Genes encoding insecticidal proteins

FIELD OF THE INVENTION
The present invention is drawn to methods and compositions for controlling plant and non-plant pests.
BACKGROUND OF THE INVENTION
Insect pests are a major factor in the loss of the world's commercially important agricultural crops. Broad spectrum chemical pesticides have been used extensively to control or eradicate pests of agricultural importance. There is, however, substantial interest in developing effective alternative pesticides.
Microbial pesticides have played an important role as alternatives to chemical pest control. The most extensively used microbial product is based on the bacterium Bacillus thuringiensis (Bt). Bt is a gram-positive spore forming Bacillus which produces an insecticidal crystal protein (ICP) during sporulation.
Numerous varieties of Bt are known that produce more than 25 different but related ICP's. The majority of ICP's made by Bt are toxic to larvae of certain insects in the orders epidoptera, Diptera and Coleoptera. In general, when an ICP is ingested by a susceptible insect the crystal is solubilized and transformed into a toxic moiety by the insect gut proteases. None of the ICP's active against coleopteran larvae such as Colorado potato beetle (Leptinotarsa decemlineata) or Yellow mealworm (Tenebrio molitor) have demonstrated significant effects on members of the genus Diabrotica particularly Diabrotica virgifera virgifera, the western corn rootworm (WCRW) or Diabrotica longicornis barberi, the northern corn rootworm.
Bacillus cereus (Bc) is closely related to Bt. A major distinguishing characteristic is the absence of a parasporal crystal in Bc. Bc is a widely distributed bacterium that is commonly found in soil and has been isolated from a variety of foods and drugs. The organism has been implicated in the spoilage of food.
Although Bt has been very useful in controlling insect pests, there is a need to expand the number of potential biological control agents.

BRIEF DESCRIPTION OF THE FIGURE
FIG. 1: Characterization of pCIB6022. Boxed regions represent the extent of VIP1A(a) and VIP2A(a). White box represents the portion of VIP1 encoding the 80 kDa peptide observed in Bacillus. Dark box represents the N-terminal `propeptide` of VIP1A(a) predicted by DNA sequence analysis. Stippled box represents the VIP2A(a) coding region. Large `X` represents the location of the frameshift mutation introduced into VIP1A(a). Arrows represent constructs transcribed by the beta-galactosidase promoter. Restriction Sites: C-Cla I; X-Xba I; S-Sca I; RI- Eco RI; B-Bgl II; RV-Eco RV.

SUMMARY OF THE INVENTION
The present invention is drawn to compositions and methods for controlling plant and non-plant pests. Particularly, new pesticidal proteins are disclosed which are isolatable from the vegetative growth stage of Bacillus. Bacillus strains, proteins, and genes encoding the proteins are provided.
The methods and compositions of the invention may be used in a variety of systems for controlling plant and non-plant pests.
DETAILED DESCRIPTION OF THE INVENTION
Compositions and methods for controlling plant pests are provided. In particular, novel pesticidal proteins are provided which are produced during vegetative growth of Bacillus strains. The proteins are useful as pesticidal agents.
The present invention recognizes that pesticidal proteins are produced during vegetative growth of Bacillus strains. To date, all of the identified pesticidal proteins of the invention are secreted from the cell. Prior to the present invention, there was no recognition in the art that a class or classes of pesticidal proteins are produced during vegetative growth of Bacillus. The only report was of a single mosquitocidal toxin from Bacillus sphaericus SSII-1 by Myers and Yousten in Infect. Immun., 19:1047-1053 (1978). Having recognized that such a class exists, the present invention embraces all vegetative insecticidal proteins, hereinafter referred to as VIPs, except for the mosquitocidal toxin from B. sphaericus.
The present VIPs are not abundant after sporulation and are particularly expressed during log phase growth before stationary phase. For the purpose of the present invention vegetative growth is defined as that period of time before the onset of sporulation. Genes encoding such VIPs can be isolated, cloned and transformed into various delivery vehicles for use in pest management programs.
For purposes of the present invention, pests include but are not limited to insects, fungi, bacteria, nematodes, mites, ticks, protozoan pathogens, animal-parasitic liver flukes, and the like. Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera and Lepidoptera.
Tables 1-10 gives a list of pests associated with major crop plants and pests of human and veterinary importance. Such pests are included within the scope of the present invention.
TABLE 1______________________________________Lepidoptera (Butterflies and Moths)______________________________________Maize Sunflower Ostrinia nubilalis, European Suleima helianthana, sunflower corn borer bud moth Agrotis ipsilon, black cutworm Homoeosoma electellum, sunflower moth Helicoverpa zea, corn earworm Spodoptera frugiperda, fall Cotton armyworm Heliothis virescens, cotton Diatraea grandiosella, southwestern boll worm corn borer Helicoverpa zea, cotton bollworm Elasmopalpus lignosellus, lesser Spodoptera exigua, beet cornstalk armyworm borer Pectinophora gossypiella, pink Diatraea saccharalis, sugarcane bollworm borer Rice Sorghum Diatraea saccharalis, sugarcane Chilo partellus, sorghum borer borer Spodoptera frugiperda, fall Spodoptera frugiperda, fall armyworm armyworm Helicoverpa zea, corn earworm Helicoverpa zea, corn earworm Soybean Elasmopalpus lignosellus, lesser Pseudoplusia includens, cornstalk borer soybean looper Feltia subterranea, granulate Anticarsia gemmatalis, cutworm velvetbean caterpillar Wheat Plathypena scabra, green Pseudaletia unipunctata, cloverworm army worm Ostrinia nubilalis, European corn borer Spodoptera frugiperda, fall Agrotis ipsilon, black cutworm armyworm Spodoptera exigua, beet Elasmopalpus lignosellus, lesser armyworm cornstalk borer Heliothis virescens, cotton boll worm Agrotis orthogonia, pale western Helicoverpa zea, cotton bollworm cutworm Elasmopalpus lignosellus, lesser Barley cornstalk borer Ostrinia nubilalis, European corn borer Agrotis ipsilon, black cutworm______________________________________
TABLE 2______________________________________Coleoptera (Beetles)______________________________________Maize Diabrotica virgifera virgifera, western corn rootworm Diabrotica longicornis barberi, northern corn rootworm Diabrotica undecimpunctata howardi, southem corn rootworm Melanotus spp., wireworms Cyclocephala borealis, northern masked chafer (white grub) Cyclocephala immaculata, southern masked chafer (white grub) Popillia japonica, Japanese beetle Chaetocnema pulicaria, corn flea beetle Sphenophorus maidis, maize billbug Sorghum Phyllophaga crinita, white grub Eleodes, Conoderus, and Aeolus spp., wireworms Oulema melanopus, cereal leaf beetle Chaetocnema pulicaria, corn flea beetle Sphenophorus maidis, maize billbug Wheat Oulema melanopus, cereal leaf beetle Hypera punctata, clover leaf weevil Diabrotica undecimpunctata howardi, southern corn rootworm Sunflower Zygogramma exclamationis, sunflower beetle Bothyrus gibbosus, carrot beetle Cotton Anthonomus grandis, boll weevil Rice Colaspis brunnea, grape colaspis Lissorhoptrus oryzophilus, rice water weevil Sitophilus oryzae, rice weevil Soybean Epilachna varivestis, Mexican bean beetle______________________________________
TABLE 3______________________________________Homoptera (Whiteflies, Aphids etc..)______________________________________Maize Rhopalosiphum maidis, corn leaf aphid Anuraphis maidiradicis, corn root aphid Sorghum Rhopalosiphum maidis, corn leaf aphid Sipha flava, yellow sugarcane aphid Wheat Russian wheat aphid Schizaphis graminum, greenbug Macrosiphum avenae, English grain aphid Cotton Aphis gossypii, cotton aphid Pseudatomoscelis seriatus, cotton fleahopper Trialeurodes abutilonea, bandedwinged whitefly Rice Nephotettix nigropictus, rice leafhopper Soybean Myzus persicae, green peach aphid Empoasca fabae, potato leafhopper Barley Schizaphis graminum, greenbug Oil Seed Rape Brevicoryne brassicae, cabbage aphid______________________________________
TABLE 4______________________________________Hemiptera (Bugs)______________________________________Maize Blissus leucopterus leucopterus, chinch bug Sorghum Blissus leucopterus leucopterus, chinch bug Cotton Lygus lineolaris, tarnished plant bug Rice Blissus leucopterus leucopterus, chinch bug Acrostenum hilare, green stink bug Soybean Acrostenum hilare, green stink bug Barley Blissus leucopterus leucopterus, chinch bug Acrostenum hilare, green stink bug Euschistus servus, brown stink bug______________________________________
TABLE 5______________________________________Orthontera (Grasshoppers, Crickets, and Cockroaches)______________________________________Maize Melanoplus femurrubrum, redlegged grasshopper Melanoplus sanguinipes, migratory grasshopper Wheat Melanoplus femurrubrum, redlegged grasshopper Melanoplus differentialis, differential grasshopper Melanoplus sanguinipes, migratory grasshopper Cotton Melanoplus femurrubrum, redlegged grasshopper Melanoplus differentialis, differential grasshopper Soybean Melanoplus femurrubrum, redlegged grasshopper Melanoplus differentialis, differential grasshopper Structural/Household Periplaneta americana, American cockroach Blattella germanica, German cockroach Blatta orientalis, oriental cockroach______________________________________
TABLE 6______________________________________Diptera (Flies and Mosquitoes)______________________________________Maize Hylemya platura, seedcorn maggot Agromyza parvicornis, corn blotch leafminer Sorghum Contarinia sorghicola, sorghum midge Wheat Mayetiola destructor, Hessian fly Sitodiplosis mosellana, wheat midge Meromyza americana, wheat stem maggot Hylemya coarctata, wheat bulb fly Sunflower Neolasioptera murtfeldtiana, sunflower seed midge Soybean Hylemya platura, seedcorn maggot Barley Hylemya platura, seedcorn maggot Mayetiola destructor, Hessian fly Insects attacking humans and animals and disease carriers Aedes aegypti, yellowfever mosquito Aedes albopictus, forest day mosquito Phlebotomus papatasii, sand fly Musca domestica, house fly Tabanus atratus, black horse fly Cochliomyia hominivorax, screwworm fly______________________________________
TABLE 7______________________________________Thysanoptera (Thrips)______________________________________ Maize Anaphothrips obscurus, grass thrips Wheat Frankliniella fusca, tobacco thrips Cotton Thrips tabaci, onion thrips Frankliniella fusca, tobacco thrips Soybean Sericothrips variabilis, soybean thrips Thrips tabaci, onion thrips______________________________________
TABLE 8______________________________________Hymenoptera (Sawflies, Ants, Wasps, etc.)______________________________________ Maize Solenopsis milesta, thief ant Wheat Cephus cinctus, wheat stem sawfly______________________________________
TABLE 9______________________________________Other Orders and Representative Species______________________________________Dermaptera (Earwigs) Forficula auricularia, European earwig Isoptera (Termites) Reticulitermes flavipes, eastern subterranean termite Mallophaga (Chewing Lice) Cuclotogaster heterographa, chicken head louse Bovicola bovis, cattle biting louse Anoplura (Sucking Lice) Pediculus humanus, head and body louse Siphonaptera (Fleas) Ctenocephalides felis, cat flea______________________________________
TABLE 10______________________________________Acari (Mites and Ticks)______________________________________Maize Tetranychus urticae, twospotted spider mite Sorghum Tetranychus cinnabarinus, carmine spider mite Tetranychus urticae, twospotted spider mite Wheat Aceria tulipae, wheat curl mite Cotton Tetranychus cinnabarinus, carmine spider mite Tetranychus urticae, twospotted spider mite Soybean Tetranychus turkestani, strawberry spider mite Tetranychus urticae, twospotted spider mite Barley Petrobia latens, brown wheat mite Important human and animal Acari Demacentor variabilis, American dog tick Argas persicus, fowl tick Dermatophagoides farinae, American house dust mite Dermatophagoides pteronyssinus, European house dust mite______________________________________
Now that it has been recognized that pesticidal proteins can be isolated from the vegetative growth phase of Bacillus, other strains can be isolated by standard techniques and tested for activity against particular plant and non-plant pests. Generally Bacillus strains can be isolated from any environmental sample, including soil, plant, insect, grain elevator dust, and other sample material, etc., by methods known in the art. See, for example, Travers et al. (1987) Appl. Environ. Microbiol. 53:1263-1266; Saleh et al. (1969) Can J. Microbiol. 15:1101-1104; DeLucca et al. (1981) Can. J. Microbiol. 27:865-870; and Norris, et al. (1981) "The genera Bacillus and Sporolactobacillus," In Starr et al. (eds.), The Prokaryotes: A Handbook on Habitats, Isolation, and Identification of Bacteria, Vol. II, Springer-Verlog Berlin Heidelberg. After isolation, strains can be tested for pesticidal activity during vegetative growth. In this manner, new pesticidal proteins and strains can be identified.
Such Bacillus microorganisms which find use in the invention include Bacillus cereus and Bacillus thuringiensis, as well as those Bacillus species listed in Table 11.
TABLE 11______________________________________List of Bacillus species Unassigned Strains______________________________________Morphological Group 1 B. megaterium Subgroup A B. cereus* B. apiarus* B. cereus var. mycoides B. filicolonicus B. thuringiensis* B. thiaminolyticus B. licheniformis B. alcalophilus B. subtilis* B. pumilus Subgroup B B. firmus* B. cirroflagellosus B. coagulans B. chitinosporus B. lentus Morphological Group 2 B. polymyxa Subgroup C B. macerans B. badius B. circulans B. aneurinolyticus B. stearothermophilus B. macroides B. alvei* B. freundenreichii B. laterosporus* B. brevis Subgroup D B. pulvifaciens B. pantothenticus B. popilliae* B. epiphytus B. lentimorbus* B. larvae* Subgroup E1 B. aminovorans Morphological Group 3 B. globisporus B. sphaericus* B. insolitus B. pasteurii B. psychrophilus Subgroup E2 B. psychrosaccharolyticus B. macquariensis______________________________________ * = Those Bacillus strains that have been previously found associated wit insects
Grouping according to Parry, J. M. et al. (1983) Color Atlas of Bacillus species, Wolfe Medical Publications, London.
In accordance with the present invention, the pesticidal proteins produced during vegetative growth can be isolated from Bacillus. In one embodiment, insecticidal proteins produced during vegetative growth, can be isolated. Methods for protein isolation are known in the art. Generally, proteins can be purified by conventional chromatography, including gel-filtration, ion-exchange, and immunoaffinity chromatography, by high-performance liquid chromatography, such as reversed-phase high-performance liquid chromatography, ion-exchange high-performance liquid chromatography, size-exclusion high-performance liquid chromatography, high-performance chromatofocusing and hydrophobic interaction chromatography, etc., by electrophoretic separation, such as one-dimensional gel electrophoresis, two-dimensional gel electrophoresis, etc. Such methods are known in the art. See for example Current Protocols in Molecular Biology, Vols. 1 and 2, Ausubel et al. (eds.), John Wiley & Sons, NY (1988). Additionally, antibodies can be prepared against substantially pure preparations of the protein. See, for example, Radka et al. (1983) J. Immunol. 128:2804; and Radka et al. (1984) Immunogenetics 19:63. Any combination of methods may be utilized to purify protein having pesticidal properties. As the protocol is being formulated, pesticidal activity is determined after each purification step.
Such purification steps will result in a substantially purified protein fraction. By "substantially purified" or "substantially pure" is intended protein which is substantially free of any compound normally associated with the protein in its natural state. "Substantially pure" preparations of protein can be assessed by the absence of other detectable protein bands following SDS-PAGE as determined visually or by densitometry scanning. Alternatively, the absence of other amino-terminal sequences or N-terminal residues in a purified preparation can indicate the level of purity. Purity can be verified by rechromatography of "pure" preparations showing the absence of other peaks by ion exchange, reverse phase or capillary electrophoresis. The terms "substantially pure" or "substantially purified" are not meant to exclude artificial or synthetic mixtures of the proteins with other compounds. The terms are also not meant to exclude the presence of minor impurities which do not interfere with the biological activity of the protein, and which may be present, for example, due to incomplete purification.
Once purified protein is isolated, the protein, or the polypeptides of which it is comprised, can be characterized and sequenced by standard methods known in the art. For example, the purified protein, or the polypeptides of which it is comprised, may be fragmented as with cyanogen bromide, or with proteases such as papain, chymotrypsin, trypsin, lysyl-C endopeptidase, etc. (Oike et al. (1982) J. Biol. Chem. 257:9751-9758; Liu et al. (1983) Int. J. Pept. Protein Res. 21:209-215). The resulting peptides are separated, preferably by HPLC, or by resolution of gels and electroblotting onto PVDF membranes, and subjected to amino acid sequencing. To accomplish this task, the peptides are preferably analyzed by automated sequenators. It is recognized that N-terminal, C-terminal, or internal amino acid sequences can be determined. From the amino acid sequence of the purified protein, a nucleotide sequence can be synthesized which can be used as a probe to aid in the isolation of the gene encoding the pesticidal protein.
It is recognized that the pesticidal proteins may be oligomeric and will vary in molecular weight, number of protomers, component peptides, activity against particular pests, and in other characteristics. However, by the methods set forth herein, proteins active against a variety of pests may be isolated and characterized.
Once the purified protein has been isolated and characterized it is recognized that it may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the pesticidal proteins can be prepared by mutations in the DNA. Such variants will possess the desired pesticidal activity. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.
In this manner, the present invention encompasses the pesticidal proteins as well as components and fragments thereof. That is, it is recognized that component protomers, polypeptides or fragments of the proteins may be produced which retain pesticidal activity. These fragments include truncated sequences, as well as N-terminal, C-terminal, internal and internally deleted amino acid sequences of the proteins.
Most deletions, insertions, and substitutions of the protein sequence are not expected to produce radical changes in the characteristics of the pesticidal protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing. so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays.
