# Patent

## Title
Lepidopteran-active Bacillus thuringiensis δ-endotoxin polynucleotides, compositions, and methods of use

## Abstract

Disclosed are Bacillus thuringiensis strains comprising novel crystal proteins which exhibit insecticidal activity against lepidopteran insects. Also disclosed are novel B. thuringiensis genes and their encoded crystal proteins, as well as methods of making and using transgenic cells comprising the novel nucleic acid sequences of the invention.

## Background
This application is a continuation application of Ser. No. 10/428,961 filed May 2, 2003 (now U.S. Pat. No. 7,078,509) which is a divisional application of Ser. No. 09/661,322 filed Sep. 13, 2000 (now U.S. Pat. No. 6,593,293, which claims benefit of claims the benefit of 60/153,995 filed Sep. 15, 1999, the entire of contents of which is hereby specifically incorporated by reference.


1.0 BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention relates generally to the fields of molecular biology. More particularly, certain embodiments concern methods and compositions comprising DNA segments, and proteins derived from bacterial species. More particularly, it concerns novel genes from Bacillus thuringiensis encoding lepidopteran-toxic crystal proteins. Various methods for making and using these DNA segments, DNA segments encoding synthetically-modified Cry proteins, and native and synthetic crystal proteins are disclosed, 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. Also disclosed are methods of making and using nucleic acid segments in the development of transgenic plant cells containing the DNA segments disclosed herein.
1.2 Description of the Related Art
Almost all field crops, plants, and commercial farming areas are susceptible to attack by one or more insect pests. Particularly problematic are Coleopteran and Lepidoptern pests. For example, vegetable and cole crops such as artichokes, kohlrabi, arugula, leeks, asparagus, lentils, beans, lettuce (e.g., head, leaf, romaine), beets, bok choy, malanga, broccoli, melons (e.g., muskmelon, watermelon, crenshaw, honeydew, cantaloupe), brussels sprouts, cabbage, cardoni, carrots, napa, cauliflower, okra, onions, celery, parsley, chick peas, parsnips, chicory, peas, chinese cabbage, peppers, collards, potatoes, cucumber, pumpkins, cucurbits, radishes, dry bulb onions, rutabaga, eggplant, salsify, escarole, shallots, endive, soybean, garlic, spinach, green onions, squash, greens, sugar beets, sweet potatoes, turnip, swiss chard, horseradish, tomatoes, kale, turnips, and a variety of spices are sensitive to infestation by one or more of the following insect pests: alfalfa looper, armyworm, beet armyworm, artichoke plume moth, cabbage budworm, cabbage looper, cabbage webworm, corn earworm, celery leafeater, cross-striped cabbageworm, european corn borer, diamondback moth, green cloverworm, imported cabbageworm, melonworm, omnivorous leafroller, pickleworm, rindworm complex, saltmarsh caterpillar, soybean looper, tobacco budworm, tomato fruitworm, tomato hornworm, tomato pinworm, velvetbean caterpillar, and yellowstriped armyworm. Likewise, pasture and hay crops such as alfalfa, pasture grasses and silage are often attacked by such pests as armyworm, beef armyworm, alfalfa caterpillar, European skipper, a variety of loopers and webworms, as well as yellowstriped armyworms.
Fruit and vine crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, quince almonds, chestnuts, filberts, pecans, pistachios, walnuts, citrus, blackberries, blueberries, boysenberries, cranberries, currants, loganberries, raspberries, strawberries, grapes, avocados, bananas, kiwi, persimmons, pomegranate, pineapple, tropical fruits are often susceptible to attack and defoliation by achema sphinx moth, amorbia, armyworm, citrus cutworm, banana skipper, blackheaded fireworm, blueberry leafroller, cankerworm, cherry fruitworm, citrus cutworm, cranberry girdler, eastern tent caterpillar, fall webworm, fall webworm, filbert leafroller, filbert webworm, fruit tree leafroller, grape berry moth, grape leaffolder, grapeleaf skeletonizer, green fruitworm, gummosos-batrachedra commosae, gypsy moth, hickory shuckworm, hornworms, loopers, navel orangeworm, obliquebanded leafroller, omnivorous leafroller. omnivorous looper, orange tortrix, orangedog, oriental fruit moth, pandemis leafroller, peach twig borer, pecan nut casebearer, redbanded leafroller, redhumped caterpillar, roughskinned cutworm, saltmarsh caterpillar, spanworm, tent caterpillar, thecla-thecla basillides, tobacco budworm, tortrix moth, tufted apple budmoth, variegated leafroller, walnut caterpillar, western tent caterpillar, and yellowstriped armyworm.
Field crops such as canola/rape seed, evening primrose, meadow foam, corn (field, sweet, popcorn), cotton, hops, jojoba, peanuts, rice, safflower, small grains (barley, oats, rye, wheat, etc.), sorghum, soybeans, sunflowers, and tobacco are often targets for infestation by insects including armyworm, asian and other corn borers, banded sunflower moth, beet armyworm, bollworm, cabbage looper, corn rootworm (including southern and western varieties), cotton leaf perforator, diamondback moth, european corn borer, green cloverworm, headmoth, headworm, imported cabbageworm, loopers (including Anacamptodes spp.), obliquebanded leafroller, omnivorous leaftier, podworm, podworm, saltmarsh caterpillar, southwestern corn borer, soybean looper, spotted cutworm, sunflower moth, tobacco budworm, tobacco hornworm, velvetbean caterpillar.
Bedding plants, flowers, ornamentals, vegetables and container stock are frequently fed upon by a host of insect pests such as armyworm, azalea moth, beet armyworm, diamondback moth, ello moth (hornworm), Florida fern caterpillar, lo moth, loopers, oleander moth, omnivorous leafroller, omnivorous looper, and tobacco budworm.
Forests, fruit, ornamental, and nut-bearing trees, as well as shrubs and other nursery stock are often susceptible to attack from diverse insects such as bagworm, blackheaded budworm, browntail moth, california oakworm, douglas fir tussock moth, elm spanworm, fall webworm, fruittree leafroller, greenstriped mapleworm, gypsy moth, jack pine budworm, mimosa webworm, pine butterfly, redhumped caterpillar, saddleback caterpillar, saddle prominent caterpillar, spring and fall cankerworm, spruce budworm, tent caterpillar, tortrix, and western tussock moth. Likewise, turf grasses are often attacked by pests such as armyworm, sod webworm, and tropical sod webworm.
Because crops of commercial interest are often the target of insect attack, environmentally-sensitive methods for controlling or eradicating insect infestation are desirable in many instances. This is particularly true for farmers, nurserymen, growers, and commercial and residential areas which seek to control insect populations using eco-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. B. thuringiensis is a Gram-positive bacterium that produces crystal proteins or inclusion bodies which are specifically toxic to certain orders and species of insects. Many different strains of B. thuringiensis have been shown to produce insecticidal crystal proteins. Compositions including B. thuringiensis strains which produce insecticidal proteins have been commercially-available and used as environmentally-acceptable insecticides because they are quite toxic to the specific target insect, but are harmless to plants and other non-targeted organisms.
1.2.1 B. Thuringienses Crystal Proteins
1.2.1 δ-Endotoxins
δ-endotoxins are used to control a wide range of leaf-eating caterpillars and beetles, as well as mosquitoes. These proteinaceous parasporal crystals, also referred to as insecticidal crystal proteins, crystal proteins, Bt inclusions, crystaline inclusions, inclusion bodies, and Bt toxins, are a large collection of insecticidal proteins produced by B. thuringiensis that are toxic upon ingestion by a susceptible insect host. Over the past decade research on the structure and function of B. thuringiensis toxins has covered all of the major toxin categories, and while these toxins differ in specific structure and function, general similarities in the structure and function are assumed. Based on the accumulated knowledge of B. thuringiensis toxins, a generalized mode of action for B. thuringiensis toxins has been created and includes: ingestion by the insect, solubilization in the insect midgut (a combination stomach and small intestine), resistance to digestive enzymes sometimes with partial digestion actually “activating” the toxin, binding to the midgut cells, formation of a pore in the insect cells and the disruption of cellular homeostasis (English and Slatin, 1992).
One of the unique features of B. thuringiensis is its production of crystal proteins during sporulation which are specifically toxic to certain orders and species of insects. Many different strains of B. thuringiensis have been shown to produce insecticidal crystal proteins. Compositions including B. thuringiensis strains which produce proteins having insecticidal activity against lepidopteran and dipteran insects have been commercially available and used as environmentally-acceptable insecticides because they are quite toxic to the specific target insect, but are harmless to plants and other non-targeted organisms.
The mechanism of insecticidal activity of the B. thuringiensis crystal proteins has been studied extensively in the past decade. It has been shown that the crystal proteins are toxic to the insect only after ingestion of the protein by the insect. The alkaline pH and proteolytic enzymes in the insect mid-gut solubilize the proteins, thereby allowing the release of components which are toxic to the insect. These toxic components disrupt the mid-gut cells, cause the insect to cease feeding, and, eventually, bring about insect death. For this reason, B. thuringiensis has proven to be an effective and environmentally safe insecticide in dealing with various insect pests.
As noted by Höfte and Whiteley (1989), the majority of insecticidal B. thuringiensis strains are active against insects of the order Lepidoptera, i.e., caterpillar insects. Other B. thuringiensis strains are insecticidally active against insects of the order Diptera, i.e., flies and mosquitoes, or against both lepidopteran and dipteran insects. In recent years, a few B. thuringiensis strains have been reported as producing crystal proteins that are toxic to insects of the order Coleoptera, i.e., beetles (Krieg et al., 1983; Sick et al., 1990; Donovan et al., 1992; Lambert et al., 1992a; 1992b).
1.2.2 Genes Encoding Crystal Proteins
Many of the δ-endotoxins are related to various degrees by similarities in their amino acid sequences. Historically, the proteins and the genes which encode them were classified based largely upon their spectrum of insecticidal activity. The review by Höfte and Whiteley (1989) discusses the genes and proteins that were identified in B. thuringiensis prior to 1990, and sets forth the nomenclature and classification scheme which has traditionally been applied to B. thuringiensis genes and proteins. cryI genes encode lepidopteran-toxic CryI proteins. cryII genes encode CryII proteins that are toxic to both lepidopterans and dipterans. cryIII genes encode coleopteran-toxic CryIII proteins, while cryIV genes encode dipteran-toxic CryIV proteins. Based on the degree of sequence similarity, the proteins were further classified into subfamilies; more highly related proteins within each family were assigned divisional letters such as CryIA, CryIB, CryIC, etc. Even more closely related proteins within each division were given names such as CryIC1, CryIC2, etc.
Recently, a new nomenclature was developed which systematically classified the Cry proteins based upon amino acid sequence homology rather than upon insect target specificities (Crickmore et al., 1998). The classification scheme for many known toxins, including allelic variations in individual proteins, is summarized and regularly updated at http://www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/. The information was most recently updated as of Apr. 27, 1999 and is herein incorporated by reference.
1.2.3 Crystal Proteins Toxic to Lepidopteran Insects
2.0 SUMMARY OF THE INVENTION
The recent review by Schnepf et al. (1998) describes the enormous diversity of insecticidal crystal proteins derived from B. thuringiensis. Cry proteins of the Cry1, Cry2, and Cry9 classes are particularly known for their toxicity towards lepidopteran larvae, however, the degree of toxicity varies significantly depending on the target lepidopteran pest (Höfte and Whiteley, 1989). For instance, Cry1Ac shows poor toxicity towards the armyworm, Spodoptera littoralis, but strong toxicity towards the tobacco budworm, Heliothis virescens. In addition, slight variations in amino acid sequence within a Cry protein class can dramatically impact insecticidal activity (see Schnepf et al., 1998 and references therein). The Cry3Ba and Cry3Bb genes, for instance, share 94% amino acid sequence identity, but only Cry3Bb exhibits significant toxicity towards the Southern corn rootworm, Diabrotica undecimpunctata howardi (Donovan et al., 1992). Similarly, Cry2Aa and Cry2Ab share 87% amino acid sequence identity, yet only Cry2Aa displays toxicity towards mosquitos (Widner and Whiteley, 1990). Von Tersch et al. (1991) demonstrated that Cry1Ac proteins varying by only seven amino acids (>99% sequence identity) nevertheless show significant differences in insecticidal activity. Lee et al. (1996) reported that Cry1Ab alleles differing at only two amino acid positions exhibited a 10-fold difference in toxicity towards the gypsy moth, Lymantria dispar. Thus, even Cry proteins that are considered to be alleles of known Cry proteins or to belong to a Cry protein subclass (Crickmore et al., 1998) may have unique and useful insecticidal properties. International Patent Application Publication No. WO 98/00546 and WO 98/40490 describe a variety of Cry1-, Cry2-, and Cry9-related crystal proteins obtained from B. thuringiensis. 
2.1 Cry DNA Segments
The present invention concerns nucleic acid segments, that can be isolated from virtually any source, that are free from total genomic DNA and that encode the novel peptides disclosed herein. Nucleic acid segments encoding these polypeptides may encode active proteins, peptides or peptide fragments, polypeptide subunits, functional domains, or the like of one or more crystal proteins. In addition the invention encompasses nucleic acid segments which may be synthesized entirely in vitro using methods that are well-known to those of skill in the art which encode the novel Cry polypeptides, peptides, peptide fragments, subunits, or functional domains disclosed herein.
As used herein, the term “nucleic acid segment” refers to a polynucleotide molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a nucleic acid segment encoding an endotoxin polypeptide refers to a nucleic acid segment that comprises one or more crystal protein-encoding sequences yet is isolated away from, or purified free from, total genomic DNA of the species from which the nucleic acid segment is obtained, which in the instant case is the genome of the Gram-positive bacterial genus, Bacillus, and in particular, the species of Bacillus known as B. thuringiensis. Included within the term “nucleic acid segment”, are polynucleotide segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phages, viruses, and the like.
Similarly, a DNA segment comprising an isolated or purified crystal protein-encoding gene refers to a DNA segment which may include in addition to peptide encoding sequences, certain other elements such as, regulatory sequences, isolated substantially away from other naturally occurring genes or protein-encoding sequences. In this respect, the term “gene” is used for simplicity to refer to a functional protein-, polypeptide- or peptide-encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, operon sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides. Also, the term includes an expression cassette comprising at least a promoter operably linked to one or more protein coding sequences, operably linked to at least a transcriptional termination sequence.
“Isolated substantially away from other coding sequences” means that the gene of interest, in this case, a nucleic acid segment or gene encoding all or part of a bacterial insecticidal crystal protein, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional nucleic acid segments or 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 later added to the segment by the hand of man.
In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that encode a Cry peptide species that includes within its amino acid sequence an amino acid 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, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO. 63.
The term “a sequence essentially as set forth in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6,” for example, means that the sequence substantially corresponds to a portion of the sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 and has relatively few amino acids that are not identical with, 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 further defined in detail herein (e.g., see Illustrative Embodiments). Accordingly, sequences that have from about 70% to about 80%, or more preferably about 81, 82, 83, 84, 85, 86, 87, 88, 89, or about 90%, or even more preferably about 91, 92, 93, 94, 95, 96, 97, 98, or about 99% amino acid sequence identity or functional equivalence to the 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, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO.63 will be 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, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO:63.”
In addition, sequences that have from about 70% to about 80%, or more preferably about 81, 82, 83, 84, 85, 86, 87, 88, 89, or about 90%, or even more preferably about 91, 92, 93, 94, 95, 96, 97, 98, or about 99% nucleic acid sequence identity or functional equivalence to the nucleic acids 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, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:62 will be 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, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:62.”
It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5′ or 3′ sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5′ or 3′ portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments may be prepared that include a short contiguous stretch encoding any of the peptide sequences disclosed 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, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO:63, or that are identical with or complementary to DNA sequences which encode any of the peptides disclosed 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, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO:63, and particularly those DNA segments disclosed 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, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:62. For example, DNA sequences such as about 18 nucleotides, and that are up to about 10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, about 100, about 50, and about 14 base pairs in length (including all intermediate lengths) are also contemplated to be useful.
It will be readily understood that “intermediate lengths”, in these contexts, means any length between the quoted ranges, such as 18, 19, 20, 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers in the ranges of from about 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 10,00 or so nucleotides and the like.
It will also be understood that this invention is not limited to the particular nucleic acid sequences which encode peptides of the present invention, or which encode the amino acid sequences 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, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO:63, including those DNA sequences which are particularly disclosed 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, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:62. Recombinant vectors and isolated DNA segments may therefore variously include the peptide-coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include these peptide-coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.
The DNA segments of the present invention encompass biologically-functional, equivalent peptides. Such sequences may arise as a consequence of codon degeneracy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally-equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test mutants in order to examine activity at the molecular level.
If desired, one may also prepare fusion proteins and peptides, e.g., where the peptide-coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively).
Recombinant vectors form further aspects of the present invention. Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length protein or smaller peptide, is positioned under the control of a promoter. The promoter may be in the form of the promoter that is naturally associated with a gene encoding peptides of the present invention, as may be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCRTM technology, in connection with the compositions disclosed herein.
2.2 Cry DNA Segments as Hybridization Probes and Primers
In addition to their use in directing the expression of crystal proteins or peptides of the present invention, the nucleic acid sequences contemplated herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that nucleic acid segments that comprise a sequence region that consists of at least a 14 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 14 nucleotide long contiguous DNA segment 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, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:62 will find particular utility. Longer contiguous identical or complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000, 2000, 5000 bp, etc. (including all intermediate lengths and up to and including the full-length gene sequences encoding each polypeptide will also be of use in certain embodiments.
The ability of such nucleic acid probes to specifically hybridize to crystal protein-encoding sequences will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
Nucleic acid molecules having sequence regions consisting of contiguous nucleotide stretches of about 14 to about 17 or so, 18-25, 26-35, 36-50, or even up to and including sequences of about 100-200 nucleotides or so, identical or complementary to DNA sequences 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, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:62, are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 10-14 and about 100 to 200 or so nucleotides, but larger contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect.
Of course, fragments may also be obtained by other techniques such as, e.g., by mechanical shearing or by restriction enzyme digestion. Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCRTM technology of U.S. Pat. Nos. 4,683,195 and 4,683,202 (each incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNA fragments. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50° C. to about 70° C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating crystal protein-encoding DNA segments. Detection of DNA segments via hybridization is well-known to those of skill in the art, and the teachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 (each incorporated herein by reference) are exemplary of the methods of hybridization analyses. Teachings such as those found in the texts of Maloy et al., 1990; Maloy 1994; Segal, 1976; Prokop, 1991; and Kuby, 1991, are particularly relevant.
Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate crystal protein-encoding sequences from related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. to about 55° C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining 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, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This 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 G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantitated, by means of the label.
2.3 Vectors and Methods for Recombinant Expression of Cry Polypeptides
In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a DNA segment encoding a crystal protein or peptide in its natural environment. Such promoters may include promoters normally associated with other genes, and/or promoters isolated from any bacterial, viral, eukaryotic, or plant cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al., 1989. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides. Appropriate promoter systems contemplated for use in high-level expression include, but are not limited to, the Pichia expression vector system (Pharmacia LKB Biotechnology).
In connection with expression embodiments to prepare recombinant proteins and peptides, it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire peptide sequence being most preferred. However, it will be appreciated that the use of shorter DNA segments to direct the expression of crystal peptides or epitopic core regions, such as may be used to generate anti-crystal protein antibodies, also falls within the scope of the invention. DNA segments that encode peptide antigens from about 8 to about 50 amino acids in length, or more preferably, from about 8 to about 30 amino acids in length, or even more preferably, from about 8 to about 20 amino acids in length are contemplated to be particularly useful. Such peptide epitopes may be amino acid sequences which comprise contiguous amino acid sequences from 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, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO:63.
2.4 Cry Transgenes and Transgenic Plants Expressing Cry Polypeptides
In yet another aspect, the present invention provides methods for producing a transgenic plant which expresses a nucleic acid segment encoding the novel polypeptides and endotoxins of the present invention. The process of producing transgenic plants is well-known in the art. In general, the method comprises transforming a suitable host cell with a DNA segment which contains a promoter operatively linked to a coding region that encodes one or more CryET31, CryET40, CryET43, CryET44, CryET45, CryET46, CryET47, CryET49, CryET51, CryET52, CryET53, CryET54, CryET55, CryET56, CryET57, CryET59, CryET60, CryET61, CryET62, CryET63, CryET64, CryET66, CryET67, CryET68, CryET72, CryET73, and CryET83 polypeptides. Such a coding region is generally operatively linked to a transcription-terminating region, whereby the promoter is capable of driving the transcription of the coding region in the cell, and hence providing the cell the ability to produce the polypeptide in vivo. Alternatively, in instances where it is desirable to control, regulate, or decrease 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 of controlling or decreasing 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 which express a gene or gene segment encoding one or more of the novel polypeptide compositions disclosed herein. As used herein, the term “transgenic plant” is intended to refer to a plant that has incorporated DNA sequences, including but not limited to genes which are perhaps not normally present, DNA sequences not normally transcribed into RNA or translated into a protein (“expressed”), or any other genes or DNA sequences which one desires to introduce into the non-transformed plant, such as genes which may normally be present in the non-transformed plant but which one desires to either genetically engineer or to have altered expression.
It is contemplated that in some instances either the nuclear or plastidic genome, or both, of a transgenic plant of the present invention will have been augmented through the stable introduction of one or more cryET31, cryET40, cryET43, cryET44, cryET45, cryET46, cryET47, cryET49, cryET51, cryET52, cryET53, cryET54, cryET55, cryET56, cryET57, cryET59, cryET60, cryET61, cryET62, cryET63, cryET64, cryET66, cryET67, cryET68, cryET72, cryET73, and cryET83 transgenes, either native, synthetically modified, or mutated. In some instances, more than one transgene will be incorporated into one or more genomes of the transformed host plant cell. Such is the case when more than one crystal protein-encoding DNA segment is incorporated into the genome of such a 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.
A preferred gene which may be introduced includes, for example, a crystal protein-encoding DNA sequence from bacterial origin, and particularly one or more of those described herein which are obtained from Bacillus spp. Highly preferred nucleic acid sequences are those obtained from B. thuringiensis, or any of those sequences which have been genetically engineered to decrease or increase the insecticidal activity of the crystal protein in such a transformed host cell.
Means for transforming a plant cell and the preparation of a transgenic cell line are well-known in the art, and are discussed herein. Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) and DNA segments for use in transforming such cells will, of course, generally comprise either the operons, genes, or gene-derived sequences of the present invention, either native, or synthetically-derived, and particularly those encoding the disclosed crystal proteins. These DNA constructs can further include structures such as promoters, enhancers, polylinkers, or even gene sequences which have positively- or negatively-regulating activity upon the particular genes of interest as desired. The DNA segment or gene may encode either a native or modified crystal protein, which will be expressed in the resultant recombinant cells, and/or which will impart an improved phenotype to the regenerated plant.
Such transgenic plants may be desirable for increasing the insecticidal resistance of a monocotyledonous or dicotyledonous plant, by incorporating into such a plant, a transgenic DNA segment encoding one or more CryET31, CryET40, CryET43, CryET44, CryET45, CryET46, CryET47, CryET49, CryET51, CryET52, CryET53, CryET54, CryET55, CryET56, CryET57, CryET59, CryET60, CryET61, CryET62, CryET63, CryET64, CryET66, CryET67, CryET68, CryET72, CryET73, and CryET83 polypeptides which are toxic to a lepidopteran insect. Particularly preferred plants include turf grasses, kapok, sorghum, cotton, corn, soybeans, oats, rye, wheat, flax, tobacco, rice, tomatoes, potatoes, or other vegetables, ornamental plants, fruit trees, and the like.
In a related aspect, the present invention also encompasses a seed produced by the transformed plant, a progeny from such seed, and a seed produced by the progeny of the original transgenic plant, produced in accordance with the above process. Such progeny and seeds will have a crystal protein-encoding transgene stably incorporated into their genome, and such progeny plants will inherit the traits afforded by the introduction of a stable transgene in Mendelian fashion. All such transgenic plants having incorporated into their genome transgenic DNA segments encoding one or more CryET31, CryET40, CryET43, CryET44, CryET45, CryET46, CryET47, CryET49, CryET51, CryET52, CryET53, CryET54, CryET55, CryET56, CryET57, CryET59, CryET60, CryET61, CryET62, CryET63, CryET64, CryET66, CryET67, CryET68, CryET72, CryET73, and CryET83 crystal proteins or polypeptides are aspects of this invention. As well-known to those of skill in the art, a progeny of a plant is understood to mean any offspring or any descendant from such a plant, but in this case means any offspring or any descendant which also contains the transgene.
2.5 Site-specific Mutagenesis
Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. The technique of site-specific mutagenesis is well known in the art, as exemplified by various publications.
In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
The preparation of sequence variants of the endotoxin-encoding nucleic acid segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
2.6 Antibody Compositions and Methods of Making
In particular embodiments, the inventors contemplate the use of antibodies, either monoclonal (mAbs) or polyclonal which bind to one or more of the polypeptides disclosed herein. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Harlow and Lane, 1988; incorporated herein by reference). mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference.
2.7 Enzyme-Linked Immuno Sorben Assay and Immunoprecipitation
ELISAs may be used in conjunction with the invention. Many different protocols exist for performing ELISAs. These are well known to those of ordinary skill in the art. Examples of basic ELISA protocols may be found in any standard molecular biology laboratory manual (e.g. Sambrook, Fritsch, and Maniatis, eds. Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory, 1989).
2.8 Western Blots
The compositions of the present invention will find great use in immunoblot or western blot analysis. Methods of performing immunoblot and western blot analysis are well known to those of skill in the are (see Sambrook, et al, ibid). Immunologically-based detection methods for use in conjunction with Western blotting include enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety are considered to be of particular use in this regard.
2.9 Crystal Protein Screening and Detection Kits
The present invention contemplates methods and kits for screening samples suspected of containing crystal protein polypeptides or crystal protein-related polypeptides, or cells producing such polypeptides. A kit may contain one or more antibodies of the present invention, and may also contain reagent(s) for detecting an interaction between a sample and an antibody of the present invention. The provided reagent(s) can be radio-, fluorescently- or enzymatically-labeled or even epitope or ligand tagged. The kit can contain a known radiolabeled agent capable of binding or interacting with a nucleic acid or antibody of the present invention.
The reagent(s) of the kit can be provided as a liquid solution, attached to a solid support or as a dried powder. Preferably, when the reagent(s) are provided in a liquid solution, the liquid solution is an aqueous solution. Preferably, when the reagent(s) provided are attached to a solid support, the solid support can be chromatograph media, a test plate having a plurality of wells, or a microscope slide. When the reagent(s) provided are a dry powder, the powder can be reconstituted by the addition of a suitable solvent, that may be provided.
In still further embodiments, the present invention concerns immunodetection methods and associated kits. It is proposed that the crystal proteins or peptides of the present invention may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention, may be employed to detect crystal proteins or crystal protein-related epitope-containing peptides. In general, these methods will include first obtaining a sample suspected of containing such a protein, peptide or antibody, contacting the sample with an antibody or peptide in accordance with the present invention, as the case may be, under conditions effective to allow the formation of an immunocomplex, and then detecting the presence of the immunocomplex.
In general, the detection of immunocomplex formation is quite well known in the art and may be achieved through the application of numerous approaches. For example, the present invention contemplates the application of ELISA, Radioimmunoassay, immunoblot (e.g., dot blot), indirect immunofluorescence techniques and the like. One may find additional advantages through 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 assaying purposes, it is proposed that virtually any sample suspected of comprising either a crystal protein or peptide or a crystal protein-related peptide or antibody sought to be detected, as the case may be, may be employed. It is contemplated that such embodiments may have application in the titering of antigen or antibody samples, in the selection of hybridomas, and the like. In related embodiments, the present invention contemplates the preparation of kits that may be employed 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 suspected of containing crystal proteins or peptides.
Generally speaking, kits in accordance with the present invention will include a suitable crystal protein, peptide or an antibody directed against such a protein or peptide, together with an immunodetection reagent and a means for containing the antibody or antigen and reagent. The immunodetection reagent will typically comprise a label associated with the antibody or antigen, or associated with a secondary binding ligand. Exemplary ligands might include a secondary antibody directed against the first antibody or antigen or a biotin or avidin (or streptavidin) ligand having an associated label. Of course, as noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention.
The container will generally include a vial into which the antibody, antigen or detection reagent may be placed, and preferably suitably aliquotted. The kits of the present invention will also typically include a means for containing the antibody, antigen, and reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
2.10 Epitopic Core Sequences
The present invention is also directed to protein or peptide compositions, free from total cells and other peptides, which comprise a purified protein or peptide which incorporates an epitope that is immunologically cross-reactive with one or more anti-crystal protein antibodies. In particular, the invention concerns epitopic core sequences derived from Cry proteins or peptides.
As used herein, the term “incorporating an epitope(s) that is immunologically cross-reactive with one or more anti-crystal protein antibodies” is intended to refer to a peptide or protein antigen which includes a primary, secondary or tertiary structure similar to an epitope located within a crystal protein or polypeptide. The level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against the crystal protein or polypeptide will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein antigen. Various immunoassay methods may be employed in conjunction with such antibodies, such as, for example, Western blotting, ELISA, RIA, and the like, all of which are known to those of skill in the art. The identification of Cry immunodominant epitopes, and/or their functional equivalents, suitable for use in vaccines is a relatively straightforward matter (e.g. U.S. Pat. No. 4,554,101; Jameson and Wolf, 1988; Wolf et al., 1988; U.S. Pat. No. 4,554,101). The amino acid sequence of these “epitopic core sequences” may then be readily incorporated into peptides, either through the application of peptide synthesis or recombinant technology.
Preferred peptides for use in accordance with the present invention will generally be on the order of about 8 to about 20 amino acids in length, and more preferably about 8 to about 15 amino acids in length. It is proposed that particular advantages of the present invention may be realized through the preparation of synthetic peptides which include modified and/or extended epitopic/immunogenic core sequences which result in a “universal” epitopic peptide directed to crystal proteins, and in particular CryET31, CryET40, CryET43, CryET44, CryET45, CryET46, CryET47, CryET49, CryET51, CryET52, CryET53, CryET54, CryET55, CryET56, CryET57, CryET59, CryET60, CryET61, CryET62, CryET63, CryET64, CryET66, CryET67, CryET68, CryET72, CryET73, CryET83 and related sequences. These epitopic core sequences are identified herein in particular aspects as hydrophilic regions of the particular polypeptide antigen.
Computerized peptide sequence analysis programs (e.g., DNAStar® software, DNAStar, Inc., Madison, Wis.) may also be useful in designing synthetic peptides in accordance with the present disclosure.
Syntheses of epitopic sequences, or peptides which include an antigenic epitope within their sequence, are readily achieved using conventional synthetic techniques such as the solid phase method (e.g., through the use of commercially available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer).
2.11 Biological Functional Equivalents
Modification and changes may be made in the structure of the peptides of the present invention and DNA segments which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics. The following is a discussion based upon 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 to be useful for increasing the insecticidal activity of the protein, and consequently increasing the insecticidal activity and/or expression of the recombinant transgene in a plant cell. The amino acid changes may be achieved by changing the codons of the DNA sequence, according to the codons given in Table 1.








