strains of B. thuringiensis producing 40-50 kDa crystal proteins were identified by SDS-PAGE analysis of parasporal crystals produced by sporulant cultivod. Total DNA was extracted from these strains following the procedures described above, digested with the restriction endonuclease HindIII, and the restriction fragments were resolved by agarose gel electrophoresis and subjected to Southern blot analysis on nitrocellulose filters. PCR ^ M was used to amplify a segment of the cryET39 gene to be used as a hybridization probe to identify and clone related toxin genes from these strains of B. thuringiensis. The PCRTM fragment extended from nucleotide 176 of the sequence
encoder of cryET39 at approximately 200-bp 3 'towards the end of the gene and were generated using the opossum intruders mr13 and mr24 and the plasmid pEG1337 as a template. mr13: 5 '- TGACACAGCTATGGAGC-3' (SEQ ID NO: 33) mr24: 5"- ATGATTGCCGGAATAGAAGC-3 '(SEQ ID NO: 34) 20 The amplified DNA fragment was radioactively labeled using ALFA-32P-dATP and a kit of random primer primer (Prime-a-Gene® Labeling System, Promega Corporation, Madison, Wl) After incubation with the cryET39-specific hybridization probe, the filters were washed under moderately adverse conditions (e.g. X-1.0X SSC at 55 C), and were exposed to X-ray film to obtain an autoradiogram that identified DNA fragments containing sequences related to cryET 39. Several strains yielded hybridization patterns that differed from EG 5899. Three strains, designated as EG4100, EG4851 and EG9444 respectively were selected for further characterization The cloning and expression of cry genes from strains EG4100, EG4851 and EG9444 were achieved using procedures described in Section 5.4, Section-5. 5 and Section 5.6. The DNA was prepared from these strains and partially digested with the restriction enzyme? Ol, resulting in a distribution of essentially chemi- cal DNA fragments. The Mbol fragments were resolved on an agarose gel and fragments in the 6-10-kb size range were purified. The purified Mbol fragments were then ligated to a shuttle vector of B. thuringiensis / E.coli, pHT315, previously digested with ßamHI and treated with alkaline phosphatase. The ligation mixture was then used to transform E. coli to resistance to ampicillin, thus constructing a library of cloned fragments representing the genome of each respective B. thuringiensis strain. The E. coli libraries were placed on LB agar containing 50 ug / ml of ampicillin and the colonies were transferred to nitrocellulose filters. To identify sequences related to cryET39, filters were tested with radiolabeled oligonucleotide wd271 (library EG9444), as described in Section 5.4 and Section 5.5, or with the cryET39-specific hybridization probe described above (EG4100 and EG4851 libraries) . The plasmid DNA was isolated from hybridizing E. coli colonies and used to transform a host strain of B. acrylostaliferous B. thuringiensis to erythromycin resistant. The recombinant B. thuringiensis clones were cultured for sporulation in a C2 medium and the crystal proteins were analyzed by SDS-PAGE as described in Section 5.6. A cloned fragment identified in the manner described above from the EG4100 library encoded a crystal protein of approximately 60-kDa, designated CryET69 (SEQ ID NO: 14). DNA sequence analysis revealed that the cryET69 gene (SEQ ID NO: 13) encoded a protein of 520 amino acid residues. The CryET69 protein showed a sequence identity of ~ 23% for CryET39. The strain of ß. Recombinant thuringiensis expressing CryET69 was designated EG11647 and the recombinant plasmid containing the cryET69 gene was designated pEG1820. EG4100 and EG11647 were deposited in the ARS Patent Crop Depot and given the access numbers of NRRL B-21786 and B-21787, respectively. A cloned fragment isolated from the EG9444 library as described above encoded a crystal protein of approximately 45-kDa, designated as CryET71, which was related to CryET39, and a crystal protein of approximately 14-kDa designated as CryET79, which was related to with CryET74. DNA sequence analysis revealed that the cryET71 gene (SEQ ID NO: 11) encodes a protein of 397 amino acids and that the cryET79 gene (SEQ ID NO: 9) encodes a 123 amino acid protein. The CryET71 protein (SEQ ID NO: 12) showed a Sequence Identity of 78% at CryET39 while the CryET79 protein (SEQ ID NO: 10) showed an 80% sequence identity at CryET74. The strain of ß. Recombinant thuringiensis expressing CryET71 and CryET79 was designated EG11648 and the recombinant plasmid containing cryET71 and cryET79 genes was designated pEG1821 (Table 9). EG11648 was toxic to WCRW larvae. By analogy with the related CryET39 and CryET74 proteins, it was assumed that CryET71 and CryET79 were required for complete toxicity to WCRW. EG9444 and EG11648 had been deposited in the ARS Patent Crop Depot and given the access numbers of NRRL B-21785 and B-21788, respectively. An isolated clone fragment of the EG4851 library as described above encoded a crystal protein of approximately 44-kDa, designated CryET76, which was related to CryET39, and an approximately 15-kDa crystal protein designated CryETdO, which was related to with CryET74. DNA sequencing analysis revealed that the cryET76 gene (SEQ ID NO: 1) encodes a protein of 387 amino acids and that the cryET80 gene (SEQ ID NO: 3) encodes a protein of 132 amino acids. The CryET76 protein (SEQ ID NO: 2) showed a sequence identity of 61% at CryET39 while the CryET80 protein (SEQ ID NO: 4) showed a sequence identity of 52% at CryET74. The strain of ß. Recombinant thuringiensis expressing CryET76 and CryET80 was designated EG11658 and the recombinant plasmid containing genes cryET76 and cryET80 was designated pEG1823 (Table 9). EG11658 was toxic to WCRW larvae. By analogy with the related CryET39 and CryET74 5 proteins, it was assumed that CryET76 and CryETdO were required for complete toxicity to WCRW. EG4851 and EG11658 had been deposited in the ARS Patent Crop Depot and given the access numbers of NRRL B-21915 and B-21916, respectively. Based on these results, the inventors contemplate that the use of procedures similar to those described herein will lead to the detachment and isolation of adidonal crystal protein toxins from B. thuringiensis. DNA probes, based on the novel sequences described herein, can be prepared from oligonucleotides, PCR ™ products, or restriction fragments and are used to identify additional genes
reladonados with those described above. These new genes can also be cloned, characterized by DNA sequencing, and their encoded proteins can be evaluated in a bioassay on a variety of insect pests using methods described herein. Innovative genes, in turn, can therefore give rise to the identification of new families of
related genes, as seen in the previous examples.
EXAMPLE 14 Sequencing of related Cry genes
. 14.1 CryET71 A nested nucleotide sequence for the cryET71 gene was obtained using the oligonucleotide wd271 as a sequence primer and the procedures described in Section 5.7. Successive oligonucleotides to be used for primer sequencing reactions were designed from the sequencing data of the above reaction set to obtain the complete sequence of the cryET71 gene. The DNA sequence of the CryET71 gene is represented by SEQ ID NO: 11, and encodes the amino acid sequence of the CryET71 polypeptide, represented by SEQ ID NO: 12.
. 14.1.4 Characteristics of the CryET71 polypeptide The CryET71 polypeptide comprises a sequence of 397 amino acids, has a calculated molecular mass of 45.576 Da, and has a calculated pi equal to 4.75. The amino acid composition of the CryET71 polypeptide is given in Table 19.
CUÁDR < 0 19 Composition of amino acids of CrvET71
No. of% of% of% det
Total amino acid residues Amino acids Total waste Wing 11 2.7 Leu 31 7.8 Arg 8 2.0 Lys 30 7.5 Asn 38 9.5 Met d 2.0 Asp 28 7.0 Phe 6 1.5 Cys 2 0.5 Pro 16 4.0 Gin 25 6.2 Ser 29 7.3 Glu 22 5.5 Thr 33 8.3 Gly 19 4.7 Trp 7 '1.7 His 5 1.2 Tyr 24 6.0 He 41 10.3 Val 14 3.5 Acid (Asp + Glu) 50 Basic (Arg + Lys) 38 Aromatic (Phe + Tf + Tyr) 37 Hydrophobic (Aromatic + He + Leu + Met + Val) 131
. 14.2 CrvET79
An initial sequence of the cryET79 gene towards the 5 'end was obtained
using an oligonucleotide primer designed from the cryET71 sequence
completed DNA samples were sequenced using a chemistry team from
Abl PRISM ™ sequencing DyeDeoxy (Applied Biosystems) according to the
manufacturer's protocol. The completed reactions were run on an automated DNA sequencer ÁBI 377. The DNA sequence data analyzed using Sequencher v3.0 DNA analysis software (Gene Codes Corp.). Successive oligonucleotides to be used for primer sequencing reactions were designed from the sequencing data of the above reaction set to obtain the complete cryET79 gene sequence.
. 14.2.3 Characteristics of the CryET79 polypeptide The CryET79 polypeptide comprises a sequence of 123 s? Or amino acids, has a calculated molecular mass of 13.609 Da, and has a pl- calculated equal to 6.32. The amino acid composition of the CryET79 polypeptide is given in Table 20.
TABLE 20
Amino acid composition of CryET79
No. of% of% of% of
Amino acid total waste Amino acid total waste
Wing 5 4.0 Leu 4 3.2
Arg 4 3.2 Lys 6 4.8
Asn 12 9.7 Met 2 1.6
Asp 5 4.0 Phe 3 2.4
Cys 0 0 Pro 3 2.4
Gln 6 4.8 Ser 13 10.5
Glu 7 5.6 Thr 13 10.5
Gly 13 10.5 Trp 1 0.8
His 6 4.8 Tyr 8 6.5
He 6 4.8 Val 6 4.8
Addo (Asp + Glu) 12
Basic (Arg + Lys) 10
Aromatic (Phe + Trp + Tyr) 12
Hydrophobic (Aromatic + He + Leu + Met + Val) 30
. 14.3 CrvET69 The NH2-terminal amino acid sequence of the isolated CryET69 protein was determined using the procedures described in Section 5.3. The NH2-terminal sequence of the isolated protein was: 1 2 3 4 5 6 7 8 9 10 11 Met Asn Val Asn His Gly Met Ser Cys Gly Cys (SEQ ID NO: 29) An oligonucleotide primer based on the amino acid sequence NH2-terminal protein CryET69 was designed to be used in the sequencing of cryET69. The oligo nucleotide, designated as crc12, has the following sequence: 5'- ATGAATGTAAATCATGGGATGWSNTGT - 3 '(SEQ ID NO: 30) where W = A and T and S = C and G. A nested nucleotide sequence was obtained using crc12 as a sequencing primer and the procedures described in Section 5.7. Successive oligonucleotides to be used for initiation sequencing reactions were designed from the sequencing data of the above reaction set. The completion of the sequence was achieved using automated sequencing. DNA samples were sequenced using a ABI PRISM DyeDeoxy sequencing chemistry kit (Applied Biosystems) according to the manufacturer's protocol. The completed reactions were run on an ABL 377 automated DNA sequencer. The DNA sequence data was analyzed using DNA analysis software.
Sequencher v3.0 (Gene Codes Corp.).
. 14.3.3 Characteristics of the CryET69 polypeptide The CryET69 polypeptide comprises a sequence of 520 amino acids, has a calculated molecular mass of 58,609 Da, and has a calculated pl equal to 5.84. The amino acid composition of the CryET69 polypeptide is given in Table 21.
TABLE 21
Amino acid composition of CryET69
No. of% of% of% of
Total amino acid residues Total amino acid residues Wing 24 4.6 Leu 31 5.9 Arg 30 5.7 Lys 15 2.8 Asn 60 11.5 Met 10 1.9 Asp 27 5.1 Phe 20 3.8 Cys 9 1.7 Pro 24 4.6 Gln 32 6.1 Ser 39 7.5 Glu 24 4.6 Thr 48 9.2 Gly 32 6.1 Trp 6 1.1 His 9 1.7 Tyr 22 4.2 He 24 4.6 Val 34 6.5 Acid (Asp + Glu) 51 Basic (Arg + Lys) 45 Aromatic (Phe + Trp + Tyr) 48 Hydrophobic (Aromatic + He + Leu + Met + Val ) 147
EXAMPLE 16
Search in database for proteins related to CryET69
The amino acid sequence deduced for CryET69 was used to challenge the SWISS-PROT ALL databases and nr using FASTA and BLASTP as described for CryET39 in Section 5.8, except that the blosumdO comparison matrix was used for the search in FASTA The search results on FASTA indicated that CryET69 showed a sequence identity of approximately -32% in a region of 338 amino acids with the 42-kDa ß 42-kDa mosquitocidal crystal protein. sphaericus and a sequence identity of -30% in a region of 440 amino acids with the 51-kDa crystal protein of B. sphaericus.