The proteins or other component polypeptides described herein may be used alone or in combination. That is, several proteins may be used to control different insect pests.
Some proteins are single polypeptide chains while many proteins consist of more than one polypeptide chain, i.e., they are oligomeric. Additionally, some VIPs are pesticidally active as oligomers. In these instances, additional protomers are utilized to enhance the pesticidal activity or to activate pesticidal proteins. Those protomers which enhance or activate are referred to as auxiliary proteins. Auxiliary proteins activate or enhance a pesticidal protein by interacting with the pesticidal protein to form an oligomeric protein having increased pesticidal activity compared to that observed in the absence of the auxiliary protein.
Auxiliary proteins activate or increase the activity of pesticidal proteins such as the VIP1 protein from AB78. Such auxiliary proteins are exemplified by, but not limited to, the VIP2 protein from AB78. As demonstrated in the Experimental section of the application, auxiliary proteins can activate a number of pesticidal proteins. Thus, in one embodiment of the invention, a plant, Parent 1, can be transformed with an auxiliary protein. This Parent 1 can be crossed with a number of Parent 2 plants transformed with one or more pesticidal proteins whose pesticidal activities are activated by the auxiliary protein.
The pesticidal proteins of the invention can be used in combination with Bt endotoxins or other insecticidal proteins to increase insect target range. Furthermore, the use of the VIPs of the present invention in combination with Bt .delta.-endotoxins or other insecticidal principles of a distinct nature has particular utility for the prevention and/or management of insect resistance. Other insecticidal principles include protease inhibitors (both serine and cysteine types), lectins, .alpha.-amylase and peroxidase. In one preferred embodiment, expression of VIPs in a transgenic plant is accompanied by the expression of one or more Bt .delta.-endotoxins. This co-expression of more than one insecticidal principle in the same transgenic plant can be achieved by genetically engineering a plant to contain and express all the genes necessary. Alternatively, a plant, Parent 1, can be genetically engineered for the expression of VIPs. A second plant, Parent 2, can be genetically engineered for the expression of Bt .delta.-endotoxin. By crossing Parent 1 with Parent 2, progeny plants are obtained which express all the genes introduced into Parents 1 and 2. Particularly preferred Bt .delta.-endotoxins are those disclosed in U.S. Pat. No. 5,625,136, herein incorporated by reference.
A substantial number of cytotoxic proteins, though not all, are binary in action. Binary toxins typically consist of two protein domains, one called the A domain and the other called the B domain (see Sourcebook of Bacterial Protein Toxins, J. E. Alouf and J. H. Freer eds.(1991) Academic Press). The A domain possesses a potent cytotoxic activity. The B domain binds an external cell surface receptor before being internalized. Typically, the cytotoxic A domain must be escorted to the cytoplasm by a translocation domain. Often the A and B domains are separate polypeptides or protomers, which are associated by a protein-protein interaction or a di-sulfide bond. However, the toxin can be a single polypeptide which is proteolytically processed within the cell into two domains as in the case for Pseudomonas exotoxin A. In summary binary toxins typically have three important domains, a cytotoxic A domain, a receptor binding B domain and a translocation domain. The A and B domain are often associated by protein-protein interacting domains.
The receptor binding domains of the present invention are useful for delivering any protein, toxin, enzyme, transcription factor, nucleic acid, chemical or any other factor into target insects having a receptor recognized by the receptor binding domain of the binary toxins described in this patent. Similarly, since binary toxins have translocation domains which penetrate phosopholipid bilayer membranes and escort cytotoxins across those membranes, such translocation domains may be useful in escorting any protein, toxin, enzyme, transcription factor, nucleic acid, chemical or any other factor across a phospholipid bilayer such as the plasma membrane or a vesicle membrane. The translocation domain may itself perforate membranes, thus having toxic or insecticidal properties. Further, all binary toxins have cytotoxic domains; such a cytotoxic domain may be useful as a lethal protein, either alone or when delivered into any target cell(s) by any means.
Finally, since binary toxins comprised of two polypeptides often form a complex, it is likely that there are protein-protein interacting regions within the components of the binary toxins of the invention. These protein-protein interacting domains may be useful in forming associations between any combination of toxins, enzymes, transcription factors, nucleic acids, antibodies, cell binding moieties, or any other chemicals, factors, proteins or protein domains.
Toxins, enzymes, transcription factors, antibodies, cell binding moieties or other protein domains can be fused to pesticidal or auxiliary proteins by producing in frame genetic fusions which, when translated by ribosomes, would produce a fusion protein with the combined attributes of the VIP and the other component used in the fusion. Furthermore, if the protein domain fused to the VIP has an affinity for another protein, nucleic acid, carbohydrate, lipid, or other chemical or factor, then a three-component complex can be formed. This complex will have the attributes of all of its components. A similar rationale can be used for producing four or more component complexes. These complexes are useful as insecticidal toxins, pharmaceuticals, laboratory reagents, and diagnostic reagents, etc. Examples where such complexes are currently used are fusion toxins for potential cancer therapies, reagents in ELISA assays and immunoblot analysis.
One strategy of altering pesticidal or auxiliary proteins is to fuse a 15-amino-acid "S-tag" to the protein without destroying the insect cell binding domain(s), translocation domains or protein-protein interacting domains of the proteins. The S-tag has a high affinity (K.sub.d =10.sup.-9 M) for a ribonuclease S-protein, which, when bound to the S-tag, forms an active ribonuclease (See F. M. Richards and H. W. Wyckoff (1971) in "The Enzymes", Vol. IV (Boyer, P.D. ed.). pp. 647-806. Academic Press, New York). The fusion can be made in such a way as to destroy or remove the cytotoxic activity of the pesticidal or auxiliary protein, thereby replacing the VIP cytotoxic activity with a new cytotoxic ribonuclease activity. The final toxin would be comprised of the S-protein, a pesticidal protein and an auxiliary protein, where either the pesticidal protein or the auxiliary protein is produced as translational fusions with the S-tag. Similar strategies can be used to fuse other potential cytotoxins to pesticidal or auxiliary proteins including (but not limited to) ribosome inactivating proteins, insect hormones, hormone receptors, transcription factors, proteases, phosphatases, Pseudomonas exotoxin A, or any other protein or chemical factor that is lethal when delivered into cells. Similarly, proteins can be delivered into cells which are not lethal, but might alter cellular biochemistry or physiology.
The spectrum of toxicity toward different species can be altered by fusing domains to pesticidal or auxiliary proteins which recognize cell surface receptors from other species. Such domains might include (but are not limited to) antibodies, transferrin, hormones, or peptide sequences isolated from phage displayed affinity selectable. libraries. Also, peptide sequences which are bound to nutrients, vitamins, hormones, or other chemicals that are transported into cells could be used to alter the spectrum of toxicity. Similarly, any other protein or chemical which binds a cell surface receptor or the membrane and could be internalized might be used to alter the spectrum of activity of VIP1 and VIP2.
The pesticidal proteins of the present invention are those proteins which confer a specific pesticidal property. Such proteins may vary in molecular weight, having component polypeptides at least a molecular weight of 30 kDa or greater, preferably about 50 kDa or greater.
The auxiliary proteins of the invention may vary in molecular weight, having at least a molecular weight of about 15 kDa or greater, preferably about 20 kDa or greater; more preferably, about 30 kDa or greater. The auxiliary proteins themselves may have component polypeptides.
It is possible that the pesticidal protein and the auxiliary protein may be components of a multimeric, insecticidal protein. Such an insecticidal protein which includes the auxiliary proteins as one or more of its component polypeptides may vary in molecular weight, having at least a molecular weight of 50 kDa up to at least 200 kDa, preferably about 100 kDa to 150 kDa.
An auxiliary protein may be used in combination with the pesticidal proteins of the invention to enhance activity or to activate the pesticidal protein. To determine whether the auxiliary protein will affect activity, the pesticidal protein can be expressed alone and in combination with the auxiliary protein and the respective activities compared in feeding assays for pesticidal activity.
It may be beneficial to screen strains for potential pesticidal activity by testing activity of the strain alone and in combination with the auxiliary protein. In some instances an auxiliary protein in combination with the native proteins of the strains yields pesticidal activity where none is seen in the absence of an auxiliary protein.
The auxiliary protein can be modified, as described above, by various methods known in the art. Therefore, for purposes of the invention, the term "Vegetative Insecticidal Protein" (VIP) encompasses those proteins produced during vegetative growth which alone or in combination can be used for pesticidal activity. This includes pesticidal proteins, auxiliary proteins and those proteins which demonstrate activity only in the presence of the auxiliary protein or the polypeptide components of these proteins.
It is recognized that there are alternative methods available to obtain the nucleotide and amino acid sequences of the present proteins. For example, to obtain the nucleotide sequence encoding the pesticidal protein, cosmid clones, which express the pesticidal protein, can be isolated from a genomic library. From larger active cosmid clones, smaller subclones can be made and tested for activity. In this manner, clones which express an active pesticidal protein can be sequenced to determine the nucleotide sequence of the gene. Then, an amino acid sequence can be deduced for the protein. For general molecular methods, see, for example, Molecular Cloning, A Laboratory Manual, Second Edition, Vols. 1-3, Sambrook et al. (eds.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1999), and the references cited therein.
The present invention also encompasses nucleotide sequences from organisms other than Bacillus, where the nucleotide sequences are isolatable by hybridization with the Bacillus nucleotide sequences of the invention. Proteins encoded by such nucleotide sequences can be tested for pesticidal activity. The invention also encompasses the proteins encoded by the nucleotide sequences. Furthermore, the invention encompasses proteins obtained from organisms other than Bacillus wherein the protein cross-reacts with antibodies raised against the proteins of the invention. Again the isolated proteins can be assayed for pesticidal activity by the methods disclosed herein or others well-known in the art.
Once the nucleotide sequences encoding the pesticidal proteins of the invention have been isolated, they can be manipulated and used to express the protein in a variety of hosts including other organisms, including microorganisms and plants.
The pesticidal genes of the invention can be optimized for enhanced expression in plants. See, for example U.S. Pat. No. 5,605,136; EPA 0359472; EPA 0385962; WO 91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al. (1989) Nucleic Acids Research 17: 477-498. In this manner, the genes can be synthesized utilizing plant preferred codons. That is the preferred codon for a particular host is the single codon which most frequently encodes that amino acid in that host. The maize preferred codon, for example, for a particular amino acid may be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants is found in Murray et al. (1989), Nucleic Acids Research 17:477-498, the disclosure of which is incorporated herein by reference. Synthetic genes can also be made based on the distribution of codons a particular host uses for a particular amino acid.
In this manner, the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used.
In like manner, the nucleotide sequences can be optimized for expression in any microorganism. For Bacillus preferred codon usage, see, for example U.S. Pat. No. 5,024,837 and Johansen et al. (1988) Gene 65:293-304.
Methodologies for the construction of plant expression cassettes as well as the introduction of foreign DNA into plants are described in the art. Such expression cassettes may include promoters, terminators, enhancers, leader sequences, introns and other regulatory sequences operably linked to the pesticidal protein coding sequence. It is further recognized that promoters or terminators of the VIP genes can be used in expression cassettes.
Generally, for the introduction of foreign DNA into plants Ti plasmid vectors have been utilized for the delivery of foreign DNA as well as direct DNA uptake, liposomes, electroporation, micro-injection, and the use of microprojectiles. Such methods had been published in the art. See, for example, Guerche et al., (1987) Plant Science 52:111-116; Neuhause et al., (1987) Theor. Appl. Genet. 75:30-36; Klein et al., (1987) Nature 327: 70-73; Howell et al., (1980) Science 208:1265; Horsch et al., (1985) Science 227: 1229-1231; DeBlock et al., (1989) Plant Physiology 91:694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press, Inc. (1988); and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc. (1989). See also abandoned U.S. patent application Ser. No. 08/008,374 herein incorporated by reference. See also, EPA 0193259 and EPA 0451878A1. It is understood that the method of transformation will depend upon the plant cell to be transformed.
It is further recognized that the components of the expression cassette may be modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications may be employed. See, for example Perlak et al. (1991) Proc. Natl. Acad. Sci. USA 88:3324-3328; Murray et al., (1989) Nucleic Acids Research 17:477-498; and WO 91/16432.
The construct may also include any other necessary regulators such as terminators, (Guerineau et al., (1991), Mol. Gen. Genet., 226:141-144; Proudfoot, (1991), Cell, 64:671-674; Sanfacon et al., (1991), Genes Dev., 5:141-149; Mogen et al., (1990), Plant Cell, 2:1261-1272; Munroe et al., (1990), Gene, 91:151-158; Ballas et al et al., (1989), Nucleic Acids Res., 17:7891-7903; Joshi et al., (1987), Nucleic Acid Res., 15:9627-9639); plant translational consensus sequences (Joshi, C. P., (1987), Nucleic Acids Research, 15:6643-6653), introns (Luehrsen and Walbot, (1991), Mol. Gen. Genet., 225:81-93) and the like, operably linked to the nucleotide sequence. It may be beneficial to include 5' leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translational leaders are known in the art and include:
Picornavirus leaders, for example, EMCV leader (encephalomyocarditis 5' noncoding region) (Elroy-Stein, O., Fuerst, T. R., and Moss, B. (1989) PNAS USA 86:6126-6130);
Potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al., (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology, 154:9-20), and
Human immunoglobulin heavy-chain binding protein (BiP), (Macejak, D. G., and Sarnow, P., (1991), Nature, 353:90-94;
Untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke, L., (1987), Nature, 325:622-625;
Tobacco mosaic virus leader (TMV), (Gallie, D. R. et al., (1989), Molecular Biology of RNA, pages 237-256; and
Maize Chlorotic Mottle Virus leader (MCMV) (Lommel, S. A. et al., (1991), Virology, 81:382-385. See also, Della-Cioppa et al., (1987), Plant Physiology, 84:965-968.
A plant terminator may be utilized in the expression cassette. See, Rosenberg et al., (1987), Gene, 56:125; Guerineau et al., (1991), Mol. Gen. Genet., 226:141-144; Proudfoot, (1991), Cell, 64:671-674; Sanfacon et al., (1991), Genes Dev., 5:141-149; Mogen et al., (1990), Plant Cell, 2:1261-1272; Munroe et al., (1990), Gene, 91:151-158; Ballas et al., (1989), Nucleic Acids Res., 17:7891-7903; Joshi et al., (1987), Nucleic Acid Res., 15:9627-9639.
For tissue specific expression, the nucleotide sequences of the invention can be operably linked to tissue specific promoters. See, for example, U.S. Pat. No. 5,625,136 herein incorporated by reference.
It is recognized that the genes encoding the pesticidal proteins can be used to transform insect pathogenic organisms. Such organisms include Baculoviruses, fungi, protozoa, bacteria and nematodes.
The Bacillus strains of the invention may be used for protecting agricultural crops and products from pests. Alternatively, a gene encoding the pesticide may be introduced via a suitable vector into a microbial host, and said host applied to the environment or plants or animals. Microorganism hosts may be selected which are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops of interest. These microorganisms are selected so as to be capable of successfully competing in the particular environment with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.
Such microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonasfluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces rosues, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms.
A number of ways are available for introducing a gene expressing the pesticidal protein into the microorganism host under conditions which allow for stable maintenance and expression of the gene. For example, expression cassettes can be constructed which include the DNA constructs of interest operably linked with the transcriptional and translational regulatory signals for expression of the DNA constructs, and a DNA sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system which is functional in the host, whereby integration or stable maintenance will occur.
Transcriptional and translational regulatory signals include but are not limited to promoter, transcriptional initiation start site, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See, for example, U.S. Pat. No. 5,039,523; U.S. Pat. No. 4,853,331; EPO 0480762A2; Sambrook et al. supra; Molecular Cloning, a Laboratory Manual, Maniatis et al. (eds) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982); Advanced Bacterial Genetics, Davis et al. (eds.) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1980); and the references cited therein.
Suitable host cells, where the pesticide-containing cells will be treated to prolong the activity of the toxin in the cell when the then treated cell is applied to the environment of the target pest(s), may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxin is unstable or the level of application sufficiently low as to avoid any possibility of toxicity to a mammalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi. Illustrative prokaryotes, both Gram-negative and -positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such a Saccharomyces and Schizosaccharromyces; and Basidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.
Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the protein gene into the host, availability of expression systems, efficiency of expression, stability of the protein in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.
Host organisms of particular interest include yeast, such as Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.; phylloplane organisms such as Pseudomonas sp., Erwinia sp. and Flavobacterium sp.; or such other organisms as Escherichia, LactoBacillus sp., Bacillus sp., and the like. Specific organisms include Pseudomonas aeurginosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis, and the like.
VIP genes can be introduced into micro-organisms that multiply on plants (epiphytes) to deliver VIP proteins to potential target pests. Epiphytes can be gram-positive or gram-negative bacteria for example.
Root colonizing bacteria, for example, can be isolated from the plant of interest by methods known in the art. Specifically, a Bacillus cereus strain which colonizes roots could be isolated from roots of a plant (for example see J. Handelsman, S. Raffel, E. Mester, L. Wunderlich and C. Grau, Appl. Environ. Microbiol. 56:713-718, (1990)). VIP1 and/or VIP2 could be introduced into a root colonizing Bacillus cereus by standard methods known in the art.
Specifically, VIP1 and/or VIP2 derived from Bacillus cereus strain AB78 can be introduced into a root colonizing Bacillus cereus by means of conjugation using standard methods (J. Gonzalez, B. Brown and B. Carlton, Proc. Natl. Acad. Sci. 79:6951-6955, (1982)).