TABLE 1





Amino Acids
Codons



















Alanine
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
G
GGA GGC GGG GGU


 


Histidine
His
H
CAC CAU


 


Isoleucine
Ile
I
AUA AUC AUU


 


Lysine
Lys
K
AAA AAG


 


Leucine
Leu
L
UUA UUG CUA CUC CUG CUU


 


Methionine
Met
M
AUG


 


Asparagine
Asn
N
AAC AAU


 


Proline
Pro
P
CCA CCC CCG CCU


 


Glutamine
Gln
Q
CAA CAG


 


Arginine
Arg
R
AGA AGG CGA CGC CGG CGU


 


Serine
Ser
S
AGC AGU UCA UCC UCG UCU


 


Threonine
Thr
T
ACA ACC ACG ACU


 


Valine
Val
V
GUA GUC GUG GUU


 


Tryptophan
Trp
W
UGG


 


Tyrosine
Tyr
Y
UAC UAU









For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.
In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+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 may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+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 (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).
It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
2.12 Insecticidal Compositions and Methods of Use
The inventors contemplate that the crystal protein compositions disclosed herein will find particular utility as insecticides for topical and/or systemic application to field crops, grasses, fruits and vegetables, and ornamental plants. In a preferred embodiment, the bioinsecticide composition comprises an oil flowable suspension of bacterial cells which expresses a novel crystal protein disclosed herein. Preferably the cells are B. thuringiensis NRRL B-21921, NRRL B-21922, NRRL B-21923, NRRL B-21924, NRRL B-21925, NRRL B-21926, NRRL B-21927, NRRL B-21928, NRRL B-21929, NRRL B-21930, NRRL B-21931, NRRL B-21932, NRRL B-21933, NRRL B-21934, NRRL B-21935, NRRL B-21936, NRRL B-21937, NRRL B-21938, NRRL B-21939, NRRL B-21940, NRRL B-21941, NRRL B-21942, NRRL B-21943, and NRRL B-21944, however, any such bacterial host cell expressing the novel nucleic acid segments disclosed herein and producing a crystal protein is contemplated to be useful, such as B. thuringiensis, B. megaterium, B. subtilis, E. coli, or Pseudomonas spp.
In another important embodiment, the bioinsecticide composition comprises a water dispersible granule. This granule comprises bacterial cells which expresses a novel crystal protein disclosed herein. Preferred bacterial cells are B. thuringiensis NRRL B-21921, NRRL B-21922, NRRL B-21923, NRRL B-21924, NRRL B-21925, NRRL B-21926, NRRL B-21927, NRRL B-21928, NRRL B-21929, NRRL B-21930, NRRL B-21931, NRRL B-21932, NRRL B-21933, NRRL B-21934, NRRL B-21935, NRRL B-21936, NRRL B-21937, NRRL B-21938, NRRL B-21939, NRRL B-21940, NRRL B-21941, NRRL B-21942, NRRL B-21943, and NRRL B-21944, however, bacteria such as B. thuringiensis, B. megaterium, B. subtilis, E. coli, or Pseudomonas spp. cells transformed with a DNA segment disclosed herein and expressing the crystal protein are also contemplated to be useful.
In a third important embodiment, the bioinsecticide composition comprises a wettable powder, dust, pellet, or collodial concentrate. This powder comprises bacterial cells which expresses a novel crystal protein disclosed herein. Preferred bacterial cells are B. thuringiensis NRRL B-21921, NRRL B-21922, NRRL B-21923, NRRL B-21924, NRRL B-21925, NRRL B-21926, NRRL B-21927, NRRL B-21928, NRRL B-21929, NRRL B-21930, NRRL B-21931, NRRL B-21932, NRRL B-21933, NRRL B-21934, NRRL B-21935, NRRL B-21936, NRRL B-21937, NRRL B-21938, NRRL B-21939, NRRL B-21940, NRRL B-21941, NRRL B-21942, NRRL B-21943, and NRRL B-21944 cells, however, bacteria such as B. thuringiensis, B. megaterium, B. subtilis, E. coli, or Pseudomonas spp. cells transformed with a DNA segment disclosed herein and expressing the crystal protein are also contemplated to be useful. Such dry forms of the insecticidal compositions may be formulated to dissolve immediately upon wetting, or alternatively, dissolve in a controlled-release, sustained-release, or other time-dependent manner.
In a fourth important embodiment, the bioinsecticide composition comprises an aqueous suspension of bacterial cells such as those described above which express the crystal protein. Such aqueous suspensions may be provided as a concentrated stock solution which is diluted prior to application, or alternatively, as a diluted solution ready-to-apply.
For these methods involving application of bacterial cells, the cellular host containing the crystal protein gene(s) may be grown in any convenient nutrient medium, where the DNA construct provides a selective advantage, providing for a selective medium so that substantially all or all of the cells retain the B. thuringiensis gene. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.
When the insecticidal compositions comprise intact B. thuringiensis cells expressing the protein of interest, such bacteria may be formulated in a variety of ways. They may be employed as wettable powders, granules or dusts, by mixing with various diluents, inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, suspensions, emulsifiable concentrates, or the like. The ingredients may include Theological agents, surfactants, emulsifiers, dispersants, or polymers.
Alternatively, the novel insecticidal polypeptides may be prepared by native or recombinant bacterial expression systems in vitro and isolated for subsequent field application. Such protein may be either in crude cell lysates, suspensions, colloids, etc., or alternatively may be purified, refined, buffered, and/or further processed, before formulating in an active biocidal formulation. Likewise, under certain circumstances, it may be desirable to isolate crystals and/or spores from bacterial cultures expressing the crystal protein and apply solutions, suspensions, or collodial preparations of such crystals and/or spores as the active bioinsecticidal composition.
Regardless of the method of application, the amount of the active component(s) is applied at an insecticidally-effective amount, which will vary depending on such factors as, for example, the specific coleopteran insects to be controlled, the specific plant or crop to be treated, the environmental conditions, and the method, rate, and quantity of application of the insecticidally-active composition.
The insecticide compositions described may be made by formulating either the bacterial cell, crystal and/or spore suspension, or isolated protein component with the desired agriculturally-acceptable carrier. The compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, dessicated, or in an aqueous carrier, medium or suitable diluent, such as saline or other buffer. The formulated compositions may be in the form of a dust or granular material, or a suspension in oil (vegetable or mineral), or water or oil/water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for agricultural application. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term “agriculturally-acceptable carrier” covers all adjuvants, E. coli, inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in insecticide formulation technology; these are well known to those skilled in insecticide formulation. The formulations may be mixed with one or more solid or liquid adjuvants and prepared by various means, E. coli, by homogeneously mixing, blending and/or grinding the insecticidal composition with suitable adjuvants using conventional formulation techniques.
The insecticidal compositions of this invention are applied to the environment of the target lepidopteran insect, typically onto the foliage of the plant or crop to be protected, by conventional methods, preferably by spraying. The strength and duration of insecticidal application will be set with regard to conditions specific to the particular pest(s), crop(s) to be treated and particular environmental conditions. The proportional ratio of active ingredient to carrier will naturally depend on the chemical nature, solubility, and stability of the insecticidal composition, as well as the particular formulation contemplated.
Other application techniques, including dusting, sprinkling, soaking, soil injection, seed coating, seedling coating, spraying, aerating, misting, atomizing, and the like, are also feasible and may be required under certain circumstances such as e.g., insects that cause root or stalk infestation, or for application to delicate vegetation or ornamental plants. These application procedures are also well-known to those of skill in the art.
The insecticidal composition of the invention may be employed in the method of the invention singly or in combination with other compounds, including and not limited to other pesticides. The method of the invention may also be used in conjunction with other treatments such as surfactants, detergents, polymers or time-release formulations. The insecticidal compositions of the present invention may be formulated for either systemic or topical use.
The concentration of insecticidal composition which is used for environmental, systemic, or foliar application will vary widely depending upon the nature of the particular formulation, means of application, environmental conditions, and degree of biocidal activity. Typically, the bioinsecticidal composition will be present in the applied formulation at a concentration of at least about 1% by weight and may be up to and including about 99% by weight. Dry formulations of the polypeptide compositions may be from about 1% to about 99% or more by weight of the protein composition, while liquid formulations may generally comprise from about 1% to about 99% or more of the active ingredient by weight. Formulations which comprise intact bacterial cells will generally contain from about 104 to about 107 cells/mg.
The insecticidal formulation may be administered to a particular plant or target area in one or more applications as needed, with a typical field application rate per hectare ranging on the order of from about 50 g to about 500 g of active ingredient, or of from about 500 g to about 1000 g, or of from about 1000 g to about 5000 g or more of active ingredient.