EXAMPLE 17 Searches in database for proteins related to CryET71 and CryET79
The amino acid sequences deduced for CryET71 and CryET79 were used to interrogate the SWISS-PROT ALL and nr databases using FASTA and BLASTP as described for CryET39 in Section 5.8. FASTA search results indicated that CryET71 showed a sequence identity of -25% in a region of 323 amino acids with the 42-kDa mosquitodda crystal protein from B. sphaericus and a sequence identity of -25% in one 38d amino acid region with the 51-kDa crystal protein of B. sphaericus. Searches in FASTA and BLASTP did not identify proteins with significant sequence identity for CryET79.
EXAMPLE 18 Sequencing of the CryET76 and CryET80 genes
A partial DNA sequence of genes donated in pEG1823 5 was determined following the procedures of DNA sequencing of established dideoxy chain termination (Sanger et al, 1977). The preparation of the double stranded plasmid template DNA was achieved using a Wizard® SV Miniprep Kit (Promega Corp.) following the manufacturer's procedures or a Qiagen Plasmid Kit (Qiagen Inc.) following the manufacturer's procedures , followed by an extrusion with phenol: chloroform: isoamyl alcohol (50: 48: 2) and then an extraction with doroform: isoamyl alcohol (24: 1). Sequencing reactions were performed using the Sequenase ™ DNA Version 2.0 sequencing kit (United States Biochemical / Amersham Life Science Inc.) following the
manufacturer's procedures and using 35S- [dATP] as the labeling isotope (DuPont NEN® Research Products). Denaturing gel electrophoresis of the reactions was performed on a 6% (w / v) acrylamide sequencing gel, 42% (w / v) urea or a 6% acrylamide sequencing gel of CastAway ™ Precast ( Stratagene). The dried gel was exposed to lightning film
XKodak X-OMAT AR (Eastman Kodak) overnight at room temperature to obtain an autoradiogram.
A pardal sequence of DNA for the genes cryET76 and xkcryETdO in pEG1823 was obtained using the procedures described above. The oligonucleotide specific for cryET39 mr18 was used as the initial sequencing primer. The sequencing of mr18 is: 5 5'-GTACCAGAAGTAGGAGG-3 '(SEQ ID NO: 31) Successive oligonucleotides can be used for primer sequencing reactions where it is designed from the sequencing data of the previous set of reactions. The completion of the sequence was achieved using automated sequencing. DNA samples were sequenced using the ABl PRISM DyeDeoxy sequencing chemistry kit (Applied Biosystems) according to the protocol suggested by the manufacturer. The completed reactions were run on an automated ABL 377 DNA sequencer. The DNA sequence data was analyzed using Sequencher v3.0 DNA analysis software (Gene Codes Corp.). The DNA sequence of cryET76 (SEQ ID NO: 1) and cryETβO (SEQ ID NO: 3) is shown below. The amino acid sequence of the protein CryET76 (SEQ ID NO: 2) and the protein CryET80 (SEQ ID NO: 4) is also shown below. The entire sequenced region is shown in (SEQ ID NO: 17).
. 18.1 CryET76
The DNA sequence of the CryET76 gene is represented by SEQ
ID NO: 1, and encodes the amino acid sequence of the CryET76 polypeptide,
represented by SEQ ID NO: 2.
. 18.1.3 Characteristics of the CryET76 polypeptide
The CryET76 polypeptide comprises a sequence of 387
amino acids, has a calculated molecular mass of 43,812 Da, and has a
calculated equal to 5.39. The amino acid composition of the CryET76 polypeptide is given in Table 22.
TABLE 22
Amino acid composition of CryET76
No. of% of% of Amino acid total waste Amino acid total waste
Wing 14 3.6 Leu 34 8.7
Arg 7 1.8 Lys 27 6.9
Asn 39 10.0 Met 5 1.2 Asp 17 4.3 Phe 8 2.0
Cys 1 0.2 Pro 10 2.5
Gln 17 4.3 Ser 30 7.7 No. of% of% of% of
Amino acids total residues Amino acids total waste Gly 22 5.6 Trp 8 2.0 His 4 1.0 Tyr 24 6.2 lie 31 8.0 Val 20 5.1
Acid (Asp + Giu) 39 Basic (Arg + Lys) 34 Aromatic (Phe + Trp + Tyr) 40 Hydrophobic (Aromatic + He + Leu + Met + Val) 130
. 18.2 CryETdO The DNA sequence of the CryETdO gene is represented by SEQ ID NO: 3, and encodes the amino acid sequence of the CryETdO polypeptide, represented by SEQ ID NO: 4.
. 16.1.3 Characteristics of the CryETdO polypeptide The CryETdO polypeptide comprises a sequence of 132 amino acids, has a calculated molecular mass of 14, d39 Da, and has a calculated pl equal to 6.03. The amino acid composition of the CryETdO polypeptide is given in Table 23.
TABLE 23
Amino acid composition of CryETdO
No. of% of% of%
Amino acid total waste Amino acid total waste
Wing 5.3 Leu 4.5
Arg d 6.0 Lys 4 3.0
Asn 13 9.6 Met 2 1.5
Asp d 6.0 Phe 2 1.5
Cys 1 0.7 Pro 3 2.2
Gln 3 2.2 Ser • 11 3.3
Glu 6 6.0 Thr 11 3.3
Gly 9 6.8 Trp 1 0.7
His d 6.0 Tyr 6 4.5
lie 13 9.8 Val 8 6.0
Acid (Asp + Glu) 16
Basic (Arg + Lys) 12
Aromatic (Phe + Trp + Tyr) 9
Hydrophobic (Aromatic + He + 1 Leu + Met + Val) 38
. 18.3 Characteristics of Aenes CrvET76. CrvETdO and CrvET84 Insulated from EG4851 (SEQ ID NO: 17) The DNA sequence of the complete operon of three genes containing the coding regions CryET76, CryET80 and CryET84 is represented by SEQ ID NO: 17. In strain EG4651, the cryET84 gene is located immediately 5 'towards the genes cryET80 and cryET76. The cryET84 gene starts at nucleotide 656 and ends at nucleotide 167d. The c ?? ET80 gene starts at nucleotide 1773 and ends at nucleotide 2166. The cryET76 gene starts at nucleotide 2264 and ends at nucleotide 3424.
EXAMPLE 19 Analysis of sequence homologies
. 19.1 Database searches for proteins related to CrvET76 v CryETdO The amino acid sequences of the CryET76 and CryETdO proteins, deduced by translation of the nucleotide sequence, were used to challenge sequence databases for related protein sequences. The SWISS-PROT ALL database was questioned using FASTA version 3.15 (Pearson and Lipman, 19dd) provided by the FASTA server at the European Institute of Biotechnology (http://www.ebi.ac.uk) using the following parameters (library = swall, array = pam150, ktup = 2, gapcost = -12, gapxcost = -2). The amino acid sequences of CryET76 and CryETdO were also used to challenge the non-redundant database (nr) of the National Center Biotechnology Information (NCBI) using BLASTP version 2.0 (Altschuk et al, 1997) with the following parameters: matrix = blosum62, space alignment, other parameters = default bindings. The results of the FASTA analysis revealed that CryET76 showed sequence identity -27% in a region of 320 amino acids with the 42-kDa mosquictocidal crystal protein of B. sphaericus while CryETdO showed no significant sequence similarity with sequences in SWISS- PROT ALL. The results of the search in BLASTP were in general agreement with those of the search in FASTA. No proteins with significant sequence similarity to CryETdO were identified.
. 19.2 Comparison of additional sequence with Cry proteins Sequence alignments were made to compare the sequences of CryET39, CryET74, CryET75, CryET71, CryET79, CryET76,
CryETdO and CryET69 with sequences of newly published patent applications.
The alignments were made using PALIGN in the sequence analysis package PC / GENE version 6.d5 (Intelligenetics Corp. Mountain View, CA). The alignments in pairs were made using the following parameters: comparison matrix = unit, open space cost = 3, space cost per unit = 1.
. 19.2.2 CryET39 Sequence alignment comparing the sequence of CryET39 with the newly published patent application sequencies revealed a sequence similarity between CryET39 and the proteins identified by sequence identifiers 11, 33 and 43 of the Patent Application Publication International No. WO 97/40162. CryET39 showed a sequence identity of 99.2% with the sequence number 11, a sequence identity of 78.6% with the sequence number 38 and a sequence identity of 79.9% with the sequence number 43.
. 19.2.2 CryET74 The sequence alignment that compared the sequence of
CryET74 with sequences of newly published patent applications revealed a sequence similarity between CryET74 and sequence identifier numbers 32, 36 and 41 of International Patent Application Publication No. WO 97/40162. CryET74 showed a sequence identity of 100% with the sequence identifier number 32, a sequence identity of 80.7% with the sequence identifier number 36 and a sequence identity of 77.3% with the sequence identifier number 41 of this request.
. 19.2.3 CrvET75 Sequence alignment comparing CryET75 with newly published patent application sequencies revealed an identity of
Sequence of approximately 26% between CryET75 and CryET33, a toxic protein for Coleoptera, described in the Patent Application Publication
Intemal on No. WO 97/17600.
. 19.2.4 CrvET71 * ío The sequence alignment that compared the sequence of
CryET71 with sequences of newly published patent applications revealed a sequence similarity between CryET71 and Sequence Identifier numbers 11, 33 and 43 of International Patent Application Publication No. WO 97/40162. CryET71 showed a sequence identity of 78.4% with the sequence identifier number 11, a sequence identity of 91.9% with the sequence identifier number 38 and a sequence identity of 97.4% with the sequence identifier number. 43
. 19.2.5 CrvET79 20 The sequence alignment that compared the sequence of
CryET79 with sequences of published patent applications revealed a sequence similarity between CryET79 and sequence identifier numbers 32, 36 and 41 of International Patent Solidtud Publication No. WO 97/40162. CryET79 showed a sequence identity of 79.8% with the sequence identifier number 32; a sequence identity of 95.9% with the sequence identifier number 36; and a sequence identity of 5 91% with the sequence identifier number 41.
. 19.2.6 CrvET76 Sequence alignment comparing the sequence of CryET76 with sequences of published patent applications revealed a semblance similarity between CryET76 and the sequence identifier numbers 11, 38 and 43 of the Application Publication of International Patent No. WO 97/40162. CryET76 showed a sequence identity of 60.8% with the sequence identifier number 11; a sequence identity of 61.6% with the sequence identifier number 38; and a sequence identity of 15 61.9% with the sequence identifier number 43.
. 19.2.4 CrvETdO Sequence alignment comparing the sequence of CryETdO with sequences of newly published patent applications revealed a
The sequence similarity between CryETdO and the sequence identifier numbers 32, 36 and 41 of the Intemational Patent Application Publication No. WO 97/40162. CryETdO showed a sequence identity of 51.2% with the sequence identifier number 32; a sequence identity of 56.1% with the sequence identifier number 36 and a sequence identity of 54.5% > with the sequence identifier number 41.
. 19.2.8 CryET69 Sequence alignment comparing the CryET69 sequence revealed only 23-25% sequence identity between CryET69 and sequence identifier numbers 11, 33 and 43 of Intemadonal Patent Application Publication No. WO 97 / 40162. The crystal protein showed a greater degree of homology with the ß-mosquitocidal crystal proteins. sphaericus than the crystal proteins of B. thuringiensis.
. 19.3 Summary of analyzes These analyzes demonstrated that the amino acid sequences of CryET69, CryET75, CryET76 and CryETdO are markedly different from the above-described insecticidal crystal protein sequences. Using the nomendatura established by the crystal proteins of B. thuringiensis (Crickmore ef al, 1998), CryET76 and CryETdO would be assigned a new secondary category and CryET69 and CryET75 would be assigned a new primary category.
EXAMPLE 20 Expression of recombinant CryET76 and CryETdO polypeptides
To characterize the properties of CryET76 proteins and
CryETdO, it was necessary to express the cryET76 and cry ET80 genes donated in a B. thuringiensis strain that did not produce other crystal proteins (ie, a Cry strain).) The plasmid containing the cryET76 and cry ET80 cloned genes, pEG1d23 , contains an origin of replication of ß.thuringiensis as well as an origin that directs the replication of E. coli, as described above, and pEG1323 was used to transform the B. thuringiensis strain Cry-EG10650 for erythromycin resistance (EmR ) by electroporation (Macaluso and Mettus, 1991).