Also, VIP1 and/or VIP2 or other VIPs of the invention can be introduced into the root colonizing Bacillus by means of electro-transformation. Specifically, VIPs can be cloned into a shuttle vector, for example, pHT3101 (D. Lereclus et al., FEMS Microbiol. Letts., 60:211-218 (1989)) as described in Example 10. The shuttle vector pHT3101 containing the coding sequence for the particular VIP can then be transformed into the root colonizing Bacillus by means of electroporation (D. Lereclus et al. 1989, FEMS Microbiol. Letts. 60:211-218).
Expression systems can be designed so that VIP proteins are secreted outside the cytoplasm of gram negative bacteria, E. coli, for example. Advantages of having VIP proteins secreted are (1) it avoids potential toxic effects of VIP proteins expressed within the cytoplasm and (2) it can increase the level of VIP protein expressed and (3) can aid in efficient purification of VIP protein.
VIP proteins can be made to be secreted in E. coli, for example, by fusing an appropriate E. coli signal peptide to the amino-terminal end of the VIP signal peptide or replacing the VIP signal peptide with the E. coli signal peptide. Signal peptides recognized by E. coli can be found in proteins already known to be secreted in E. coli, for example the OmpA protein (J. Ghrayeb, H. Kimura, M. Takahara, Y. Masui and M. Inouye, EMBO J., 3:2437-2442 (1984)). OmpA is a major protein of the E. coli outer membrane and thus its signal peptide is thought to be efficient in the translocation process. Also, the OmpA signal peptide does not need to be modified before processing as may be the case for other signal peptides, for example lipoprotein signal peptide (G. Duffaud, P. March and M. Inouye, Methods in Enzymology 153:492 (1987)).
Specifically, unique BamHil restriction sites can be introduced at the amino-terminal and carboxy-terminal ends of the VIP coding sequences using standard methods known in the art. These BamHI fragments can be cloned, in frame, into the vector pIN-III-ompA1, A2 or A3 (J. Ghrayeb, H. Kimura, M. Takahara, H. Hsiung, Y. Masui and M. Inouye, EMBO J., 3:2437-2442 (1984)) thereby creating ompA:VIP fusion gene which is secreted into the periplasmic space. The other restriction sites in the polylinker of pIN-III-ompA can be eliminated by standard methods known in the art so that the VIP amino-terminal amino acid coding sequence is directly after the ompA signal peptide cleavage site. Thus, the secreted VIP sequence in E. coli would then be identical to the native VIP sequence.
When the VIP native signal peptide is not needed for proper folding of the mature protein, such signal sequences can be removed and replaced with the ompA signal sequence. Unique BamHI restriction sites can be introduced at the amino-ternini of the proprotein coding sequences directly after the signal peptide coding sequences of VIP and at the carboxy-termini of VIP coding sequence. These BamHI fragments can then be cloned into the pIN-III-ompA vectors as described above.
General methods for employing the strains of the invention in pesticide control or in engineering other organisms as pesticidal agents are known in the art. See, for example U.S. Pat. No. 5,039,523 and EP 0480762A2.
VIPs can be fermented in a bacterial host and the resulting bacteria processed and used as a microbial spray in the same manner that Bacillus thuringiensis strains have been used as insecticidal sprays. In the case of a VIP(s) which is secreted from Bacillus, the secretion signal is removed or mutated using procedures known in the art. Such mutations and/or deletions prevent secretion of the VIP protein(s) into the growth medium during the fermentation process. The VIPs are retained within the cell and the cells are then processed to yield the encapsulated VIPs. Any suitable microorganism can be used for this purpose. Psuedomonas has been used to express Bacillus thuringiensis endotoxins as encapsulated proteins and the resulting cells processed and sprayed as an insecticide. (H. Gaertner et al. 1993, In Advanced Engineered Pesticides, L. Kim ed.)
Various strains of Bacillus thuringiensis are used in this manner. Such Bt strains produce endotoxin protein(s) as well as VIPs. Alternatively, such strains can produce only VIPs. A sporulation deficient strain of Bacillus subtilis has been shown to produce high levels of the CryIIIA endotoxin from Bacillus thuringiensis (Agaisse, H. and Lereclus, D., "Expression in Bacillus subtilis of the Bacillus thuringiensis CryIIIA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spoOA mutant", J. Bacteriol., 176:4734-4741 (1994)). A similar spoOA mutant can be prepared in Bacillus thuringiensis and used to produce encapsulated VIPs which are not secreted into the medium but are retained within the cell.
To have VIPs maintained within the Bacillus cell the signal peptide can be disarmed so that it no longer functions as a secretion signal. Specifically, the putative signal peptide for VIP 1 encompasses the first 31 amino acids of the protein with the putative consensus cleavage site, Ala-X-Ala, at the C-terminal portion of this sequence (G. von Heijne, J. Mol. Biol. 184:99-105 (1989)) and the putative signal peptide for VIP2 encompasses the first 40 amino acids of the protein with the putative cleavage site after Ala40. The cleavage sites in either VIP 1 or VIP2 can be mutated with methods known in the art to replace the cleavage site consensus sequence with alternative amino acids that are not recognized by the signal peptidases.
Alternatively, the signal peptides of VIP1, VIP2 and/or other VIPs of the invention can be eliminated from the sequence thereby making them unrecognizable as secretion proteins in Bacillus. Specifically, a methionine start site can be engineered in front of the proprotein sequence in VIP1, starting at Asp32, or the proprotein sequence in VIP2, starting at Glu41 using methods known in the art.
VIP genes can be introduced into micro-organisms that multiply on plants (epiphytes) to deliver VIP proteins to potential target pests. Epiphytes can be gram-positive or gram-negative bacteria for example.
The Bacillus strains of the invention or the microorganisms which have been genetically altered to contain the pesticidal gene and protein may be used for protecting agricultural crops and products from pests. In one aspect of the invention, whole, i.e., unlysed, cells of a toxin (pesticide)-producing organism are treated with reagents that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s).
Alternatively, the pesticides are produced by introducing a heterologous gene into a cellular host. Expression of the heterologous gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. These cells are then treated under conditions that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s). The resulting product retains the toxicity of the toxin. These naturally encapsulated pesticides may then be formulated in accordance with conventional techniques for application to the environment hosting a target pest, e.g., soil, water, and foliage of plants. See, for example EPA 0192319, and the references cited therein.
The active ingredients of the present invention are normally applied in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with other compounds. These compounds can be both fertilizers or micronutrient donors or other preparations that influence plant growth. They can also be selective herbicides, insecticides, fungicides, bactericides, nematicides, mollusicides or mixtures of several of these preparations, if desired, together with further agriculturally acceptable carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.
Preferred methods of applying an active ingredient of the present invention or an agrochemical composition of the present invention which contains at least one of the pesticidal proteins produced by the bacterial strains of the present invention are leaf application, seed coating and soil application. The number of applications and the rate of application depend on the intensity of infestation by the corresponding pest.
In one embodiment of the invention a Bacillus cereus microorganism has been isolated which is capable of killing Diabrotica virgifera virgifera, and Diabrotica longicornis barberi. The novel B. cereus strain AB78 has been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, IL 61604, USA and given Accession No. NRRL B-21058.
A fraction protein has been substantially purified from the B. cereus strain. This purification of the protein has been verified by SDS-PAGE and biological activity. The protein has a molecular weight of about 60 to about 100 kDa, particularly about 70 to about 90 kDa, more particularly about 80 kDa, hereinafter VIP.
Amino-terminal sequencing has revealed the N-terminal amino-acid sequence to be: NH.sub.2 -Lys-Arg-Glu-Ile-Asp-Glu-Asp-Thr-Asp-Thr-Asx-Gly-Asp-Ser-Ile-Pro-(SEQ ID NO:8) where Asx represents either Asp or Asn. The entire amino acid sequence is given in SEQ ID NO:7. The DNA sequence which encodes the amino acid sequence of SEQ ID NO:7 is disclosed in SEQ ID NO:6.
An oligonuleotide probe for the region of the gene encoding amino acids 3-9 of the NH.sub.2 -terminus has been generated. The probe was synthesized based on the codon usage of a Bacillus thuringiensis (Bt) .delta.-endotoxin gene. The nucleotide sequence of the oligonucleotide is probe used for Southern hybridizations was as follows:
5'-GAA ATT GAT CAA GAT ACN GAT-3' (SEQ ID NO:9)
where N represents any base.
In addition, the DNA probe for the Bc AB78 VIP1 gene described herein, permits the screening of any Bacillus strain or other organisms to determine whether the VIP1 gene (or related gene) is naturally present or whether a particular transformed organism includes the VIP1 gene.
The invention now being generally described, the same will be better understood by reference to the following detailed examples that are provided for the purpose of illustration and are not to be considered limiting of the invention unless so specified.
A standard nomenclature has been developed based on the sequence identity of the proteins encompassed by the present invention. The gene and protein names for the detailed examples which follow and their relationship to the names used in the parent application are shown below.
______________________________________Gene/ Protein Name under Standard Gene/Protein Nomenclature Name in Parent Description of Protein______________________________________VIP1A(a) VIP1 VIP1 from strain AB78 as disclosed in SEQ ID NO:5. VIP2A(a) VIP2 VIP2 from strain AB78 as disclosed in SEQ ID NO:2. VIP1A(b) VIP1 homolog VIP1 from Bacillus thuringiensis var. tenebrionis as disclosed in SEQ ID NO:21. VIP2A(b) VIP2 homolog VIP2 from Bacillus thuringiensis var. tenebrionis as disclosed in SEQ ID NO:20. VIP3A(a) -- VIP from strain AB88 as disclosed in SEQ ID NO:28 of the present application VIP3A(b) -- VIP from strain AB424 as disclosed in SEQ ID NO:31 of the present application______________________________________
EXPERIMENTAL
EXAMPLE 1
AB78 ISOLATION AND CHARACTERIZATION
Bacillus cereus strain AB78 was isolated as a plate contaminant in the laboratory on T3 edia (per liter: 3 g tryptone, 2 g tryptose, 1.5 g yeast extract, 0.05 M sodium phosphate (pH 6.8), and 0.005 g MnCl.sub.2 ; Travers, R. S. 1983). During log phase growth, AB78 gave significant activity against western corn rootworm. Antibiotic activity against gram-positive Bacillus spp. was also demonstrated (Table 12).
TABLE 12______________________________________Antibiotic activitv of AB78 culture supernatant Zone of inhibition(cm)Bacteria tested AB78 Streptomycin______________________________________E. coli 0.0 3.0 B. megaterium 1.1 2.2 B. mycoides 1.3 2.1 B. cereus CB 1.0 2.0 B. cereus 11950 1.3 2.1 B. cereus 14579 1.0 2.4 B. cereus AB78 0.0 2.2 Bt var. israelensis 1.1 2.2 Bt var. tenebrionis 0.9 2.3______________________________________
Morphological characteristics of AB78 are as follows:
Vegetative rods straight, 3.1-5.0 mm long and 0.5-2.0 mm wide. Cells with rounded ends, single in short chains. Single subterminal, cylindrical-oval, endospore formed per cell. No parasporal crystal formed. Colonies opaque, erose, lobate and flat. No pigments produced. Cells motile. Flagella present.
Growth characteristics of AB78 are as follows:
Facultative anaerobe with optimum growth temperature of 21-30.degree. C. Will grow at 15, 20, 25, 30 and 37.degree. C. Will not grow above 40.degree. C. Grows in 5-7% NaCl.
Table 13 provides the biochemical profile of AB78.
TABLE 13______________________________________Biochemical characteristics of B. cereus strain AB78.______________________________________Acid from L-arabinose - Methylene blue reoxidized + Gas from L-arabinose - Nitrate reduced + Acid from D-xylose - NO.sub.3 reduced to NO.sub.2 + Gas from D-xylose - VP + Acid from D-glucose + H.sub.2 O.sub.2 decomposed + Gas from D-glucose - Indole - Acid from lactose - Tyrosine decomposed + Gas from lactose - Dihydroxiacetone - Acid from sucrose - Litmus milk acid - Gas from sucrose - Litmus milk coagulated - Acid from D-mannitol - Litmus milk alkaline - Gas from D-mannitol - Litmus milk peptonized - Proprionate utilization + Litmus milk reduced - Citrate utilization + Casein hydrolyzed + Hippurate hydrolysis w Starch hydrolyzed + Methylene blue reduced + Gelatin liquidified + Lecithinase produced w______________________________________ w = weak reaction
EXAMPLE 2
BACTERIAL CULTURE
A subculture of Bc strain AB78 was used to inoculate the following medium, known as TB broth:
______________________________________Tryptone 12 g/l Yeast Extract 24 g/l Glycerol 4 ml/l KH.sub.2 PO.sub.4 2.1 g/l K.sub.2 HPO.sub.4 14.7 g/l pH 7.4______________________________________
The potassium phosphate was added to the autoclaved broth after cooling. Flasks were incubated at 30.degree. C. on a rotary shaker at 250 rpm for 24 h.-36 h, which represents an early to mid-log growth phase.
The above procedure can be readily scaled up to large fermentors by procedures well known in the art.
During vegetative growth, usually 24-36 h. after starting the culture, which represents an early to mid-log growth phase, AB78 bacteria were centrifuged from the culture supernatant. The culture supernatant containing the active protein was used in bioassays.
EXAMPLE 3
INSECT BIOASSAYS
B. cereus strain AB78 was tested against various insects as described below.
Western, Northern and Southern corn rootworm, Diabrotica virgifera virgifera, D. longcornis barberi and D. undecempunctata howardi, respectively: dilutions were made of AB78 culture supernatant grown 24-36 h., mixed with molten artificial diet (Marrone et al. 1985) J. of Economic Entomology 78:290-293) and allowed to solidify. Solidified diet was cut and placed in dishes. Neonate larvae were placed on the diet and held at 30.degree. C. Mortality was recorded after 6 days.
E. coli clone bioassay: E. coli cells were grown overnight in broth containing 100 .mu.g/ml ampicillin at 37.degree. C. Ten ml culture was sonicated 3.times. for 20 sec each. 500 .mu.l of sonicated culture was added to molten western corn rootworm diet.
Colorado potato beetle, Leptinotarsa decemlineata: dilutions in Triton X-100 (to give final concentration of 0.1% TX-100) were made of AB78 culture supernatant grown 24-36 h. Five cm.sup.2 potato leaf pieces were dipped into these dilutions, air dried, and placed on moistened filter paper in plastic dishes. Neonate larvae were placed on the leaf pieces and held at 30.degree. C. Mortality was recorded after 3-5 days.
Yellow mealworm, Tenebrio molitor: dilutions were made of AB78 culture supernatant grown 24-36 h., mixed with molten artificial diet (Bioserv #F9240) and allowed to solidify. Solidified diet was cut and placed in plastic dishes. Neonate larvae were placed on the diet and held at 30.degree. C. Mortality was recorded after 6-8 days.
European corn borer, black cutworm, tobacco budworm, tobacco hornworm and beet armyworm; Ostrinia nubilalis, Agrotis epsilon, Heliothis virescens, Manduca sexta and Spodoptera exigua, respectively: dilutions, in TX-100 (to give final concentration of 0.1% TX-100), were made of AB78 culture supernatant grown 24-36 hrs. 100 .mu.l was pipetted onto the surface of 18 cm.sup.2 of solidified artificial diet (Bioserv #F9240) and allowed to air dry. Neonate larvae were then placed onto the surface of the diet and held at 30.degree. C. Mortality was recorded after 3-6 days.
Northern house mosquito, Culex pipiens:-dilutions were made of AB78 culture supernatant grown 24-36 h. 100 .mu.l was pipetted into 10 ml water in a 30 ml plastic cup. Third instar larvae were added to the water and held at room temperature. Mortality was recorded after 24-48 hours. The spectrum of entomocidal activity of AB78 is given in Table 14.
TABLE 14______________________________________Activity of AB78 culture supernatant against various insect species Insect species tested to date Order Activity______________________________________Western corn rootworm Col +++ (Diabrotica virgifera virgifera) Northern corn rootworm Col +++ (Diabrotica longicornis barberi) Southern corn rootworm Col - (Diabrotica undecimpunctata howardi) Colorado potato beetle Col - (Leptinotarsa decemlineata) Yellow mealworm Col - (Tenebrio molitor) European corn borer Lep - (Ostrinia nubilalis) Tobacco budworm Lep - (Heliothis virescens) Tobacco hornworm Lep - (Manduca sexta) Beet armyworm Lep - (Spodoptera exigua) Black cutworm Lep - (Agrotis ipsilon) Northern house mosquito Dip - (Culex pipiens)______________________________________
The newly discovered B. cereus strain AB78 showed a significantly different spectrum of insecticidal activity as compared to known coleopteran active .delta.-endotoxins from Bt. In particular, AB78 showed more selective activity against beetles than known coleopteran-active Bt strains in that it was specifically active against Diabrotica W. More specifically, it was most active against D. virgifera virgifera and D. longicornis barberi but not D. undecimpunctata howardi.
A number of Bacillus strains were bioassayed for activity during vegetative growth (Table 15) against western corn rootworm. The results demonstrate that AB78 is unique in that activity against western corn rootworm is not a general phenomenon.
TABLE 15______________________________________Activity of culture supernatants from various Bacillus spp. against western corn rootworm Percent Bacillus strain WCRW mortality______________________________________B. cereus AB78 (Bat.1) 100 B. cereus AB78 (Bat.2) 100 B. cereus (Carolina Bio.) 12 B. cereus ATCC 11950 12 B. cereus ATCC 14579 8 B. mycoides (Carolina Bio.) 30 B. popilliae 28 B. thuringiensis HD135 41 B. thuringiensis HD191 9 B. thuringiensis GC91 4 B. thuringiensis isrealensis 24 Water Control 4______________________________________
Specific activity of AB78 against western corn rootworm is provided in Table 16.
TABLE 16______________________________________Activity of AB78 culture supernatant against neonate western corn rootworm Culture supernatant Percent concentration (.mu.l/ml) WCRW mortality______________________________________100 100 25 87 10 80 5 40 2.5 20 1 6 0 0______________________________________
The LC.sub.50 was calculated to be 6.2 .mu.l of culture supernatant per ml of western corn rootworm diet.