5.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
5.1 Some Advantages of the Invention
The use of B. thuringiensis insecticidal crystal protein genes for in planta production of insecticidal proteins, thereby conferring insect resistance on important agronomic plants, is rapidly gaining commercial acceptance in the United States and abroad. The need for new insecticidal traits does not diminish, however, with the successful deployment of a handful of cry genes in plants. Concerns over the potential for insect resistance development, for instance, makes it imperative that an arsenal of insecticidal proteins (i.e. cry genes) be assembled to provide the genetic material necessary for tomorrow's insecticidal traits. In addition, transgenic plants producing a B. thuringiensis Cry protein may still be susceptible to damage from secondary insect pests, thus prompting the search for additional Cry proteins with improved efficacy towards these pests. The B. thuringiensis crystal proteins of the present invention represent a diverse collection of insecticidal proteins, including several that are toxic towards a lepidopteran colony exhibiting resistance to certain types of Cry1 proteins. Bioassays against a wide range of lepidopteran pests confirm that these proteins possess insecticidal activity and, furthermore, that these proteins vary in efficacy against this array of target insects. This variation in the spectrum of insects affected by the toxin proteins suggests differing modes of action that may be important for future insect resistance management strategies. In planta expression of the cry genes of the present invention can confer insect resistance to the host plant as has been demonstrated for other cry genes from B. thuringiensis. 
5.2 Probes and Primers
In another aspect, DNA sequence information provided by the invention allows for the preparation of relatively short DNA (or RNA) sequences having the ability to specifically hybridize to gene sequences of the selected polynucleotides disclosed herein. In these aspects, nucleic acid probes of an appropriate length are prepared based on a consideration of a selected crystal protein gene sequence, e.g., a sequence such as that shown 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, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:62. The ability of such DNAs and nucleic acid probes to specifically hybridize to a crystal protein-encoding gene sequence lends them particular utility in a variety of embodiments. Most importantly, the probes may be used in a variety of assays for detecting the presence of complementary sequences in a given sample.
In certain embodiments, it is advantageous to use oligonucleotide primers. The sequence of such primers is designed using a polynucleotide of the present invention for use in detecting, amplifying or mutating a defined segment of a crystal protein gene from B. thuringiensis using PCRTM technology. Segments of related crystal protein genes from other species may also be amplified by PCRTM using such primers.
To provide certain of the advantages in accordance with the present invention, a preferred nucleic acid sequence employed for hybridization studies or assays includes sequences that are complementary to at least a 14 to 30 or so long nucleotide stretch of a crystal protein-encoding sequence, such as that shown 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, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:62. A size of at least about 14 or so nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than about 14 or so bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of about 14 to about 20 or so nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCRTM technology of U.S. Pat. Nos. 4,683,195, and 4,683,202, herein incorporated by reference, or by excising selected DNA fragments from recombinant plasmids containing appropriate inserts and suitable restriction sites.
5.3 Expression Vectors
The present invention contemplates an expression vector comprising a polynucleotide of the present invention. Thus, in one embodiment an expression vector is an isolated and purified DNA molecule comprising a promoter operatively linked to an coding region that encodes a polypeptide of the present invention, which coding region is operatively linked to a transcription-terminating region, whereby the promoter drives the transcription of the coding region.
As used herein, the term “operatively linked” means that a promoter is connected to an coding region in such a way that the transcription of that coding region is controlled and regulated by that promoter. Means for operatively linking a promoter to a coding region are well known in the art.
In a preferred embodiment, the recombinant expression of DNAs 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 B. thuringiensis host cells being highly preferred. Promoters that function in bacteria are well-known in the art. An exemplary and preferred promoter for the Bacillus crystal proteins include any of the known crystal protein gene promoters, including the cryET31, cryET40, cryET43, cryET44, cryET45, cryET46, cryET47, cryET49, cryET51, cryET52, cryET53, cryET54, cryET55, cryET56, cryET57, cryET59, cryET60, cryET61, cryET62, cryET63, cryET64, cryET66, cryET67, cryET68, cryET72, cryET73, and cryET83 gene promoters. Alternatively, mutagenized or recombinant crystal protein-encoding gene promoters may be engineered by the hand of man and used to promote expression of the novel gene segments disclosed herein.
In an alternate embodiment, the recombinant expression of DNAs encoding the crystal proteins of the present invention is performed using a transformed Gram-negative bacterium such as an E. coli or Pseudomonas spp. host cell. Promoters which 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 an expression vector of the present invention is to be used to transform a plant, a promoter is selected that has the ability to drive expression in plants. Promoters that function in plants are also well known in the art. Useful in expressing the polypeptide in plants are promoters that are inducible, viral, synthetic, constitutive as described (Poszkowski et al., 1989; Odell et al., 1985), and temporally regulated, spatially regulated, and spatio-temporally regulated (Chau et al., 1989).
A promoter is also selected for its ability to direct the transformed plant cell's or transgenic plant's transcriptional activity to the coding region. Structural genes can be driven by a variety of promoters in plant tissues. Promoters can be near-constitutive, such as the CaMV 35S promoter, or tissue-specific or developmentally specific promoters affecting dicots or monocots.
Where the promoter is a near-constitutive promoter such as CaMV 35S, increases in polypeptide expression are found in a variety of transformed plant tissues (e.g., callus, leaf, seed and root). Alternatively, the effects of transformation can be directed to specific plant tissues by using plant integrating vectors containing a tissue-specific promoter.
An exemplary 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 (Le1) that is only expressed during seed maturation and accounts for about 2 to about 5% of total seed mRNA. The lectin gene and seed-specific promoter have been fully characterized and used to direct seed specific expression in transgenic tobacco plants (Vodkin et al., 1983; Lindstrom et al., 1990.)
An expression vector containing a coding region that encodes a polypeptide of interest is engineered to be under control of the lectin promoter and that vector is introduced into plants using, for example, a protoplast transformation method (Dhir et al., 1991). The expression of the polypeptide is directed specifically to the seeds 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 tissue specific promoter to produce a hybrid transgenic plant that shows the effects of transformation in more than one specific tissue.
Exemplary tissue-specific promoters are corn sucrose synthetase 1 (Yang et al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989), corn light harvesting complex (Simpson, 1986), corn heat shock protein (Odell et al., 1985), pea small subunit RuBP carboxylase (Poulsen et al., 1986; Cashmore et al., 1983), Ti plasmid mannopine synthase (Langridge et al., 1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petunia chalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein 1 (Keller et al., 1989), CaMV 35s transcript (Odell et al., 1985) and Potato patatin (Wenzler et al., 1989). Preferred promoters include a cauliflower mosaic virus (CaMV 35S) promoter, a S-E9 small subunit RuBP carboxylase promoter, a rice actin promoter, a maize histone promoter, a fused CaMV 35S-Arabidopsis histone promoter, a CaMV 35S promoter, a CaMV 19S promoter, a nos promoter, an Adh promoter, an actin promoter, a histone promoter, a ribulose bisphosphate carboxylase promoter, an R-allele promoter, a root cell promoter, an α-tubulin promoter, an ABA-inducible promoter, a turgor-inducible promoter, a rbcS promoter, a corn sucrose synthetase 1 promoter, a corn alcohol dehydrogenase 1 promoter, a corn light harvesting complex promoter, a corn heat shock protein promoter, a pea small subunit RuBP carboxylase promoter, a Ti plasmid mannopine synthase promoter, a Ti plasmid nopaline synthase promoter, a petunia chalcone isomerase promoter, a bean glycine rich protein 1 promoter, a CaMV 35s transcript promoter, a potato patatin promoter, a cab promoter, a PEP-Carboxylase promoter and an S-E9 small subunit RuBP carboxylase promoter.
The choice of which expression vector and ultimately to which promoter a polypeptide coding region is operatively linked depends directly on the functional properties desired, e.g., the location and timing of protein expression, and the host cell to be transformed. These are well known limitations inherent in the art of constructing recombinant DNA molecules. However, a vector useful in practicing the present invention is capable of directing the expression of the polypeptide coding region to which it is operatively linked.
Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described (Rogers et al., 1987). However, several other plant integrating vector systems are known to function in plants including pCaMVCN transfer control vector described (Fromm et al., 1985). Plasmid pCaMVCN (available from Pharmacia, Piscataway, N.J.) includes the cauliflower mosaic virus CaMV 35S promoter.
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. One preferred drug resistance marker is the gene whose expression results in kanamycin resistance; i.e., the chimeric gene containing the nopaline synthase promoter, Tn5 neomycin phosphotransferase II (nptII) and nopaline synthase 3′ non-translated region described (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 downstream of the polyadenylation site serve to terminate transcription. Those DNA sequences are referred to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA).
Means for preparing expression vectors are well known in the art. Expression (transformation vectors) used to transform plants and methods of making those vectors are described in U.S. Pat. Nos. 4,971,908, 4,940,835, 4,769,061 and 4,757,011, the disclosures of which are incorporated herein by reference. Those vectors can be modified to include a coding sequence in accordance with the present invention.
A variety of methods has been developed to operatively link DNA to vectors via complementary cohesive termini or blunt ends. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted and to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
A coding region that encodes a polypeptide having the ability to confer insecticidal activity to a cell is preferably a CryET31, CryET40, CryET43, CryET44, CryET45, CryET46, CryET47, CryET49, CryET51, CryET52, CryET53, CryET54, CryET55, CryET56, CryET57, CryET59, CryET60, CryET61, CryET62, CryET63, CryET64, CryET66, CryET67, CryET68, CryET72, CryET73, and CryET83 polypeptide-encoding gene.
5.7 Nomenclature of the Novel Polypeptides
The inventors have arbitrarily assigned the designation CryET31, CryET40, CryET43, CryET44, CryET45, CryET46, CryET47, CryET49, CryET51, CryET52, CryET53, CryET54, CryET56, CryET57, CryET59, CryET60, CryET61, CryET62, CryET63, CryET64, CryET66, CryET67, CryET68, CryET72, CryET73, and CryET83 to the polypeptides of this invention. Likewise, the arbitrary designations of cryET31, cryET40, cryET43, cryET44, cryET45, cryET46, cryET47, cryET49, cryET51, cryET52, cryET53, cryET54, cryET56, cryET57, cryET59, cryET60, cryET61, cryET62, cryET63, cryET64, cryET66, cryET67, cryET68, cryET72, cryET73, and cryET83 have been assigned to the novel nucleic acid sequence which encodes these polypeptides, respectively. 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 B. thuringiensis crystal proteins. The inventors contemplate that the arbitrarily assigned designations of the present invention will be superceded by the official nomenclature assigned to these sequences.
5.8 Transformed Host Cells and Transgenic Plants
Methods and compositions for transforming a bacterium, a yeast cell, a plant cell, or an entire plant with one or more expression vectors comprising a crystal protein-encoding gene segment are further aspects of this disclosure. A transgenic bacterium, yeast cell, plant cell or plant derived from such a transformation process or the progeny and seeds from such a transgenic plant are also further embodiments of the invention.
Means for transforming bacteria and yeast cells are well known in the art. Typically, means of transformation are similar to those well known means used to transform other bacteria or yeast such as E. coli or Saccharomyces cerevisiae. Methods for DNA transformation of plant cells include Agrobacterium-mediated plant transformation, protoplast transformation, gene transfer into pollen, injection into reproductive organs, injection into immature embryos and particle bombardment. Each of these methods has distinct advantages and disadvantages. Thus, one particular method of introducing genes into a particular plant strain may not necessarily be the most effective for another plant strain, but it is well known which 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 virtually any method by which DNA can be introduced into a cell, such as by Agrobacterium infection, direct delivery of DNA such as, for example, by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993), by desiccation/inhibition-mediated DNA uptake, by electroporation, by agitation with silicon carbide fibers, by acceleration of DNA coated particles, etc. In certain embodiments, acceleration methods are preferred and include, for example, microprojectile bombardment and the like.
Technology for introduction of DNA into cells is well-known to those of skill in the art. Four general methods for delivering a gene into cells have been described: (1) chemical methods (Graham and van der Eb, 1973; Zatloukal et al., 1992); (2) physical methods such as microinjection (Capecchi, 1980), electroporation (Wong and Neumann, 1982; Fromm et al., 1985; U.S. Pat. No. 5,384,253) and the gene gun (Johnston and Tang, 1994; Fynan et al., 1993); (3) viral vectors (Clapp, 1993; Lu et al., 1993; Eglitis and Anderson, 1988a; 1988b); and (4) receptor-mediated mechanisms (Curiel et al., 1991; 1992; Wagner et al., 1992).
5.8.3 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, thereby bypassing the need for regeneration of an intact plant from a protoplast. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art. See, for example, the methods described (Fraley et al., 1985; Rogers et al., 1987). Further, the integration of the Ti-DNA is a relatively precise process resulting in few rearrangements. The region of DNA to be transferred is defined by the border sequences, and intervening DNA is usually inserted into the plant genome as described (Spielmann et al., 1986; Jorgensen et al., 1987).
Modem Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations as described (Klee et al., 1985). Moreover, recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate construction of vectors capable of expressing various polypeptide coding genes. The vectors described (Rogers et al., 1987), have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes. In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant strains where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.
Agrobacterium-mediated transformation of leaf disks and other tissues such as cotyledons and hypocotyls appears to be limited to plants that Agrobacterium naturally infects. Agrobacterium-mediated transformation is most efficient in dicotyledonous plants. Few monocots appear to be natural hosts for Agrobacterium, although transgenic plants have been produced in asparagus using Agrobacterium vectors as described (Bytebier et al., 1987). Therefore, commercially important cereal grains such as rice, corn, and wheat must usually be transformed using alternative methods. However, as mentioned above, the transformation of asparagus using Agrobacterium can also be achieved (see, for example, Bytebier et al., 1987).
A transgenic plant formed using Agrobacterium transformation methods typically contains a single gene on one chromosome. Such transgenic plants can be referred to as being heterozygous for the added gene. However, inasmuch as use of the word “heterozygous” usually implies the presence of a complementary gene at the same locus of the second chromosome of a pair of chromosomes, and there is no such gene in a plant containing one added gene as here, it is believed that a more accurate name for such a plant is an independent segregant, because the added, exogenous gene segregates independently during mitosis and meiosis.
More preferred is a transgenic plant that is homozygous for the added structural gene; i.e., a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains a single added gene, germinating some of the seed produced and analyzing the resulting plants produced for enhanced carboxylase activity relative to a control (native, non-transgenic) or an independent segregant transgenic plant.
It is to be understood that two different transgenic plants can also be mated to produce offspring that contain two independently segregating added, exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both added, exogenous genes that encode a polypeptide of interest. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated.
Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, e.g., Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1985; Uchimiya et al., 1986; Callis et al., 1987; Marcotte et al., 1988).
Application of these systems to different plant strains depends upon the ability to regenerate that particular plant strain from protoplasts. Illustrative methods for the regeneration of cereals from protoplasts are described (Fujimura et al., 1985; Toriyama et al., 1986; Yamada et al., 1986; Abdullah et al., 1986).
5.8.4 Other Transformation Methods
Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, e.g., Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1985; Uchimiya et al., 1986; Callis et al., 1987; Marcotte et al., 1988).
Illustrative methods for the regeneration of cereals from protoplasts are described (Fujimura et al., 1985; Toriyama et al., 1986; Yamada et al., 1986; Abdullah et al., 1986).
5.8.5 Gene Expression in Plants
Although great progress has been made in recent years with respect to preparation of transgenic plants which express bacterial proteins such as B. thuringiensis crystal proteins, the results of expressing native bacterial genes in plants are often disappointing. In recent years, however, several potential factors have been implicated as responsible in varying degrees for the level of protein expression from a particular coding sequence. For example, scientists now know that maintaining a significant level of a particular mRNA in the cell is indeed a critical factor. Unfortunately, the causes for low steady state levels of mRNA encoding foreign proteins are many. First, full length RNA synthesis may not occur at a high frequency. This could, for example, be caused by the premature termination of RNA during transcription or due to unexpected mRNA processing during transcription. Second, full length RNA may be produced in the plant cell, but then processed (splicing, polyA addition) in the nucleus in a fashion that creates a nonfunctional mRNA. If the RNA is not properly synthesized, terminated and polyadenylated, it cannot move to the cytoplasm for translation. Similarly, in the cytoplasm, if mRNAs have reduced half lives (which are determined by their primary or secondary sequence) insufficient protein product will be produced. In addition, there is an effect, whose magnitude is uncertain, of translational efficiency on mRNA half-life. In addition, every RNA molecule folds into a particular structure, or perhaps family of structures, which is determined by its sequence. The particular structure of any RNA might lead to greater or lesser stability in the cytoplasm. Structure per se is probably also a determinant of mRNA processing in the nucleus. It is likely that dramatically changing the sequence of an RNA will have a large effect on its folded structure It is likely that structure per se or particular structural features also have a role in determining RNA stability.
To overcome these limitations in foreign gene expression, researchers have identified particular sequences and signals in RNAs that have the potential for having a specific effect on RNA stability. In certain embodiments of the invention, therefore, there is a desire to optimize expression of the disclosed nucleic acid segments in planta. One particular method of doing so, is by alteration of the bacterial gene to remove sequences or motifs which decrease expression in a transformed plant cell. The process of engineering a coding sequence for optimal expression in planta is often referred to as “plantizing” a DNA sequence.
Particularly problematic sequences are those which are A+T rich. Unfortunately, since B. thuringiensis has an A+T rich genome, native crystal protein gene sequences must often be modified for optimal expression in a plant. The sequence motif ATTTA (or AUUUA as it appears in RNA) has been implicated as a destabilizing sequence in mammalian cell mRNA (Shaw and Kamen, 1986). Many short lived mRNAs have A+T rich 3′ untranslated regions, and these regions often have the ATTTA sequence, sometimes present in multiple copies or as multimers (e.g., ATTTATTTA . . . ). Shaw and Kamen showed that the transfer of the 3′ end of an unstable mRNA to a stable RNA (globin or VA 1) decreased the stable RNA's half life dramatically. They further showed that a pentamer of ATTTA 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 also appear to be important in determining whether they function as destabilizing sequences. Shaw and Kamen showed that a trimer of ATTTA had much less effect than a pentamer on mRNA stability and a dimer or a monomer had no effect on stability (Shaw and Kamen, 1987). Note that multimers of ATTTA such as a pentamer automatically create an A+T rich region. This was shown to be a cytoplasmic effect, not nuclear. In other unstable mRNAs, the ATTTA sequence may be present in only a single copy, but it is often contained in an A+T rich region. From the animal cell data collected to date, it appears that ATTTA at least in some contexts is important in stability, but it is not yet possible to predict which occurrences of ATTTA are destabiling elements or whether any of these effects are likely to be seen in plants.
Some studies on mRNA degradation in animal cells also indicate that RNA degradation may begin in some cases with nucleolytic attack in A+T rich regions. It is not clear if these cleavages occur at ATTTA sequences. There are also examples of mRNAs that have differential stability depending on the cell type in which they are expressed or on the stage within the cell cycle at which they are expressed. For example, histone mRNAs are stable during DNA synthesis but unstable if DNA synthesis is disrupted. The 3′ end of some histone mRNAs seems to be responsible for this effect (Pandey and Marzluff, 1987). It does not appear to be mediated by ATTTA, nor is it clear what controls the differential stability of this mRNA. Another example is the differential stability of IgG mRNA in B lymphocytes during B cell maturation (Genovese and Milcarek, 1988). A final example is the instability of a mutant β-thallesemic globin mRNA. In bone marrow cells, where this gene is normally expressed, the mutant mRNA is unstable, while the wild-type mRNA is stable. When the mutant gene is expressed in HeLa or L cells in vitro, the mutant mRNA shows no instability (Lim et al., 1988). These examples all provide evidence that mRNA stability can be mediated by cell type or cell cycle specific factors. Furthermore 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 may act differentially depending on the nature of the cell in which the RNA is present. Shaw and Kamen (1987) have reported that activation of protein kinase C can block degradation mediated by ATTTA.
The addition of a polyadenylate string to the 3′ end is common to most eukaryotic mRNAs, both plant and animal. The currently accepted view of polyA addition is that the nascent transcript extends beyond the mature 3′ terminus. Contained within this transcript are signals for polyadenylation and proper 3′ end formation. This processing at the 3′ end involves cleavage of the mRNA and addition of polyA to the mature 3′ end. By searching for consensus sequences near the polyA tract in both plant and animal mRNAs, it has been possible to identify consensus sequences that apparently are involved in polyA addition and 3′ end cleavage. The same consensus sequences seem to be important to both of these processes. These signals are typically a variation on the sequence AATAAA. In animal cells, some variants of this 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 of these consensus sequences are variations on AATAAA, they all are A+T rich sequences. This sequence is typically found 15 to 20 bp before the polyA tract in a mature mRNA. Studies in animal cells indicate that this sequence is involved in both polyA addition and 3′ maturation. Site directed mutations in this sequence can disrupt 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 polyA signal are also required; i.e., a gene that has a normal AATAAA but has been replaced or disrupted downstream does not get properly polyadenylated (Gil and Proudfoot, 1984; Sadofsky and Alwine, 1984; McDevitt et al., 1984). That is, the polyA signal itself is not sufficient for complete and proper processing. It is not yet known what specific downstream sequences are required in addition to the polyA signal, or if there is a specific sequence that has this function. Therefore, sequence analysis can only identify potential polyA signals.
In naturally occurring mRNAs that are normally polyadenylated, it has been observed that disruption of this process, either by altering the polyA signal or other sequences in the mRNA, profound effects can be obtained in the level of functional mRNA. This has been observed in several naturally occurring mRNAs, with results that are gene-specific so far.
It has been shown that in natural mRNAs proper polyadenylation is important in mRNA accumulation, and that disruption of this process can effect mRNA levels significantly. However, insufficient knowledge exists to predict the effect of changes in a normal gene. In a heterologous gene, it is 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 proper polyA sites, but instead function as aberrant sites that give rise to unstable mRNAs.
In animal cell systems, AATAAA is by far the most common signal identified in mRNAs upstream of the polyA, but at least four variants have also been found (Wickens and Stephenson, 1984). In plants, not nearly so much analysis has been done, but it is clear that multiple sequences similar to AATAAA can be used. The plant sites in Table 2 called major or minor refer only to the study of Dean et al. (1986) which analyzed only three 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 hard to predict with any certainty that a site designated major or minor is more or less likely to function partially or completely when found in a heterologous gene such as those encoding the crystal proteins of the present invention.