Transformed EMR cells were selected by overnight incubation on LB agar plates containing 25 μg / ml erythromycin. One EmR colony of each transformation was selected for further analysis. An isolated
was designated EG11658. EG11658 was cultured in a C2 sporulation medium containing 25 μg / ml erythromycin for four days at 25 ° C, at which time the sporulation and lysis of the cell had occurred. Microscopic examination of the sporulated cultures showed that the recombinant strain was reproducing parasporal indusions. The sporulated culture of EG11658 was harvested by centrifugation, washed and resuspended to one tenth of the original volume in H20. The crystal protein in the suspension was characterized by SDS-PAGE analysis which revealed the protein production of approximately 44 and 15 kDa.
EXAMPLE 21 Toxicity of CryET76 and CryETdO to insects
The toxicity of protein CryET76 and CryETdO fair WCRW was determined. EG11658 was cultured in a C2 medium at 25 ° C for four days until sporulation and cell lysis had occurred. The culture was harvested by centrifugation, washed in approximately 2.5 times the original volume with distilled H20 and resuspended in Triton X-100® at 0.005% at one tenth of the original volume. For comparison with EG11658, strains of ß. recombinant thuringiensis, EG11529 which produced the toxic proteins for WCRW CryET39 and CryET74, and EG11646, which produced the WCRW toxic proteins CryET71 and CryET79, were harvested and harvested in the same manner. The toxin proteins of the samples were quantified by SDS-PAGE as described (Brussock and Currier, 1990). The procedure was modified to eliminate the neutralization step with 3M HEPES. Larvae of WCRW were subjected to bioassay for surface contamination of an artificial diet (20 g of agar, 50 g of wheat germ, 39 g of sucrose, 32 g of casein, 14 gr of fiber, 9 gr of Wesson salt mixture, 1 gr of methyl paraben, 0.5 gr of ascorbic acid, 0.06 gr of cholesterol, 9 gr of Vanderzant's vitamin mixture, 0.5 ml of oil Flaxseed, 2.5 ml phosphoric acid / propionic acid per 1 liter). Each bioassay of EG1165d (CryET76 and CryETdO), EG11529 (CryET39 and CryET74) and EG11648 (CryET71 and CryET79) consisted of eight aqueous dilutions with aliquots applied to the surface of the diet. After the diluent (an aqueous solution of Triton X-100® at 0.005%) had dried, the neonate larvae were placed in the diet and incubated at 28 ° C. Thirty-two larvae were tested per dose. Mortality was evaluated after seven days. The data from the replicated bioassays were stored for test analysis (Daum, 1970), mortality being corrected for the control of death, the control being only diluent (Abbott, 1925). The results are reported as the amount of crystal protein per well (175 mm ^ of diet surface) resulting in an LC50, concentrating killing 50% of the test insects. We also reported 95% confidence intervals for LC50 values (Table 24).
TABLE 24 Insecticidal activity for Cry proteins to WCRW larvae
LC50 protein (95 μg LC95 (μg of protein / well crystal sample) of I.C. protein / well)
EG11653 CryET76 10.7 2.2-13.9 46
CryETdO
EG1164d CryET71 5.3 1.9-10.1 27
- CryET79
s 10 EG11936 CryET39 12.3 12.5-14.3 32
CryET74
The results shown in Table 24 showed that the
CryET76 and CryETdO proteins had significant activity on larvae of 15 WCRW.
EXAMPLE 22
Toxicity of CryET69 to insects
The toxicity of CryET69 towards WCRW was demonstrated using
procedures described in Section 5.21. The results are reported as the amount of crystal proteins per well (175 mm2 of the diet surface) resulting in an LC50, concentrating by killing 50% of the test insects. 95% confidence intervals are also reported for LC50 values (Table 25).
TABLE 25 Insecticidal activity of CryET69 to WCRW larvae
LC50 protein (95 μg LC95 (μg of
Sample crystal protein / well) of I.C. protein / well)
EG11204 Cry3b2 13.8 3.2-30.1 502 Eg11647 Cryet69 147.3 73-1292 6190
Control Mortality = 22% These results demonstrated that CryET69 was significantly less active than Cry3B2 against WCRW. However, the crystal protein apparently represents a new class of d-endotoxin toxic to Coleoptera.
EXAMPLE 23 Construction of Strains EG12156 and EG12158
Strains of ß were constructed. recombinant thuringiensis that produce either CryET76 or CryETdO. A frame change mutation was introduced into the cryET76 coding sequence on pEg1d23 to generate a recombinant plasmid capable of directing the production of CryETdO alone. A single Age restriction site within the cryET76 coding sequence was identified by computer analysis of the determined cryET76 nudeotide sequence. Subsequent digestion of pEG1823 with Agel confirmed that this restriction site was unique for the plasmid. To generate a frame change mutation on this site, pEG1823 was digested with > 4gel and the DNA ends were shaved with T4 polymerase in the presence of dNTPs. The linear DNA fragment was subsequently resolved by electrophoresis on a 1% agarose gel, the DNA band was cut with a shaving blade, and the DNA was purified using Qiagen gel extraction equipment. The purified DNA was self-ligated using T4 ligase and used to transform E. coli strain DH5a for ampicillin resistance. Restriction enzyme analysis of DNA recovered from several ampicillin-resistant clones confirmed the alteration of the > 4gel over pEG1623. The recombinant plasmid of one of those clones was designated pEG2206. pEG2206 was subsequently used to transform, by electroporadón, the strain of B. thuringiensis acrylostaliferous EG10650 for resistance to erythromycin. The recombinant B. thuringiensis strain containing pEG2206 was designated EG12156. A deletion mutation was introduced into the cryEt80 coding sequence on pEG1323 to generate a recombinant plasmid capable of directing the production of CryET76 alone. A single Dralll restriction site within the cryET80 coding sequence was identified by computer analysis of the determined cryET80 nucleotide sequence. Subsequent digestion of pEG1823 with Dralll confirmed that this restriction site was unique for the plasmid. To generate a mutation at the site, pEG1823 was digested with Dralll and the DNA ends were shaved with the T4 pollmerase in the presence of dNTPs. The linear DNA fragment was subsequently resolved by electrophoresis on a 1% agarose gel, the DNA band was cut with a shaving blade, and the DNA was purified using the Qiagen gel extraction equipment. The purified DNA was self-ligated using T4 ligase and used to transform E. coli strain DH5a for ampicillin resistance. Restriction enzyme analysis of DNA recovered from several ampicillin-resistant clones confirmed the alteration of the Dralll site on pEG1823. The recombinant plasmid of one of those gifts was designated pEG2207. pEG2207 was subsequently used to transform, by electroporation, strain EG10650 of ß. Acrylostaliferous thuringiensis for resistance to erythromycin. The recombinant strain containing pEG2207 was designated EG12158. Strains EG1165d, EG12156 and EG12158 were used to inoculate 100 ml of C2 broth culture containing 10 μg / ml erythromycin. The broth cultures were grown with shaking in 500 ml flasks with deviations at 28-30 ° C for three days, at which time the cultures were fully sporulated and the sporangias were used. The spores and crystals were compressed by centrifugation at 8,000 rpm (-9800 x g) in a JA14 rotor for 20 minutes at 4 ° C. The tablets were suspended in 50 ml of 10 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 0.001% Triton X-100 * (pH 7.0). The spores and the crystals were again compressed by centrifugation at 3750 rpm (-3200 x g) in a Beckman GPR centrifuge for 1 hour at 4 ° C. The tablets were resuspended in 10 ml of 10 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 0.005% Triton X-100® (pH 7.0) and stored at 4-d ° C. The crystal proteins produced by these cultures were detected by SDA-PAGE and subsequent staining of the SDA gels with Coomassie brilliant blue R-250 as described in Section 5.11. The results of this analysis confirmed that strain EG12156 produced CryETdO, but not CryET76, while strain EG12156 produced CryET76, but not CryETdO (figure 3). Therefore, each crystal protein could be produced independently of the other crystal protein. The function of each crystal protein to produce toxicity towards WCRW larvae can now be studied in more detail. The SDS-PAGE analysis described in Figure 3 also revealed the presence of an adidonal protein present in both crystal preparations EG12156 and EG12158. The protein showed an apparent molecular mass of approximately 35 kDa and was designated CryET84. Additional DNA sequence analysis of the insert cloned into pEG1d23 revealed a third open reading frame sufficient to encode a ~ 3d-kDa protein (SEQ ID NO: 19). The coding region is located immediately 5 * to the cryEtdO gene. Therefore, cryEt84, cryEtβO and cryEt76 may comprise an operon. the CryET84 protein isolated from EG4851 comprises a sequence of 341 amino acids, and has a calculated molecular mass of approximately 37, dd4 Da. CryET84 has a calculated isoelectric constant (pl) equal to 5.5. SDA-PAGE analysis of the EG11658 crystal proteins used for the WCRW bioassay described in Section 5.21 did not detect the CryET84 protein band. Apparently, subtle differences in the cultivation of the strain or in the harvesting and washing of the spore-crystal suspension can result in the loss of CryET84. Sequence comparisons using Blast 2.0 and FASTA 3, as described in Example 8, revealed that there was no significant sequence similarity between CryET84 and all crystal proteins of B. thuringiensis.
EXAMPLE 24 Preparation of transgenic plants resistant to insects
. 24.1 Construction of plant transgene Expression of the plant transgene that exists in the form of double-stranded DNA involves the transcription of messenger RNA (mRNA) from a DNA strand by RNA polymerase enzyme, and the sub-sequent processing of the primary transcript of mRNA within of the nucleus. This processing involves a 3 'untranslated region that adds polyadenylate nucleotides to the 3' end of the RNA. The transcription of DNA into mRNA is regulated by a region of DNA generally known as the "promoter". The promoter region contains a base sequence that directs the RNA polymerase to associate with the DNA and to indent the transcription of mRNA using one of the DNA strands as a template to make a corresponding strand of RNA. The number of promoters that are active in plant cells has been described in the literature. Such promoters can be obtained from plants or plant viruses and include, but are not limited to, the nopaline synthase (NOS) and octopinasynthase (OCS) promoters (which are carried in the tumor-inducing plasmids of Agrobacterium tumefaciens), the promoters. of cauliflower mozaic virus (CaMV) 19S and 35S, the light-inducible promoter of a small subunit of ribulose 1, 5-bisphosphatecarboxylase (ssRUBISCO, a very abundant plant polypeptide), and the 35S promoter of the scrofularia mosaic virus (FMV) All of those promoters have been used to create various types of DNA constructs that have been expressed in plants (see, for example, U.S. Patent No. 5,463,175, specifically incorporated herein by reference). The particular promoter selected must be capable of causing sufficient expression of the enzyme coding sequence to result in the promotion of an effective amount of protein. A set of preferred promoters are constitutive promoters such as CaMV35S or FMV35S that produce high levels of expression in most plant organs (U.S. Patent No. 5,378,619, specifically incorporated herein by reference). Another set of specific promoters are root specific promoters or enhancers such as the 4 as-1 promoter derived from CaMV or the POX1 promoter from wheat (US Patent No. 5,023,179, specifically incorporated herein by reference, Hertig et al., 1991). . Specific or increased root promoters should be particularly preferred for control of corn rootworm (Diabroticus spp.) In transgenic maize plants. The promoters used in the DNA constructs of the present invention can be modified, if desired,. to affect its control characteristics. For example, CaMV35S promoters can be linked to the portion of the ssRUBISCO gene that represses the expression of ssRUBISCO in the absence of light, to create a promoter that is active in the leaves but not in the roots. The resulting chimeric promoters can be used as described herein. For purposes of this description, the phrase "CaMV35S" promoter therefore includes variations of CaMV35S promoter, for example promoters derived by means of ligation with operator regions, random or controlled mutagenesis, etc. In addition, the promoters can be altered to contain multiple "enhancer sequences" to help elevate gene expression. The RNA produced by a DNA construct of the present invention also contains a 5 'untranslated leader sequence. This sequence can be derived from the promoter selected to express the gene, and can be specifically modified to increase translation of the mRNA. The 5 'untranslated regions can also be obtained from viral RNAs, from suitable eukaryotic genes or from synthetic gene sequences. The present invention is not limited to constructs where the untranslated region is derived from the 5 'untranslated sequence that accompanies the promoter sequence. For optimized expression in monocotyledonous plants such as corn, an intron can also be induced in the expression construct of the DNA. This intron will typically be positioned near the 5 'end of the mRNA in untranslated sequence. This intron could be obtained from, but not limited to, a set of introns that consist of the corn hsp70 intron (U.S. Patent No. 5,424,412).; specifically incorporated here by reference) or the Intron Act? of rice (McEIroy ef al, 1990). As indicated above, the 3 'untranslated region of the chimeric plant genes of the present invention contains a polyadenylate signal that fondona in plants to produce the addition of adenylate nucleotides to the 3' end of the RNA. Examples of the preferred 3 'regions are (1) of the 3' transcribed untranslated regions containing the signal of
polyadenylate of Agrobacterium tumor-inducing plasmids (Ti) genes, such as the nopalinasynthase gene (NOS) and (2) plant genes such as the ssRUBISCO E9 gene of the pea (Wong et al., 1992).