The cell pellet was also bioassayed and had no activity against WCRW. Thus, the presence of activity only in the supernatant indicates that this VIP is an exotoxin.
EXAMPLE 4
ISOLATION AND PURIFICATION OF CORN ROOTWORM ACTIVE PROTEINS FROM AB78.
Culture media free of cells and debris was made to 70% saturation by the addition of solid ammonium sulfate (472 g/L). Dissolution was at room temperature followed by cooling in an ice bath and centrifugation at 10,000.times.g for thirty minutes to pellet the precipitated proteins. The supernatant was discarded and the pellet was dissolved in 1/10 the original volume of 20 mM TRIS-HCl at pH 7.5. The dissolved pellet was desalted either by dialysis in 20 mM TRIS-HCl pH 7.5, or passing through a desalting column.
The desalted material was titrated to pH 3.5 using 20 mM sodium citrate pH 2.5. Following a thirty minute room temperature incubation the solution was centrifuged at 3000.times.g for ten minutes. The supernatant at this stage contained the greatest amount of active protein.
Following neutralization of the pH to 7.0 the supernatant was applied to a Mono-Q, anion exchange, column equilibrated with 20 mM TRIS pH 7.5 at a flow rate of 300 mL/min. The column was developed with a stepwise and linear gradient employing 400 mM NaCl in 20 mM TRIS pH 7.5.
Bioassay of the column fractions and SDS-PAGE analysis were used to confirm the active fractions. SDS-PAGE analysis identified the biologically active protein as having components of a molecular weight in the range of about 80 kDa and 50 kDa.
EXAMPLE 5
SEQUENCE ANALYSIS OF THE CORN ROOTWORM ACTIVE PROTEIN
The 80 kDa component isolated by SDS-PAGE was transferred to PVDF membrane and was subjected to amino-terminal sequencing as performed by repetitive Edman cycles on an ABI 470 pulsed-liquid sequencer. Transfer was carried out in 10 mM CAPS buffer with 10% methanol pH 11.0 as follows:
Incubation of the gel following electrophoresis was done in transfer buffer for five minutes. ProBlott PVDF membrane was wetted with 100% MeOH briefly then equilibrated in transfer buffer. The sandwich was arranged between foam sponges and filter paper squares with the configuration of cathode-gel-membrane-anode.
Transfer was performed at 70 V constant voltage for 1 hour.
Following transfer, the membrane was rinsed with water and stained for two minutes with 0.25% Coomassie Blue R-250 in 50% MeOH.
Destaining was done with several rinses with 50% MeOH 40% water 10% acetic acid.
Following destaining the membrane was air dried prior to excision of the bands for sequence analysis. A BlottCartridge and appropriate cycles were utilized to achieve maximum efficiency and yield. Data analysis was performed using model 610 Sequence Analysis software for identifying and quantifying the PTH-amino acid derivatives for each sequential cycle.
The N-terminal sequence was determined to be: NH2-Lys-Arg-Glu-Ile-Asp-Glu-Asp-Thr-Asp-Thr-Asx-Gly-Asp-Ser-Ile-Pro-(SEQ ID NO:8) where Asx represents Asp or Asn. The complete amino acid sequence for the 80 kDa component is disclosed in SEQ ID NO:7. The DNA sequence which encodes SEQ ID NO:7 is disclosed in SEQ ID NO:6.
EXAMPLE 6
CONSTRUCTION OF DNA PROBE
An oligonucleotide probe for the region of the gene encoding amino acids 3-9 of the N-terminal sequence (Example 5) was generated. The probe was synthesized based on the codon usage of a Bacillus thuringiensis (Bt) .delta.-endotoxin gene. The nucleotide sequence
5'-GAA ATT GAT CAA GAT ACN GAT-3' (SEQ ID NO:9)
was used as a probe in Southern hybridizations. The oligonucleotide was synthesized using standard procedures and equipment.
EXAMPLE 7
ISOELECTRIC POINT DETERMINATION OF THE CORN ROOTWORM ACTIVE PROTEIN
Purified protein from step 5 of the purification process was analyzed on a 3-9 pI isoelectric focusing gel using the Phastgel electrophoresis system (Pharmacia). Standard operating procedures for the unit were followed for both the separation and silver staining development procedures. The pI was approximated at about 4.9.
EXAMPLE 8
PCR DATA ON AB78
PCR analysis (See, for example U.S. patent application Ser. No. 08/008,006; and, Carozzi et al. (1991) Appl. Environ. Microbiol. 57(11):3057-306 1, herein incorporated by reference.) was used to verify that the B. cereus strain AB78 did not contain any insecticidal crystal protein genes of B. thuringiensis or B. sphaericus (Table 17).
TABLE 17______________________________________Bacillus insecticidal crystal protein gene primers tested by PCR against AB78 DNA. Primers Tested Product Produced______________________________________2 sets specific for CryIIIA Negative CryIIIB Negative 2 sets specific for CryIA Negative CryIA(a) Negative CryIA(b) specific Negative CryIB Negative CryIC specific Negative CryIE specific Negative 2 sets specific for B. sphaericus Negative 2 sets specific for CryIV Negative Bacillus control (PI-PLC) Positive______________________________________
EXAMPLE 9
COSMID CLONING OF TOTAL DNA FROM B. CEREUS STRAIN AB78
The VIP1A(a) gene was cloned from total DNA prepared from strain AB78 as follows:
Isolation of AB78 DNA was as follows:
1. Grow bacteria in 10 ml L-broth overnight. (Use 50 ml sterile centrifuge tube)
2. Add 25 ml of fresh L-broth and ampicillin (30 .mu.g/ml).
3. Grow cells 2-6 h. at 30.degree. C. with shaking.
4. Spin cells in a 50 ml polypropylene orange cap tube in IEC benchtop clinical centrifuge at 3/4 speed.
5. Resuspend cell pellet in 10 ml TES (TES=50 mM TRIS pH 8.0, 100 mM EDTA, 15 mM NaCl).
6. Add 30 mg lysozyme and incubate 2 hrs at 37.degree. C.
7. Add 200 .mu.l 20% SDS and 400 .mu.l Proteinase K stock (20 mg/ml). Incubate at 37.degree. C.
8. Add 200 .mu.l fresh Proteinase K. Incubate 1 hr. at 55.degree. C. Add 5 ml TES to make 15 ml final volume.
9. Phenol extract twice (10 ml phenol, spin at room temperature at 3/4 speed in an IEC benchtop clinical centrifuge). Transfer supernatant (upper phase) to a clean tube using a wide bore pipette.
10. Extract once with 1:1 vol. phenol:chloroform/isoamyl alcohol (24:1 ratio).
11. Precipitate DNA with an equal volume of cold isopropanol; Centrifuge to pellet DNA.
12. Resuspend pellet in 5 ml TE.
13. Precipitate DNA with 0.5 ml 3M NaOAc pH 5.2 and 11 ml 95% ethanol. Place at -20.degree. C. for 2h.
14. "Hook" DNA from tube with a plastic loop, transfer to a microfuge tube, spin, pipette off excess ethanol, dry in vacuo.
15. Resuspend in 0.5 ml TE. Incubate 90 min. at 65.degree. C. to help get DNA back into solution.
16. Determine concentration using standard procedures.
Cosmid Cloning of AB78
All procedures, unless indicated otherwise, were performed according to Stratagene Protocol, Supercos 1 Instruction Manual, Cat. No. 251301.
Generally, the steps were as follows:
A. Sau 3A partial digestion of the AB78 DNA.
B. Preparation of vector DNA
C. Ligation and packaging of DNA
D. Tittering the cosmid library
1. Start a culture of HB101 cells by placing 50 ml of an overnight culture in 5 mls of TB with 0.2% maltose. Incubate 3.5 hrs. at 37.degree. C.
2. Spin out cells and resuspend in 0.5 ml 10 mM MgSO.sub.4.
3. Add together:
100 .mu.l cells
100 .mu.l diluted packaging mixture
100 .mu.l 10 mM MgSO.sub.4
30 .mu.l TB
4. Adsorb at room temperature for 30 minutes with no shaking.
5. Add 1 ml TB and mix gently. Incubate 30 minutes at 37.degree. C.
6. Plate 200 .mu.l onto L-amp plates. Incubate at 37.degree. C. overnight.
At least 400 cosmid clones were selected at random and screened for activity against western corn rootworm as described in Example 3. DNA from 5 active clones and 5 non-active clones were used in Southern hybridizations. Results demonstrated that hybridization using the above described oligonucleotide probe correlated with western corn rootworm activity (Table 18).
Cosmid clones P3-12 and P5-4 have been deposited with the Agricultural Research Service Patent Culture Collection (NRRL) and given Accession Nos. NRRL B-21061 and NRRL B-21059 respectively.
TABLE 18______________________________________Activity of AB78 cosmid clones against western corn rootworm. Mean Clone percent mortality (N = 4)______________________________________Clones which hybridize with probe P1-73 47 P1-83 64 P2-2 69 P3-12 85 P5-4 97 Clones which do not hybridize with probe P1-2 5 P3-8 4 P3-9 12 P3-18 0 P4-6 9______________________________________
EXAMPLE 10
IDENTIFICATION OF A 6 KB REGION ACTIVE AGAINST WESTERN CORN ROOTWORM.
DNA from P3-12 was partially digested with restriction enzyme Sau 3A, and ligated into the E. coli vector pUC 19 and transformed into E. coli. A DNA probe specific for the 80 kDa VIP1A(a) protein was synthesized by PCR amplification of a portion of P3-12 DNA. Oligonucleotides MK113 and MK117, which hybridize to portions of VIP1A(a), were synthesized using the partial amino acid sequence of the 80 kDa protein. Plasmid subclones were identified by colony hybridization to the PCR-generated probe, and tested for activity against western corn rootworm. One such clone, PL2, hybridized to the PCR-generated fragment, and was active against western corn rootworm in the assay previously described.
A 6 kb Cla I restriction fragment from pL2 was cloned into the Sma I site of the E. coli-Bacillus shuttle vector pHT 3101 (Lereclus, D. et al., FEMS Microbiology Letters 60:211-218 (1989)) to yield pCIB6201. This construct confers anti-western corn rootworm activity upon both Bacillus and E. coli strains, in either orientation. pCIB6022 contains this same 6 kb Cla I fragment in pBluescript SK(+) (Stratagene), produces equivalent VIP1A(a) protein (by western blot), and is also active against western corn rootworm.
The nucleotide sequence of pCIB6022 was determined by the dideoxy termination method of Sanger et al., Proc. Natl. Acad. Sci. USA, 74:5463-5467 (1977), using PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kits and PRISM Sequenase.RTM. Terminator Double-Stranded DNA Sequencing Kit and analyzed on an ABI 373 automatic sequencer. The sequence is given in SEQ ID NO:1. The 6 kb fragment encodes both VIP1A(a) and VIP2A(a), as indicated by the open reading frames described in SEQ ID NO:1. The sequence encoding VIP1A(a) is further disclosed in SEQ ID NO:4. The relationship between VIP1A(a) and VIP2A(a) within the 6 kb fragment found in pCIB6022 is depicted in FIG. 1. pCIB6022 was deposited with the Agricultural Research Service, Patent Culture Collection, (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Ill. 61604, USA, and given the Accession No. NRRL B-21222.
EXAMPLE 11
FUNCTIONAL DISSECTION OF THE VIP1A(a) DNA REGION.
To confirm that the VIP1A(a) open reading frame (ORF) is necessary for insecticidal activity a translational frameshift mutation was created in the gene. The restriction enzyme Bgl II recognizes a unique site located 857 bp into the coding region of VIP1A(a). pCIB6201 was digested with Bgl II, and the single-stranded ends filled-in with DNA polymerase (Klenow fragment) and dNTPS. The plasmid was re-ligated and transformed into E. coli. The resulting plasmid, pCIB6203, contains a four nucleotide insertion in the coding region of VIP1A(a). pCIB6203 does not confer WCRW insecticidal activity, confirming that VIP1A(a) is an essential component of western corn rootworm activity.
To further define the region necessary to encode VIP1A(a), subclones of the VIP1A(a) and VIP2A(a) (auxiliary protein) region were constructed and tested for their ability to complement the mutation in pCIB6203. pCIB6023 contains the 3.7 kb Xba I-EcoRV fragment in pBluescript SK(+) (Stratagene). Western blot analysis indicates that pCIB6023 produces VIP1A(a) protein of equal size and quantity as clones PL2 and pCIB6022. pCIB6023 contains the entire gene encoding the 80 kD protein. pCIB6023 was deposited with the Agricultural Research Service, Patent Culture Collection, (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Ill. 61604, USA, and given the Accession No. NRRL B-21223N. pCIB6206 contains the 4.3 kb Xba I-Cla I fragment from pCIB6022 in pBluescript SK(+) (Stratagene). pCIB6206 was also deposited with the Agricultural Research Service, Patent Culture Collection, (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Ill. 61604, USA, and given the Accession No. NRRL B-21321.
pCIB6023, pCIB6206, and pCIB6203 do not produce detectable western corn rootworm activity when tested individually. However, a mixture of cells containing pCIB6203 (VIP1A(a)-mutated, plus VIP2A(a)) and cells containing pCIB6023 (only VIP1A(a)) shows high activity against western corn rootworm. Similarly, a mixture of cells containing pCIB6206 and cells containing pCIB6203 shows high activity against western corn rootworm.
To further define the limits of VIP2A(a), we constructed pCIB6024, which contains the entirety of VIP2A(a), but lacks most of the VIP1A(a) coding region. pCIB6024 was constructed by gel purifying the 2.2 kb Cla I-Sca I restriction fragment from pCIB6022, filling in the single-stranded ends with DNA polymerase (Kienow fragment) and dNTPs, and ligating this fragment into pBluescript SK(+) vector (Stratagene) digested with the enzyme Eco RV. Cells containing pCIB6024 exhibit no activity against western corn rootworm. However, a mixture of cells containing pCIB6024 and cells containing pCIB6023 shows high activity against western corn rootworm (See FIG. 1).
Thus, pCIB6023 and pCIB6206 must produce a functional VIP1A(a) gene product, while pCIB6203 and pCIB6024 must produce a functional VIP2A(a) gene product. These results suggest a requirement for a gene product(s) from the VIP2A(a) region, in combination with VIP1A(a), to confer maximal western corn rootworm activity. (See FIG. 1)
EXAMPLE 12
AB78 ANTIBODY PRODUCTION
Antibody production was initiated in 2 Lewis rats to allow for both the possibility of moving to production of hybridoma cell lines and also to produce enough serum for limited screening of genomic DNA library. Another factor was the very limited amount of antigen available and the fact that it could only be produced to purity by PAGE and subsequent electrotransfer to nitrocellulose.
Due to the limited availability of antigen on nitrocellulose, the nitrocellulose was emulsified in DMSO and injected into the hind footpads of the animals to elicit B-cell production in the popliteal lymph nodes just upstream. A strong reacting serum was produced as judged by western blot analysis with the first production bleed. Several subsequent injections and bleeds produced enough serum to accomplish all of the screening required.
Hybridoma production with one of the rats was then initiated. The popliteal lymph node was excised, macerated, and the resulting cells fused with mouse myeloma P3.times.63Ag8.653. Subsequent cell screening was accomplished as described below. Four initial wells were selected which gave the highest emulsified antigen reaction to be moved to limited dilution cloning. An additional 10 wells were chosen for expansion and cryoperservation.
Procedure to Emulsify AB78 on nitrocellulose in DMSO for ELISA screening:
After electrotransfer of AB78 samples run on PAGE to nitrocellulose, the reversible strain Ponceau S is used to visualize all protein transferred. The band corresponding to AB78 toxin, previously identified and N-terminal sequenced, was identified and excised from nitrocellulose. Each band is approximately 1 mm.times.5 mm in size to minimize the amount of nitrocellulose emulsified. A single band is placed in a microfuge tube with 250 .mu.l of DMSO and macerated using a plastic pestle (Kontes, Vineland, N.J.). To aid in emulsification, the DMSO mixture is heated for 2-3 minutes at 37.degree. C.-45.degree. C. Some further maceration might be necessary following heating; however, all of the nitrocellulose should be emulsified. Once the AB78 sample is emulsified, it is placed on ice. In preparation for microtiter plate coating with the emulsified antigen, the sample must be diluted in borate buffered saline as follows: 1:5, 1:10, 1:15, 1:20, 1:30, 1:50, 1:100, and 0. The coating antigen must be prepared fresh immediately or to use.
ELISA protocol:
1. Coat with AB78/DMSO in BBS. Incubate overnight at 4.degree. C.
2. Wash plate 3.times. with 1.times. ELISA wash buffer.
3. Block (1% BSA & 0.05% Tween 20 in PBS) for 30 minutes at Room Temperature.
4. Wash plate 3.times. with 1.times. ELISA wash buffer.
5. Add rat serum. Incubate 1.5 hours at 37.degree. C.
6. Wash plate 3.times. with 1.times. ELISA wash buffer.
7. Add goat anti-rat at a concentration of 2 .mu.g/ml in ELISA diluent. Incubate 1 hr. at 37.degree. C.
8. Wash plate 3.times. with 1.times. ELISA wash buffer.
9. Add rabbit anti-goat alkaline phosphatase at 2 .mu.g/ml in ELISA diluent. Incubate 1 hr. at 37.degree. C.
10. Wash 3.times. with 1.times. ELISA wash buffer.
11. Add Substrate. Incubate 30 minutes at room temperature.
12. Stop with 3N NaOH after 30 minutes.
Preparation of VIP2A(a) Antisera
A partially purified AB78 culture supernatant was separated by discontinuous SDS PAGE (Novex) following manufacturer's instructions. Separated proteins were electrophoresed to nitrocellulose (S&S #21640) as described by Towbin et al., (1979). The nitrocellulose was stained with Ponceau S and the VIP2A(a) band identified. The VIP2A(a) band was excised and emulsified in DMSO immediately prior to injection. A rabbit was initially immunized with emulsified VIP2A(a) mixed approximately 1:1 with Freund's Complete adjuvant by intramuscular injection at four different sites. Subsequent immunizations occurred at four week intervals and were identical to the first, except for the use of Freund' Incomplete adjuvant. The first serum harvested following immunization reacted with VIP2A(a) protein. Western blot analysis of AB78 culture supernatant using this antisera identifies predominately full length VIP2A(a) protein.