TABLE 2





POLYADENYLATION SITES IN PLANT GENES


















PA
AATAAA
Major consensus site



 


P1A
AATAAT
Major plant site


 


P2A
AACCAA
′′


 


P3A
ATATAA
′′


 


P4A
AATCAA
′′


 


P5A
ATACTA
′′


 


P6A
ATAAAA
′′


 


P7A
ATGAAA
′′


 


P8A
AAGCAT
′′


 


P9A
ATTAAT
′′


 


P10A
ATACAT
′′


 


P11A
AAAATA
′′


 


P12A
ATTAAA
Minor animal site


 


P13A
AATTAA
′′


 


P14A
AATACA
′′


 


P15A
CATAAA
′′









The present invention provides a method for preparing synthetic plant genes which genes express their protein product at levels significantly higher than the wild-type genes which were commonly employed in plant transformation heretofore. In another aspect, the present invention also provides novel synthetic plant genes which encode non-plant proteins.
As described above, the expression of native B. thuringiensis genes in plants is often problematic. The nature of the coding sequences of B. thuringiensis genes distinguishes them from plant genes as well as many other heterologous genes expressed in plants. In particular, B. thuringiensis genes are very rich ( ̃62%) in adenine (A) and thymine (T) while plant genes and most other bacterial genes which have been expressed in plants are on the order of 45-55% A+T.
Due to the degeneracy of the genetic code and the limited number of codon choices for any amino acid, most of the “excess” A+T of the structural coding sequences of some Bacillus species are found in the third position of the codons. That is, genes of some Bacillus species have A or T as the third nucleotide in many codons. Thus A+T content in part can determine codon usage bias. 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 from one organism, where they may play no role except to code for a particular stretch of amino acids, have the potential to be recognized as gene control elements in another organism (such as transcriptional promoters or terminators, polyA addition sites, intron splice sites, or specific mRNA degradation signals). It is perhaps surprising that such misread signals are not a more common feature of heterologous gene expression, but this can be explained in part by the relatively homogeneous A+T content ( ̃50%) of many organisms. This A+T content plus the nature of the genetic code put clear constraints on the likelihood of occurrence of any particular oligonucleotide sequence. Thus, a gene from E. coli with a 50% A+T content is much less likely to contain any particular A+T rich segment than a gene from B. thuringiensis. 
Typically, to obtain high-level expression of the S-endotoxin genes in plants, existing structural coding sequence (“structural gene”) which codes for the S-endotoxin are modified by removal of ATTTA sequences and putative polyadenylation signals by site directed mutagenesis of the DNA comprising the structural gene. It is most preferred that substantially all the polyadenylation signals and ATTTA sequences are removed although enhanced expression levels are observed with only partial removal of either of the above identified sequences. Alternately if a synthetic gene is prepared which codes for the expression of the subject protein, 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, AATTAA, AATACA and CATAAA. In replacing the ATTTA sequences and polyadenylation signals, codons are preferably utilized which avoid the codons which are rarely found in plant genomes.
The selected DNA sequence is scanned to identify regions with greater than four consecutive adenine (A) or thymine (T) nucleotides. The A+T regions are scanned for potential plant polyadenylation signals. Although the absence of five or more consecutive A or T nucleotides eliminates most plant polyadenylation signals, if there are 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 about 15 to about 30 or so nucleotide residues surrounding the A+T rich region identified in step one. If the A+T content of the surrounding region is less than 80%, the region should be examined for polyadenylation signals. Alteration of the region based on polyadenylation signals is dependent upon (1) the number of polyadenylation signals present and (2) presence of a major plant polyadenylation signal.
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 which are also removed by mutagenesis.
It is also preferred that regions comprising many consecutive A+T bases or G+C bases are disrupted since these regions are predicted to have a higher likelihood to form hairpin structure due to self-complementarity. Therefore, insertion of heterogeneous base pairs would reduce the likelihood of self-complementary secondary structure formation which are known to inhibit transcription and/or translation in some organisms. In most cases, the adverse effects may be minimized by using sequences which do not contain more than five consecutive A+T or G+C.
5.8.6 Synthetic Oligonculeotides for Mutagenesis
When oligonucleotides are used in the mutagenesis, it is desirable to maintain the proper amino acid sequence and reading frame, without introducing common restriction sites such as BglII, HindIII, SacI, KpnI, EcoRI, NcoI, PstI and SalI into the modified gene. These restriction sites are found in poly-linker insertion sites of many cloning vectors. Of course, the introduction of new polyadenylation signals, ATTTA sequences or consecutive stretches of more than five A+T or G+C, should also be avoided. The preferred size for the oligonucleotides is about 40 to about 50 bases, but fragments ranging from about 18 to about 100 bases have been utilized. In most cases, a minimum of about 5 to about 8 base pairs of homology to the template DNA on both ends of the synthesized fragment are maintained to insure proper hybridization of the primer to the template. The oligonucleotides should avoid sequences longer than five base pairs A+T or G+C. Codons used in the replacement of wild-type codons should preferably avoid the TA or CG doublet wherever possible. Codons are selected from a plant preferred codon table (such as Table 3 below) so as to avoid codons which are rarely found in plant genomes, and efforts should be made to select codons to preferably adjust the G+C content to about 50%.
Regions with many consecutive A+T bases or G+C bases are predicted to have a higher likelihood to form hairpin structures due to self-complementarity. Disruption of these regions by the insertion of heterogeneous base pairs is preferred and should reduce the likelihood of the formation of self-complementary secondary structures such as hairpins which are known in some organisms to inhibit transcription (transcriptional terminators) and translation (attenuators).
Alternatively, a completely synthetic gene for a given amino acid sequence can be prepared, with regions of five or more consecutive A+T or G+C nucleotides being avoided. Codons are selected avoiding the TA and CG doublets in codons whenever possible. Codon usage can be normalized against a plant preferred codon usage table (such as Table 3) and the G+C content preferably adjusted to about 50%. The resulting sequence should be examined to ensure that there are minimal putative plant polyadenylation signals and ATTTA sequences.