. 24.2 Transformation and expression in plants A transgene containing a d-endotoxin coding sequence of the present invention can be inserted into the genome of a plant by any suitable method such as those described herein. Suitable plant transformation vectors include those derived from a Ti plasmid of A tumefaciens, as well as those described, for example by Herrera-Estrella (1983), Bevan et al. (1983), Klee (1985) and European Patent Application Publication No. EP0120516. In addition to the transformation vectors of plants derived from Ti plasmids or root inducers (Rí), from A. tumefaciens, alternative methods can be used to insert the DNA constructs of this invention into plant cells. Such methods may involve, for example, the use of liposomes, electroporadons, chemical compounds that increase DNA uptake, delivery of free DNA by bombardment of microprojectiles, and transformation using viruses or pollen (Fromm et al., 1986; Fromm et al. ., 1990). These methods are described in detail in Section 4.0.
524. 3 Construction of plant expression vectors For the efficient expression of the polynucleotides described herein in transgenic plants, the selected sequence regions that code for the insecticidal polypeptides must have a suitable sequencing composition (Diehn et al., 1996). For example, to place one or more of the cry genes described herein in a vector suitable for expression in monocotyledonous plants (eg, under the control of the 35S promoter of cauliflower mozaic virus and bind to the hsp70 intron followed by a nopaiinasynthase polyadenylation site as in U.S. Patent No. 5,424,412, specifically incorporated herein by reference), the vector can be digested with appropriate enzymes such as Ncol and EcoRI. The larger vector band of approximately 4.6 kb is then subjected to electrophoresis, purified and ligated with T4 DNA ligase to the appropriate restriction fragment containing the plated cry gene. The ligation mixture is then transformed to plasmid DNA and recovered from E. coli carbenicillin-resistant stains recovered by DNA mini-cleavage procedures. The DNA can then be subjected to restriction endonuclease analysis with enzymes such as Ncol and EcoRi (together), Nofí and Pst to identify colonies containing the coding sequence of the cry fused gene to the hsp70 intron under the control of the increased CaMV35S promoter. To place the d-endotoxin gene in a vector suitable for the recovery of stably transformed and insect-resistant plants, the restriction fragment of pMON33708 containing the lysine oxidase coding sequence fused to the hsp70 intron under the control of the increased CaMV35S promoter can be isolated by gel electrophoresis and purification. This fragment can then be ligated with a vector such as pMON30460 treated with Noti and calf intestinal alkaline phosphatase (pMON30460 containing the coding sequence of neomycin phosphotransferase under the control of the CaMV35S promoter). The kanamycin-resistant colonies can then be obtained by transforming the ligation mixture into E. coli and colonies containing the resulting plasmid can be identified by restriction endonuclease digestion of plasmid miniprepradiated DNA. Restriction enzymes such as Notl, EcoRV, Hincin, Ncol, EcoR1 and BglII can be used to identify the appropriate clones containing the appropriately inserted restriction fragment in the corresponding site of pMON30460, in the orientation such that both genes are in tandem (is dedr, the 3 'end of the cry gene expression cassette is ligated to the 5' end of the nptll expression cassette). The expression of the Cry proteins by the resulting vector is then confirmed in plant protoplasts by electroporation of the vector into protoplasts followed by protein blotting and ELISA analysis. The vector can be introduced into the genomic DNA of plant embryos such as corn by particle bombardment followed by selection of paromomycin to obtain maize plants that express the cry gene essentially as described in US Pat. No. 5,424,412, specifically incorporated herein by reference. In this example, the vector was introduced by cobombard with a hygromidine resistance conferencing plasmid in immature embryo screener (IES) of corn, followed by hygromycin selection and regeneration.
The lines of transgenic plants that express the selected cry protein are
identified later by ELISA analysis. The progeny seed of these
events can be subsequently tested for power protection by
susceptible insects.
EXAMPLE 25
Modification of bacterial genes for expression in plants
Many wild-type genes that encode crystal proteins
Bacteria are known to express poorly in plants as a full-length gene or a truncated gene. Typically, the G + C content of a
cry gene is low (37%) and often contains many regions rich in A + T,
potential polyadenylation sites and numerous ATTTA sequences. He
Table 26 shows a list of potential polyadenylation sequences that are
they should avoid when preparing the "planted" gene construct.
TABLE 26 List of potential polyadenylation signal sequences
AATAAA * AAGCAT AATAAT * ATTAAT AACCAA ATACAT
ATATAA AAAATA
AATCAA ATTAAA ** ATACTA AATTAA ** ATAAAA AATACA ** ATGAAA CATAAA * * indicates a polyadenylation site of a potendal major plant. ** indicates a potential minor animal polyadenylate site. All others are polyadenylation sites of smaller plants. The regions for mutagenesis can be selected as follows. All regions of the DNA sequence of the cry gene containing five or more consecutive base pairs that were A or T were identified. These were classified in terms of length and highest percentage of A + T in the surrounding sequence in a region. of 20-30 base pairs. The DNA is analyzed for regions that may contain polyadenylation sites or ATTTA sequences. The oligonucleotides are then designed to maximize the removal of consecutive A + T regions containing one or more polyadenylation sites or ATTTA sequences. Two potential plant polyadenylate sites have also been shown to be more critical based on published reports. Codons are selected that increase the G + C content, but do not generate restriction sites for enzymes useful for cloning and assembling the modified gene (e.g., ßamHI, BglII, Sacl, Ncol, EcoRV, etc.). Also, codons containing TA or GC doublets that have been reported to be codons not frequently found in plants are avoided. Although the CaMV35S promoter is generally a high-level constitutive promoter in most plant tissues, the level of expression of the genes that activate the CaMV35S promoter is low in floral tissue in relation to the levels seen in leaf tissue. Because the economically important targets damaged by some insects are floral parts or derived from floral parts (for example, cotton pods and capsules, tobacco shoots, tomato shoots and tomato fruit), it is often advantageous to increase the expression of the crystal proteins in these tissues over those obtained with the CaMV35S promoter. The 35S promoter of the scopolaria mozaico virus (FMV) is analogous to the CaMV35S promoter. This promoter has been isolated and genetically manipulated in a plant transformation vector. In relation to the CaMV promoter, the FMV 35S promoter is highly expressed in the floral tissue, while still providing similar high levels of gene expression in other tissues such as leaves. It can be considered a plant transformation vector in which one or more genes encoding full length native or planted d-endotoxin is driven by the FMV 35S promoter. For example, tobacco plants can be transformed with a vector of this type and compared for expression of the crystal protein (s) by Western blot or ELISA immunoassay in foliar and / or floral tissue. The FMV promoter has been used to produce relatively high levels of crystal protein in floral tissue compared to the CaMV promoter.
EXAMPLE 26 Expression of native or planted cry genes with ssRUBISCO promoters and transient chloroplast peptides
The genes in plants that encode the small subunit of
RUBISCO (SSU) are often highly expressed, are regulated by light and sometimes show tissue specificity. These expression properties are largely due to the promoter sequences of these genes. It has been possible to use SSU promoters to express heterologous genes in transformed plants. Typically, a plant will contain multiple SSU genes, and the levels of expression and tissue stiffness of different SSU genes will be different. The SSU proteins are encoded in the nucleus and synthesized in the cytoplasm as precursors containing an NH2-terminal extension known as the doroplast transition peptide (CTP). CTP directs the precursor to the chloroplast and promotes the absorption of SSU protein in the doroplast. In this process, the CTP is cut off from the SSU protein. These CTP sequences have been used to direct heterologous proteins in doroplasts of transformed plants. The SSU promoters could have several advantages for the expression of heterologous genes in plants. Some SSU promoters are highly expressed and could give rise to expression levels also higher or higher than those observed with the CaMV35S promoter. The tissue distribution of the expression of SSU promoters is different from that of the CaMV35S promoter, so that to control some of the insect pests, it may be advantageous to direct the expression of crystal proteins to those cells in which SSU is expressed more highly For example, although it is relatively constitutive, in the promoter sheet CaMV35S is more highly expressed in vascular tissue than in some other parts of the leaf, whereas most SSU promoters are more highly expressed in leaf mesophilic cells. Some SSU promoters are also more highly tissue specific, so it might be possible to use an SU promoter to express the protein of the present invention only in a subset of plant tissues, if for example the expression of said protein in certain cells were found. deleterious to those cells. For example, to control the Colorado potato beetle in potato, it may be advantageous to use SSU promoters to direct the expression of crystal protein to the leaves but not to the edible tubers. The use of SSU CTP sequences to locate crystal proteins for chloroplasts could also be advantageous. The localization of crystal proteins from B. thuringiensis to the doroplast could protect these against proteases found in the cytoplasm. This could stabilize the proteins and lead to higher levels of accumulation of active toxin. The cry genes containing CTP can be used in combination with the SSU promoter or with other promoters such as CaMV35S.
EXAMPLE 27 Direction of d-endotoxin polypeptides to the extracellular space or vacuoles using signal peptides
The B. thuringiensis d-endotoxin polypeptides described herein can be located primarily towards the dtoplasma of a transformed plant cell, and this cytoplasmic localization can result in plants that are resistant to insecticides. However, in some embodiments, it may be advantageous to direct the localization or production of the B. thuringiensis peptide (s) to one or more compartments of a plant, or to particular types of plant cells. The localization of ß proteins. thuringiensis in compartments other than the cytoplasm may result in less exposure of B. thuringiensis proteins to cytoplasmic proteases that lead to increased accumulation of the protein producing increased insecticidal activity. The extracellular localization could lead to more efficient exposure of certain insects to B. thuringiensis proteins leading to greater efficacy. If a B. thuringiensis protein were found to be deleterious to the function of the plant cell, then the location to a non-cytoplasmic compartment could protect these cells against protein toxicity. In plants as well as in other eukaryotes, proteins that are intended to be localized either extracellularly or in several specific compartments are typically synthesized with an NH2-terminal amino acid extension known as the signal peptide. This signal peptide directs the protein to enter the compartmentalization pathways, and is typically cut off from the mature protein as an early step in the compartmentali / ration. For an extracellular protein, the secretory pathway typically involves the insertion of co -duction within the endoplasmic reticulum, at which point the segmentation of the signal peptide occurs. The mature protein then passes through the Golgi apparatus into vesicles that fuse with the plasma membrane, thereby releasing the protein into the extracellular space. The proteins destined for other compartments follow a similar route. For example, proteins that are destined for the endoplasmic reticulum or the Golgi apparatus follow this scheme, but are specifically retained in the appropriate compartment. In plants, some proteins are also directed to the vacuole, another membrane attached to the compartment in the dtoplasma of many plant cells. Proteins directed to vacuoles diverge from the anterior pathway in the Golgi apparatus where they enter vesicles that fuse with the vacuole. A common feature of this protein directron is the signal peptide that initiates the compartmentalization process. The fusion of a signal peptide to a protein will in many cases lead to directing that protein to the endoplasmic reticulum. The timing of this step may depend on the sequence of the same mature protein as well. The signals that direct a protein to a specific compartment instead of to an extracellular space have not been clearly defined. It appears that many of the signals that direct the protein to specific compartments are contained within the amino acid sequence of the mature protein. This has been shown for some proteins directed to vacuoles, but it is not yet possible to define these sequences with prediction. It appears that secretion into the extracellular space is the "default" pathway for a protein that contains a signal sequence but no other compartmentalization signals. Therefore, a strategy to direct B. thuringiensis proteins towards the exterior of the dtoplasma is to fuse the genes for ß genes. synthetic thuringiensis to DNA sequences encoding signal peptides of known plants. These fusion genes will give rise to B. thuringiensis proteins that enter the secretory pathway, and lead to extracellular sequestration or direction towards the vacuole or other compartments. Signal sequences for several plant genes have been described. One such sequence is for the PR1b protein related to tobacco pathogenesis described above (Comelissen et al., 1986). The PR1b protein is normally localized to the extracellular space. Another type of signal peptide is contained in legume seed storage proteins. These proteins are located towards the protein body of the seeds, which is a compartment in the form of a vacuole found in the seeds. A signal peptide DNA sequence for the β subunit of the 7S storage protein of common bean (Phaseolus vulgaris) PvuB has been described (Doyle et al., 1986). Based on these published sequences, the genes can be synthesized chemically using oligonucleotides that encode the signal peptides for PR1b and PvuB. In some cases, to achieve secretion or compartmentalization of heterologous proteins, it may be necessary to include some amino acid sequence beyond the normal cut-off site of the signal peptide. This may be necessary to ensure proper cleavage of the signal peptide.