EXAMPLE 13
ACTIVATION OF INSECTICIDAL ACTIVITY OF NON-ACTIVE BT STRAINS WITH AB78 VIP CLONES.
Adding pCIB6203 together with a 24 h culture (early to mid-log phase) supernatant from Bt strain GC91 produces 100% mortality in Diabrotica virgifera virgifera. Neither pCIB6203 not GC91 is active on Diabrotica virgifera virgifera by itself. Data are shown below:
______________________________________Test material Percent Diabrotica mortality______________________________________pCIB6203 0 GC91 16 pCIB6203 + GC91 100 Control 0______________________________________
EXAMPLE 14
ISOLATION AND BIOLOGICAL ACTIVITY OF B. CEREUS AB81
A second B. cereus strain, designated AB81, was isolated from grain bin dust samples by standard methodologies. A subculture of AB81 was grown and prepared for dioassay as described in Example 2. Biological activity was evaluated as described in Example 3. The results are as follows:
______________________________________Insect species Percent tested Mortality______________________________________Ostrinia nubilalis 0 Agrotis ipsilon 0 Diabrotica virgifera virgifera 55______________________________________
EXAMPLE 15
ISOLATION AND BIOLOGICAL ACTIVITY OF B. THURINGIENSIS AB6.
A B. thuringiensis strain, designated AB6, was isolated from grain bin dust samples by standard methods known in the art. A subculture of AB6 was grown and prepared for bioassay as described in Example 2. Half of the sample was autoclaved 15 minutes to test for the presence of .beta.-exotoxin.
Biological activity was evaluated as described in Example 3. The results are as follows:
______________________________________Insect species Percent tested Mortality______________________________________Ostrinia nubilalis 0 Agrotis ipsilon 100 Agrotis ipsilon (autoclaved sample) 0 Diabrotica virgifera virgifera 0______________________________________
The reduction of insecticidal activity of the culture supernatant to insignificant levels by autoclaving indicates that the active principle is not .beta.-exotoxin.
Strain AB6 has been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Ill. 61604, USA, and given Accession No. NRRL B-21060.
EXAMPLE 16
ISOLATION AND BIOLOGICAL CHARACTERIZATION OF B. THURINGIENSIS AB88.
A Bt strain, designated AB88, was isolated from grain bin dust samples by standard methodologies. A subculture of AB88 was grown and prepared for bioassay as described in Example 2. Half of the sample was autoclaved 15 minutes to test for the presence of .beta.-exotoxin. Biological activity was evaluated against a number of insect species as described in Example 3. The results are as follows:
______________________________________ Percent mortality of culture supernatantInsect species tested Order Non-autoclaved Autoclaved______________________________________Agrotis ipsilon Lepidoptera 100 5 Ostrinia nubilalis Lepidoptera 100 0 Spodoptera Lepidoptera 100 4 frugiperda Helicoverpa zea Lepidoptera 100 12 Heliothis virescens Lepidoptera 100 12 Leptinotarsa Coleoptera 0 0 decemlineata Diabrotica virgifera Coleoptera 0 5 virgifera______________________________________
The reduction of insecticidal activity of the culture supernatant to insignificant levels by autoclaving indicates that the active principle is not .beta.-exotoxin.
Delta-endotoxin crystals were purified from strain AB88 by standard methodologies. No activity from pure crystals was observed when bioassayed against Agrotis ipsilon.
EXAMPLE 17
PURIFICATION OF VIPS FROM STRAIN AB88:
Bacterial liquid culture was grown overnight at 30.degree. C. in TB media. Cells were spun out and the supernatant retained. Proteins were precipitated with ammonium sulfate (70% saturation), centrifuged and the pellet retained. The pellet was resuspended in the original volume of 20 mM Tris pH 7.5 and dialyzed against the same buffer. AB88 dialysate was more turbid than comparable material from AB78. AB88 proteins have been separated by several different methods following clarification including isoelectric focusing (Rotofor, BioRad, Hercules, Calif.), precipitation at pH 4.5, ion-exchange chromotography, size exclusion chromatography and ultrafiltration.
European corn borer (ECB)-active protein remained in the pellet obtained by pH 4.5 precipitation of dialysate. When preparative IEF was done on the dialysate using pH 3-10 ampholytes, ECB insecticidal activity was found in all fractions with pH of 7 or greater. SDS-PAGE analysis of these fractions showed protein bands of MW .about.60 kDa and .about.80 kDa. The 60 kDa and 80 kDa bands were separated by anion exchange HPLC on a Poros-Q column (PerSeptive Biosystems, Cambridge, Mass.). N-terminal sequence was obtained from two fractions containing proteins of slightly differing MW, but both of approximately 60 kDa in size. The sequences obtained were similar to each other and to some .delta.-endotoxins. anion exchange fraction 23 (smaller): xEPFVSAxxxQxxx (SEQ ID NO:10) anion exchange fraction 28 (larger): xEYENVEPFVSAx (SEQ ID NO:11)
When the ECB-active pH 4.5 pellet was further separated by anion exchange on a Poros-Q column, activity was found only in fractions containing a major band of .about.60 kDa.
Black cutworm-active protein also remained in the pellet when AB88 dialysate was brought down to pH 4.5. In preparative IEF using pH 3-10 ampholytes, activity was not found in the ECB-active IEF fractions; instead, it was highest in a fraction of pH 4.5-5.0. Its major components have molecular weights of .about.35 and .about.80 kDa.
The pH 4.5 pellet was separated by anion exchange HPLC to yield fractions containing only the 35 kDa material and fractions containing both 35 kDa and 80 kDa bands.
EXAMPLE 18
CHARACTERIZATION OF AB88 VIP.
Fractions containing the various lepidopteran active vegetative proteins were generated as described in Example 17. Biological analysis of fractions demonstrated that different VIPs were responsible for the different lepidopteran species activity.
The Agrotis ipsilon activity is due to an 80 kDa and/or a 35 kDa protein, either delivered singly or in combination. These proteins are not related to any .delta.-endotoxins from Bt as evidenced by the lack of sequence homology of known Bt .delta.-endotoxm sequences. Also, these proteins are not found in the AB88 .delta.-endotoxin crystal. N-terminal sequences of the major .delta.-endotoxin proteins were compared with the N-terminal sequences of the 80 kDa and 35 kDa VIP and revealed no sequence homology. A summary of the results follows:
______________________________________ N-terminal sequence of major .delta.- Agrotis VIP N-terminal sequences endotoxin proteins______________________________________ 130 kDa MDNNPNINE (SEQ ID NO:14) 80 kDa 80 kDa MNKNNTKLPTRALP MDNNPNINE (SEQ ID NO:15) (SEQ ID NO:12) 60 kDa MNVLNSGRTTI (SEQ ID NO:16) 35 kDa ALSENTGKDGGYIVP (SEQ ID NO:13)______________________________________
The Ostrinia nubilalis activity is due to a 60 kDa VIP and the Spodoptera frugiperda activity is due to a VIP of unknown size.
Bacillus thuringiensis strain AB88 has been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Ill. 61604, USA and given the Accession No. NRRL B-21225.
EXAMPLE 18A
ISOLATION AND BIOLOGICAL ACTIVITY OF B. THURINGIENSIS AB424
A B. thuringiensis strain, designated AB424, was isolated from a moss covered pine cone sample by standard methods known in the art. A subculture of AB424 was grown and prepared for bioassay as described in Example 2.
Biological activity was evaluated as described in Example 3. The results are as follows:
______________________________________Insect species tested Percent mortality______________________________________Ostrinia nubilalis 100 Agrotis ipsilon 100 Diabrotica virgifera 0 virgifera______________________________________
Strain AB424 has been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Ill. 61604, USA, and given Accession No. NRR B-21439.
EXAMPLE 18B
CLONING OF THE VIP3A(a) and VIP3A(b) GENES WHICH ENCODE PROTEINS ACTIVE AGAINST BLACK CUTWORM.
DNA from isolates AB88 and AB424 was digested with the restriction enzymes XbaI and EcoRI respectively, ligated into pBluescript vector previously linearized with the same enzymes and dephosphorylated, and transformed into E. coli DH5.alpha. strain. Recombinant clones were blotted onto nitrocellulose filters which were subsequently probed with a 33-bases long oligonucleotide corresponding to the 11-N terminal amino acids of the 80 kDa protein active against Agrotis epsilon (black cutworm). Four out of 400 recombinant clones were positive. Insect bioassays of the positive recombinants exhibited toxicity to black cutworm larvae comparable to that of AB88 or AB424 supernantants.
The nucleotide sequence of pCIB7104, a positive recombinant clone from AB88, and of pCIB7107, a positive recombinant clone from AB424, was determined by the dideoxy termination method of Sanger et al., Proc. Natl. Acad. Sci. USA, 74: 5463-5467 (1977), using PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kits and PRISM Sequenase.RTM. Terminator Double-Stranded DNA Sequencing Kit and analysed on an ABI 373 automatic sequencer.
The clone pCIB7104 contains the VIP3A(a) gene whose coding region is disclosed in SEQ ID NO:28 and the encoded protein sequence is disclosed in SEQ ID NO:29. A synthetic version of the coding region designed to be highly expressed in maize is given in SEQ ID NO:30. Any number of synthetic genes can be designed based on the amino acid sequence given in SEQ ID NO:29.
The clone pCIB7107 contains the VIP3A(b) gene whose coding region is disclosed in SEQ ID NO:31 and the encoded protein is disclosed in SEQ ID NO:32. Both pCIB7104 and pCIB7107 have been deposited with the Agricultural Research Service Patent Culture Collection (NRRL) and given Accession Nos. NRRL B-21422 and B-21423, respectively.
EXAMPLE 18C.
IDENTIFICATION OF NOVEL VIP3-LIKE GENES BY HYBRIDIZATION
To identify Bacillus containing genes related to the VIP3A(a) from isolate AB88, a collection of Bacillus isolates was screened by hybridization. Cultures of 463 Bacillus strains were grown in microtiter wells until sporulation. A 96-pin colony stampel was used to transfer the cultures to 150 mm plates containing L-agar. Inoculated plates were kept at 30.degree. C. for 10 hours, then at 4.degree. C. overnight. Colonies were blotted onto nylon filters and probed with a 1.2Kb HindIII VIP3A(a) derived fragment. Hybridization was performed overnight at 62.degree. C. using hybridization conditions of Maniatis et al. Molecular Cloning: A Laboratory Manual (1982). Filters were washed with 2.times.SSC/0.1% SDS at 62.degree. C. and exposed to X-ray film.
Of the 463 Bacillus strains screened, 60 contain VIP3-like genes that could detected by hybridization.
EXAMPLE 18D.
CHARACTERIZATION OF A B. thuringiensis STRAIN M2194 CONTAINING A CRYPTIC VIP3-LIKE GENE
A B. thuringiensis strain, designated M2194, was shown to contain VIP3-like gene(s) by colony hybridization as described in Example 18C. The M2194 VIP3 like gene is considered cryptic since no expression can be detected throughout the bacterial growth phases either by immunoblot analysis using polyclonal antibodies raised against the VIP3A(a) protein isolated from AB88 or by bioassay as described in Example 3.
The M2194 VIP3-like gene was cloned into pKS by following the protocol described in Example 9, which created pCIB7108. E. coli containing pCIB7108 which comprises the M2194 VIP3 gene were active against black cutworm demonstrating that the gene encodes a functional protein with insecticidal activity. The plasmid pCIB7108 has been deposited with the Agricultural Research Service Patent Culture Collection (NRRL) and given Accession No. NRRL B-21438.
EXAMPLE 19
ISOLATION AND BIOLOGICAL ACTIVITY OF OTHER BACILLUS SP.
Other Bacillus species have been isolated which produce proteins with insecticidal activity during vegetative growth. These strains were isolated from environmental samples by standard methodologies. Isolates were prepared for bioassay and assayed as described in Examples 2 and 3 respectively. Isolates which produced insecticidal proteins during vegetative growth with activity against Agrotis ipsilon in the bioassay are tabulated below. No correlation was observed between the presence of a .delta.-endotoxin crystal and vegetative insecticidal protein production.
______________________________________ Presence of .delta.-endotoxin Bacillus isolate crystal Percent mortality______________________________________AB6 + 100 AB53 - 80 AB88 + 100 AB195 - 60 AB211 - 70 AB217 - 83 AB272 - 80 AB279 - 70 AB289 + 100 AB292 + 80 AB294 - 100 AB300 - 80 AB359 - 100______________________________________
Isolates AB289, AB294 and AB359 have been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria Ill. 61604, USA and given the Accession Numbers NRRL B-21227, NRRL B-21229, and NRRL B-21226 respectively.
Bacillus isolates which produce insecticidal proteins during vegetative growth with activity against Diabrotica virgifera virgifera are tabulated below.
______________________________________ Presence of .delta.-endotoxin Bacillus isolate crystal Percent mortality______________________________________AB52 - 50 AB59 - 71 AB68 + 60 AB78 - 100 AB122 - 57 AB218 - 64 AB256 - 64______________________________________
Isolates AB59 and AB256 have been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria Ill. 61604, USA, and given the Accession Numbers NRRL B-21228 and NRRL B-21230, respectively.
EXAMPLE 20
IDENTIFICATION OF NOVEL VIP1JVIP2 LIKE GENES BY HYBRIDIZATION
To identify strains containing genes related to those found in the VIP1A(a)/VIP2A(a) region of AB78, a collection of Bacillus strains was screened by hybridization. Independent cultures of 463 Bacillus strains were grown in wells of 96 well microtiter dishes (five plates total) until the cultures sporulated. Of the strains tested, 288 were categorized as Bacillus thuringiensis, and 175 were categorized as other Bacillus species based on the presence or absence of 8-endotoxin crystals. For each microtiter dish, a 96-pin colony stamper was used to transfer approximately 10 .mu.l of spore culture to two 150 mm plates containing L-agar. Inoculated plates were grown 4-8 hours at 30.degree. C., then chilled to 4.degree. C. Colonies were transferred to nylon filters, and the cells lysed by standard methods known in the art. The filters were hybridized to a DNA probe generated from DNA fragments containing both VIP1A(a) and VIP2A(a) DNA sequences. Hybridization was performed overnight at 65.degree. C. using the hybridization conditions of Church and Gilbert (Church, G. M., and W. Gilbert, PNAS, 81:1991-1995 (1984)). Filters were washed with 2.times.0 SSC containing 0.1% SDS at 65.degree. C. and exposed to X-Ray film.
Of the 463 Bacillus strains screened, 55 strains were identified that hybridized to the VIP1A(a)/VIP2A(a) probe. DNA was isolated from 22 of these strains, and analyzed using a Southern blot with VIP1A(a)/VIP2A(a) DNA as probes. These strains were grouped into 8 classes based on their Southern blot pattern. Each class differed in Southern blot pattern from AB78. One class had a pattern identical to that of the VIP1A(a)/VIP2A(a) homologs from Bacillus thuringiensis var tenebrionis (see below). Each of the 22 strains was tested for activity against western corn rootworm (WCRW). Three strains, AB433, AB434, and AB435 were found to be active on WCRW. Western blot analysis using VIP2A(a) antisera revealed that strains AB6, AB433, AB434, AB435, AB444, and AB445 produce a protein(s) of equivalent size to VIP2A(a).
Notable among the strains identified was Bacillus thuringiensis strain AB6, (NRRL B-1060) which produced a VIP active against black cutworm (Agrotis ipsilon) as described in Example 15. Western blot analysis with polyclonal antisera to VIP2A(a) and polyclonal antisera to VIP1A(a) suggests that AB6 produces proteins similar to VIP2A(a) and VIP1A(a). Thus, AB6 may contain VIPs similar to VIP1A(a) and VIP2A(a), but with a different spectrum of insecticidal activity.
EXAMPLE 21
CLONING OF A VIP1A(a)/VIP2A(a) HOMOLOG FROM BACILLUS THURINGIENSIS VAR. TENEBRIONIS.
Several previously characterized Bacillus strains were tested for presence of DNA similar to VIP1A(a)/VIP2A(a) by Southern blot analysis. DNA from Bacillus strains AB78, AB88, GC91, HD-1 and ATCC 10876 was analyzed for presence of VIP1A(a)/VIP2A(a) like sequences. DNA from Bt strains GC91 and HD-1, and the Bc strain ATCC 10876 did not hybridize to VIP2A(a)/VIP1A(a) DNA, indicating they lack DNA sequences similar to VIP1A(a)/VIP2A(a) genes. Similarly, DNA from the insecticidal strain AB88 (Example 16) did not hybridize to VIP1A(a)/VIP2A(a) DNA region, suggesting that the VIP activity produced by this strain does not result from VIP1A(a)/VIP2A(a) homologs. In contrast, Bacillus thuringiensis var. tenebrionis (Btt) contained sequences that hybridized to the VIP1A(a)/VIP2A(a) region. Further analysis confirmed that Btt contains VIP1A(a)/VIP2A(a) like sequences.