TABLE 3







PREFERRED CODON USAGE IN PLANTS











Amino Acid
Codon
Percent Usage in Plants















ARG
CGA
7




CGC
11




CGG
5




CGU
25




AGA
29




AGG
23



SER
UCA
14




UCC
26




UCG
3




UCU
21




AGC
21




AGU
15



THR
ACA
21




ACC
41




ACG
7




ACU
31



PRO
CCA
45




CCC
19




CCG
9




CCU
26



HIS
CAC
65




CAU
35



GLU
GAA
48




GAG
52



ASP
GAC
48




GAU
52



TYR
UAC
68




UAU
32



CYS
UGC
78




UGU
22



LEU
CUA
8




CUC
20




CUG
10




CUU
28




UUA
5




UUG
30



ALA
GCA
23




GCC
32




GCG
3




GCU
41



GLY
GGA
32




GGC
20




GGG
11




GGU
37



ILE
AUA
12




AUC
45




AUU
43



VAL
GUA
9




GUC
20




GUG
28




GUU
43



LYS
AAA
36




AAG
64



ASN
AAC
72




AAU
28



GLN
CAA
64




CAG
36



PHE
UUC
56




UUU
44



MET
AUG
100



TRP
UGG
100










Restriction sites found in commonly used cloning vectors are also preferably avoided. However, placement of several unique restriction sites throughout the gene is useful for analysis of gene expression or construction of gene variants.
5.8.7 “Plantized” Gene Constructs
The expression of a plant gene which exists in double-stranded DNA form involves transcription of messenger RNA (mRNA) from one strand of the DNA by RNA polymerase enzyme, and the subsequent processing of the mRNA primary transcript inside the nucleus. This processing involves a 3′ non-translated region which adds polyadenylate nucleotides to the 3′ end of the RNA. Transcription of DNA into mRNA is regulated by a region of DNA usually referred to as the “promoter.” The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA and to initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding strand of RNA.
A number of promoters which are active in plant cells have been described in the literature. These include the nopaline synthase (NOS) and octopine synthase (OCS) promoters (which are carried on tumor-inducing plasmids of A. tumefaciens), the Cauliflower Mosaic Virus (CaMV) 19S and 35S promoters, the light-inducible promoter from the small subunit of ribulose bis-phosphate carboxylase (ssRUBISCO, a very abundant plant polypeptide) and the mannopine synthase (MAS) promoter (Velten et al., 1984; Velten and Schell, 1985). All of these promoters have been used to create various types of DNA constructs which have been expressed in plants (see e.g., Intl. Pat. Appl. Publ. Ser. No. WO 84/02913).
Promoters which are known or are found to cause transcription of RNA in plant cells can be used in the present invention. Such promoters may be obtained from plants or plant viruses and include, but are not limited to, the CaMV35S promoter and promoters isolated from plant genes such as ssRUBISCO genes. As described below, it is preferred that the particular promoter selected should be capable of causing sufficient expression to result in the production of an effective amount of protein.
The promoters used in the DNA constructs (i.e. chimeric plant genes) of the present invention may be modified, if desired, to affect their control characteristics. For example, the CaMV35S promoter may be ligated to the portion of the ssRUBISCO gene that represses the expression of ssRUBISCO in the absence of light, to create a promoter which is active in leaves but not in roots. The resulting chimeric promoter may be used as described herein. For purposes of this description, the phrase “CaMV35S” promoter thus includes variations of CaMV35S promoter, e.g., promoters derived by means of ligation with operator regions, random or controlled mutagenesis, etc. Furthermore, the promoters may be altered to contain multiple “enhancer sequences” to assist in elevating gene expression.
The RNA produced by a DNA construct of the present invention also contains a 5′ non-translated leader sequence. This sequence can be derived from the promoter selected to express the gene, and can be specifically modified so as to increase translation of the mRNA. The 5′ non-translated regions can also be obtained from viral RNA's, from suitable eukaryotic genes, or from a synthetic gene sequence. The present invention is not limited to constructs, as presented in the following examples. Rather, the non-translated leader sequence can be part of the 5′ end of the non-translated region of the coding sequence for the virus coat protein, or part of the promoter sequence, or can be derived from an unrelated promoter or coding sequence. In any case, it is preferred that the sequence flanking the initiation site conform to the translational consensus sequence rules for enhanced translation initiation reported by Kozak (1984).
The cry DNA constructs of the present invention may also contain one or more modified or fully-synthetic structural coding sequences which have been changed to enhance the performance of the cry gene in plants. The structural genes of the present invention may optionally encode a fusion protein comprising an amino-terminal chloroplast transit peptide or secretory signal sequence.
The DNA construct also contains a 3′ non-translated region. The 3′ non-translated region contains a polyadenylation signal which functions in plants to cause the addition of polyadenylate nucleotides to the 3′ end of the viral RNA. Examples of suitable 3′ regions are (1) the 3′ transcribed, non-translated regions containing the polyadenylation signal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopaline synthase (NOS) gene, and (2) plant genes like the soybean storage protein (7S) genes and the small subunit of the RuBP carboxylase (E9) gene.
5.9 Methods for Producing Insect-resistant Transgenic Plants
By transforming a suitable host cell, such as a plant cell, with a recombinant cryET31, cryET40, cryET43, cryET44, cryET45, cryET46, cryET47, cryET49, cryET51, cryET52, cryET53, cryET54, cryET56, cryET57, cryET59, cryET60, cryET61, cryET62, cryET63, cryET64, cryET66, cryET67, cryET68, cryET72, cryET73, and cryET83 gene-containing segment, the expression of the encoded crystal protein (i.e., a bacterial crystal protein or polypeptide having insecticidal activity against coleopterans) can result in the formation of insect-resistant plants.
By way of example, one may utilize an expression vector containing a coding region for a B. thuringiensis crystal protein and an appropriate selectable marker to transform a suspension of embryonic plant cells, such as wheat or corn cells using a method such as particle bombardment (Maddock et al., 1991; Vasil et al., 1992) to deliver the DNA coated on microprojectiles into the recipient cells. Transgenic plants are then regenerated from transformed embryonic calli that express the insecticidal proteins.
The formation of transgenic plants may also be accomplished using other methods of cell transformation which 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; Luo et al., 1988), by injection of the 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 desiccated embryos (Neuhaus et al., 1987; Benbrook et al., 1986).
The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach and Weissbach, 1988). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.
The development or regeneration of plants containing the foreign, exogenous gene that encodes a polypeptide of interest introduced by Agrobacterium from leaf explants can be achieved by methods well known in the art such as described (Horsch et al., 1985). In this procedure, transformants are cultured in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant strain 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. Shoots that rooted in the presence of the selective agent to form plantlets are then transplanted to soil or other media to allow the production of roots. These procedures vary depending upon 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 discussed before. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important, preferably inbred lines. Conversely, pollen from plants of those important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art.
A transgenic plant of this invention thus has an increased amount of a coding region (e.g., a cryET31, cryET40, cryET43, cryET44, cryET45, cryET46, cryET47, cryET49, cryET51, cryET52, cryET53, cryET54, cryET56, cryET57, cryET59, cryET60, cryET61, cryET62, cryET63, cryET64, cryET66, cryET67, cryET68, cryET72, cryET73, and cryET83 gene) that encodes one or more CryET31, CryET40, CryET43, CryET44, CryET45, CryET46, CryET47, CryET49, CryET51, CryET52, CryET53, CryET54, CryET56, CryET57, CryET59, CryET60, CryET61, CryET62, CryET63, CryET64, CryET66, CryET67, CryET68, CryET72, CryET73, and CryET83 polypeptides. 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 of its offspring on sexual mating. Seed from a transgenic plant may be grown in the field or greenhouse, and resulting sexually mature transgenic plants are self-pollinated to generate true breeding plants. The progeny from these plants become true breeding lines that are evaluated for, by way of example, increased insecticidal capacity 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 turf grasses, wheat, corn, rice, barley, oats, a variety of ornamental plants and vegetables, as well as a number of nut- and fruit-bearing trees and plants.
5.10 Definitions
The following words and phrases have the meanings set forth 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.
Identity or percent identity: refers to the degree of similarity between two nucleic acid or protein sequences. An alignment of the two sequences is performed by a suitable computer program. A widely used and accepted computer program for performing sequence alignments is CLUSTALW v1.6 (Thompson, et al. Nuc. Acids Res., 22: 4673-4680, 1994). The number of matching bases or amino acids is divided by the total number of bases or amino acids, and multiplied by 100 to obtain a percent identity. For example, if two 580 base pair sequences had 145 matched bases, they would be 25 percent identical. If the two compared sequences are of different lengths, the number of matches is divided by the shorter of the two lengths. For example, if there were 100 matched amino acids between 200 and a 400 amino acid proteins, they are 50 percent identical with respect to the shorter sequence. If the shorter sequence is less than 150 bases or 50 amino acids in length, the number of matches are divided by 150 (for nucleic acid bases) or 50 (for amino acids), and multiplied by 100 to obtain a percent identity.
Promoter: A recognition site on a DNA sequence or group of DNA sequences that provide an expression control element for a structural gene and to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene.
Regeneration: The process of growing a plant from a plant cell (e.g., plant protoplast or explant).
Structural gene: A polynucleotide sequence that encodes a polypeptide, that is expressed to produce a polypeptide, or which is cryptic or incapable of expression in its natural host cell but which can be isolated and purified and operably linked to at least a promoter functional in one or more host cell types to express the encoded polypeptide.
Transformation: A process of introducing an exogenous DNA sequence (e.g., 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 the introduction of an exogenous DNA molecule into that cell.
Transgenic cell: Any cell derived or regenerated from a transformed cell or derived from a transgenic cell. Exemplary transgenic cells include plant calli derived from a transformed plant cell and particular cells such as leaf, root, stem, e.g., somatic cells, or reproductive (germ) cells obtained from a transgenic plant.
Transgenic plant: A plant or progeny thereof derived from a transformed plant cell or protoplast, wherein the plant DNA contains an introduced exogenous DNA molecule not originally present in a native, non-transgenic plant of the same strain. The terms “transgenic plant” and “transformed plant” have sometimes been used in the art as synonymous terms to define a plant whose DNA contains an exogenous DNA molecule. However, it is thought more scientifically correct to refer to a regenerated plant or callus obtained from a transformed plant cell or protoplast as being a transgenic plant, and that usage will be followed herein.
Vector: A DNA molecule capable of replication in a host cell and/or to which another DNA segment can be operatively linked so as to bring about replication of the attached segment. A plasmid is an exemplary vector.
5.11 Isolating Homologous Gene and Gene Fragments
The genes and δ-endotoxins according to the subject invention include not only the full length sequences disclosed herein but also fragments of these sequences, or fusion proteins, which retain the characteristic insecticidal activity of the sequences specifically exemplified herein.
It should be apparent to a person skill in this art that insecticidal δ-endotoxins can be identified and obtained through several means. The specific genes, or portions thereof, may be obtained from a culture depository, or constructed synthetically, for example, by use of a gene machine. Variations of these genes may be readily constructed using standard techniques for making point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Also, genes which code for active fragments may be obtained using a variety of other restriction enzymes. Proteases may be used to directly obtain active fragments of these δ-endotoxins.
Equivalent δ-endotoxins and/or genes encoding these equivalent δ-endotoxins can also be isolated from Bacillus strains and/or DNA libraries using the teachings provided herein. For example, antibodies to the δ-endotoxins disclosed and claimed herein can be used to identify and isolate other δ-endotoxins from a mixture of proteins. Specifically, antibodies may be raised to the portions of the δ-endotoxins which are most constant and most distinct from other B. thuringiensis δ-endotoxins. These antibodies can then be used to specifically identify equivalent δ-endotoxins with the characteristic insecticidal activity by immunoprecipitation, enzyme linked immunoassay (ELISA), or Western blotting.
A further method for identifying the δ-endotoxins and genes of the subject invention is through the use of oligonucleotide probes. These probes are nucleotide sequences having a detectable label. As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming a strong bond between the two molecules, it can be reasonably assumed that the probe and sample are essentially identical. The probe's detectable label provides a means for determining in a known manner whether hybridization has occurred. Such a probe analysis provides a rapid method for identifying formicidal δ-endotoxin genes of the subject invention.
The nucleotide segments which are used as probes according to the invention can be synthesized by use of DNA synthesizers using standard procedures. In the use of the nucleotide segments as probes, the particular probe is labeled with any suitable label known to those skilled in the art, including radioactive and non-radioactive labels. Typical radioactive labels include 32P, 125I, 35S, or the like. A probe labeled with a radioactive isotope can be constructed from a nucleotide sequence complementary to the DNA sample by a conventional nick translation reaction, using a DNase and DNA polymerase. The probe and sample can then be combined in a hybridization buffer solution and held at an appropriate temperature until annealing occurs. Thereafter, the membrane is washed free of extraneous 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, or the various chemiluminescers such as luciferin, or fluorescent compounds like fluorescein and its derivatives. The probe may also be labeled at both ends with different types of labels for ease of separation, as, for example, by using an isotopic label at the end mentioned above and a biotin label at the other end.
Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid, and, as noted above, a certain degree of mismatch can be tolerated. Therefore, the probes of the subject invention include mutations (both single and multiple), deletions, insertions of the described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given polynucleotide sequence in many ways, by methods currently known to an ordinarily skilled artisan, and perhaps by other methods which may become known in the future.
The potential variations in the probes listed is due, in part, to the redundancy of the genetic code. Because of the redundancy of the genetic code, i.e., more than one coding nucleotide triplet (codon) can be used for most of the amino acids used to make proteins. Therefore different nucleotide sequences can code for a particular amino acid. Thus, the amino acid sequences of the B. thuringiensis δ-endotoxins and peptides can be prepared by equivalent nucleotide sequences encoding the same amino acid sequence of the protein or peptide. Accordingly, the subject invention includes such equivalent nucleotide sequences. Also, inverse or complement sequences are an aspect of the subject invention and can be readily used by a person skilled in this art. In addition it has been shown that proteins of identified structure and function may be constructed by changing the amino acid sequence if such changes do not alter the protein secondary structure (Kaiser and Kezdy, 1984). Thus, the subject invention includes mutants of the amino acid sequence depicted herein which do not alter the protein secondary structure, or if the structure is altered, the biological activity is substantially retained. Further, the invention also includes mutants of organisms hosting all or part of a δ-endotoxin encoding a gene of the invention. Such mutants can be made by techniques well known to persons skilled in the art. For example, UV irradiation can be used to prepare mutants of host organisms. Likewise, such mutants may include asporogenous host cells which also can be prepared by procedures well known in the art.
6.0 EXAMPLES
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
6.1 Example 1
Identification of B. Thuringiensis Strains Containing Novel δ-Endotoxins
Wild-type B. thuringiensis strains containing novel insecticidal protein genes were identified by Southern blot hybridization studies employing specific DNA probes. Twenty-four unique cry genes were discovered that are related to B. thuringiensis genes in the cry1, cry2, or cry9 classes of toxin genes.
Various methods were employed to clone the novel genes and express them in a crystal protein-negative (Cry−) strain of B. thuringiensis. These methods include PCRTM amplification of the region of cry1-related genes that encodes the active portion of the toxin gene. The PCRTM product is then joined to a fragment from the cry1Ac gene encoding the C-terminal region of the protoxin. This gene fusion was then expressed in a B. thuringiensis recombinant strain to produce a hybrid protoxin. In this instance, it is recognized that the sequence of the amplified DNA can be used to design hybridization probes to isolate the entire coding sequence of the novel cry gene from the wild-type B. thuringiensis strain.
Wild-type B. thuringiensis strains were screened in a bioassay to identify strains that are toxic to larvae of lepidopteran insects (procedure described in Example 10). Active strains were then examined genetically to determine if they contain novel toxin genes. The method used to make this determination is described below and includes isolation of genomic DNA from the B. thuringiensis strain, restriction enzyme digestion, Southern blot hybridization, and analysis of the hybridizing restriction fragments to determine which genes are present in a strain.
Total genomic DNA was extracted by the following procedure. Vegetative cells were resuspended in a lysis buffer 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 h. Following incubation, the suspension was extracted once with an equal volume of phenol, then once with an equal volume of phenol:chloroform:isoamyl alcohol (50:48:2), and once with an equal volume of chloroform:isoamyl alcohol (24:1). The DNA was precipitated from the aqueous phase by the addition of one-tenth volume 3 M sodium acetate and two volumes of 100% ethanol. The precipitated DNA was collected by centrifugation, washed with 70% ethanol and resuspended in distilled water.
The DNA samples were digested with the restriction enzymes ClaI and PstI. The combination of these two enzymes give a digestion pattern of fragments that, when hybridized with the probe: wd207 (described below), allows the identification of many of the known cry1-related toxin genes. Hybridizing fragments that did not correspond to the fragment sizes expected for the known genes were classified as unknown and were candidates for cloning and characterization.
The digested DNA was size fractionated by electrophoresis through a 1.0% agarose gel in 1×TBE (0.089 M Tris-borate, 0.089 M boric acid, 0.002 M EDTA) overnight at 2 V/cm of gel length. The fractionated DNA fragments were then transferred to a Millipore Immobilon-NC® nitrocellulose filter (Millipore Corp., Bedford, Mass.) according to the method of Southern (1975). The DNA fragments were fixed to the nitocellulose by baking the filter at 80° C. in a vacuum oven.
To identify the DNA fragment(s) containing the sequences related to cry1 genes, the oligonucleotide wd207 was radioactively labeled at the 5′ end and used as a hybridization probe. To radioactively label the probe, 1-5 pmoles of wd207 were added to a reaction (20 ul total volume) containing 3 ul [γ-32P]ATP (3,000 Ci/mmole at 10 mCi/ml), 70 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 5 mM DTT, and 10 units T4 polynucleotide kinase (Promega Corp., Madison, Wis.). The reaction was incubated for 20 min at 37° C. to allow the transfer of the radioactive phosphate to the 5′-end of the oligonucleotide, thus making it useful as a hybridization probe.
The oligonucleotide probe used in this analysis, designated wd207, has the following sequence:








(SEQ ID NO:51)









5′-TGGATACTTGATCAATATGATAATCCGTCACATCTGTTTTTA-3′







This oligonucleotide was designed to specifically hybridize to a conserved region of cry1 genes downstream from the proteolyic activation site in the protoxin. Table 4 lists some of the B. thuringiensis toxin genes and their identities with wd207. The orientation of the wd207 sequence is inverted and reversed relative to the coding sequences of the cry genes.









TABLE 4





cry Gene
% Identity to wd207
Nucleotide Position in coding sequence







cry1Aa
 100%
1903-1944


cry1Ba
95.2%
1991-2032


cry1Ca
97.6%
1930-1971


cry1Da
97.6%
1858-1899


cry1Ea
97.6%
1885-1926









The labeled probe was then incubated with the nitrocellulose filter overnight at 45° C. in 3×SSC (1×SSC=0.15 M NaCl, 0.015 M sodium citrate), 0.1% SDS, 10× Denhardt's reagent (0.2% BSA, 0.2% polyvinylpyrrolidone, 0.2% Ficoll), and 0.2 mg/ml heparin. Following this incubation period, the filter was washed in several changes of 3×SSC, 0.1% SDS at 45° C. The filter was blotted dry and exposed to Kodak X-OMAT AR X-ray film (Eastman Kodak Co., Rochester, N.Y.) overnight at −70° C. with an intensifying screen to obtain an autoradiogram.
The autoradiograms were analyzed to determine which wild-type B. thuringiensis strains contained cry1 genes that could be novel. Since the probe was only 42 nucleotides, it is unlikely that recognition sites for the restriction endonucleases ClaI and PstI would occur within the hybridizing region of the cry1-related genes. Therefore, it was assumed that each hybridizing restriction fragment represented one cry1-related gene. The sizes, in kilobases (kb), of the hybridizing restriction fragments were determined based on the migration of the fragment in the agarose gel relative to DNA fragments of known size. The size of a fragment could be used to determine if that fragment represented a known cry1 gene. For example, from the DNA sequence of the cry1Ac gene it was known that wd207 would hybridize to a 0.43 kb fragment after digestion of cry1Ac DNA with ClaI and PstI. If the Southern blot analysis of a strain showed a 0.43 kb hybrizing fragment, that strain was assigned a probable genotype of cry1Ac. Fragments that could not be easily assigned a probable genotype were selected as candidates for further analysis. Because many cry1-containing strains have more than one cry1-related gene, all fragments were given a putative designation.







TABLE 5







SUMMARY OF GENES AND PROTEINS
















Polypeptide
Polypeptide
Polynucleotide
WT-
Recomb.
Gene
Cloning
DNA
Cloning



Designation
Seq. ID No.:
Seq ID No.:
Strain
Strain
Family
Method1
Probe2
Vector
Plasmid



















Cry ET31
2
1
EG6701
EG11562
cry2
MboI
cry2a
pHT315
pEG1331


Cry ET40
4
3
EG5476
EG11901
cry1
PCR TM
—
pEG1064
pEG1901


Cry ET43
6
5
EG2878
EG7692
cry1
PCR TM
—
pEG1064
pEG1806


Cry ET44
8
7
EG3114
EG11629
cry1
PCR TM
—
pEG1064
pEG1807


Cry ET45
10
9
EG3114
EG7694
cry1
PCR TM
—
pEG1064
pEG1808


Cry ET46
12
11
EG6451
EG7695
cry1
PCR TM
—
pEG1064
pEG1809


Cry ET47
14
13
EG6451
EG7696
cry1
PCR TM
—
pEG1064
pEG1810


Cry ET49
16
15
EG6451
EG11630
cry1
PCR TM
—
pEG1064
pEG1812


Cry ET51
18
17
EG5391
EG11921
cry1
MboI
wd207
pHT315
pEG1912


Cry ET52
20
19
EG10475
EG11584
cry1
BamHI
wd207
pEG290
pEG1340


Cry ET53
22
21
EG3874
EG11906
cry1
MboI
cry1Aa
pHT315
pEG1904


Cry ET54


EG3874
EG11907
cry1
MboI
cry1Aa
pHT315
pEG1905


Cry ET56
24
23
EG3874
EG11909
cry1
MboI
cry1Aa
pHT315
pEG1907


Cry ET57
26
25
EG3874
EG11910
cry1
MboI
cry1Aa
pHT315
pEG1908


Cry ET59
28
27
EG9290
EG12102
cry9
MboI
pr56,
pHT315
pEG945









cryET59


Cry ET60
30
29
EG9290
EG12103
cry9
MboI
pr56,
pHT315
pEG946









cryET59


Cry ET61
32
31
EG4612
EG11634
cry1
MboI
wd207
pHT315
pEG1813


Cry ET62
34
33
EG6831
EG11635
cry1
MboI
wd207
pHT315
pEG1814


Cry ET63
36
35
EG4623
EG11636
cry1
MboI
wd207
pHT315
pEG1815


Cry ET64
38
37
EG4612
EG11638
cry1
MboI
wd207
pHT315
pEG1816


Cry ET66
40
39
EG5020
EG11640
cry1
MboI
wd207
pHT315
pEG1817


Cry ET67
42
41
EG4869
EG11642
cry1
MboI
wd207
pHT315
pEG1818


Cry ET68
44
43
EG5020
EG11644
cry1
MboI
wd207
pHT315
pEG1819


Cry ET72
46
45
EG4420
EG11440
cry2
HindIII
cry2Aa
pEG597
pEG1260


Cry ET73
48
47
EG3874
EG11465
cry2
HindIII
cry2Aa
pEG597
pEG1279


Cry ET83
50
49
EG6346
EG11785
cry9
MboI
cryET59,
pHT315
pEG397









cryET83





1Methods include the construction of genomic libraries containing partial MboI fragments (Example 4), the construction of genomic libraries containing size-selected BamHI or HindIII restriction fragments (Example 5), the amplification of novel cry sequences by PCR TM and the construction of novel cry gene fusions (Example 6).


2Hybridization probes included the 700 base pair EcoRI fragment obtained from digestion of the cry1Aa gene, gene fragments from the cry2Aa, cryET59, and cryET83 genes, and synthetic oligonucleotides (wd207, pr56).






6.2 Example 2 
Identification of B. Thuringienses Strains Containing Novel Cry2-Related Genes
Proteins encoded by the cry2 class of B. thuringiensis class of toxin genes have activity on the larvae of lepidopteran and diopteran insects. Southern blot hybridization analysis of DNA extracted from lepidopteran-active strains was utilized to identify novel cry2-related genes. Total genomic DNA was isolated as described in Section 6.1. The DNA was digested with the restriction endonuclease Sau3A and run on a 1.2% agarose gel as described. The digested DNA was transferred to nitrocellulose filters to be probed with a DNA fragment containing the cry2Aa gene. Hybridizations were performed at 55° C. and the filters washed and exposed to X-ray film to obtain an autoradiogram.
Sau3A digestion followed by hybridization with the cry2Aa gene gave characteristic patterns of hybridizing fragments allowing the identification of the cry2Aa, cry2Ab, and cry2Ac genes. Hybridizing fragments that differed from these patterns indicated the presence of a novel cry2-related gene in that strain.
Once a strain was identified as containing one or more novel cry2-related genes, an additional Southern blot hybridization was performed. The procedures were the same as those already described above, except another restriction enzyme, usually HindIII, was used. Since an enzyme like HindIII (a “six base cutter”) cuts DNA less frequently than does Sau3A or MboI, it was more likely to generate a restriction fragment containing the entire cry2-related gene which could then be readily cloned.
6.3 Example 3
Identification of B. Thuringienses Strains Containing Novel Cry9-Type Genes
A cry9-specific oligonucleotide, designated pr56, was designed to facilitate the identification of strains harboring cry9-type genes. This oligonucleotide corresponds to nucleotides 4349-4416 of the gene (GenBank Accession No. Z37527). The sequence of pr56 was as follows:








(SEQ ID NO:52)









5′-AGTAACGGTGTTACTATTAGCGAGGGCGGTCCATTCTTTAAAGGTCG



 


TGCACTTCAGTTAGC-3′.