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He Arg Thr Gln He Thr Glu Glu Leu Lys Val Glu Tyr Ser Ser Glu 275 280 285 Asn Lys Glu Met Arg Lys Tyr Lys Gln Ser Phe Asp Val Asp Asn Leu 290 295 300 Asn Tyr Asp Glu Wing Leu Asn Wing Val Gly Phe He Val Glu Thr Ser 305 310 315 320
Phe Glu Leu Tyr Arg Met Asn Gly Asn Val Leu He Thr Ser He Lys 325 330 335 Thr Thr Asn Lys Asp Thr Tyr Asn Thr Val Thr Tyr Pro Asn His Lys 340 345 350 Glu Val Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Thr Ala 355 360 365 Leu Thr Gly He Ser Lys Glu Arg Leu Gln Asn Leu Lys Asn Asn Trp 370 375 380 Lys Lys Arg 385
< 210 > 3 < 211 > 396 < 212 > DNA < 213 > Bacillus thuringiensis < 400 > 3 atgtcagcac gtgaagtaca ataaatcata cattgaaata caggtcatac cttacaaatg 60 ctagacttgc gataaaagaa acatggtgaa tggattatta cacccgtgaa tgttccaaat 120 atttatttca aattcttctg agcaggttct gatggagttt tgacaggagt agaaggaata 180 ataatttata ctataaatgg agaaatagaa attaccttac tccttatgca attttgacaa 240 ggttctaata aatattctgg acgttctagt gatgatgatt ataaagttat aactgaagca 300 agagcagaac atagagctaa taatcatgat catgtaacat atacagttca aagaaacata 360 tcacgatata ccaataaatt atgttctaat aactcc 396
< 210 > 4 < 211 > 132 < 212 > PRT < 213 > Bacillus thuringiensis < 400 > 4 Met Ser Wing Arg Glu Val His He Glu He He Asn His Thr Gly His 1 5 10 15 Thr Leu Gln Met Asp Lys Arg Thr Arg Leu Wing His Gly Glu Trp He 20 25 30 He Thr Pro Val Asn Val Pro Asn Asn Ser Being Asp Leu Phe Gln Wing 35 40 45 Gly Being Asp Gly Val Leu Thr Gly Val Glu Gly He He He Tyr Thr 50 55 60 He Asn Gly Glu He Glu He Thr Leu His Phe Asp Asn Pro Tyr Ala 65 70 75 80
Giy Ser Asn Lys Tyr Ser Gly Arg Ser As Asp Asp Asp Tyr Lys Val 85 90 95 He Thr Glu Wing Arg Wing Glu His Arg Wing Asn Asn His Asp His Val 100 105 110 Thr Tyr Thr Val Gln Arg Asn He Ser Arg Tyr Thr Asn Lys Leu Cys 115 120 125 Ser Asn Asn Ser 130
< 210 > 5 < 211 > 402 < 212 > DNA < 213 > Bacillus thuringiensis < 400 > 5 aaggaacata catataaaaa ggggaaacct accgaaaaat attatcattt ttttaagtta 60 aatacataca ttaatttagt atctgtaaaa acattaattt tatggaggtt gatatttatg 120 tcagctcgcg aagtacacat tgaaataaac aataaaacac gtcatacatt acaattagag 180 gataaaacta aacttagcgg aggtagatgg cgaacatcac ctacaaatgt tgctcgtgat 240 acaattaaaa catttgtagc agaatcacat ggttttatga caggagtaga aggtattata 300 tattttagtg taaacggaga cgcagaaatt agtttacatt ttgacaatcc ttatatagtt 360 ctaataaatg tgatggttct tctgatagac ctgaatatga 402 ag
< 210 > 6 < 211 > 119 < 212 > PRT < 213 > Bacillus thuringiensis < 400 > 6 Met Being Wing Arg Glu Val His He Glu He Asn Asn Lys Thr Arg His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Ser Gly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Wing Arg Asp Thr He Lys Thr Phe Val Wing 35 40 45 Glu Ser His Gly Phe Met Thr Gly Val Glu Gly He He Tyr Phe Ser 50 55 60 Val Asn Gly Asp Ala Glu He Ser Leu His Phe Asp Asn Pro Tyr He 65 70 75 80 Gly Ser Asn Lys Cys Asp Gly Ser Ser Asp Lys Pro Glu Tyr Glu Val 85 90 95 He Thr Gln Ser Gly Ser Gly Asp Lys Ser His Val Thr Tyr Thr He 100 105 110 Gln Thr Val Ser Leu Arg Leu 115 <; 210 > 7 < 211 > 1155 < 212 > DNA < 213 > Bacillus thuringiensis < 400 > 7 atgttagata ctaataaagt ttatgaaata agcaatcttg ctaatggatt atatacatca 60 gtcttgatga acttatttaa agtttaatga ttcaggtgtt gtaaaaagga tgaagatatt 120 atttaaaatg gatgattaca cctattgata gtttttattt tattattaca ataatcaata 180 agctatggag ctaataattg taaagtttgg aatgttaaaa atgataaaat aaatgtttca 240 caacaaactc acttattctt tgtacaaaaa tggcaaataa aagctaaaga ttcttcatat 300 ataatacaaa gtgataatgg aaaggtctta acagcaggag taggtgaatc tcttggaata 360 gtacgcctaa ctgatgaatt tccagagaat tctaaccaac aatggaattt aactcctgta 420 caaacaattc aactcccaca atagatgaaa aaaacctaaa aattaaaaga tcatcctgaa 480 tattcagaaa ccggaaatat aaatcctaaa acaactcctc aattaatggg atggacatta 540 ttatggtaaa gtaccttgta tgattcagga atagataaaa acactcaaat taaaactact 600 tttttaaaaa ccatattata atataaatac tggaatctag caaaaggaag taatgtatct 660 ttacttccac atcaaaaaag atcatatgat tatgaatggg gtacagaaaa aaatcaaaaa 720 acatctatta ttaatacagt aggattgcaa attaatatag attcaggaat gaaatttgaa 780 gtaccagaag taggaggagg tacagaagac ataaaaacac aattaactga agaattaaaa 840 gttgaatata gcactgaa ac caaaataatg acgaaatatc aagaacactc agagatagat 900 atcaaccaat aatccaacta ggacttctta gaattctata 'tttatacttc tttagaatta 960 acggtacaga tatcgatata aattaagata atggacatag aaacttcaga tcatgatact 1020 tacactctta cttcttatcc aaatcataaa gaagcattat tacttctcac aaaccattcg 1080 tatgaagaag tagaagaaat cctaagcata aacaaaaata attgaaaaaa cacttataaa cattatttta aaaaa 1140 1155
< 210 > 8 < 211 > 385 < 212 > PRT < 213 > Bacillus thuringiensis < 400 > 8 Met Leu Asp Thr Asn Lys Val Tyr Glu He Ser Asn Leu Wing Asn Gly 1 5 10 15 Leu Tyr Thr Ser Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met Ser Lys Lys Asp Glu Asp He Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45
Leu Phe Pro He Asp Asn Asn Gln Tyr He He Thr Ser Tyr Gly Ala
50 55 60 Asn Asn Cys Lys Val Trp Asn Val Lys Aßn Asp Lys He Asn Val Ser
65 70 75 80
Thr Tyr Ser Ser Thr Asn Ser Val Gln Lys Trp Gln He Lys Wing Lys 85 90 95
Asp Being Ser Tyr He He Gln Ser Asp Asn Gly Lys Val Leu Thr Ala
100 105 110 Gly Val Gly Glu Ser Leu Gly He Val Arg Leu Thr Asp Glu Phe Pro
"115 120 125 Glu Asn Ser Asn Gln Gln Trp Asn Leu Thr Pro Val Gln Thr He Gln
130 135, 140 Leu Pro Gln Lys Pro Lys He Asp Glu Lys Leu Lys Asp His Pro Glu
145 150 155 160
Tyr Ser Glu Thr Gly Asn He Asn Pro Lys Thr Thr Pro Gln Leu Met 165 170 175
Gly Trp Thr Leu Val Pro Cys He Met Val Asn Asp Ser Gly He Asp 180 185 190 Lys Asn Thr Gln He Lys Thr Thr Pro Tyr Tyr He Phe Lys Lys Tyr 195 200 205 Lys Tyr Trp Asn Leu Wing Lys Gly Ser Asn Val Ser Leu Leu Pro His 210 215 220 Gln Lys Arg Ser Tyr Asp Tyr Glu Trp Gly Thr Glu Lys Asn Gln Lys 225 230 235 240
Thr Ser He He Asn Thr Val Gly Leu Gln He Asn He Asp Ser Gly 245 250 255
Met Lys Phe Glu Val Pro Glu Val Gly Gly Gly Thr Glu Asp He Lys 260 265 270 Thr Gln Leu Thr Glu Glu Leu Lys Val Glu Tyr Ser Thr Glu Thr Lys 275 280 285 He Met Thr Lys Tyr Gln Glu His Ser Glu He Asp Asn Pro Thr Asn 290 295 300 Gln Pro Met Asn Be He Gly Leu Leu He Tyr Thr Ser Leu Glu Leu
305 310 315 320
Tyr Arg Tyr Asn Gly Thr Glu He Lys He Met Asp He Glu Thr Ser 325 330 335 Asp His Asp Thr Tyr Thr Leu Thr Ser Tyr Pro Asn His Lys Glu Wing 340 345 350 Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu He Thr 355 360 365 Lys He Pro Lys His Thr Leu He Lys Leu Lys Lys His Tyr Phe Lys 370 375 380 Lys 385
< 210 > 9 < 211 > 372 < 212 > DNA < 213 > Bacillus thuringiensis < 400 > 9 gtgaagtaca atgtcagcac cattaatgta aataataaga attacaatta caggtcatac 60 caaaacttga gaagataaaa tggtggtaga tggcgaacat cacctacaaa tgttgctaat 120 gatcaaatta aaacatttgt agcagaatca catggtttta tgacaggtac agaaggtcat 180 atatattata gtataaatgg agaagcagaa attagtttat attttgataa tccttattca 240 ggttctaata aatatgatgg gcattccaat aaacctcaat atgaagttac tacccaagga 300 atcaatctca ggatcaggaa tgttacgtat actattcaaa ctgcatcttc acgatatggg aataactcat 360 aa 372
< 210 > 10 < 211 > 123 < 212 > PRT < 213 > Bacillus thuringiensis < 400 > 10 Met Ser Wing Arg Glu Val His He Asn Val Asn Asn Lys Thr Gly His 1 5 10 15 T r Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly aly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Asn Asp Gln He Lys Thr Phe Val Wing 35 40 45 Glu Ser His Gly Phe Met Thr Gly Thr Glu Gly His He Tyr Tyr Ser 50 55 60 He Asn Gly Glu Wing Glu He Ser Leu Tyr Phe Asp Asn Pro Tyr Ser 65 70 75 80
Gly Being Asn Lys Tyr Asp Gly His Being Asn Lys Pro Gln Tyr Glu Val 85 90 95 Thr Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr He 100 Gln Thr Wing Being Ser Arg Tyr Gly Asn Asn Ser 115 120 < 210 > 11 < 211 > 1152 < 212 > ADH < 213 > Bacillus thuringiensis < 400 > 11 atgttagata ctaataaagt ttatgaaata agtaatcatg ctaatggact atatgcagca 60 gtttagatga acttatttaa ttcaggtgtt agtttaatga ataaaaatga tgatgatatt 120 gatgattata acttaaaatg gtttttattt cctattgatg tattattaca atgatcaata 180 agctatgcag caaataattg taaagtttgg aatgttaata atgataaaat aaatgtttcg 240 acttattctt caacaaattc aatacaaaaa tggcaaataa aagctaatgg ttcttcatat 300 gtaatacaaa gtgataatgg aaaagtctta acagcaggaa ccggtcaagc tcttggattg 360 atacgtttaa ctgatgaatc ctcaaataat cccaatcaac aatggaattt aacttctgta 420 caaacaattc aacttccaca atagatacaa aaaacctata ttatcccaaa aattaaaaga 480 tattcaccaa ctggaaatat agataatgga acatctcctc aattaatggg atggacatta 540 ttatggtaaa gtaccttgta tgatccaaat atagataaaa atactcaaat taaaactact 600 ttttaaaaaa ccatattata atatcaatat tggcaacgag cagtaggaag taatgtagct 660 ttacgtccac atgaaaaaaa atcatatact tatgaatggg gaacagaaat agatcaaaaa 720
acaacaatca taaatacatt aggatttcaa atcaatatag attcaggaat gaaatttgat 780 ataccagaag taggtggagg tacagatgaa ataaaaacac aactaaatga agaattaaaa 840 atagaatata gtcgtgaaac taaaataatg gaaaaatatc aagaacaatc tgaaatagat 900 aatccaactg atcaaccaat gaattctata ggatttctta ctattacttc tttagaatta 960 atggctcaga tatagatata aattcgtata atgcaaattc aaacctcaga taatgatact 1020 tataatgtta cttcttatcc agatcatcaa caagctttat tacttcttac aaatcattca 1080 tatgaagaag tagaagaaat aacaaatatt cctaaaagta attaaaaaaa cactaaaaaa 1140 tattattttt aa 1152
< 210 > 12 < 211 > 383 < 212 > PRT < 213 > Bacillus thuringiensis < 400 > 12 Met Leu Asp Thr Asn Lys Val Tyr Glu He Ser Asn His Wing Asn Gly 1 5 10 15 Leu Tyr Wing Wing Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met Asn Lys Asn Asp Asp Asp Asp Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro He Asp Asp Asp Gln Tyr He He Thr Ser Tyr Ala Wing 50 55 60 Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys He Asn Val Ser 65 70 75 80
Thr Tyr Being Ser Thr Asn Being He Gln Lys Trp Gln He Lys Wing Asn 85 90 95