To characterize the Btt homologs of VIP2A(a) and VIP1A(a), the genes encoding these proteins were cloned. Southern blot analysis identified a 9.5 kb Eco RI restriction fragment likely to contain the coding regions for the homologs. Genomic DNA was digested with Eco RI, and DNA fragments of approximately 9.5 kb in length were gel-purified. This DNA was ligated into pBluescript SK(+) digested with Eco RI, and transformed into E. coli to generate a plasmid library. Approximately 10,000 colonies were screened by colony hybridization for the presence of VIP2A(a) homologous sequences. Twenty eight positive colonies were identified. All twenty eight clones are identical, and contain VIP1A(a)/VIP2A(a) homologs. Clone pCIB7100 has been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria Ill. 61604, USA, and given the Accession Number B-21322. Several subclones were constructed from pCIB7100. A 3.8 kb Xba I fragment from pCIB7100 was cloned into pBluescript SK(+) to yield pCIB7101. A 1.8 kb Hind III fragment and a 1.4 kb Hind III fragment from pCIB7100 were cloned into pBluescript SK(+) to yield pCIB7102 and pCIB7103, respectively. Subclones pCIB7101, pCIB7102 and pCIB7103 have been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria Ill. 61604, USA, and given the Accession Numbers B-21323, B-21324 and B-21325 respectively.
The DNA sequence of the region of pCIB7100 containing the VIP2A(a)/VIP1A(a) homologs was determined by the dideoxy chain termination method (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74:5463-5467). Reactions were performed using PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kits and PRISM Sequenase.RTM. Terminator Double-Stranded DNA Sequencing Kits, and analyzed on an ABI model 373 automated sequencer. Custom oligonucleotides were used as primers to determine the DNA sequence in certain regions. The DNA sequence of this region is shown in SEQ ID NO:19.
The 4 kb region shown in SEQ ID NO:19 contains two open readings frames (ORFs), which encode proteins with a high degree of similarity to VIP1A(a) and VIP2A(a) proteins from strain AB78. The amino acid sequence of the VIP2A(a) homolog, designated as VIP2A(b) using the standardized nomenclature, is found at SEQ ID NO:20 and the amino acid sequence of the VIP1A(a) homolog, designated as VIP1A(b) using the standardized nomenclature, is disclosed at SEQ ID NO:21. The VIP2A(b) protein exhibits 91% amino acid identity to VIP2A(a) from AB78. An alignment of the amino acid sequences of the two VIP2 proteins is provided in Table 19. The VIP1A(b) protein exhibits 77 % amino acid identity to VIP1A(a) from AB78. An alignment of these two VIP1 proteins is provided in Table 20. The alignment shown in Table 20 discloses the similarity between VIP1A(b) and VIP1A(a) from AB78. This alignment reveals that the amino terminal regions of the two VIP1 proteins share higher amino acid identity in the amino-terminal region than in the carboxy terminal region. In fact, the amino terminal two thirds (up to aa 618 of the VIP1A(b) sequence shown in Table 20 ) of the two proteins exhibit 91% identity, while the carboxy-terminal third (from aa 619-833 of VIP1A(b)) exhibit only 35% identity.
Western blot analysis indicated that Bacillus thuringiensis var. tenebrionis (Btt) produces both VIP1A(a) like and VIP2A(a) like proteins. However, these proteins do not appear to have activity against western corn rootworm. Bioassay for activity against western corn rootworm was performed using either a 24 h culture supernatant from Btt or E. coli clone pCIB7100 (which contains the entire region of the VIP1A(a)/VIP2A(a) homologs). No activity against western corn rootworm was detected in either case.
Given the similarity between the VIP2 proteins from Btt and AB78, the ability of VIP2A(b) from Btt to substitute for VIP2A(a) from AB78 was tested. Cells containing pCIB6206 (which produces AB78 VIP1A(a) but not VIP2A(a) protein) were mixed with Btt culture supernatant, and tested for activity against western corn rootworm. While neither Btt culture supernatant nor cells containing pCIB6206 had activity on WCRW, the mixture of Btt and pCIB6206 gave high activity against WCRW. Furthermore, additional bioassay showed that the Btt clone pCIB7100, which contains the Btt VIP 1 A(b)VIP2A(b) genes in E. coli, also confers activity against WCRW when mixed with pCIB6206. Thus, the VlP2A(b) protein produced by Btt is functionally equivalent to the VIP2A(a) protein produced by AB78.
Thus, the ability to identify new strains with insecticidal activity by using VIP DNA as hybridization probes has been demonstrated. Furthermore, Bacillus strains that contain VIP1A(a)/VIP2A(a) like sequences, produce VIP1A(a)/VIP2A(a) like protein, yet demonstrate toxicity toward different insect pests. Similar methods can identify many more members of the VIP1/VIP2 family. Furthermore, use of similar methods can identify homologs of other varieties of VIPs (for example, the VIPs from AB88).
TABLE 19__________________________________________________________________________Alignment of VIP2 Amino Acid Sequences from Bacillus thuringiensis var. tenebrionis (VIP2A(b)) vs. AB78 (VIP2A(a))__________________________________________________________________________ Btt 1 MQRMEGKLFVVSKTLQVVTRTVLLSTVYSITLLNNVVIKADQLNINSQSK 50 SEQ ID NO:20 .vertline...vertline..vertline..vertline..vertline..vertline..v ertline..vertline.:.vertline..vertline..vertline...vertline..vertline ..vertline..vertline..vertline.:.vertline..vertline..vertline..vertli ne..vertline..vertline..vertline.:.vertline..vertline...vertline..ver tline..vertline..vertline. .vertline..vertline..vertline..vertline.:. vertline..vertline..vertline..vertline..vertline..vertline..vertline. .vertline..vertline. AB78 1 MKRMEGKLFMVSKKLQVVTKTVLLSTVFSISLLNNEVIKAEQLNINSQSK 50 SEQ ID NO:2 - 51 YTNLQNLKIPDNAEDFKEDKGKAKEWGKEKGEEWRPPATEKGEMNNFLDN 100 .vertline..vertline..vertline..vertline..vertline..vertline ..vertline..vertline..vertline...vertline....vertline..vertline..vert line..vertline..vertline..vertline..vertline.:.vertline..vertline..ve rtline..vertline..vertline..vertline..vertline..vertline..vertline.:. .vertline..vertline.: ..vertline..vertline..vertline..vertline..vertl ine...vertline..vertline..vertline..vertline..vertline..vertline..ver tline. 51 YTNLQNLKITDKVEDFKEDKEKAKEWGKEKEKEWKLTATEKGKMNNFLDN 100 - 101 KNDIKTNYKEITFSMAGSCEDEIKDLEEIDKIFDKANLSSSIITYKNVEP 150 .vertline..vertline..vertline..vertline. .vertline..vertlin e..vertline..vertline..vertline..vertline..vertline..vertline..vertli ne..vertline..vertline..vertline..vertline. .vertline..vertline..vert line..vertline..vertline..vertline..vertline...vertline..vertline..ve rtline..vertline.:.vertline..vertline..vertline...vertline..vertline. .vertline...vertline..vertline..vertline..vertline..vertline..vertlin e..vertline..vertline..vertline..vertline. 101 KNDIXTNYKEITFSMAGSFEDEIKDLKEIDKMFDKTNLSNSIITYKNVEP 150 - 151 ATIGFNKSLTEGNTINSDAMAQFKEQFLGKDMKFDSYLDTHLTAQQVSSK 200 ..vertline..vertline..vertline..vertline..vertline..vertlin e..vertline..vertline..vertline..vertline..vertline..vertline..vertli ne..vertline..vertline..vertline..vertline..vertline..vertline..vertl ine..vertline..vertline..vertline..vertline..vertline..vertline..vert line.::.vertline.:.vertline..vertline..vertline..vertline..vertline.. vertline..vertline..vertline..vertline..vertline..vertline..vertline. .vertline..vertline..vertline..vertline..vertline..vertline. 151 TTIGFNKSLTEGNTINSDAMAQFKEQFLDRDIKFDSYLDTHLTAQQVSSK 200 - 201 KRVILKVTVPSGKGSTTPTKAGVILNNNEYKMLIDNGYVLHVDKVSKVVK 250 ..vertline..vertline..vertline..vertline..vertline..vertlin e..vertline..vertline..vertline..vertline..vertline..vertline..vertli ne..vertline..vertline..vertline..vertline..vertline..vertline..vertl ine..vertline..vertline..vertline..vertline..vertline..vertline...ver tline..vertline..vertline..vertline..vertline..vertline..vertline..ve rtline..vertline..vertline.::.vertline..vertline..vertline..vertline. .vertline..vertline..vertline..vertline..vertline..vertline. 201 ERVILKVTVPSGKGSTTPTKAGVILNNSEYKMLIDNGYMVHVDKVSKVVK 250 - 251 KGMECLQVEGTLKKSLDFKNDINAEAHSWGMKIYEDWAKNLTASQREALD 300 .vertline..vertline.:.vertline..vertline..vertline..vertlin e.:.vertline..vertline..vertline..vertline..vertline..vertline..vertl ine..vertline..vertline..vertline..vertline..vertline..vertline..vert line..vertline..vertline..vertline..vertline..vertline..vertline..ver tline..vertline..vertline..vertline. .vertline..vertline.:.vertline.. vertline..vertline.:.vertline..vertline...vertline..vertline..vertlin e..vertline..vertline..vertline..vertline. 251 KGVECLQIEGTLKKSLDFKNDINAEAHSWGMKNYEEWAKDLTDSQREALD 300 - 301 GYARQDYKEINNYLRNQGGSGNEKLDAQLKNISDALGKKPIPENITVYRW 350 .vertline..vertline..vertline..vertline..vertline..vertline ..vertline..vertline..vertline..vertline..vertline..vertline..vertlin e..vertline..vertline..vertline..vertline..vertline..vertline..vertli ne..vertline..vertline..vertline..vertline..vertline..vertline..vertl ine..vertline.:.vertline..vertline..vertline..vertline..vertline..ver tline..vertline..vertline..vertline..vertline..vertline..vertline..ve rtline..vertline..vertline..vertline..vertline..vertline..vertline..v ertline..vertline. 301 GYARQDYKEINNYLRNQGGSGNEKLDAQIKNISDALGKKPIPENITVYRW 350 - 351 CGMPEFGYQISDPLPSLKDFEEQFLNTIKEDKGYMSTSLSSERLAAFGSR 400 .vertline..vertline..vertline..vertline..vertline..vertline ..vertline..vertline..vertline..vertline..vertline..vertline..vertlin e..vertline..vertline..vertline..vertline..vertline..vertline..vertli ne..vertline..vertline..vertline..vertline..vertline..vertline..vertl ine..vertline..vertline..vertline..vertline..vertline..vertline..vert line..vertline..vertline..vertline..vertline..vertline..vertline..ver tline..vertline..vertline..vertline..vertline..vertline..vertline..ve rtline..vertline..vertline. 351 CGMPEFGYQISDPLPSLKDFEEQFLNTIKEDKGYMSTSLSSERLAAFGSR 400 - 401 KIILRLQVPKGSTGAYLSAIGGFASEKEILLDKDSKYHIDKATEVIIKGV 450 .vertline..vertline..vertline..vertline..vertline..vertline ..vertline..vertline..vertline..vertline..vertline..vertline..vertlin e..vertline..vertline..vertline..vertline..vertline..vertline..vertli ne..vertline..vertline..vertline..vertline..vertline..vertline..vertl ine..vertline..vertline..vertline..vertline..vertline..vertline..vert line..vertline..vertline..vertline..vertline..vertline..vertline..ver tline...vertline..vertline..vertline..vertline..vertline..vertline..v ertline..vertline. 401 KIILRLQVPKGSTGAYLSAIGGFASEKEILLDKDSKYHIDKVTEVIIKGV 450 - 451 KRYVVDATLLTN 462 .vertline..vertline..vertline..vertline..vertline..vertline..ve rtline..vertline..vertline..vertline..vertline..vertline. 451 KRYVVDATLLTN 462__________________________________________________________________________
TABLE 20__________________________________________________________________________Alignment of VIP1 Amino Acid Sequences from Bacillus thuringiensis var. tenebrionis (VIP1A(b)) vs. AB78 (VIP1A(a))__________________________________________________________________________ Btt 1 MKNMKKKLASVVTCMLLAPMFLNGNVNAVNADSKINQISTTQENQQKEMD 50 SEQ ID NO:21 .vertline..vertline..vertline..vertline..vertline..vertline..ve rtline..vertline..vertline..vertline..vertline..vertline..vertline..v ertline. .vertline..vertline..vertline..vertline..vertline..vertline. .vertline..vertline..vertline..vertline..vertline..vertline..vertline ..vertline. .vertline..vertline..vertline..vertline...vertline..vertl ine..vertline..vertline..vertline..vertline..vertline...vertline..ver tline..vertline..vertline..vertline..vertline..vertline. Ab78 1 MKNMKKKLASVVTCTLLAPMFLNGNVNAVYADSKTNQISTTQKNQQKEMD 50 SEQ ID NO:5 - 51 RXGLLGYYFKGKDFNNLTMFAPTRDNTLMYDQQTANALLDKKQQEYQSIR 100 .vertline..vertline..vertline..vertline..vertline..vertline ..vertline..vertline..vertline..vertline..vertline..vertline..vertlin e..vertline...vertline..vertline..vertline..vertline..vertline..vertl ine..vertline..vertline..vertline..vertline...vertline..vertline.:.ve rtline..vertline..vertline..vertline..vertline..vertline..vertline. .vertline..vertline..vertline..vertline..vertline..vertline..vertline ..vertline..vertline..vertline..vertline..vertline..vertline. 51 RKGLLGYYFKGKDFSNLTMFAPTRDSTLIYDQQTANKLLDKKQQEYQSIR 100 - 101 WIGLIQRKETGDFTFNLSKDEQAIIEIDGKIISNKGKEKQVVHLEKEKLV 150 .vertline..vertline..vertline..vertline..vertline..vertline ...vertline..vertline..vertline..vertline..vertline..vertline..vertli ne..vertline..vertline..vertline..vertline...vertline..vertline..vert line..vertline..vertline..vertline..vertline..vertline.:.vertline..ve rtline..vertline..vertline..vertline..vertline..vertline..vertline..v ertline..vertline..vertline..vertline..vertline..vertline..vertline.. vertline..vertline..vertline.:.vertline..vertline..vertline. 101 WIGLIQSKETGDFTFNLSEDEQAIIEINGKIISNKGKEKQVVHLEKGKLV 150 - 151 PIKIEYQSDTKFNIDSKTFKELKLFKIDSQNQSQQVQ...LRNPEFNKKE 197 .vertline..vertline..vertline..vertline..vertline..vertline ..vertline..vertline..vertline..vertline..vertline..vertline..vertlin e..vertline..vertline..vertline..vertline..vertline..vertline..vertli ne..vertline..vertline..vertline..vertline..vertline..vertline..vertl ine..vertline..vertline..vertline..vertline..vertline...vertline..ver tline..vertline..vertline. .vertline..vertline..vertline..vertline. .vertline..vertline..vertline..vertline..vertline..vertline. 151 PIKIEYQSDTKFNIDSKTFKELKLFKIDSQNQPQQVQQDELRNPEFNKKE 200 - 198 SQEFLAKASKTNLFKQKMKRDIDEDTDTDGDSIPDLWEENGYTIQNKVAV 247 .vertline..vertline..vertline..vertline..vertline..vertline ..vertline.:.vertline..vertline...vertline..vertline..vertline...vert line..vertline..vertline..vertline..vertline.:.vertline..vertline..ve rtline..vertline..vertline..vertline..vertline..vertline..vertline..v ertline..vertline..vertline..vertline..vertline..vertline..vertline.. vertline..vertline..vertline..vertline..vertline..vertline..vertline. .vertline..vertline.::.vertline..vertline. 201 SQEFLAKPSKINLFTQKMKREIDEDTDTDGDSIPDLWEENGYTIQNRIAV 250 - 248 KWDDSLASKGYTKFVSNPLDSHTVGDPYTDYEKAARDLDLSNAKETFNPL 297 .vertline..vertline..vertline..vertline..vertline..vertline ..vertline..vertline..vertline..vertline..vertline..vertline..vertlin e..vertline..vertline..vertline..vertline..vertline..vertline.:.vertl ine..vertline..vertline..vertline..vertline..vertline..vertline..vert line..vertline..vertline..vertline..vertline..vertline..vertline..ver tline..vertline..vertline..vertline..vertline..vertline..vertline..ve rtline..vertline..vertline..vertline..vertline..vertline..vertline..v ertline..vertline. 251 KWDDSLASKGYTKFVSNPLESHTVGDPYTDYEKAARDLDLSNAKETFNPL 300 - 298 VAAFPSVNVSMEKVILSPNENLSNSVESHSSTNWSYTNTEGASIEAGGGP 347 .vertline..vertline..vertline..vertline..vertline..vertline ..vertline..vertline..vertline..vertline..vertline..vertline..vertlin e..vertline..vertline..vertline..vertline..vertline..vertline..vertli ne..vertline..vertline..vertline..vertline..vertline..vertline..vertl ine..vertline..vertline..vertline..vertline..vertline..vertline..vert line..vertline..vertline..vertline..vertline..vertline..vertline..ver tline..vertline..vertline.:.vertline..vertline..vertline. .vertline.. vertline. 301 VAAFPSVNVSMEKVILSPNENLSNSVESHSSTNWSYTNTEGASVEAGIGP 350 - 348 LGLSFGVSVTYQHSETVAQEWGTSTGNTSQFNTASAGYLNAVVRYNNVGT 397 .vertline.:.vertline..vertline..vertline..vertline..vertli ne..vertline...vertline..vertline..vertline..vertline..vertline..vert line..vertline..vertline..vertline..vertline..vertline..vertline..ver tline..vertline..vertline..vertline..vertline..vertline..vertline..ve rtline..vertline..vertline..vertline..vertline..vertline..vertline..v ertline..vertline..vertline..vertline..vertline..vertline..vertline.. vertline..vertline..vertline..vertline..vertline..vertline..vertline. 351 KGISFGVSVNYQHSETVAQEWGTSTGNTSQFNTASAGYLNANVRYNNVGT 400 - 398 GAIYDVKPTTSFVLNNNTIATITAKSNSTALRISPGDSYPEIGENAIAIT 447 .vertline..vertline..vertline..vertline..vertline..vertline ..vertline..vertline..vertline..vertline..vertline..vertline..vertlin e..vertline..vertline..vertline.:.vertline..vertline..vertline..vertl ine..vertline..vertline..vertline..vertline..vertline..vertline..vert line..vertline..vertline..vertline...vertline..vertline..vertline..ve rtline.:.vertline..vertline..vertline.. .vertline.:.vertline.:.vertli ne..vertline..vertline..vertline. 401 GAIYDVKPTTSFVLNNDTIATITAKSNSTALNISPGESYPKKGQNGIAIT 450 - 448 SMDDFNSHPITLNKQQVNQLINNKPIMLETDQTDGGYKIRDTHGNIVTGG 497 .vertline..vertline..vertline..vertline..vertline..vertline ..vertline..vertline..vertline..vertline..vertline..vertline..vertlin e..vertline...vertline..vertline.:..vertline.:.vertline..vertline..ve rtline..vertline.:.vertline..vertline..vertline..vertline.:.vertline. .vertline..vertline..vertline..vertline..vertline..vertline..vertline .:.vertline..vertline..vertline..vertline..vertline..vertline..vertli ne..vertline..vertline..vertline. 