B. thuringiensis isolates were spotted or “patched” on SGNB plates, with no more than 50 isolates per plate, and grown overnight at 25° C. The B. thuringiensis colonies were transferred to nitrocellulose filters and the filters placed, colony side up, on fresh SGNB plates for overnight growth at 30° C. Subsequently, the filters were placed, colony side up, on Whatman paper soaked in denaturing solution (1.5 M NaCl, 0.5 N NaOH) for 20 min. After denaturation, the filters were placed on Whatman paper soaked in neutralizing solution (3 M NaCl, 1.5 M Tris-HCl, pH 7.0) for 20 min. Finally, the filters were washed in 3×SSC (1×SSC=0.15 M NaCl and 0.015 M sodium citrate) to remove cellular debris and baked in a vacuum oven at 80° C. for 90 min.
The cry9-specific oligonucleotide pr56 ( ̃10 pmoles) was end-labeled with [γ-32P]ATP using T4 polynucleotide kinase. The labeling reaction was carried out at 37° C. for 20 min and terminated by incubating the reaction at 100 C for 3 min. After ethanol precipitation, the labeled oligonucleotide was resuspended in 100 μl distilled H2O.
The filters were incubated with the cry9-specific probe in 6×SSC, 10× Denhardt's solution, 0.5% glycine, 0.2% SDS at 47° C. overnight. The filters were washed twice in 3×SSC, 0.1% SDS for 15 min at 47° C. and twice in 1×SSC, 0.1% SDS for 15 min at 47° C. The dried filters were exposed to X-OMAT XAR-5 film (Eastman Kodak Co.) at −70° C. using an intensifying screen. The developed autoradiogram revealed 24 isolates of B. thuringiensis containing DNA that hybridized to the cry9 probe.
To identify cry9C-type genes among these strains, two opposing oligonucleotide primers specific for the cry9C gene (GenBank Accession No. Z37527) were designed for polymerase chain reaction (PCRTM) analyses. The sequence of pr58 is:









5′-CGACTTCTCCTGCTAATGGAGG-3′.
(SEQ ID NO:53)







The sequence of pr59 is:










5′-CTCGCTAATAGTAACACCGTTACTTGCC-3′.
(SEQ ID NO:54)







Plasmid DNAs were isolated from the isolates of B. thuringiensis believed to contain cry9-type genes. B. thuringiensis isolates were grown overnight at 30° C. on Luria agar plates and 2 loopfuls of cells from each isolate were suspended in 50 mM glucose, 10 mM Tris-HCl, 1 mM EDTA (1×GTE) containing 4 mg/ml lysozyme. After a 10 min incubation at room temperature, plasmid DNAs were extracted using a standard alkaline lysis procedure (Maniatis et al., 1982). The plasmid DNAs were resuspended in 20 μl of 1×TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). Two microliters of the plasmid DNA preparations were used in the PCRTM reactions. Amplifications were performed in 100 μl volumes with a Perkin-Elmer DNA Thermocycler (Perkin-Elmer Cetus, Foster City, Calif.) using materials and methods provided in the Perkin-Elmer GeneAmpTM kit. Conditions for the PCRTM were as follows: 95° C. for 30 sec, 46° C. for 30 sec, 70° C. for 1 min; 30 cycles. A PCRTM using these primers and the cry9C gene as a template should yield a DNA fragment of  ̃970 bp. Of twenty-four strains found to hybridize to the cry9 probe (SEQ ID NO:XX), only one strain, EG9290, yielded the predicted amplified DNA fragment.

6.4 Example 4
Cloning of B. Thuringienses Toxin Genes by Constructing MboI Partial Digest Libraries
The restriction endonuclease MboI was utilized in the construction of genomic DNA libraries because it has a recognition sequence of four base pairs which occurs frequently in long stretches of DNA. Total genomic DNA was isolated from B. thuringiensis strains as described in Section 6.1. The DNA was digested under conditions allowing limited cleavage of a DNA strand. The method of establishing these conditions has been described (Maniatis et al., 1982). Digestion of DNA in this manner created a set of essentially randomly cleaved, overlapping fragments which were used to create a library representative of the entire genome.
The digested DNA fragments were separated, according to size, by agarose gel electrophoresis through a 0.6% agarose, 1×TBE gel, overnight at 2 volts/cm of gel length. The gel was stained with ethidium bromide so that the digested DNA could be visualized when exposed to long-wave UV light. A razor blade was used to excise a gel slice containing DNA fragments of approximately 9-kb to 12-kb in size. The DNA fragments were removed from the agarose by placing the slice in a dialysis bag with enough TE (10 mM Tris-HCl, 1 mM EDTA) to cover the slice. The bag was then closed and placed in a horizontal electrophoresis apparatus filled with 1×TBE buffer. The DNA was electroeluted from the slice into the TE at 100 volts for 2 h. The TE was removed from the bag, extracted with phenol:chloroform (1:1), followed by extraction with chloroform. The DNA fragments are then collected by the standard technique of ethanol precipitation (see Maniatis et al., 1982).
To create a library in E. coli of the partially-digested DNA, the fragments were ligated into the shuttle vector, pHT315 (Arantes and Lereclus, 1991). This plasmid contains replication origins for E. coli and B. thuringiensis, genes for resistance to the antibiotics erythromycin and ampicillin, and a multiple cloning site. The MboI fragments were mixed with BamHI-digested pHT315 that had been treated with calf intestinal, or bacterial, alkaline phosphatase (GibcoBRL, Gaithersburg, Md.) to remove the 5′-phosphates from the digested plasmid, preventing re-ligation of the vector to itself. After purification, T4 ligase and a ligation buffer (Promega Corp., Madison, Wis.) were added to the reaction containing the digested vector and the MboI fragments. These were incubated overnight at 15° C., or at room temperature for 1 h, to allow the insertion and ligation of the MboI fragments into the pHT315 vector DNA.
The ligation mixture was then introduced into transformation-competent E. coli SURE® cells (Stratagene Cloning Systems, La Jolla, Calif.), following procedures described by the manufacturer. The transformed E. coli cells were then plated on LB agar plates containing 50-75 μg/ml ampicillin and incubated overnight at 37° C. The growth of several hundred ampicillin-resistant colonies on each plate indicated the presence of recombinant plasmid in the cells of each of those colonies.
To isolate the colonies harboring sequences encoding toxin genes, the colonies were first transferred to nitrocellulose filters. This was accomplished by simply placing a circular nitrocellulose filter (Millipore HATF 08525, Millipore Corp., Bedford, Mass.) directly on top of the LB-ampicillin agar plates containing the transformed colonies. When the filter was slowly peeled off of the plate the colonies stick to the filter giving an exact replica of the pattern of colonies from the original plate. Enough cells from each colony were left on the plate that 5 to 6 h of growth at 37° C. restored the colonies. The plates were then stored at 4° C. until needed. The nitrocellulose filters with the transferred colonies are then placed, colony-side up, on fresh LB-ampicillin agar plates and allowed to grow at 37° C. until they reached an approximate 1 mm diameter.
To release the DNA from the recombinant E. coli cells the nitrocellulose filters were placed, colony-side up, on 2-sheets of Whatman 3MM chromatography paper (Whatman International Ltd., Maidstone, England) soaked with 0.5 N NaOH, 1.5 M NaCl for 15 min. This treatment lysed the cells and denatured the released DNA allowing it to stick to the nitrocellulose filter. The filters were then neutralized by placing the filters, colony-side up, on 2 sheets of Whatman paper soaked with 1 M NH4-acetate, 0.02 M NaOH for 10 min. The filters were rinsed in 3×SSC, air dried, and baked for 1 h at 80° C. in a vacuum oven. The filters were then ready for use in hybridization studies using probes to identify different classes of B. thuringiensis genes, as described in the above examples.
In order to identify colonies containing cloned cry1-related genes, the cry1-specific oligonucleotide wd207 was labeled at the 5′-end with [γ-32P]ATP and T4 polynucleotide kinase. The labeled probe was added to the filters in 3×SSC, 0.1% SDS, 10× Denhardt's reagent (0.2% BSA, 0.2% polyvinylpyrrolidone, 0.2% Ficoll), 0.2 mg/ml heparin and incubated overnight at 47° C. These conditions allowed hybridization of the labeled oligonucleotide to related sequences present on the nitrocellulose blots of the transformed E. coli colonies. Following incubation the filters were washed in several changes of 3×SSC, 0.1% SDS at 45° C. The filters were blotted dry and exposed to Kodak X-OMAT AR X-ray film (Eastman Kodak Co., Rochester, N.Y.) overnight at −70° C. with an intensifying screen.
Colonies that contain cloned cry1-related sequences were identified by aligning signals on the autoradiogram with the colonies on the original transformation plates. The isolated colonies were then grown in LB-ampicillin liquid medium from which the cells could be harvested and recombinant plasmid prepared by the standard alkaline-lysis miniprep procedure (Maniatis et al., 1982). The plasmid DNA was then used as a template for DNA sequencing reactions necessary to confirm that the cloned gene was novel. If the cloned gene was novel, the plasmid was then introduced into a crystal protein-negative strain of B. thuringiensis (Cry−) so that the encoded protein could be expressed and characterized. These procedures are described in detail in the following sections.
6.5 Example 5
Cloning of Specific Endonuclease Restriction Fragments
The identification of a specific restriction fragment containing a novel B. thuringiensis gene has been described for cry2-related genes in Section 2. The procedure for cloning a restriction fragment of known size was essentially the same as described for cloning an MboI fragment. The DNA was digested with a restriction enzyme (e.g., HindIII), and run through an agarose gel to separate the fragments by size. Fragments of the proper size, identified by Southern blot analysis (Example 2), were excised with a razor blade and electroeluted from the gel slice into TE buffer from which they could be precipitated. The isolated restriction fragments were then ligated into an E. coli/B. thuringiensis shuttle vector and transformed into E. coli to construct a size-selected library. The library could then be hybridized with a specific gene probe, as described in Example 4, to isolate the colony containing the cloned novel gene.
6.6 Example 6
Cloning of PCRTM-Amplified Fragments
A rapid method for cloning and expressing novel cry1 gene fragments from B. thuringiensis was developed using the polymerase chain reaction. Flanking primers were designed to anneal to conserved regions 5′ to and within cry1 genes. With the exception of certain cry3 genes, most B. thuringiensis cry genes are transcriptionally regulated, at least in part, by RNA polymerases containing the mother cell-specific σE or sigE, sigma factor. These σE-regulated cry genes possess 5′ promoter sequences that are recognized by σE. Alignment of these promoter sequences reveals considerable sequence variation, although a consensus sequence can be identified (Baum and Malvar, 1995). A primer, designated “sigE”, containing a sequence identical to the cry1Ac σE promoter sequence, was designed that would anneal to related σE promoter sequences 5′ to uncharacterized cry genes. The sigE primer also includes a BbuI site (isoschizimer: SphI) to facilitate cloning of amplified fragments. The sequence of the sigE primer is shown below:








(SEQ ID NO:55)









5′-ATTTAGTAGCATGCGTTGCACTTTGTGCATTTTTTCATAAGATGAGT



 


CATATGTTTTAAAT-3′.






The opposing primer, designated KpnR, anneals to a 3′-proximal region of the cry1 gene that is generally conserved. This primer incorporates an Asp718 site (isoschizimer: KpnI) conserved among the cry1A genes to facilitate cloning of the amplified fragment and to permit the construction of fusion proteins containing a carboxyl-terminal portion of the Cry1Ac protein. The sequence of the KpnR primer is shown below:









5′-GGATAGCACTCATCAAAGGTACC-3′
(SEQ ID NO:56)







PCRTMs were carried out using a Perkin Elmer DNA thermocycler and the following parameters: 94° C., 2 min.; 40 cycles consisting of 94° C., 30 sec; 40° C., 2 min; 72° C., 3 min; and a 10 second extension added to the 72° C. incubation after 20 cycles. The standard PCRTM buffer (100 μl volume) was modified to include 1× Taq Extender buffer, 25 μM each of the sigE and KpnR primers, and 0.5-1.0 μl of Taq Extender (Stratagene Inc.) in addition to 0.5-1.0 μl of Taq polymerase. Typically, 1-2 μl of the DNA preparations from novel B. thuringiensis isolates were included in the PCRTMs. PCRTMs with cry genes incorporating these primers resulted in the amplification of a  ̃2.3-kb DNA fragment flanked by restriction sites for BbuI and Asp718.
For the cloning and expression of these gene fragments, the cry1Ac shuttle vector pEG1064 was used. This plasmid is derived from the cry1Ac shuttle vector pEG857 (Baum et al., 1990), with the following modifications. A frameshift mutation was generated at a unique NcoI site within the cry1Ac coding region by cleaving pEG857 with the restriction endonuclease NcoI, blunt-ending the NcoI-generated ends with Klenow polymerase and ligating the blunt ends with T4 ligase. In similar fashion, an Asp718 site located in the multiple cloning site 3′ to the cry1Ac gene was removed, leaving only the single Asp718 site contained within the cry1Ac coding sequence. The resulting plasmid, pEG1064, cannot direct the production of crystal protein when introduced into an acrystalliferous (Cry−) strain of B. thuringiensis because of the frameshift mutation For cloning and expression of unknown cry genes, pEG1064 was cleaved with BbuI and Asp718 and the vector fragment purified following gel electrophoresis. Amplified fragments of unknown cry genes, obtained by PCRTM amplification of total B. thuringiensis DNA, were digested with the restriction endonucleases BbuI and Asp718 and ligated into the BbuI and Asp718 sites of the pEG1064 vector fragment. The ligation mixture was used to transform the Cry− B. thuringiensis strains, EG10368 or EG10650, to chloramphenicol resistance using an electroporation protocol previously described (Mettus and Macaluso, 1990) Chloramphenicol-resistant (CmR) isolates were evaluated for crystal protein production by phase-contrast microscopy. Crystal forming (Cry+) isolates were subsequently grown in C2 liquid broth medium (Donovan et al., 1988) to obtain crystal protein for SDS-PAGE analysis and insect bioassay.
Because of the frameshift mutation within the cry1Ac gene, the crystal proteins obtained from the transformants could not be derived from the vector pEG1064. The Cry+ transformants thus contained unknown cry gene fragments fused, at the Asp718 site, to a 3′-portion of the cry1Ac gene. Transcription of these gene fusions in B. thuringiensis was presumably directed from the σE promoter incorporated into the amplified cry gene fragment. The fusion proteins, containing the entire active toxin region of the unknown Cry protein, were capable of producing crystals in B. thuringiensis. 
6.7 Example 7
Cloning of Cry9-Related Genes
Total DNA was isolated from B. thuringiensis strain EG9290 for cloning studies. EG9290 was grown overnight at 30° C. in 1× brain heart infusion, 0.5% glycerol (BHIG). In the morning, 500 μl of the overnight growth was suspended in 50 ml BHIG and the culture incubated at 30° C. with agitation until the culture reached a Klett reading of 150 (red filter). The cells were harvested by centrifugation, suspended in 5 ml 1×GTE buffer containing 4 mg/ml lysozyme and 100 μg/ml Rnase A, and incubated at 37° C. for 20 min. The cells were lysed by the addition of 0.5 ml of 20% SDS. The released DNA was precipitated by the addition of 2.5 ml 7.5 M ammonium acetate and 7 ml of isopropanol. The precipitated DNA was spooled out of the mixture using a glass micropipette and washed in 80% ethanol. The DNA was resuspended in 10 ml 1×TE, extracted with one volume each of buffered phenol and chloroform:isoamyl alcohol (24:1), and precipitated as before. The spooled DNA was washed in 80% ethanol, allowed to air dry for several min, and suspended in 600 μl 1×TE. The DNA concentration was estimated at 500 μg/ml.
A library of EG9290 total DNA was constructed using partially digested MboI fragments of EG9290 DNA and the general methods described herein. The partial MboI fragments were inserted into the unique BamHI site of cloning vector pHT315. The ligation mixture was used to transform E. coli SureTM cells to ampicillin resistance by electroporation employing electrocompetent cells and protocols provided by Stratagene (La Jolla, Calif.) and the BioRad Gene PulserTM apparatus (Bio-Rad Laboratories, Hercules, Calif.). Recombinant clones harboring cry9-type genes were identified by colony blot hybridization using a 32P-labeled probe consisting of the putative cry9C fragment generated by amplification of EG9290 DNA with primers pr58 and pr59. Plasmid DNAs were extracted from the E. coli clones using a standard alkaline lysis procedure.
Plasmid DNAs from the E. coli recombinant clones were used to transform B. thuringiensis strain EG10368 to erythromycin resistance using the electroporation procedure described by Mettus and Macaluso (1990). Cells were plated onto starch agar plates containing 20 μg/ml erythromycin and incubated at 30° C. After six days, colonies with a more opaque appearance were recovered from the plates and streaked out onto fresh starch agar plates containing 20 μg/ml erythromycin to isolate single colonies. Colonies exhibiting a more opaque appearance were observed to produce large parasporal inclusions/crystals by phase-contrast microscopy.
Recombinant EG10368 clones producing parasporal inclusion/crystals were evaluated for crystal protein production in broth culture. Single colonies were inoculated into C2 medium containing 10 μg/ml erythromycin and grown at 30° C. for 3 days at 28-30° C., at which time the cultures were fully sporulated and lysed. Spores and crystals were pelleted by centrifugation and resuspended in 20 mM Tris-HCl, 1 mM EDTA, pH 7.0. Aliquots of this material were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Two EG10368 recombinant clones, initially identified as 9290-2 and 9290-3, were observed to produce distinct proteins of  ̃130 kDa. 9290-2 was designated EG12102 and 9290-3 was designated EG12103. The EG12102 protein was designated CryET59 while the EG12103 protein was designated CryET60.
Plasmid DNAs were prepared from EG12102 and EG12103 using a standard alkaline lysis procedure. Digestion of the plasmids with the restriction endonuclease XbaI confirmed that the two strains harbored distinct cry genes. The cry plasmids of EG12102 and EG12103, designated pEG945 and pEG946, respectively, were used to transform E. coli SureTM cells to ampicillin resistance by electroporation, employing electrocompetent cells and protocols provided by Stratagene Inc. The E. coli recombinant strain containing pEG945 was designated EG12132, and the E. coli recombinant strain containing pEG946 was designated EG12133. pEG945 and pEG946 were purified from the E. coli recombinant strains using the QIAGEN midi-column plasmid purification kit and protocols (QIAGEN Inc., Valencia, Calif.).
The cryET83 gene was cloned from B. thuringiensis strain EG6346 subspecies aizawai using similar methods. Southern blot analysis of genomic DNA from EG6346 revealed a unique restriction fragment that hybridized to the cryET59 probe. A series of degenerate oligonucleotide primers, pr95, pr97, and pr98, were designed to amplify cry9-related sequences from genomic DNA. The sequences of these primers are as shown:









pr95:




5′-GTWTGGACSCRTCGHGATGTGG-3′
(SEQ ID NO:57)


 


pr97:








(SEQ ID NO:58)









5′-TAATTTCTGCTAGCCCWATTTCTGGATTTAATTGTTGATC-3′



 


pr98:









5′-ATWACNCAAMTWCCDTTRG-3′
(SEQ ID NO:59)







where D=A, G; H=A, C, T; M=A, C; N=A, C, T; R=A, G; S=C, G; and W=A, T.