Gly Ser Ser Tyr Val He Gln Ser Asp Asn Gly Lys Val Leu Thr Wing 100 105 110 Gly Thr Gly sln Wing Leu Gly Leu He Arg Leu Thr Asp Glu Ser 115 115 125 Asn Asn Pro Asn Gln sln Trp Asn Leu Thr Ser Val sln Thr He Gln 130 135 140 Leu Pro Gln Lys Pro He He Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155 160
Tyr Ser Pro Thr sly Asn He Asp Asn sly Thr Ser Pro Gln Leu Met 165 170 175
Gly Trp Thr Leu Val Pro Cys He Met Val Asn Asp Pro Asn He Asp 180 185 190 Lys Asn Thr sln He Lys Thr Thr Pro Tyr Tyr He Leu Lys Lys Tyr 195 200 205 Gln Tyr Trp Gln Arg Wing Val Gly Ser Asn Val Wing Leu Arg Pro His
210 215 220 Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu He Asp Gln Lys 225 230 235 240
Thr Thr He He Asn Thr Leu Gly Phe Gln He Asn He Asp Ser Gly 245 250 255
Met Lys Phe Asp He Pro Glu Val Gly Gly Gly Thr Asp Glu He Lys 260 265 270 Thr Gln Leu Asn Glu Glu Leu Lys He alu Tyr Ser Arg Glu Thr Lys 275 280 285 He Met slu Lys Tyr Gln Glu Gln Ser Glu He Asp Asn Pro Thr Asp 290 295 300 Gln Pro Met Asn Be He Gly Phe Leu Thr He Thr Ser Leu Glu Leu 305 310 315 320
Tyr Arg Tyr Asn Gly Ser Glu He Arg He Met Gln He Gln Thr Ser 325 330 335 Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asp His Gln Gln Wing 340 345 350 Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val slu slu He Thr 355 360 365 Asn He Pro Lys Ser Thr Leu Lys Lys Leu Lys Lys Tyr Tyr Phe 370 375 380
< 210 > 13 < 211 > 1952 < 212 > DNA < 213 > Bacillus thuringiensis < 400 > 13 aaaatctttt acatatattt gttaggaagc atgaaaataa aaatagatta tatagaagga 60 tgaatgtaaa gtgaaataga tcacggtatg tcttgtggat gtggttgcca gcaaggtaaa 120 acgattatca gaagaatata tgtgtcaaat gaatataggg acgaaaatcc tagtacaact 180 tgtaattctc aacaaggtaa ttatgagtac gaacaaagta aagaaacata taacaatgat 240 tatcaatcat atgaatacaa tcaacaaaat tataatactt gcggaaggaa tcaaggaacg 300 atggaacagg agtcgatgca aaaggatagg aattgggaga atgcaaatta tagtggatat 360 gtccaaatca gatggatgta gttgaatgca ctaaatttac cagatgaaag tactaggttt 420 caaaaaataa ctaatgtaaa tactcgtgat agtcatcgtg ttttagacat gatggacgtt 480 ctaggcttga cctagtggaa cctcctattt tactcgtgta cgaatttaca gtagtcaaac 540 aatacggtta gtaatgaatt agtttccacg aatcatgata cacaattttt aattttttat 600 caaacagatg atagttcatt tattattggg aatcgaggaa atggtcgagt tttagatgtt 660 tttcctagta atagaaatgg ttatacaata gtttcaaatg tgtatagtgg ttcaaggaat 720 aatcagcgtt ttcgtatgaa taaagcatct aataatcaat ttagtttaca aaccattttt 780 aaggacagag taaatatatg tggtcatatt cacaatttta acgcgataat tacagctact 840 actttaggtg agaatga tag taatgcttta tttcaagtac aatcttccac aaatataaca 900 ctacctacat taccacctag gacaacatta gaaccaccaa gagcattaac aaatataaat 960 gatacaggtg attctccagc gcaagcacct cgagcggtag aaggaagtgt tcttatcccc 1020 gcaatagcgg taaatgatgt cattccggta gcgcaaagaa tgcaagaaag tccgtattat 1080 ataatacata gtgttaacat ttggcataga gttatttcag caatactacc aggtagtggg 1140 caaactacaa ggttcgatgt aaacttacca ggtcctaatc aaagtacaat ggtagatgta 1200 ttagatacag caattactgc agattttaga ttacaatttg ttggaagtgg acgaacaaat 1260 gtatttcaac aacaaattag aaatggatta aatatattaa attctacaac gtctcatcgt 1320 ttaggagatg aaacacgtaa ttgggatttt acaaatagag gtgctcaagg aagattagcg 1380 aagcacatga ttttttgtaa gtttgtatta acacgtgcga atggaacacg agtaagtgat 1440 ccatgggtgg cattagatcc gaatgttaca gctgctcaaa catttggagg agtattactt 1500 acattagaaa aagaaaaaat agtatgtgca agtaatagtt ataatttatc agtatggaaa 1560 aaataaagaa acaccaatgg tatacaaaaa tggaaaaatt tacaaaacca atgaatggaa 1620 aactacaaat aaacaaaatg attctgttga aaaacaaaaa caagtttgaa ttggtttgca 1680 aaatatggtt ccggtgcaaa aattccaaaa tgattgaaaa ggatttatca aacttgtcca 1740 tactggtact actacttaaa aaaggtgtgt gattagtatg ggaccagaaa atttatttaa 1800
gtggaaacat tatcaaccag atattatttt atcaac gta cgttggtacc tacggtacaa 1860 cttaagtttt cgtgatttgg tagaaatgat ggaggaacga ggnttatctt tggctcatac 1920 aaccattatg cngttgggtt catcaatatg gt 1952
< 210 > 14 < 211 > 520 < 212 > PRT < 213 > Bacillus thuringiensis < 400 > 14 Met Asn Val Asn His Gly Met Ser Cys Gly Cys Gly Cys Gln Gln Gly 1 5 10 15 Lys Glu Glu Tyr Asn Asp Tyr His Val Ser Asn slu Tyr Arg Asp Glu 20 25 30 Asn Pro Ser Thr Thr Cys Asn Ser sln Gln Gly Asn Tyr Glu Tyr Glu 35 40 45 Gln Ser Lys slu Thr Tyr Asn Asn Asp Tyr Oln Ser Tyr Glu Tyr Asn 50 55 60 Gln Oln Asn Tyr Asn Thr Cys Gly Arg Asn Gln sly Thr Met alu aln 65 70 75 80 slu Ser Met Gln Lys Asp Arg Asn Trp Glu Asn Wing Asn Tyr Ser Gly 85 90 95 Tyr Asp Gly Cys Ser Pro Asn Gln Leu Asn Wing Leu Asn Leu Pro Asp 100 105 110 Glu Be Thr Arg Phe Gln Lys He Thr Asn Val Asn Thr Arg Asp Ser 115 120 125 is Arg Val Leu Asp Met Met Asp Val Pro Ser Gly Thr Arg Leu Asp 130 135 140 Thr Arg Val Pro Pro He Cys Ser Gln Thr Glu Phe Thr Asn Thr Val 145 150 155 160 Ser Asn Glu Leu Val Being Thr Asn His Asp Thr Gln Phe Leu He Phe 165 170 175 Tyr Gln Thr Asp Asp Being Ser Phe He Gly Asn Arg Gly Asn Gly 180 185 190 Arg Val Leu Asp Val Phe Pro Ser Asn Arg Asn Gly Tyr Thr He Val 195 200 205
Ser Asn Val Tyr Ser Gly Ser Arg Asn Asn Gln Arg Phe Arg Met Asn 210 215 220 Lys Wing Being Asn Asn Gln Phe Ser Leu Gln Thr He Phe Lys Asp Arg 225 230 235 240
Val Asn lie Cys sly His He His Asn Phe Asn Ala He He Thr Ala 245 250 255
Thr Thr Leu Gly Glu Asn Asp Ser Asn Wing Leu Phe Gln Val Gln Ser 260 265 270 Being Thr Asn He Thr Leu Pro Thr Leu Pro Pro Arg Thr Thu Leu Glu 275 280 285 Pro Pro Arg Ala Leu Thr Asn He Asn Asp Thr Gly Asp Ser Pro Ala 290 295 300 Gln Ala Pro Arg Ala Val Glu sly Ser Val Leu He Pro Ala He Ala 305 310 315 320
Val Asn Asp Val He Pro Val Wing Oln Arg Met Oln Glu Ser Pro Tyr 325 330 335 Tyr Val Leu Thr Tyr Asn Thr Tyr Trp His Arg Val He Ser Wing He 340 345 350 Leu Pro Gly Ser Gly Gln Thr Thr Arg Phe Aep Val Asn Leu Pro Gly 355 360 365 Pro Asn Gln Be Thr Met Val Asp Val Leu Asp Thr Wing He Thr Wing 370 375 380 Asp Phe Arg Leu Gln Phe Val Gly Ser Gly Arg Thr Asn Val Phe Gln 385 390 395 400 sln Gln He Arg Asn Gly Leu Asn He Leu Asn Ser Thr Thr Ser His 405 410 415
Arg Leu Gly Asp Glu Thr Arg Asn Trp Asp Phe Thr Asn Arg Gly Wing 420 425 430 Gln Gly Arg Leu Wing Phe Phe Val Lys Wing His Glu Phe Val Leu Thr 435 440 445 Arg Wing Asn Gly Thr Arg Val Ser Asp Pro Trp Val Wing Leu Asp Pro 450 455 460 Asn Val Thr Wing Wing Gln Thr Phe Oly Gly Val Leu Leu Thr Leu Glu 465 470 475 480
Lys Glu Lys He Val Cys Wing Ser Asn Ser Tyr Asn Leu Ser Val Trp 485 490 495 Lys Thr Pro Met Glu He Lys Asn Gly Lys He Tyr Thr Lys Asn Glu 500 505 510 Trp Asn Thr Lys Pro Asn Tyr Lys 515 520 < 210 > 15 < 211 > 1024 < 212 > DNA < 213 > Bacillus thuringiensis < 400 > 15 tttattaata agtgcgagca caatagaaat gctcacatat gtaacaacct ttagtatatt 60 taaatataag gagttgtata acttgagtat cttaaatctt caagacttat cacaaaaata 120 tatgactgca gctttaaata aaaaaaagta agataaatcc ggtactttcc attttgagga 180 accaatagta ctttcagaat cttctactcc cacacgttct gaaattgatg cccctcttaa 240 tgttatgttt cacgcttcac aagatcttga taatagaagg ggcactagtg atttaaaaca 300 aactgtttct ttttctcaaa ctcaaataaa tactgttgaa accaaaacta ctgatggtgt 360 taaaacaact aaagaacata catttagtgg tacattagaa ctaaagatta aatatgcaat 420 gtttgattta gggggagtgt caggcacata tcaatataaa aaaagtactg aaaacgatat 480 tagttcagaa aagagtaaat cgaagtcaga ttctcaaact tggtcaatat caagtgaata 540 cctggagtaa tacagttaaa aagaaactct tcatttttat attgtaggaa taaaaaaccg 600 aagtgccttt taaatatttt tgctgaattt aaactattga caaggtacta taatgtatcc 660 aatgttatgg cttatcaaga gtttataagt caagatgatg aacatataag agcatgtatg 720 aattggctaa aaagcaagta tcctgatcat ctttcaggat atacagctcc aaaggaatta 780 caagtaaagg aaagcaaata atcagtagaa tttagaggta cagcta? agc taaaataaat 840 acaggagtaa aatgtct tgt tgtagttaat ggaaaaaatt caataactgg aaaaacttat 900 tcttatatac atcctaaaac aatgttagct gatggaacca ttgaatattt agaaagtgag 960 atagatcttt tagaaagtga gatagatctt ttaactacaa gtagtatttt agtttaaaca
1020 atta 1024 < 210 > 16 < 211 > 310 < 212 > PRT < 13 > Bacillus thuringiensis < 400 > 16 Met Ser Le Le Asn Leu Gln Asp Leu Ser Gln Lys Tyr Met Thr Ala 1 5 10 15 Ala Leu Asn Lys He Asn Pro Lys Lys Val Gly Thr Phe His Phe Glu 20 25 30 Glu Pro He Val Leu Ser Glu Ser Ser Thr Pro Thr Arg Ser Glu He 35 40 45 Asp Wing Pro Leu Asn Val Met Phe His Wing Ser Gln Asp Leu Asp Asn 50 55 60 Arg Arg Gly Thr Ser Asp Leu Lys Gln Thr Val Ser Phe Ser Gln Thr 65 70 75 80
Gln He Asn Thr Val Glu Thr Lys Thr Thr Asp Gly Val Lys Thr Thr 85 90 95 Lys Glu His Thr Phe Ser Gly Thr Leu Glu Leu Lys He Lys Tyr Ala 100 105 110 Met Phe Asp Leu Gly Gly Val Ser Gly Thr Tyr Gln Tyr Lys Lys Ser 115 120 125 Thr Glu Asn Asp Be Ser Glu Lys Ser Lys Ser Lys Ser Asp Ser
130 135 140 Gln Thr Trp Ser Be Ser Olu Tyr Thr Val Lys Pro Gly Val Lys
145 150 155 160
Glu Thr Leu Asp Phe Tyr He Val Gly He Lys Thr Glu Val Pro Leu 165 170 175
Asn He Phe Wing Glu Phe Gln Gly Thr Lys Thr He Asp Asn Val Ser 180 185 190 Asn Val Met Wing Tyr Gln Glu Phe He Ser Gln Asp Asp Glu His He 195 200 205 Arg Wing Cys Met Lys Wing Ser Lys Leu Wing Asn Pro Asp His Leu Ser 210 215 220 Gly Tyr Thr Wing Pro Lys Glu Leu Lys Wing Asn Thr Ser Lys Gly Ser 225 230 235 240
Val Glu Phe Arg Gly Thr Ala He Ala Lys He Asn Thr Gly Val Lys 245 250 255
Cys Leu Val Val Val Asn Gly Lys Asn Ser lie Thr aly Lys Thr Tyr 260 265 270 Ser Tyr He His Pro Lys Thr Met Leu Wing Asp aly Thr He Glu Tyr 275 280 285 Leu Glu Ser Glu He Asp Leu Leu Glu Ser Glu He Asp Leu Leu Thr 290 295 300 Thr Ser Ser He Leu Val 305 310
< 210 > 17 < 211 > 3607 < 212 > DNA < 213 > Bacillus thuringiensis < 400 > 17