451 SMDDFNSHPITLNKKQVDNLLNNKPMMLETNQTDGVYKIKDTHGNIVTGG 500 - 498 EWNGVTQQIKAKTASIIVDDGKQVAEKRVAAKDYGHPEDKTPPLTLKDTL 547 .vertline..vertline..vertline..vertline..vertline...vertlin e..vertline..vertline..vertline..vertline..vertline..vertline..vertli ne..vertline..vertline..vertline..vertline..vertline..vertline..vertl ine....vertline..vertline..vertline..vertline..vertline..vertline..ve rtline..vertline..vertline..vertline..vertline.::.vertline..vertline. .vertline..vertline..vertline..vertline...vertline..vertline..vertlin e..vertline..vertline...vertline. 501 EWNGVIQQIKAKTASIIVDDGERVAEKRVAAKDYENPEDKTPSLTLKDAL 550 - 548 KLSYPDEIKETNGLLYYDDKPIYESSVMTYLDENTAKEVKKQINDTTGKF 597 .vertline..vertline..vertline..vertline..vertline..vertline ..vertline..vertline..vertline..vertline..:.vertline..vertline..vertl ine..vertline..vertline..:.vertline..vertline..vertline..vertline..ve rtline..vertline..vertline..vertline..vertline..vertline..vertline..v ertline..vertline..vertline..vertline..vertline..vertline..vertline.. vertline..vertline...vertline..vertline.:.vertline..vertline..vertlin e..vertline..vertline..vertline..vertline. 551 KLSYPDEIKEIEGLLYYKNKPIYESSVMTYLDENTAKEVTKQLNDTTGKF 600 - 598 KDVNHLYDVKLTPKMNFTIKMASLYDGAENNHNSLGTWYLTYNVAGGNTG 647 .vertline..vertline..vertline...vertline..vertline..vertlin e..vertline..vertline..vertline..vertline..vertline..vertline..vertli ne..vertline..vertline...vertline..vertline..vertline.:. .vertline..v ertline..vertline...vertline..vertline...vertline...vertline..vertlin e.:.vertline...vertline. .vertline. .vertline...vertline..vertline. .vertline...vertline. 601 KDVSHLYDVKLTPKMNVTIKLSILYDNAESNDNSIGKWTNTNIVSGGNNG 650 - 648 KRQYRSAHSCAHVALSSEAKKKLNQNANYYLSMYMKADSTTEPTIEVAGE 697 .vertline.:.vertline..vertline...vertline..:. .vertline.::. .vertline...:.vertline....vertline..vertline..vertline...vertline. :.vertline..vertline.:.vertline.:.vertline..vertline..vertline..:...v ertline.:...vertline..:..vertline..vertline. 651 KKQYSSNNPDANLYLNTDAQEKLNKNRDYYISLYMKSEKNTQCEITIDGE 700 - 698 KSAITSKKVKLNNQNYQRVDILVKNSERNPMDKIYIRGNGTTNVYGDDVT 747 :.vertline..vertline...vertline...vertline..:.vertline..: .vertline..vertline...vertline.:.vertline..vertline.:...vertline. ...vertline..vertline.:..:..vertline.:..vertline.:...:: .vertline..ve rtline.:. 701 IYPITTKTVNVNKDNYKRLDIIAHNIKSNPISSLHIKTNDEITLFWDDIS 750 - 748 IPEVSAINPASLSDEEIQEIFKDSTIEYGNPSFVADAVTFK......... 788 .vertline..:.vertline....vertline...vertline....vertline... vertline...vertline..vertline..:.vertline.:. ..vertline...::. ::.. ..:. 751 ITDVASIKPENLTDSEIKQTYSRYGIKLEDGILIDKKGGIHYGEFINEAS 800 - 789 .NIKPLQNYVKEYEIYHK.......SHRYEKKTVFDIMGVHYEYSIAREQ 830 .vertline..vertline...vertline..vertline..vertline..vertli ne..vertline..vertline....vertline..: .. .vertline.. ..vertline ....::. .:.:::. ... 801 FNIEPLQNYVTKYKVTYSSELGQNVSDTLESDKIYKDGTIKFDFTKYSKN 850 - 831 KKA 833 ..: 851 EQG 853__________________________________________________________________________
EXAMPLE 22
FUSION OF VIP PROTEINS TO MAKE A SINGLE POLYPEPTIDE
VIP proteins may occur in nature as single polypeptides, or as two or more interacting polypeptides. When an active VIP is comprised of two or more interacting protein chains, these protein chains can be produced as a single polypeptide chain from a gene resulting from the fusion of the two (or more) VIP coding regions. The genes encoding the two chains are fused by merging the coding regions of the genes to produce a single open reading frame encoding both VIP polypeptides. The composite polypeptides can be fused to produce the smaller polypeptide as the NH.sub.2 terminus of the fusion protein, or they can be fused to produce the larger of the polypeptides as the NH.sub.2 terminus of the fusion protein. A linker region can optionally be used between the two polypeptide domains. Such linkers are known in the art. This linker can optionally be designed to contain protease cleavage sites such that once the single fused polypeptide is ingested by the target insect it is cleaved in the linker region to liberate the two polypeptide components of the active VIP molecule.
VIP1A(a) and VIP2A(a) from B. cereus strain AB78 are fused to make a single polypeptide by fusing their coding regions. The resulting DNA has the sequence given in SEQ ID NO:22 with the encoded protein given in SEQ ID NO:23. In like manner, other fusion proteins may be produced.
The fusion of the genes encoding VIP1A(a) and VIP2A(a) is accomplished using standard techniques of molecular biology. The nucleotides deleted between the VIP1A(a) and VIP2A(a) coding regions are deleted using known mutagenesis techniques or, alternatively, the coding regions are fused using PCR techniques.
The fused VIP polypeptides can be expressed in other organisms using a synthetic gene, or partially synthetic gene, optimized for expression in the alternative host. For instance, to express the fused VIP polypeptide from above in maize, one makes a synthetic gene using the maize preferred codons for each amino acid, see for example patent application U.S. Pat. No. 5,625,136 herein incorporated by reference. Synthetic DNA sequences created according to these methods are disclosed in SEQ ID NO:17 (maize optimized version of the 100 kDa VIP1A(a) coding sequence), SEQ ID NO:18 (maize optimized version of the 80 kDa VIP1A(a) coding sequence) and SEQ ID NO:24 (maize optimized version of the VIP2A(a) coding sequence).
Synthetic VIP1 and VIP2 genes optimized for expression in maize can be fused using PCR techniques, or the synthetic genes can be designed to be fused at a common restriction site. Alternatively, the synthetic fusion gene can be designed to encode a single polypeptide comprised of both VIP1 and VIP2 domains.
Addition of a peptide linker between the VIP1 and VIP2 domains of the fusion protein can be accomplished by PCR mutagenesis, use of a synthetic DNA linker encoding the linker peptide, or other methods known in the art.
The fused VIP polypeptides can be comprised of one or more binding domains. If more than one binding domain is used in the fusion, multiple target pests are controlled using such a fusion. The other binding domains can be obtained by using all or part of other VIPs; Bacillus thuringiensis endotoxins, or parts thereof; or other proteins capable of binding to the target pest or appropriate biding domains derived from such binding proteins.
One example of a fusion construction comprising a maize optimized DNA sequence encoding a single polypeptide chain fusion having VIP2A(a) at the N-terminal end and VIP1A(a) at the C-terminal end is provided by pCIB5531. A DNA sequence encoding a linker with the peptide sequence PSTPPTPSPSTPPTPS (SEQ ID NO:47) has been inserted between the two coding regions. The sequence encoding this linker and relevant cloning sites is 5'-CCC GGG CCT TCT ACT CCC CCA ACT CCC TCT CCT AGC ACG CCT CCG ACA CCT AGC GAT ATC GGA TC C-3' (SEQ ID NO:48). Oligonucleotides were synthesized to represent both the upper and lower strands and cloned into a pUC vector following hybridization and phosphorylation using standard procedures. The stop codon in VIP2A(a) was removed using PCR and replaced by the BglII restriction site with a SmaI site. A translation fusion was made by ligating the Bam HI/PstI fragment of the VIP2A(a) gene from pCIB5522 (see Example 24), a PCR fragment containing the PstI-end fragment of the VIP2A(a) gene (identical to that used to construct pCIB5522), a synthetic linker having ends that would ligate with a blunt site at the 5' end and with BamHI at the 3' end and the modified synthetic VIP1A(a) gene from pCIB5526 described below (See SEQ ID NO:35). The fusion was obtained by a four way ligation that resulted in a plasmid containing the VIP2A(a) gene without a translation stop codon, with a linker and the VIP1A(a) coding region without the Bacillus secretion signal. The DNA sequence for this construction is disclosed in SEQ ID NO:49, which encodes the fusion protein disclosed in SEQ ID NO:50. A single polypeptide fusion where VIP1A(a) is at the N-terminal end and VIP2A(a) is at the C-terminal end can be made in a similar fashion. Furthermore, either one or both genes can be linked in a translation fusion with or without a linker at either the 5' or the 3' end to other molecules like toxin encoding genes or reporter genes.
EXAMPLE 23
TARGETING OF VIP2 TO PLANT ORGANELLES
Various mechanisms for targeting gene products are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterized in some detail. For example, the targeting of gene products to the chloroplast is controlled by a signal sequence found at the amino-terminal end of various proteins. This signal is cleaved during chloroplast import, yielding the mature protein (e.g. Comai et al. J. Biol. Chem. 263: 15104-15109 (1988)). These signal sequences can be fused to heterologous gene products such as VIP2 to effect the import of those products into the chloroplast (van den Broeck et al. Nature 313: 358-363 (1985)). DNA encoding for appropriate signal sequences can be isolated from the 5' end of the cDNAs encoding the RUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2 protein and many other proteins which are known to be chloroplast localized.
Other gene products are localized to other organelles such as the mitochondrion and the peroxisome (e.g. Unger et al. Plant Molec. Biol. 13: 411418 (1989)). The cDNAs encoding these products can also be manipulated to effect the targeting of heterologous gene products such as VIP2 to these organelles. Examples of such sequences are the nuclear-encoded ATPases and specific aspartate amino transferase isoforms for mitochondria. Similarly, targeting to cellular protein bodies has been described by Rogers et al. (Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).
By the fusion of the appropriate targeting sequences described above to coding sequences of interest such as VIP2 it is possible to direct the transgene product to any organelle or cell compartment. For chloroplast targeting, for example, the chloroplast signal sequence from the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused in frame to the amino-terminal ATG of the transgene. The signal sequence selected should include the known cleavage site and the fusion constructed should take into account any amino acids after the cleavage site which are required for cleavage. In some cases this requirement may be fulfilled by the addition of a small number of amino acids between the cleavage site and the start codon ATG, or alternatively replacement of some amino acids within the coding sequence. Fusions constructed for chloroplast import can be tested for efficacy of chloroplast uptake by in vitro translation of in vitro transcribed constructions followed by in vitro chloroplast uptake using techniques described by (Bartlett et al. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier. pp 1081-1091 (1982); Wasmann et al. Mol. Gen. Genet. 205: 446-453 (1986)). These construction techniques are well known in the art and are equally applicable to mitochondria and peroxisomes.
The above described mechanisms for cellular targeting can be utilized not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters so as to effect a specific cell targeting goal under the transcriptional regulation of a promoter which has an expression pattern different to that of the promoter from which the targeting signal derives.
A DNA sequence encoding a secretion signal is present in the native Bacillus VIP2 gene. This signal is not present in the mature protein which has the N-terminal sequence of LKITDKVEDF (amino acid residues 57 to 66 of SEQ ID NO:2). It is possible to engineer VIP2 to be secreted out of the plant cell or to be targeted to subcellular organelles such as the endoplasmic reticulum, vacuole, mitochondria or plastids including chloroplasts. Hybrid proteins made by fusion of a secretion signal peptide to a marker gene have been successfully targeted into the secretion pathway. (Itirriaga G. et al., The Plant Cell, 1: 381-390 (1989) , Denecke et al., The Plant Cell, 2:51-59 (1990). Amino-terminal sequences have been identified that are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)).
The presence of additional signals are required for the protein to be retained in the endoplasmic reticulum or the vacuole. The peptide sequence KDELIHDEL at the carboxy-terminal of a protein is required for its retention in the endoplasmic reticulum (reviewed by Pelham, Annual Review Cell Biol., 5:1-23 (1989). The signals for retention of proteins in the vacuole have also been characterized. Vacuolar targeting signals may be present either at the amino-terminal portion, (Holwerda et al., The Plant Cell, 4:307-318 (1992), Nakamura et al., Plant Physiol., 101:1-5 (1993)), carboxy-terminal portion, or in the internal sequence of the targeted protein. (Tague et al., The Plant Cell, 4:307-318 (1992), Saalbach et al., The Plant Cell, 3:695-708 (1991)). Additionally, amino-terminal sequences in conjunction with carboxy-terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)). Similarly, proteins may be targeted to the mitochondria or plastids using specific carboxy terminal signal peptide fusions (Heijne et al., Eur. J. Biochem., 180:535-545 (1989), Archer and Keegstra, Plant Molecular Biology, 23:1105-1115 (1993)).
In order to target VIP2, either for secretion or to the various subcellular organelles, a maize optimized DNA sequence encoding a known signal peptide(s) may be designed to be at the 5' or the 3' end of the gene as required. To secrete VIP2 out of the cell, a DNA sequence encoding the eukaryotic secretion signal peptide MGWSWIFLFLLSGAAGVHCL (SEQ ID NO:25) from U.S. patent application Ser. No. 08/267,641 or any other described in the literature (Itirriaga et al., The Plant Cell, 1:381-390 (1989) , Denecke, et al., The Plant Cell, 2:51-59 (1990)) may be added to the 5' end of either the complete VIP2 gene sequence or to the sequence truncated to encode the mature protein or the gene truncated to nucleotide 286 or encoding a protein to start at amino acid residue 94 (methionine). To target VIP2 to be retained in the endoplasmic reticulum, a DNA sequence encoding the ER signal peptide KDEL/HDEL, in addition to the secretion signal, can be added to the 3' end of the gene. For vacuolar targeting a DNA sequence encoding the signal peptide SSSSFADSNPIRVTDRAAST (SEQ ID NO:3; Holwerda et al., The Plant Cell, 4:307-318 (1992)) can be designed to be adjacent to the secretion signal or a sequence encoding a carboxyl signal peptide as described by Dombrowski et al., The Plant Cell, 5:587-596 (1993) or a functional variation may be inserted at the 3' end of the gene. Similarly, VIP2 can be designed to be targeted to either the mitochondria or the plastids, including the chloroplasts, by inserting sequences in the VIP2 sequence described that would encode the required targeting signals. The bacterial secretion signal present in VIP2 may be retained or removed from the final construction.
One example of a construction which incorporates a eukaryotic secretion signal fused to a coding sequence for a VIP is provided by pCIB5528. Oligonucleotides corresponding to both the upper and lower strand of sequences encoding the secretion signal peptide of SEQ ID NO:25 was synthesized and has the sequence 5'-GGATCCACC ATG GGC TGG AGC TGG ATC TTC CTG TTC CTG CTG AGC GGC GCC GCG GGC GTG CAC TGC CTGCAG-3' (SEQ ID NO:41). When hybridized, the 5' end of the secretion signal resembled "sticky-ends" corresponding to restriction sites BamHI and PstI. The oligonucleotide was hybridized and phosphorylated and ligated into pCIB5527 (construction described in Example 23A) which had been digested with BamHI/PstI using standard procedures. The resulting maize optimized coding sequence is disclosed in SEQ ID NO:42 which encodes the protein disclosed in SEQ ID NO:43. This encoded protein comprises the eukaryotic secretion signal in place of the Bacillus secretion signal.
One example of a construction which incorporates a vacuolar targetting signal fused to a coding sequence for a VIP is provided by pCIB5533. Oligonucleotides corresponding to both the upper and lower strand of sequences encoding the vacuolar targetting peptide of SEQ ID NO:3 was synthesized and has the sequence 5'-CCG CGGGCG TGC ACT GCC TCA GCA GCA GCA GCT TCG CCG ACA GCA ACC CCA TCC GCG TGA CCG ACC GCG CCG CCA GCA CCC TGC AG-3' (SEQ ID NO:44). When hybridized, the 5' end of the vacuolar targetting signal resembled "sticky-ends" corresponding to restriction sites SacII and PstI. The oligonucleotide was hybridized and phosphorylated and ligated into pCIB5528 (construction described above) which had been digested with SacII/PstI using standard procedures. The resulting maize optimized coding sequence is disclosed in SEQ ID NO:45 which encodes the protein disclosed in SEQ ID NO:46. This encoded protein comprises the vacuolar targetting peptide in addition to the eukaryotic secretion signal.