A PCRTM using Taq polymerase, Taq ExtenderTM (Stratagene, La Jolla, Calif.), the opposing primers pr95 and pr97, and total EG6346 DNA yielded a DNA fragment that was faintly visible on an ethidium bromide-stained agarose gel. This DNA served as the template for a second round of PCRTM using the opposing primers pr97 and pr98. The resulting amplified DNA fragment was suitable for cloning and served as a hybridization probe for subsequent cloning experiments. A library of EG6346 total DNA was constructed using partially digested 9-12 kb MboI fragments of EG6346 DNA ligated into the unique BamHI site of cloning vector pHT315. E. coli recombinant clones harboring the cryET83 gene were identified by colony blot hybridization using the EG6346-specific DNA fragment as a chemiluminescent hybridization probe and the CDP-StarTM nucleic acid chemiluminescent reagent kit from NENTM Life Science Products (Boston, Mass.) to prepare the hybridization probe. The recombinant plasmid harboring the cryET83 gene was designated pEG397. The E. coli recombinant stain containing pEG397 was designated EG11786. The B. thuringiensis recombinant strain containing pEG397 was designated EG11785.
6.8 Example 8
Sequencing of Cloned B. Thuringienses Toxin Genes
Partial sequences for the cloned toxin genes were determined following established dideoxy chain-termination DNA sequencing procedures (Sanger et al., 1977). Preparation of the double stranded plasmid template DNA was accomplished using a standard alkaline lysis procedure or using a QIAGEN plasmid purification kit (QIAGEN Inc., Valencia, Calif.). The sequencing reactions were performed using the SequenaseTM Version 2.0 DNA Sequencing Kit (United States Biochemical/Amersham Life Science Inc., Cleveland, Ohio) following the manufacturer's procedures and using 35S-dATP as the labeling isotope (obtained from DuPont NEN® Research Products, Boston, Mass.). Denaturing gel electrophoresis of the reactions is done on a 6% (wt./vol.) acrylamide, 42% (wt./vol.) urea sequencing gel. The dried gels are exposed to Kodak X-OMAT AR X-ray film (Eastman Kodak Company, Rochester, N.Y.) overnight at room temperature. Alternatively, some cry genes were sequenced using automated sequencing methods. DNA samples were sequenced using the ABI PRISMTM DyeDeoxy sequencing chemistry kit (Applied Biosystems, Foster City, Calif.) according to the manufacturer's suggested protocol. The completed reactions were run on as ABI 377 automated DNA sequencer. DNA sequence data were analyzed using SequencherTM v3.0 DNA analysis software (Gene Codes Corp., Ann Arbor, Mich.). Successive oligonucleotides to be used for priming sequencing reactions were designed from the sequencing data of the previous set of reactions.
The sequence determination for the cry1-related genes involved the use of the oligonucleotide probe wd207, described in Example 2, as the initial sequencing primer. This oligonucleotide anneals to a conserved region of cry1 genes, but because of the inverted and reversed orientation of wd207, it generates sequence towards the 5′-end of the coding region allowing sequence of the variable region of the gene to be read. A typical sequencing run of 250-300 nucleotides was usually sufficient to determine the identity of the gene. If additional data were necessary, one or more additional oligonucleotides could be synthesized to continue the sequence until it could be determined if the sequence was unique. In cases where wd207 did not function well as a primer, other oligonucleotides, designed to anneal to conserved regions of cry1 genes, were used. One such oligonucleotide was the KpnR primer described herein above.
The sequencing of the cloned cry2-related genes followed the same general procedures as those described for the cry1 genes, except that oligonucleotides specific for conserved regions in cry2 genes were used as sequencing primers. The two primers used in these examples were wd268 and wd269, shown below.










Primer wd268 corresponds to cry2Aa nucleotides




579-597











5′-AATGCAGATGAATGGGG-3′.
(SEQ ID NO:60)




 



Primer wd269 corresponds to cry2Aa 1740-1757



5′-TGATAATGGAGCTCGTT-3′
(SEQ ID NO:61)







The sequencing of cryET59 and cryET60 commenced with the use of primer pr56. The sequencing of cryET83 commenced with the use of primer pr98. Successive oligonucleotides to be used for priming sequencing reactions were designed from the sequencing data of the previous set of reactions.
The derived sequences were compared to sequences of known cry genes using the FSTNSCAN program in the PC/GENE sequence analysis package (Intelligenetics, Mountain View, Calif.). This analysis permitted a preliminary classification of the cloned cry genes with respect to previously-known cry genes (Table 11).







TABLE 6







HOMOLOGY COMPARISON OF DNA SEQUENCES1








Cloned Gene
DNA Sequence Identity





cryET31
90% identity with SEQ ID NO: 4 of WO 98/40490


cryET40
99% identity with cry1Aa


cryET43
88% identity with cry1Bd1


cryET44
90% identity with cry1Da/1Db


cryET45
91% identity with cry1Da/1Db


cryET46
98% identity with cry1Ga


cryET47
99% identity with cry1Ab


cryET49
95% identity with cry1Ja


cryET51
85% identity with cry1Ac


cryET52
84% identity with cry1Da/1Db


cryET53
99% identity with SEQ ID NO: 8 of



U.S. Pat. No. 5,723,758


cryET54
99.8% identity with cry1Be


cryET56
80% identity with cry1Ac


cryET57
98% identity with cry1Da


cryET59
95% identity with cry9Ca


cryET60
99.6% identity with cry9Aa


cryET61
97% identity with cry1Ha


cryET62
99% identity with cry1Ad


cryET63
93% identity with cry1Ac


cryET64
91% identity with SEQ ID NO: 9 of



U.S. Pat. No. 5,723,758


cryET66
76% identity with cry1Ga


cryET67
99% identity with SEQ ID NO: 10 of



U.S. Pat. No. 5,723,758


cryET72
98% identity with SEQ ID NO: 4 of WO 98/40490


cryET73
99% identity with SEQ ID NO: 6 of WO 98/40490


cryET83





1Ktup value set at 2 for FSTNSCAN. The cryET59 and cryET60 sequences were compared using the FASTA program (Ktup = 6) in the PC/GENE sequence analysis package.






6.9 Example 9
Expression of Cloned Toxin Genes in a B. Thuringiensis Host
Plasmid DNA was isolated from E. coli colonies identified by hybridization to a gene-specific probe. The isolated plasmid was then introduced into a crystal protein-negative (Cry−) strain of B. thuringiensis using the electroporation protocol of Mettus and Macaluso (1990). Each of the cloning vectors used (see Table 5) has a gene to confer antibiotic resistance on the cells harboring that plasmid. B. thuringiensis transformants were selected by growth on agar plates containing 25 mg/ml erythromycin (pHT315) or 5 mg/ml chloramphenicol (pEG597 and pEG1064). Antibiotic-resistant colonies were then evaluated for crystal protein production by phase-contrast microscropy. Crystal producing colonies were then grown in C2 medium (Donovan et al., 1988) to obtain cultures which were analyzed by SDS-PAGE and insect bioassay.
C2 cultures were inoculated with cells from Cry+ colonies and grown for three days at 25-30° C. in the presence of the appropriate antibiotic. During this time the culture grew to stationary phase, sporulated and lysed, releasing the protein inclusions into the medium. The cultures are harvested by centrifugation, which pellets the spores and crystals. The pellets were washed in a solution of 0.005% Triton X-100®, 2 mM EDTA and centrifuged again. The washed pellets were resuspended at one-tenth the original volume in 0.005% Triton X-100®, 2 mM EDTA.
Crystal protein were solubilized from the spores-crystal suspension by incubating the suspension in a solubilization buffer [0.14 M Tris-HCl pH 8.0, 2% (wt./vol.) sodium dodecyl sulfate (SDS), 5% (vol./vol.) 2-mercaptoethanol, 10% (vol./vol.) glycerol, and 0.1% bromphenol blue] at 100° C. for 5 min. The solubilized crystal proteins were size-fractionated by SDS-PAGE using a gel with an acrylamide concentration of 10%. After size fractionation the proteins were visualized by staining with Coomassie Brilliant Blue R-250.
The expected size for Cry1- and Cry9-related crystal proteins was approximately 130 kDa. The expected size for Cry2-related proteins was approximately 65 kDa.
6.10 Example 10
Insecticidal Activity of the Cloned B. Thuringiensis Toxin Genes
B. thuringiensis recombinant strains producing individual cloned cry genes were grown in C2 medium until the cultures were fully sporulated and lysed. These C2 cultures were used to evaluate the insecticidal activity of the crystal proteins produced. Each culture was diluted with 0.005% Triton® X-100 to achieve the appropriate dilution for two-dose bioassay screens. Fifty microliters of each dilution were topically applied to 32 wells containing 1.0 ml artificial diet per well (surface area of 175 mm2). A single lepidopteran larvae was placed in each of the treated wells and the tray was covered by a clear perforated mylar sheet. With the exception of the P. xylostella bioassays, that employed 3rd instar larvae, all the bioassays were performed with neonate larvae. Larval mortality was scored after 7 days of feeding at 28-30 ° C. and percent mortality was expressed as ratio of the number of dead larvae to the total number of larvae treated (Table 12). In some instances, severe stunting of larval growth was observed after 7 days, and the ratio of stunted/unstunted larva was also recorded. The bioassay results shown in Table 7 demonstrate that the crystal proteins produced by the recombinant B. thuringiensis strains do exhibit insecticidal activity and, furthermore,







TABLE 7





Bioassay evaluations with ET crystal proteins


















Spodoptera exigua













250 nl/well %
2500 nl/well %

Spodoptera frugiperda














mortality
mortality
# stunted/# treated
250 nl/well %
2500 nl/well
# stunted/# treated





Cry1Ac
0
5
4/32
16
53
1/32


ET31
5
12
17/32 
9
6
4/32


ET40
0
5
0
3
3
0


ET43
0
8
0
3
3
2/32


ET44
0
2
0
6
0
1/32


ET45
0
0
0
0
0
1/32


ET46
0
12
0
0
6
0


ET47
19
49
11/32 
31
81
6/32


ET49
0
8
0
0
3
0


ET51
0
0
0
0
0
0


ET52
0
0
0
3
3
0


ET53
0
0
0
3
0
0


ET54
0
66
3/32
6
34
9/32


ET56
0
0
0
0
6
0


ET57
2
15
18/32 
3
94
0


ET59
0
0
0
0
3
0


ET60
0
0
0
0
3
0


ET61
2
5
2/32
0
3
0


ET62
2
59
12/32 
0
13
0


ET63
0
12
5/32
3
0
0


ET64
0
0
0
3
6
0


ET66
0
12
1/32
3
0
1/31


ET67
29
90
0
13
61
0


ET72
0
0
0
3
94
5/31


ET73
0
2
0
0
0
0


Control
8
8
0
0
0
0













Plutella xylostella
Ostrinia nubilalis














250
2500 nl/well %

250 nl/well %
2500 nl/well %




nl/well %
mortality
# stunted/# treated
mortality
mortality
# stunted/# treated





Cry1Ac
100
100
0
100
100
0


ET31
0
2
0
100
100
0


ET40
0
68
0
0
0
2/32


ET43
5
100
0
46
100
0


ET44
0
0
0
0
0
3/32


ET45
0
0
0
0
0
4/32


ET46
0
8
0
0
0
0


ET47
100
100
0
100
100
0


ET49
0
5
0
0
0
0


ET51
0
0
0
0
0
0


ET52
2
43
0
0
14
16/32 


ET53
8
97
0
4
46
5/32


ET54
14
100
0
25
89
1/32


ET56
0
0
0
0
0
0


ET57
0
97
0
0
7
0


ET59
100
100
0
96
100
0


ET60
100
100
0
100
96
0


ET61
0
11
0
0
0
2/32


ET62
97
100
0
100
100
0


ET63
100
100
0
100
100
0


ET64
40
100
0
68
100
0


ET66
100
100
0
86
100
0


ET67
87
100
0
0
79
1/32


ET72
0
0
0
0
0
0


ET73
2
2
0
93
100
0


Control
2
2
0
0
0
0













Heliothis virescens
Helicoverpa zea














2500 nl/well %

250 nl/well %
2500 nl/well %



250 nl/well %
mortality
# stunted/# treated
mortality
mortality





Cry1Ac
100
100
0
100
100


ET31
97
97
1/32
8
81


ET40
2
5
2/32
2
5


ET43
87
97
1/32
0
2


ET44
8
5
1/32
5
8


ET45
0
11
0
8
18


ET46
12
25
0
0
8


ET47
87
100
0
83
100


ET49
8
2
0
11
15


ET51
2
15
0
5
5


ET52
0
31
1/32
93
11


ET53
22
64
2/32
90
61


ET54
15
64
5/32
2
5


ET56
0
11
0
8
0


ET57
2
0
0
11
28


ET59
28
84
4/32
2
2


ET60
56
97
1/32
31
28


ET61
5
5
0
8
5


ET62
44
87
4/32
21
64


ET63
100
100
0
100
100


ET64
0
21
0
5
0


ET66
0
8
1/32
0
5


ET67
18
93
1/32
0
68


ET72
34
64
11/32 
8
2


ET73
42
90
2/32
8
48


Control
5
5
0
5
5













Agrotis ipsilon
Trichoplusia ni














250
2500 nl/well %

250 nl/well %
2500 nl/well %




nl/well %
mortality
# stunted/# treated
mortality
mortality
# stunted/# treated





Cry1Ac
94
100

100
100
0


ET31
6
6

90
100
0


ET40
0
6

13
32
0


ET43
0
45

100
100
0


ET44
6
13

16
26
0


ET45
0
6

13
39
0


ET46
0
0

29
74
0


ET47
0
34

97
100
0


ET49
3
0

13
81
0


ET51
0
0

3
19
0


ET52
0
28

81
100
0


ET53
25
81

74
100
0


ET54
3
6

100
100
0


ET56
3
3

16
26
0


ET57
13
74

19
100
0


ET59
3
3

10
84
0


ET60
3
0

97
100
0


ET61
6
28

29
52
0


ET62
23
58

100
100
0


ET63
3
0

100
100
0


ET64
0
0

87
100
0


ET66
13
91

26
81
0


ET67
3
0

6
100
0


ET72
0
0

23
74
8/32


ET73
13
6

94
100
0


Control
0
0

3
3
0









that the crystal proteins exhibit differential activity towards the lepidopteran species tested.

Additional bioassays were performed with the crystal proteins designated CryET59, CryET60, CryET66, and CryET83. Crystal proteins produced in C2 medium were quantified by SDS-PAGE and densitometry using the method described by Brussock, S. M. and Currier, T. C., 1990, “Use of Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis to Quantify Bacillus thuringiensis δ-Endotoxins”, in Analytical Chemistry of Bacillus thuringiensis (L. A. Hickle and W. L. Fitch, eds.), The American Chemical Society, pp. 78-87.







TABLE 8







Bioassay Evaluation of CryET59 and CryET60


Percent mortality1

















Dose










Toxin
ng/well
AI
HV
HZ
ON
PX
rPX
SE
TN



















Control2
—
2
6
0
0
2
0
2
0


CryET59
100
2
37
0
94
100
100
2
13


CryET59
500
11
80
3
100
100
100
0
63


CryET59
5000
62
100
6
100
100
100
71
100


CryET60
500
0
93
22
100
100
100
0
100


CryET60
5000
2
100
25
100
100
100
14
100





1AI = Agrotis ipsilon, HV = Heliothis virescens, HZ = Helicoverpa zea, ON = Ostrinia nubilalis, PX = Plutella xylostella, rPX = Plutella xylostella colony resistant to Cry1A and Cry IF toxins, SE = Spodoptera exigua, TN = Trichoplusia ni.


2Control = no toxin added.






The procedure was modified to eliminate the neutralization step with 3M HEPES. Crystal proteins resolved by SDS-PAGE were quantified by densitometry using a Molecular Dynamics model 300A computing densitometer and purified bovine serum albumin (Pierce, Rockford, Ill.) as a standard.

The bioassay results shown in Table 8 demonstrate that CryET59 and CryET60 are toxic to a number of lepidopteran species, including a colony of P. xylostella that is resistant to Cry1A and Cry1F crystal proteins. Eight-dose assays with CryET66 also demonstrated excellent toxicity towards both the susceptible and resistant colonies of P. xylostella (Table 14). In this instance, eight crystal protein concentrations were prepared by serial dilution of the crystal protein suspensions in 0.005% Triton® X-100 and 50 ul of each concentration was topically applied to wells containing 1.0 ml of artificial diet. After the wells had dried, a single larvae was placed in each of the treated wells and the tray was covered by a clear perforated mylar sheet (32 larvae for each crystal protein concentration). Larval mortality was scored after 7 days of feeding at 28-30° C. Mortality data was expressed as LC50 and LC95 values, the concentration of crystal protein (ng/175 mm2 diet well) causing 50% and 95% mortality, respectively (Daum, 1970).











TABLE 9





Toxin
LC501
95% C.I.
LC952
Slope















Toxicity of CryET66 towards Plutella xylostella











Cry1Ac
8.05
5.0-15.2
52.94
2.01


Cry1C
25.06
15.7-40.6 
117.07
2.46


CryET66
0.42
0.4-0.5 
1.4
3.13







Toxicity of CryET66 towards Cry1A-resistant Plutella xylostella










Cry1Ac
*No significant mortality













Cry1C
27.32
15.4-51.1 
156.13
2.17


CryET66
1.65
1.3-2.0 
6.41
2.79





1the concentration of crystal protein, in nanograms of crystal protein per well, required to achieve 50% mortality


2the concentration of crystal protein, in nanograms of crystal protein per well, required to achieve 95% mortality.






Table 15 shows that the CryET83 protein exhibits toxicity towards a wide variety of lepidopteran pests and may exhibit improved toxicity towards S. exigua and H. virescens when compared to the other Cry9-type proteins CryET59 and CryET60.








TABLE 10







Toxicity of CryET83 towards lepidopteran larvae1















Dose2
AI3
HV
HZ
ON
PX
SE
SF
TN


















5




5





10



9


50

53


75


69


100



91


500
0
100



67

100


5000
32




100


10000


84



100





1Toxicity calculated as percent mortality among treated larvae.


2ng CryET83 crystal protein/175 mm2 diet well


3Abbreviations described in Table 8; SF = Spodoptera frugiperda






The recombinant B. thuringiensis strains listed in Table 5 were deposited with the ARS Patent Culture Collection and had been assigned the NRRL deposit numbers shown in Table 11.








TABLE 11







Biological Deposits











Polypeptide
Polypeptide
Polynucleotide
Recomb.
NRRL Deposit


Designation
Seq. ID No.:
Seq ID No.:
Strain
No.:














Cry ET31
2
1
EG11562
B-21921


Cry ET40
4
3
EG11901
B-21922


Cry ET43
6
5
EG7692
B-21923


Cry ET44
8
7
EG11629
B-21924


Cry ET45
10
9
EG7694
B-21925


Cry ET46
12
11
EG7695
B-21926


Cry ET47
14
13
EG7696
B-21927


Cry ET49
16
15
EG11630
B-21928


Cry ET51
18
17
EG11921
B-21929


Cry ET52
20
19
EG11584
B-21930


Cry ET53
22
21
EG11906
B-21931


Cry ET54
63
62
EG11907
B-21932


Cry ET56
24
23
EG11909
B-21933


Cry ET57
26
25
EG11910
B-21934


Cry ET59
28
27
EG12102
B-21935


Cry ET60
30
29
EG12103
B-21936


Cry ET61
32
31
EG11634
B-21937


Cry ET62
34
33
EG11635
B-21938


Cry ET63
36
35
EG11636
B-21939


Cry ET64
38
37
EG11638
B-21940


Cry ET66
40
39
EG11640
B-21941


Cry ET67
42
41
EG11642
B-21942


Cry ET68
44
43
EG11644
B-30137


Cry ET72
46
45
EG11440
B-21943


Cry ET73
48
47
EG11465
B-21944


CryET83
50
49
EG11785
B-30138









6.11 Example 11
Modification of Cry Genes for Expression in Plants
Wild-type cry genes are known to be expressed poorly in plants as a full length gene or as a truncated gene. Typically, the G+C content of a cry gene is low (37%) and often contains many A+T rich regions, potential polyadenylation sites and numerous ATTTA sequences. Table 12 shows a list of potential polyadenylation sequences which should be avoided when preparing the “plantized” gene construct.