gaattcttaa aaaaaataag gttttttatg gaaaattgtc ggaaagctgt atgttttgtg 60 aatagataag tatatttttt aaaattaatt tatataaaat atataatatc aacgagtgaa 120 tatatagcat tgtctaatta tagataaaag agcttatttt tttcacatat aaactactta 180 ttacgtatag tacagtgaga caatttttaa cagttgtttc atataaccct ccattcattt 240 aaaaaacaaa tataagagca cacgcttatg aaaaggaata tttgtttttc atttattatt 300 tatttcaaga aaattgaaat gtgtatatat gattaagcaa catttggagt tgtttttgat 360 ttcaaattgc tctcctctta cggagtttaa aattcaaata aatttattga tgtatattac 420 tcttctgaag atgataatct taaatattac caattgataa aagttgaatc tcattttgta 480 ttagcaaaca caaactacct gagcgtggaa gttgatgaaa aaattaaaca atagtttagt 540 catttcaaag ataaagggct ggaacagcca cgttgatatg gttaaaatcg ctatctattg 600 catatatatt tttagtaaat aactttttat tattaaaaat ataattttat aaaggatgtg 660 actatcataa tttaagtttg atatattaga ttatgcagat tcttatttaa gagctgctat 720 taaaaaatat ggaggatacc caagttctag taaagctaga ttcttatcta ctccaaaaat 780 ttcagaacca gagtggtatt accctgctaa agaatctgtt aatgcatatg aaattggtaa 840 tcgtatccta acaatctggt atcattcttc tacatctcaa aattttaatg taccaattcg 900 ttatcctgtt tccactacta gttcaacaaa aactataaat ggttttaaaa cagataaaag 960 tatttctaaa aatttaaatc ttaacttagg gataaatgca aaaataccta atataaatat 1020 tcctggtggc tttgaaattg aagttaaacc tggagctgag gtttcaagaa atgttaaaac 1080 gtagacttta gaatcaaaca gtagtacttc tgaaaaaaca caaaatacaa atgacactcc 1140 atctgacaca actcaatctt tctcttgtcc tcctaacaca aaagcaacat atatagttat 1200 ttatttcggg ggagaaccta aagtagaagt tacagctgta acagatataa taggaaatgg 1260 atctggaata ggaacagatc ctactactgg tcaagaaaaa tcgcaaagaa atgttttagc 1320 aactttagat tacagtaaag aaggtcaagc tggtaaaaaa tatactatga tggtaactgc 1380 gcaactaaaa agatcaatta taatcctcca tacctggata ccaagagtcg aacaagatcg 1440 gcattaacta tagtcataat ttcatagtga ccttatagta aatttaaaag aagattttgc 1500 atatgaaata attgtaaaat ttgaaaattt atcttattcg acacttttta atgaagatct 1560 agattcgaca ctttatttat aaaatcataa tcttcttata gaaaaaacag ttggatcatt 1620 atttgaaact aatctacatg cagatatttt ttatgaacat attgaaagtg aattagaata 1680 aaaatatttt tttaaatatg ataactc cac ttatttaaaa tcacaaaagt tttaaacaaa 1740 attaacaaaa aaattaaatg gaggttgaaa atatgtcagc acgtgaagta cacattgaaa 1800 taataaatca tacaggtcat accttacaaa tggataaaag aactagactt gcacatggtg 1860 aatggattat tacacccgtg aatgttccaa ataattcttc tgatttattt caagcaggtt 1920 tttgacagga ctgatggagt taataattta gtagaaggaa tactataaat ggagaaatag 1980 aaattacctt acattttgac aatccttatg caggttctaa taaatattct ggacgttcta 2040 gtgatgatga ttataaagtt ataactgaag caagagcaga acatagagct aataatcatg 2100 atcatgtaac atatacagtt caaagaaaca taccaataaa tatcacgata ttatgttcta 2160 ataactccta aaatttattt taattattaa aaacaaagtt ctataaattt gaataaagaa 2220 atttgaaaaa ctttgttttt ggtgtgtgaa atcacaaaaa attatgatag aaactaataa 2280 ataagcaata gatatatgaa aagctaatgg attatatgca actacttatt taagttttga 2340 taattcaggt gttagtttat taaataaaaa tgaatctgat attaatgatt ataatttgaa 2400 atggttttta tttcctattg ataataatca gtatattatt acaagttatg gagtaaataa 2460 tggactgcta aaataaggtt aataaatgtt atggtaataa acaacatatt ccgcagaaaa 2520 caatggcaaa ttcagcacaa taagaaacag ttcttctgga tatataatag aaaataataa 2580 tgggaaaatt ttaacggcag gaacaggcca atcattaggt ttattatatt taactgatga 2640 aatacctgaa gattctaatc aacaatggaa tttaacttca atacaaacaa tttcacttcc 2700 ataattgata ttcacaacca caacattagt agattaccct aaatattcaa cgaccggtag 2760 tataaattat aatggtacag cacttcaatt aatgggatgg acactc atac catgtattat 2820 ggtatacgat aaaacgatag cttctacaca cactcaaatt acaacaaccc cttattatat 2880 tttgaaaaaa tatcaacgtt gggtacttgc aacaggaagt ggtctatctg tasctgcaca 2940
tgtcaaatca actttcgaat acgaatgggg aacagacaca gatcaaaaaa ccagtgtaat 3000 ggttttcaaa aaatacatta ttaatacaga tacaaaatta aaagctactg taccagaagt 3060 acaacagata aggtggaggt taagaacaca aatcactgaa gaacttaaag tagaatatag 3120 tagtgaaaat aaagaaatgc gaaaatataa gacgtagaca acaaagcttt acttaaatta 3180 tgatgaagca ctaaatgctg taggatttat tgttgaaact tcattcgaat tatatcgaat 3240 gtccttataa gaatggaaat caagtataaa aactacaaat aaagacacct ataatacagt 3300 tacttatcca aatcataaag aagttttatt acttcttaca aatcattctt atgaagaagt 3360 aacagcacta actggcattt ccaaagaaag acttcaaaat cttaaaaaca attggaaaaa 3420 aagataaaat atatatagag ttaaaagttc cgtaaggaac ggggagtgtt tttgagaaga 3480 agtcggtttt acactaaaaa ttaattttca cctaaaggca aagacaatcc ctcagaagcg 3540 tctagaagct tgtatagagc gtttaaaagt atgtttagat aaaatactag ggaaaagtag tgaattc 3600 3607
< 210 > 18 < 211 > 1026 < 212 > DNA < 213 > Bacillus thuringiensis < 400 > 18 atgtgtttaa gtttgactat cataaatata ttagattatg cagattctta tttaagagct 60 gctattaaaa aatatggagg atacccaagt tctagtaaag ctagattctt atctactcca 120 aaaatttcag aaccagagtg gtattaccct gctaaagaat ctgttaatgc atatgaaatt 180 ggtaaacaat ctggttcgta tcctaatcat tcttctacat ctcaaaattt taatgtacca 240 attcgttatc ctgtttccac tactagttca acaaaaacta taaatggttt taaaacagat 300 aaaagtattt ctaaaaattt aaatcttaac ttagggataa atgcaaaaat acctaatata 360 • aatattcctg gtggctttga aattgaagtt aaacctggag ctgaggtttc aagaaatgtt 420 aaacagtaga aaaacgaatc acttctgaaa ctttagtagt aaacacaaaa tacaaatgac 480 acacaactca actccatctg atctttctct tgtcctccta acacaaaagc aacatatata 540 tcgggggaga gttatttatt acctaaagta gaagttacag tataatagga ctgtaacaga 600 aatggatctg gaataggaac agatcctact actggtcaag aaaaatcgca aagaaatgtt 660 ttagcaactt tagattacag taaagaaggt caagctggta aaaaatatac tatgatggta 720 actgcagatc aattagcaac taaaatacct ggatataatc ctccaccaag agtcgaacaa 780 gatcgtagtc ataatgcatt aactattcat agtgacctta tagtaaattt aaaagaagat 840 tttgcatatg aa ataattgt aaaatttgaa aatttatctt attcgacact ttttaatgaa 900 gatctcttta tttatagatt cgacaaaaat cataatcttc ttatagaaaa aacagttgga 960 tcattatttg aaactaatct acatgcagat attttttatg aacatattga aagtgaatta 1020 gaataa 1026
< 210 > 19 < 211 > 341 < 212 > PRT < 213 > Bacillus thuringiensis < 400 > 19 Met Cys Leu Ser Leu Thr He He Asn He Leu Asp Tyr Wing Asp Ser 1 5 10 15
Tyr Leu Arg Wing Wing He Lys Lys Tyr Gly Gly Tyr Pro Being Ser 20 25 30 Lys Wing Arg Phe Leu Ser Thr Pro Lys He Ser Glu Pro Glu Trp Tyr 35 40 45 Tyr Pro Wing Lys Glu Ser Val Asn Wing Tyr Glu He Gly Lys Gln Ser 50 55 60
Gly Ser Tyr Pro Asn His Ser Ser Thr Ser Gln Asn Phe Asn Val Pro 65 70 75 80
He Arg Tyr Pro Val Ser Thr Thr Ser Ser Thr Lys Thr He Asn Gly 85 90 95
Phe Lys Thr Asp Lys Ser Be Lys Asn Leu Asn Leu Asn Leu Gly 100 105 110 He Asn Wing Lys He Pro Asn He Asn He Pro Gly Gly Phe Glu He 115 120 125 Glu Val Lys Pro Gly Wing Glu Val Ser Arg Asn Val Lys Thr Asn Gln 130 135 140 Thr Val Asp Phe Ser Ser Thr Ser Glu Lys Thr Gln Asn Thr Asn Asp 145 150 155 160
Thr Pro Ser Asp Thr Thr Sln Ser Phe Ser Cys Pro Pro Asn Thr Lys 165 170 175
Wing Thr Tyr He Val He Tyr Phe Gly Gly Glu Pro Lys Val Glu Val 180 185 190 Thr Wing Val Thr Asp He He Gly Asn Gly Ser Gly He Gly Thr Asp 195 200 205 Pro Thr Thr Gly Gln Olu Lys Ser Gln Arg Asn Val Leu Ala Thr Leu 210 215 220 Asp Tyr Ser Lys Glu Gly Gln Wing Gly Lys Lys Tyr Thr Met Met Val 225 230 235 240
Thr Wing Asp Gln Leu Wing Thr Lys He Pro Gly Tyr Asn Pro Pro Pro 245 250 255
Arg Val Glu Gln Asp Arg Ser His Asn Ala Leu Thr He His As Asp 260 265 270 Leu He Val Asn Leu Lys Glu Asp Phe Wing Tyr Glu He He Val Val 277 280 285 Phe Glu Asn Leu Ser Tyr Ser Thr Leu Phe Asn Glu Asp Leu Phe He 290 295 300 Tyr Arg Phe Asp Lys Asn Hís Asn Leu Leu He Glu Lys Thr Val Gly 305 310 315 320
Being Leu Phe Glu Thr Asn Leu His Wing Asp He Phe Tyr Glu His He 325 330 335 Glu Ser Glu Leu Glu 340
< 210 > 20 < 211 > 15 < 212 > PRT < 213 > Baciílus thuringiensis < 400 > 20 Met Leu Asp Thr Asn Lys Val Tyr Glu He Ser Asn His Wing Asn 1 5 10 15
< 210 > 21 < 211 > 41 < 12 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic < 400 > 21 atgttagata caaataaagt atatgaaatt tcaaatcatg c 41
< 210 > 22 < 211 > 20 < 212 > PRT < 213 > Bacillus thuringiensis < 400 > 22 Ser He Leu Asn Leu Gln Asp Leu Ser Gln Lys Tyr Met Thr Ala Wing 1 5 10 15 Leu Asn Lys He 20 < 210 > 23 < 211 > 15 < 212 > PRT < 213 > Bacillus thuringiensis < 400 > 23 Be Ala Arg Gln Val His He Gln He Asn Asn Lys Thr Arg His 1 5 10 15 < 210 > 24 < 211 > 21 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic < 400 > 24 tcacaaaaat atatgaacag c 21
< 210 > 25 < 211 > 31 • < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic < 400 > 25 atatctatag aattcgcaat tcgtccatgt g 31
< 210 > 26 < 211 > 30 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic < 400 > 26 cagtattcat ataagcttcc tcctttaata 30
< 210 > 27 < 211 > 39 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic < 400 > 27 aaggtgaagc ttttatgtta gatactaata aagtttatg 39
< 210 > 28 < 211 > 24 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic
< 400 > 28 ccggaataga agctttgcat atgg 24
< 210 > 29 < 211 > 11 < 212 > PRT < 213 > Bacillus thuringiensis < 400 > 29 Met Asn Val Aen His Gly Met Ser Cys Gly Cys 1 5 10
< 210 > 30 < 211 > 27 < 212 > DNA < 213 > Artificial sequence < 220 > < 221 > various characteristics < 222 > (22) < 223 > W = A or T / U < 220 > < 221 > various characteristics < 222 > (23) < 223 > S = G or C < 220 > < 221 > various characteristics < 222 > (24) < 223 > N = A, C, G or T / U < 220 > < 223 > Description of the artificial sequence: synthetic < 400 > 30 atgaatgtaa atcatgggat gwsntgt 27
< 210 > 31 < 211 > 12 < 212 > RNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic < 400 > 31 uaaacaaugg cu 12
< 210 > 32 < 211 > 17 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic < 400 > 32 gtaccagaag taggagg 17
< 210 > 33 < 211 > 17 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic < 400 > 33 tgacacagct atggagc 17
< 210 > 34 < 211 > 20 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic < 400 > 34 atgattgccg gaatagaagc 20
Claims (55)
- NOVELTY OF THE INVENTION CLAIMS 1. - An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 19.
- 2. The polypeptide according to claim 1, further characterized in that it is encoded by SEQ ID NO. : 1, SEQ ID NO: 3 or SEQ ID NO: 18.
- 3. A composition containing at least one polypeptide, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ. ID NO: 4 and SEQ ID NO: 19.