TABLE 12





List of Sequences of Potential



Polyadenylation Signals


















AATAAA*




 



AATAAT*



 



AACCAA



 



ATATAA



 



AATCAA



 



ATACTA



 



ATAAAA



 



ATGAAA



 



AAGCAT



 



ATTAAT



 



ATACAT



 



AAAATA



 



ATTAAA**



 



AATTAA**



 



AATACA**



 



CATAAA**







*indicates a potential major plant polyadenylation site.



**indicates a potential minor animal polyadenylation site.



All others are potential minor plant polyadenylation sites.






The regions for mutagenesis may be selected in the following manner. All regions of the DNA sequence of the cry gene are identified which contained five or more consecutive base pairs which were A or T. These were ranked in terms of length and highest percentage of A+T in the surrounding sequence over a 20-30 base pair region. The DNA is analysed for regions which might contain polyadenylation sites or ATTTA sequences. Oligonucleotides are then designed which maximize the elimination of A+T consecutive regions which contained one or more polyadenylation sites or ATTTA sequences. Two potential plant polyadenylation sites have been shown to be more critical based on published reports. Codons are selected which increase G+C content, but do not generate restriction sites for enzymes useful for cloning and assembly of the modified gene (e.g., BamHI, BglII, SacI, NcoI, EcoRV, etc.). Likewise condons are avoided which contain the doublets TA or GC which have been reported to be infrequently-found codons in plants.
Although the CaMV35S promoter is generally a high level constitutive promoter in most plant tissues, the expression level of genes driven the CaMV35S promoter is low in floral tissue relative to the levels seen in leaf tissue. Because the economically important targets damaged by some insects are the floral parts or derived from floral parts (e.g., cotton squares and bolls, tobacco buds, tomato buds and fruit), it is often advantageous to increase the expression of crystal proteins in these tissues over that obtained with the CaMV35S promoter.
The 35S promoter of Figwort Mosaic Virus (FMV) is analogous to the CaMV35S promoter. This promoter has been isolated and engineered into a plant transformation vector. Relative 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 leaf. A plant transformation vector, may be constructed in which the full length synthetic cry gene is driven by the FMV 35S promoter. Tobacco plants may be transformed with the vector and compared for expression of the crystal protein by Western blot or ELISA immunoassay in leaf and floral tissue. The FMV promoter has been used to produce relatively high levels of crystal protein in floral tissue compared to the CaMV promoter.
6.12 Example 12
Expression of Synthetic Cry Genes with ssRUBISCO Promoters and Chloroplast Transit Peptides
The genes in plants encoding the small subunit of RUBISCO (SSU) are often highly expressed, light regulated 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 expression levels and tissue specificity of different SSU genes will be different. The SSU proteins are encoded in the nucleus and synthesized in the cytoplasm as precursors that contain an N-terminal extension known as the chloroplast transit peptide (CTP). The CTP directs the precursor to the chloroplast and promotes the uptake of the SSU protein into the chloroplast. In this process, the CTP is cleaved from the SSU protein. These CTP sequences have been used to direct heterologous proteins into chloroplasts of transformed plants.
The SSU promoters might have several advantages for expression of heterologous genes in plants. Some SSU promoters are very highly expressed and could give rise to expression levels as high or higher than those observed with the CaMV35S promoter. The tissue distribution of expression from SSU promoters is different from that of the CaMV35S promoter, so for control of some insect pests, it may be advantageous to direct the expression of crystal proteins to those cells in which SSU is most highly expressed. For example, although relatively constitutive, in the leaf the CaMV35S promoter is more highly expressed in vascular tissue than in some other parts of the leaf, while most SSU promoters are most highly expressed in the mesophyll cells of the leaf. Some SSU promoters also are more highly tissue specific, so it could be possible to utilize a specific SSU promoter to express the protein of the present invention in only a subset of plant tissues, if for example expression of such a protein in certain cells was found to be deleterious to those cells. For example, for control of Colorado potato beetle in potato, it may be advantageous to use SSU promoters to direct crystal protein expression to the leaves but not to the edible tubers.
Utilizing SSU CTP sequences to localize crystal proteins to the chloroplast might also be advantageous. Localization of the B. thuringiensis crystal proteins to the chloroplast could protect these from proteases found in the cytoplasm. This could stabilize the proteins and lead to higher levels of accumulation of active toxin. cry genes containing the CTP may be used in combination with the SSU promoter or with other promoters such as CaMV35S.
6.13 Example 13
Targeting of Cry Proteins to the Extracellular Space or Vacuole Through the use of Signal Peptides
The B. thuringiensis proteins produced from the synthetic genes described here are localized to the cytoplasm of the plant cell, and this cytoplasmic localization results in plants that are insecticidally effective. It may be advantageous for some purposes to direct the B. thuringiensis proteins to other compartments of the plant cell. Localizing B. thuringiensis proteins in compartments other than the cytoplasm may result in less exposure of the B. thuringiensis proteins to cytoplasmic proteases leading to greater accumulation of the protein yielding enhanced insecticidal activity. Extracellular localization could lead to more efficient exposure of certain insects to the B. thuringiensis proteins leading to greater efficacy. If a B. thuringiensis protein were found to be deleterious to plant cell function, then localization to a noncytoplasmic compartment could protect these cells from the protein.
In plants as well as other eukaryotes, proteins that are destined to be localized either extracellularly or in several specific compartments are typically synthesized with an N-terminal amino acid extension known as the signal peptide. This signal peptide directs the protein to enter the compartmentalization pathway, and it is typically cleaved from the mature protein as an early step in compartmentalization. For an extracellular protein, the secretory pathway typically involves cotranslational insertion into the endoplasmic reticulum with cleavage of the signal peptide occurring at this stage. The mature protein then passes through the Golgi body into vesicles that fuse with the plasma membrane thus releasing the protein into the extracellular space. Proteins destined for other compartments follow a similar pathway. For example, proteins that are destined for the endoplasmic reticulum or the Golgi body follow this scheme, but they are specifically retained in the appropriate compartment. In plants, some proteins are also targeted to the vacuole, another membrane bound compartment in the cytoplasm of many plant cells. Vacuole targeted proteins diverge from the above pathway at the Golgi body where they enter vesicles that fuse with the vacuole.
A common feature of this protein targeting is the signal peptide that initiates the compartmentalization process. Fusing a signal peptide to a protein will in many cases lead to the targeting of that protein to the endoplasmic reticulum. The efficiency of this step may depend on the sequence of the mature protein itself as well. The signals that direct a protein to a specific compartment rather than to the extracellular space are not as 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 vacuole targeted proteins, but it is not yet possible to define these sequences precisely. 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. Thus, a strategy to direct B. thuringiensis proteins out of the cytoplasm is to fuse the genes for synthetic B. thuringiensis genes to DNA sequences encoding known plant signal peptides. These fusion genes will give rise to B. thuringiensis proteins that enter the secretory pathway, and lead to extracellular secretion or targeting to the vacuole or other compartments.
Signal sequences for several plant genes have been described. One such sequence is for the tobacco pathogenesis related protein PR1b has been previously described (Cornelissen et al., 1986). The PR1b protein is normally localized to the extracellular space. Another type of signal peptide is contained on seed storage proteins of legumes. These proteins are localized to the protein body of seeds, which is a vacuole like compartment found in 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 the published these published sequences, genes may 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 cleavage site of the signal peptide. This may be necessary to insure proper cleavage of the signal peptide.
6.14 Example 14
Isolation of Transgenic Plants Resistant to Insects Using Cry Transgenes
6.64.1 Plant Gene Construction
The expression of a plant gene which exists in double-stranded DNA form involves transcription of messenger RNA (mRNA) from one strand of the DNA by RNA polymerase enzyme, and the subsequent processing of the mRNA primary transcript inside the nucleus. This processing involves a 3′ non-translated region which adds polyadenylate nucleotides to the 3′ end of the RNA. Transcription of DNA into mRNA is regulated by a region of DNA usually referred to as the “promoter”. The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA and to initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding strand of RNA.
A number of promoters which are active in plant cells have been described in the literature. Such promoters may be obtained from plants or plant viruses and include, but are not limited to, the nopaline synthase (NOS) and octopine synthase (OCS) promoters (which are carried on tumor-inducing plasmids of Agrobacterium tumefaciens), the cauliflower mosaic virus (CaMV) 19S and 35S promoters, the light-inducible promoter from the small subunit of ribulose 1,5-bisphosphate carboxylase (ssRUBISCO, a very abundant plant polypeptide), and the Figwort Mosaic Virus (FMV) 35S promoter. All of these promoters have been used to create various types of DNA constructs which have been expressed in plants (see e.g., U.S. Pat. No. 5,463,175, specifically incorporated herein by reference).
The particular promoter selected should be capable of causing sufficient expression of the enzyme coding sequence to result in the production of an effective amount of protein. One set of preferred promoters are constitutive promoters such as the CaMV35S or FMV35S promoters that yield high levels of expression in most plant organs (U.S. Pat. No. 5,378,619, specifically incorporated herein by reference). Another set of preferred promoters are root enhanced or specific promoters such as the CaMV derived 4 as-1 promoter or the wheat POX1 promoter (U.S. Pat. No. 5,023,179, specifically incorporated herein by reference; Hertig et al., 1991). The root enhanced or specific promoters would be particularly preferred for the control of corn rootworm (Diabroticus spp.) in transgenic corn plants.
The promoters used in the DNA constructs (i.e. chimeric plant genes) of the present invention may be modified, if desired, to affect their control characteristics. For example, the CaMV35S promoter may be ligated to the portion of the ssRUBISCO gene that represses the expression of ssRUBISCO in the absence of light, to create a promoter which is active in leaves but not in roots. The resulting chimeric promoter may be used as described herein. For purposes of this description, the phrase “CaMV35S” promoter thus includes variations of CaMV35S promoter, e.g., promoters derived by means of ligation with operator regions, random or controlled mutagenesis, etc. Furthermore, the promoters may be altered to contain multiple “enhancer sequences” to assist in elevating gene expression.
The RNA produced by a DNA construct of the present invention also contains a 5′ non-translated leader sequence. This sequence can be derived from the promoter selected to express the gene, and can be specifically modified so as to increase translation of the mRNA. The 5′ non-translated regions can also be obtained from viral RNA's, from suitable eucaryotic genes, or from a synthetic gene sequence. The present invention is not limited to constructs wherein the non-translated region is derived from the 5′ non-translated sequence that accompanies the promoter sequence.
For optimized expression in monocotyledenous plants such as maize, an intron should also be included in the DNA expression construct. This intron would typically be placed near the 5′ end of the mRNA in untranslated sequence. This intron could be obtained from, but not limited to, a set of introns consisting of the maize hsp70 intron (U.S. Pat. No. 5,424,412; specifically incorporated herein by reference) or the rice Act1 intron (McElroy et al., 1990). As shown below, the maize hsp70 intron is useful in the present invention.
As noted above, the 3′ non-translated region of the chimeric plant genes of the present invention contains a polyadenylation signal which functions in plants to cause the addition of adenylate nucleotides to the 3′ end of the RNA. Examples of preferred 3′ regions are (1) the 3′ transcribed, non-translated regions containing the polyadenylate signal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopaline synthase (NOS) gene and (2) plant genes such as the pea ssRUBISCO E9 gene (Fischhoff et al., 1987).
6.14.2 Plant Transformation and Expression
A plant gene containing a structural coding sequence of the present invention can be inserted into the genome of a plant by any suitable method. Suitable plant transformation vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed, e.g., by Herrera-Estrella (1983), Bevan (1983), Klee (1985) and Eur. Pat. Appl. Publ. No. EP0120516. In addition to plant transformation vectors derived from the Ti or root-inducing (Ri) plasmids of Agrobacterium, 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, electroporation, chemicals that increase free DNA uptake, free DNA delivery via microprojectile bombardment, and transformation using viruses or pollen (Fromm et al., 1986; Armstrong et al., 1990; Fromm et al., 1990).
6.14.3 Construction of Monocot Plant Expression Vectors for Cry Genes
For efficient expression of cry genes in transgenic plants, the gene must have a suitable sequence composition (Diehn et al., 1996). To place the cry gene in a vector suitable for expression in monocotyledonous plants (i.e. under control of the enhanced Cauliflower Mosaic Virus 35S promoter and link to the hsp70 intron followed by a nopaline synthase polyadenylation site as in U.S. Pat. No. 5,424,412, specifically incorporated herein by reference), a vector such as pMON19469 may be used. Such a vector is conveniently digested with NcoI and EcoRI restriction enzymes. The larger vector band of approximately 4.6 kb is then electrophoresed, purified, and ligated with T4 DNA ligase to an NcoI-EcoRI fragment which contains the synthetic cry gene. The ligation mix is then transformed into E. coli, carbenicillin resistant colonies recovered and plasmid DNA recovered by DNA miniprep procedures. The DNA is then subjected to restriction endonuclease analysis with enzymes such as NcoI and EcoRI (together), NotI, and/or PstI individually or in combination, to identify clones containing the cry coding sequence fused to an intron such as the hsp70 intron, placed under the control of the enhanced CaMV35S promoter.
To place the gene in a vector suitable for recovery of stably transformed and insect resistant plants, the 3.75-kb NotI restriction fragment from pMON33708 containing the lysine oxidase coding sequence fused to the hsp70 intron under control of the enhanced CaMV35S promoter may be isolated by gel electrophoresis and purification. This fragment is then ligated with a vector such as pMON30460 which has been previously treated with NotI and calf intestinal alkaline phosphatase (pMON30460 contains the neomycin phosphotransferase coding sequence under control of the CaMV35S promoter). Kanamycin resistant colonies may then be obtained by transformation of this ligation mix into E. coli and colonies containing the desired plasmid may be identified by restriction endonuclease digestion of plasmid miniprep DNAs. Restriction enzymes such as NotI, EcoRV, HindIII, NcoI, EcoRI, and BglII may be used to identify the appropriate clones in which the orientation of both genes are in tandem (i.e. the 3′ end of the cry expression cassette is linked to the 5′ end of the nptII expression cassette). Expression of the Cry protein by the resulting plasmid in corn protoplasts may be confirmed by electroporation of the vector DNA into protoplasts followed by protein blot and ELISA analysis. This vector may be introduced into the genomic DNA of corn embryos by particle gun bombardment followed by paromomycin selection to obtain corn plants expressing the cry gene essentially as described in U.S. Pat. No. 5,424,412, specifically incorporated herein by reference.
As an example, the vector may be introduced via cobombardment with a hygromycin resistance conferring plasmid into immature embryo scutella (IES) of maize, followed by hygromycin selection, and regeneration. Transgenic corn lines expressing the cry protein may then be identified by ELISA analysis. Progeny seed from these events may then be subsequently tested for protection from insect feeding.
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## Claims

1. An isolated polynucleotide comprising a nucleic acid sequence encoding the polypeptide of SEQ ID NO:40.

2. The isolated polynucleotide of claim 1, wherein said polynucleotide comprises SEQ ID NO:39.

3. The isolated polynucleotide of claim 2, wherein said polynucleotide is SEQ ID NO:39.

4. A vector comprising the isolated polynucleotide of claim 1.

5. The vector of claim 4, wherein said isolated polynucleotide is operably linked to a promoter.

6. The vector of claim 5, wherein said promoter is a plant-expressible promoter.

7. The vector of claim 6, wherein said plant-expressible promoter is selected from the group consisting of corn sucrose synthetase 1, corn alcohol dehydrogenase 1, corn light harvesting complex, corn heat shock protein, pea small subunit RuBP carboxylase, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, bean glycine rich protein 1, Potato patatin, lectin, CaMV 35S, and the S-E9 small subunit RuBP carboxylase promoter.

8. The vector of claim 4, wherein said vector is a plasmid, baculovirus, artificial chromosome, virion, cosmid, phagemid, phage or viral vector.

9. A transformed host cell comprising the isolated polynucleotide of claim 1.

10. The transformed host cell of claim 9, further defined as a prokaryotic or eukaryotic host cell.

11. The transformed host cell of claim 10, further defined as a bacterial cell or a plant cell.

12. The transformed host cell of claim 11, wherein said plant cell is further defined as a monocotyledonous or dicotyledonous plant cell.

13. The transformed host cell of claim 12, wherein said plant cell is selected from the group consisting of a corn, wheat, soybean, oat, cotton, rice, rye, sorghum, sugarcane, tomato, tobacco, kapok, flax, potato, barley, turf grass, pasture grass, berry, fruit, legume, vegetable, ornamental plant, shrub, cactus, succulent, and tree cell.

14. The transformed host cell of claim 13, wherein said plant cell is acorn, wheat, rice, or sugarcane cell.

15. The transformed host cell of claim 13, wherein said plant cell is a soybean, cotton, potato, tomato, or tobacco cell.

16. The transformed host cell of claim 11, wherein said bacterial cell is a Bacillus thuringiensis, Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Escherichia, Salmonella, Agrobacterium or Pseudomonas cell.

17. The transformed host cell of claim 16, wherein said bacterial cell is a Bacillus thuringiensis cell having the NRRL accession number NRRL B-21941.

18. A transgenic plant having incorporated into its genome the isolated polynucleotide of claim 1.

19. The transgenic plant of claim 18, further defined as a monocotyledonous plant.

20. The transgenic plant of claim 19, further defined as a corn, wheat, oat, rice, barley, turf grass, or pasture grass plant.

21. The transgenic plant of claim 18, further defined as a dicotyledonous plant.

22. The transgenic plant of claim 21, further defined as a legume, soybean, tobacco, tomato, potato, cotton, fruit, berry, vegetable or tree plant.

23. Progeny of any generation of the transgenic plant of claim 18, wherein said progeny comprises said polynucleotide.

24. A seed of any generation of the transgenic plant of claim 18, wherein said seed comprises said polynucleotide.

25. A seed of any generation of the progeny of claim 23, wherein said seed comprises said polynucleotide.

26. A plant of any generation of the seed of claim 24 or 25, wherein said plant comprises said polynucleotide.

27. A method of preparing an insect resistant plant, comprising: (a) transforming recipient plant cells with the isolated polynucleotide of claim 1; (b) selecting a recipient plant cell comprising said polynucleotide; and (c) regenerating a plant from the selected recipient plant cell; wherein said plant has enhanced insect resistance relative to the corresponding non-transformed plant.