- 4. The composition according to claim 3, further characterized in that the composition comprises two or more polypeptides, and two of the polypeptides are SEQ ID NO: 2 and SEQ ID NO: 4. The composition according to claim 3, further characterized by a cell extract, cell suspension, cell homogenate, cell lysate, cell supernatant, cell filtrate or Bacillus thuringiensis cell tablet EG4550, EG5899 , EG11529, NRRL B-2178 4, NRRL B-21783, NRRL B-21917, NRRL B-21786, NRRL B-21787, NRRL B-21785, NRRL B-21788, NRRL B-21915 or NRRL B-21916. 6. - The composition according to claim 5, further characterized in that the composition is a powder, fine powder, tablet, granule, spray, emulsion, colloid or solution. 7. The composition according to claim 5, further characterized in that said composition is prepared by desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation or concentration of a culture of Bacillus thuringiensis cells. 8. The composition according to claim 5, further characterized in that from 1% to 99% by weight of said polypeptide. 9. An insecticidal polypeptide prepared by a method comprising the steps of (a) culturing Bacillus thuringiensis cells EG4550, EG5899, EG11529, NRRL B-21784, NRRL B-21783, NRRL B-21917, NRRL B-21786, NRRL B-21787, NRRL B-21785, NRRL B-21788, NRRL B-21915 or NRRL B-21916 under conditions effective to produce an insecticidal polypeptide; and (b) obtaining from said cells the insecticidal polypeptide thus produced. 10. The polypeptide according to claim 9, further characterized in that said polypeptide comprises SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 19. 11.- A Bacillus thuringiensis cell having the access number of NRRL: NRRL B-21784, NRRL B-21783, NRRL B-21917, NRRL B-21786, NRRL B-21787, NRRL B-21785, NRRL B-21788, NRRL B-21915 or NRRL B-21916. 12. - An isolated polynucleotide encoding SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 19. 13. The polynucleotide according to claim 12, further characterized in that the polynucleotide encodes a polypeptide comprising the amino acid sequence SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 19. 14. The polynucleotide according to claim 13, comprising the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 18. 15. The polynucleotide according to claim 13, comprising the nucleic acid sequence of SEQ ID NO: 1 and SEQ ID NO: 3. 16. The polynucleotide according to claim 13, further characterized as RNA or DNA. 17. The polynucleotide according to claim 13, further characterized in that said isolated polynucleotide is operably linked to a first promoter. 18. The polynucleotide according to claim 17, further characterized in that said promoter is a heterologous promoter. 19. The polynucleotide according to claim 18, further characterized in that said heterologous promoter is a promoter expressible by a plant. 20. The polynucleotide according to claim 19, further characterized in that the promoter expressible by the plant can be selected from the group consisting of corn sucrose synthetase 1, corn alcohol dehydrogenase 1, maize light harvesting complex, corn heat shock protein, RuBP carboxylase of small subunit of pea, Manipin synthase of Ti plasmid, nopalin synthase of Ti plasmid, chalcone isomerase of petunia, protein 1 rich in bean glycine, potato patatin, lectin, CaMV 35S and the RuBp carboxylase promoter of small subunit S-E9. 21. A method for detecting a nucleic acid sequence encoding a d-endotoxin polypeptide, comprising the steps of: a) obtaining sample nucleic acids that are suspected of encoding a d-endotoxin polypeptide; b) contacting said sample nucleic acids with the polynucleotide of claim 14 under conditions effective to allow hybridization of substantially complementary nucleic acids; and detecting the hybridized complementary nucleic acids thus formed. 22. A nucleic acid detection equipment comprising, in a suitable container means, at least one segment of nucleic acids according to claim 13 and at least one first detection reagent. 23. A nucleic acid vector comprising at least a first region encoding one or more amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 19. 24. - The vector according to claim 23, further characterized in that said first sequence region encodes SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 19. The vector according to claim 23, further characterized in that said first sequence region encodes SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 18. 26.- The vector according to claim 23, further characterized by defining a plasmid, baculovirus, chromosome artificial, virion, cosmid, phagemid, phage or viral vector, 27.- A transformed host cell comprising a nucleic acid encoding one or more amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 19. The transformed host cell according to claim 27, further characterized in that the nucleic acid encodes SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 19. 29.- The host cell transformed in accordance with with claim 27, further characterized because the nucleic acid encodes SEQ ID NO: 2 and SEQ ID NO: 4. 30.- The transformed host cell according to claim 27, further defined as a prokaryotic or eukaryotic hoasis cell. 31. - The host cell transformed according to claim 27, further defined as a bacterial cell or a plant cell. 32. The host cell transformed according to claim 31, further characterized in that said bacterial cell is a ß cell. thuringiensis, B. subtilis, B. megaterium, B. cereus, Escherichia, Salmonella, Agrobacterium or Pseudomonas. 33. The transformed host cell according to claim 31, further characterized in that said bacterial cell is a 'l EG4550 cell, EG5899, EG11529, NRRL B-21784, NRRL B-21783, NRRL B-21917, NRRL B-21786, NRRL B-21787, NRRL B-21785, NRRL B-21788, NRRL B-21915 or NRRL B-21916. 34. The host cell transformed according to claim 31, further characterized in that said bacterial cell is a cell of Agrobacterium tumefaciens. 35.- The transformed host cell according to claim 31, further defined as a monocot or dicot plant cell. 36.- The host cell transformed in accordance with the 20 claim 35, further characterized in that said plant cell is selected from the group consisting of a cell of corn, wheat, soybean, oats, cotton, rice, rye, sorghum, sugar cane, tomato, tobacco, kapok, flax, potato, barley, peat grass, grass for pas, berries, fruits, legumes, vegetables, ornamental plants, shrubs, cactus, succulents and trees. 37.- The host cell transformed according to claim 35, further characterized in that said plant cell is a cell of corn, wheat, rice or sugar cane. 38.- The host cell transformed according to claim 35, further characterized in that said plant cell is a cell of soybean, cotton, potato, tomato or tobacco. 39.- A callus or embryo of a plant comprising a polynucleotide that encodes one or more amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 19. 40.- The callus or embryo of a plant according to claim 39, further characterized in that said polynucleotide encodes SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 19. 41.- The callus or embryo of a plant according to claim 39, characterized in addition because said polynucleotide encodes SEQ ID NO: 2 and SEQ ID NO: 4. 42.- A transgenic plant having incorporated into its genome a selected polynucleotide comprising a first sequence region encoding one or more amino acid sequences of SEQ ID NO: 2. NO: 2, SEQ ID NO: 4 or SEQ ID NO: 19. 43. - The transgenic plant according to claim 42, further characterized in that said first sequence region encodes SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 19. 44.- The transgenic plant according to claim 42, further characterized in that said first sequence region encodes SEQ ID NO: 2 and SEQ ID NO: 4. 45.- The transgenic plant according to claim 42, further characterized in that said first sequence region comprises SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 18. 46.- The transgenic plant according to claim 42, further characterized in that said first sequence region comprises SEQ ID NO: 1 and SEQ ID NO: 3. 47.- The transgenic plant according to claim 42, further defined as a monocotyledonous plant. 48. The transgenic plant according to claim 42, further defined as a plant of corn, wheat, oats, rice, barley, peat grass or pasture pasture. 49.- The transgenic plant according to claim 42, further defined as a diocotyledonous plant. 50.- The transgenic plant according to claim 42, further defined as a legume, soybean, tobacco, tomato, potato, cotton, fruits, berries, vegetables or trees. 51. - A progeny of any generation of the transgenic plant according to claim 42, further characterized in that said progeny comprises the first selected sequence region. 52. A seed of any generation according to claim 42, further characterized in that said seed comprises the first sequence region. 53. A seed of any generation according to claim 51, further characterized in that said seed comprises the first sequence region. T-T ío 54.- A plant of any seed generation according to claim 52 or 53, further characterized in that said plant comprises the first sequence region. A method for preparing an insect resistant plant comprising: (a) contacting recipient plant cells with a polynucleotide composition comprising at least one nucleic acid sequence encoding the polypeptide of claim 1; (b) selecting a recipient plant cell comprising the first nucleic acid sequence; and (c) regenerating a plant from the selected cell; wherein said plant has increased insect resistance relative to the corresponding non-transformed plant.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US60/172,240 | 1999-05-04 |
Publications (1)
Publication Number | Publication Date |
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MXPA01011313A true MXPA01011313A (en) | 2002-06-05 |
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