The VIP1 gene can also be designed to be secreted or targeted to subcellular organelles by similar procedures.
EXAMPLE 23A
REMOVAL OF BACILLUS SECRETION SIGNAL FROM VIP1A(a) AND VIP2A(a)
VIP1A(a) and VIP2A(a) are secreted during the growth of strain AB78. The nature of peptide sequences that act as secretion signals has been described in the literature (Simonen and Palva, Microbiological reviews, pg. 109-137 (1993)). Following the information in the above publication, the putative secretion signal was identified in both genes. In VIP1A(a) this signal is composed of amino acids 1-33 (See SEQ ID NO:5). Processing of the secretion signal probably occurs after the serine at amino acid 33. The secretion signal in VIP2A(a) was identified as amino acids 1-49 (See SEQ ID NO:2). N-terminal peptide analysis of the secreted mature VIP2A(a) protein revealed the N-terminal sequence LKITDKVEDFKEDK. This sequence is found beginning at amino acid 57 in SEQ ID NO:2. The genes encoding these proteins have been modified by removal of the Bacillus secretion signals.
A maize optimized VIP1A(a) coding region was constructed which had the sequences encoding the first 33 amino acids, i.e., the secretion signal, removed from its 5' end. This modification was obtained by PCR using an forward primer that contained the sequence 5'-GGA TCC ACC ATG AAG ACC AAC CAG ATC AGC-3' (SEQ ID NO:33), which hybridizes with the maize optimized gene (SEQ ID NO:26) at nucleotide position 100, and added a BamHI restriction site and a eukaryotic translation start site consensus including a start codon. The reverse primer that contained the sequence 5'-AAG CTT CAG CTC CTT G-3' (SEQ ID NO:34) hybridizes on the complementary strand at nucelotide position 507. A 527 bp amplification product was obtained containing the restriction sites BamHI at the 5' end and HindIII site at the 3' end. The amplification product was cloned into a T-vector (described in Example 24, below) and sequenced to ensure the correct DNA sequence. The BamHI/HindIII fragment was then obtained by restriction digest and used to replace the BamHI/HindIII fragment of the maize optimized VIP1A(a) gene cloned in the root-preferred promoter cassette. The construct obtained was designated pCIB5526. The maize optimized coding region for VIP1A(a) with the Bacillus secretion signal removed is disclosed as SEQ ID NO:35 and the encoded protein is disclosed as SEQ ID NO:36.
The gene encoding the processed form of VIP2A(a), i.e., a coding region with the secretion signal removed, was constructed by a procedure similar to that described for that used to construct the processed form of VIP1 A(a), above. The modification was obtained by PCR using the forward primer 5'-GGA TCC ACC ATG CTG CAG AAC CTG AAG ATC AC -3' (SEQ ID NO:37). This primer hybridizes at nucleotide position 150 of the maize optimized VIP2A(a) gene (SEQ ID NO:27). A silent mutation has been inserted at nucleotide position 15 of this primer to obtain a PstI restriction site. The reverse primer has the sequence 5'-AAG CTT CCA CTC CIT CTC-3' (SEQ ID NO:38). A 259 bp product was obtained with HindIII restriction site at the 3' end. The amplification product was cloned into a T-vector, sequenced and ligated to a BamHI/HindIII digested root-preferred promoter cassette containing the maize optimized VIP2A(a). The construct obtained was designated pCIB5527. The maize optimized coding region for VIP2A(a) with the Bacillus secretion signal removed is disclosed as SEQ ID NO:39 and the encoded protein is disclosed as SEQ ID NO:40.
EXAMPLE 24
CONSTRUCTION AND CLONING OF THE VIP1A(a) AND VIP2A(a) MAIZE OPTIMIZED GENES
Design: The maize optimized genes were designed by reverse translation of the native VIP1 A(a) and VIP2A(a) protein sequences using codons that are used most often in maize (Murray et al., Nucleic Acid Research, 17:477498 (1989)). To facilitate cloning, the DNA sequence was further modified to incorporate unique restriction sites at intervals of every 200-360 nucleotides. VIP1 A(a) was designed to be cloned in 11 such fragments and VIP2A(a) was cloned in 5 fragments. Following cloning of the individual fragments, adjacent fragments were joined using the restriction sites common to both fragments, to obtain the complete gene. To clone each fragment, oligonucleotides (50-85 nucleotides) were designed to represent both the upper and the lower strand of the DNA. The upper oligo of the first oligo pair was designed to have a 15 bp single stranded region at the 3' end which was homologous to a similar single stranded region of the lower strand of the next oligo pair to direct the orientation and sequence of the various oligo pairs within a given fragment. The oligos are also designed such that when the all the oligos representing a fragment are hybridized, the ends have single stranded regions corresponding to the particular restriction site to be formed. The structure of each oligomer was examined for stable secondary structures such as hairpin loops using the OLIGO program from NBI Inc. Whenever neccesary, nucleotides were changed to decrease the stability of the secondary structure without changing the amino acid sequence of the protein. A plant ribosomal binding site consensus sequence, TAAACAATG (Joshi et al., Nucleic Acid Res., 15:6643-6653 (1987)) or eukaryotic ribosomal binding site concensus sequence CCACCATG (Kozak, Nucleic Acid Research, 12:857-872 (1984)) was inserted at the translational start codon of the gene.
Cloning: Oligos were synthesized by IDT Inc., and were supplied as lyophilized powders. They were resuspended at a concentration of 200 .mu.M. To 30 .mu.l of each oligo formamide was added a final concentration of 25-50% and the sample was boiled for two minutes before separation on a premade 10% polyacryamide/urea gel obtained from Novex. After electrophoresis, the oligo was detected by UV shadowing by placing the gel on a TLC plate containing a fluorescent indicator and exposing it to UV light. The region containing DNA of the correct size was excised and extracted from the polyacryamide by an overnight incubation of the minced gel fragment in a buffer containing 0.4 M LiCl, 0.1 mM EDTA. The DNA was separated from the gel residue by centrifugation through a Millipore UFMC filter. The extracted DNA was ethanol precipitated by the addition of 2 volumes of absolute alcohol. After centrifugation, the precipitate was resuspended in dH.sub.2 O at a concentration of 2.5 .mu.M. Fragments were cloned either by hybridization of the oligos and ligation with the appropriate vector or by amplification of the hybridized fragment using a equimolar mixture of all the oligos for a particular fragment as a template and end-specific PCR primers.
Cloning by hybridization and ligation: Homologous double stranded oligo pairs were obtained by mixing 5 .mu.l of the upper and of the lower oligo for each oligo pair with buffer containing 1.times. polynucleotide kinase (PNK) buffer (70 mM Tris-HCl (pH 7.6), 10 mM MgCl.sub.2, 5 mM dithiothreitol (DTT)), 50 mM KCl, and 5% formamide in a final volume of 50 .mu.l. The oligos were boiled for 10 minutes and slow cooled to 37.degree. C. or room temperature. 10 .mu.l was removed for analysis on a 4% agarose in a TAE buffer system (Metaphore.RTM.; FMC). Each hybridized oligo pair was kinased by the addition of ATP at a final concentration of 1 mM, BSA at a final concentration of 100 .mu.g per ml and 200 units of polynucleotide kinase and 1 .mu.l of 10.times. PNK buffer in a volume of 10 .mu.l. Following hybridization and phosphorylation, the reaction was incubated at 37.degree. C. for 2 hours to overnight. 10 .mu.l of each of the oligo pairs for a particular fragment, were mixed in a final volume of 50 .mu.l. The oligo pairs were hybridized by heating at 80.degree. C. for 10 minutes and slow cooling to 37.degree. C. 2 ul of oligos was mixed with about 100 ng of an appropriate vector and ligated using a buffer containing 50 mM Tris-HCl (pH 7.8), 10 mM MgCl.sub.2, 10 mM DTT, 1 mM ATP. The reaction was incubated at room temp. for 2 hours to overnight and transformed into DH5.alpha. strain of E. coli, plated on L- plates containing ampicillin at a concentration of 100 .mu.g/ml using standard procedures. Positive clones were further characterized and confirmed by PCR miniscreen described in detail in U.S. Pat. No. 5/625,136 using the universal primers "Reverse" and M13 "-20" as primers. Positive clones were identified by digestion of DNA with appropriate enzymes followed by sequencing. Recombinants that had the expected DNA sequence were then selected for further work.
PCR Amplification and cloning into T-vector:
PCR amplification was carried out by using a mixture of all the oligomers that represented the upper and the lower strand of a particular fragment (final concentration 5 mM each) as template, specific end primers for the particular fragment (final concentration 2 .mu.M) 200 .mu.M of each dATP, dTTP, dCTP and dGTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl.sub.2,0.01% gelatin and 5 units of Taq polymerase in a final reaction volume of 50 .mu.l. The amplification reaction was carried out in a Perkin Elmer thermocycler 9600 by incubation at 95.degree. C. for 1 min (1 cycle ), followed by 20 cycles of 95.degree. C. for 45 sec., 50.degree. C. for 45 sec., 72.degree. C. for 30 sec. Finally the reaction was incubated for 5 min at 72.degree. C. before analyzing the product. 10.mu.l of the reaction was analyzed on a 2.5% Nusieve (FMC) agarose gel in a TAE buffer system. The correct size fragment was gel purified and used for cloning into a PCR cloning vector or T-vector. T-vector construction was as described by Marchuk et al., Nucleic Acid Research, 19:1154 (1991). pBluescriptsk+ (Stratagene.RTM., Ca.) was used as the parent vector. Transformation and identification of the correct clone was carried out as described above.
Fragments 1, 3, 4, 5, 6, 8, and 9 of VIP1A(a) and fragments 2 and 4 of VIP2A(a) were obtained by cloning of PCR amplification products; whereas, fragments 2, 7, 10 and 11 of VIP1A(a) and fragments 1, 3, and 5 of VIP2A(a) were obtained by hybridization/ligation.
Once fragments with the desired sequence were obtained, the complete gene was assembled by cloning together adjacent fragments. The complete gene was resequenced and tested for activity against WCRW before moving it into plant expression vectors containing the root preferred promoter (disclosed in U.S. Pat. No. 5,466,785, herein incorporated by reference) and the rice actin promoter.
One such plant expression vector is pCIB5521. The maize optimized VIP1A(a) coding region (SEQ ID NO:26) was cloned in a plant expression vector containing the root preferred promoter at the 5' of the gene with the PEP Carboxylase intron #9 followed by the 35S terminator at the 3' end. The plasmid also contains sequences for ampicillin resistance from the plasmid pUC19. Another plant expression vector is pCIB5522, which contains the maize optimized VIP2A(a) coding region (SEQ ID NO:27) fused to the root preferred promoter at the 5' of the gene with the PEP Carboxylase intron #9 followed by the 35S terminator at the 3' end.
EXAMPLE 25
NAD AFFINITY CHROMATOGRAPHY
A purification strategy was used based on the affinity of VIP2 for the substrate NAD. The supernatant from the pH 3.5 sodium citrate buffer treatment described in Example 4 was dialyzed in 20 mM TRIS pH 7.5 overnight. The neutralized supematant was added to an equal volume of washed NAD agarose and incubated with gentle rocking at 4.degree. C. overnight. The resin and protein solution were added to a 10 ml disposable polypropylene column and the protein solution allowed to flow out. The column was washed with 5 column volumes of 20 mM TRIS pH 7.5 then washed with 2-5 column volumes of 20 mM TRIS pH 7.5, 100 mM NaCl, followed by 2-5 column volumes of 20 mM TRIS 7.5. The VIP proteins were eluted in 20 mM TRIS pH 7.5 supplemented with 5 mM NAD. Approximately 3 column volumes of the effluent were collected and concentrated in a Centricon -10. Yield is typically about 7-15 .mu.g of protein per ml of resin.
When the purified proteins were analyzed by SDS-PAGE followed by silver staining, two polypeptides were visible, one with Mr of approximately 80,000 and one with Mr of approximately 45,000. N-terminal sequencing revealed that the Mr 80,000 protein corresponded to a proteolytically processed form of VIP1A(A) and the Mr 45,000 form corresponded to a proteolytically processed form of VIP2A(a). The co-purification of VIP1A(a) with VIP2A(a) indicates that the two proteins probably form a complex and have protein-protein interacting regions. VIP1A(a) and VIP2A(a) proteins purified in this manner were biologically active against western corn rootworm.
EXAMPLE 26
EXPRESSION OF MAIZE OPTIMIZED VIP1A(a) AND VIP2A(a)
E. coli strains containing different plasmids comprising VIP genes were assayed for expression of VIPs. E. coli strains harboring the individual plasmids were grown overnight in L-broth and expressed protein was extracted from the culture as described in Example 3, above. Protein expression was assayed by Western Blot analysis using antibodies developed using standard methods known in the art, similar to those described in Example 12, above. Also, insecticidal activity of the expressed proteins were tested against Western corn rootworm according to the method in Example 3, above. The results of the E. coli expression assays are described below.
______________________________________Expression of VIPs in E. coli Assay Assay Extract of E. coli Strain Harboring No. 1 No. 2 ProteinIndicated Plasmid % Mortality Detected______________________________________Control 0 0 no pCIB5521 (maize optimized VIP1A(a)) 47 27 yes pCIB5522 (maize optimized VIP2A(a)) 7 7 yes pCIB6024 (native VIP2A(a)) 13 13 yes pCIB6206 (native VIP1A(a)) 27 40 yes Extracts pCIB5521 + pCIB5522 combined 87 47 Extracts pCIB5521 + pCIB6024 combined 93 100 Extracts pCIBSS22 + pCIB6206 combined 100 100 Extracts pCIB6024 + pCIB6206 combined 100 100______________________________________
The DNA from these plasmids was used to transiently express the VIPs in a maize protoplast expression system. Protoplasts were isolated from maize 2717 Line 6 suspension cultures by digestion of the cell walls using Cellulase RS and Macerase R10 in appropriate buffer. Protoplasts were recovered by sieving and centrifugation. Protoplasts were transformed by a standard direct gene transfer method using approximately 75 .mu.g plasmid DNA and PEG40. Treated protoplasts were incubated overnight in the dark at room temperature. Analysis of VIP expression was accomplished on protoplast explants by Western blot analysis and insecticidal activity against Western corn rootworm as described above for the expression in E. coli. The results of the maize protoplast expression assays are described below.
______________________________________Expression of VIPs in Plant Protoplasts Assay Assay Protein No. 1 No. 2 De-Extract Tested % Mortality tected______________________________________No DNA Control 27 10 no pCIB5521 (p) (maize optimized VIP1A(a)) 20 (0) 30 yes pCIB5522 (p) (maize optmizied VIP2A(a)) 20 (0) 20 yes Extracts pCIB5521 (p) + 87 (82) 90 pCIB5522 (p) combined Extracts pCIB5521 (p) + 100 -- pCIB5522 (e) combined Extracts pCIB5522 (p) + 53 (36) -- pCIB5521 (e) combined Extracts pCIB5521 (p) + 100 -- pCIB6024 (e) combined Extracts pCIB5522 (p) + 100 -- pCIB6206 (e) combined pCIB6024 (e) (native VIP2A(a)) 0 -- yes pCIB6206 (e) (native VIP1A(a)) 20 -- yes pCIB5521 + pCIB 5522 plasmids delivered 100 100 yes by cotransformation)______________________________________ (p) = extract of protoplast culture transformed with indicated plasmid (e) = extract of E. coli strain harboring indicated plasmid
The expression data obtained with both E. coli and maize protoplasts show that the maize optimized VIP1A(a) and VIP2A(a) genes make the same protein as the native VIP1A(a) and VIP2A(a) genes, respectively, and that the proteins encoded by the maize optimized genes are functionally equivalent to the proteins encoded by the native genes.
All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The following deposits have been made at Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Ill. 61604, USA:
______________________________________ 1. E. coli PL2 Accession No. NRRL B-21221N 2. E. coli pCIB6022 Accession No. NRRL B-21222 3. E. coli pCIB6023 Accession No. NRRL B-21223N 4. Bacillus thuringiensis HD73-78VIP Accession No. NRRL B-21224 5. Bacillus thuringiensis AB88 Accession No. NRRL B-21225 6. Bacillus thuringiensis AB359 Accession No. NRRL B-21226 7. Bacillus thuringiensis AB289 Accession No. NRRL B-21227 8. Bacillus sp. AB59 Accession No. NRRL B-21228 9. Bacillus sp. AB294 Accession No. NRRL B-21229 10. Bacillus sp. AB256 Accession No. NRRL B-21230 11. E. coli P5-4 Accession No. NRRL B-21059 12. E. coli P3-12 Accession No. NRRL B-21061 13. Bacillus cereus AB78 Accession No. NRRL B-21058 14. Bacillus thuringiensis AB6 Accession No. NRRL B-21060 15. E. coli pCIB6202 Accession No. NRRL B-21321 16. E. coli pCIB7100 Accession No. NRRL B-21322 17. E. coli pCIB7101 Accession No. NRRL B-21323 18. E. coli pCIB7102 Accession No. NRRL B-21324 19. E. coli pCIB7102 Accession No. NRRL B-21325 20. E. coli pCIB7104 Accession No. NRRL B-21422 21. E. coli pCIB7107 Accession No. NRRL B-21423 22. E. coli PCIB7108 Accession No. NRRL B-21438 23. Bacillus thuringiensis AB424 Accession No. NRRL B-21439______________________________________
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
__________________________________________________________________________# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES: 50 - - - - (2) INFORMATION FOR SEQ ID NO:1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6049 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus - #cereus (B) STRAIN: AB78 (C) INDIVIDUAL ISOLATE: - #NRRL B-21058 - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1082..2467 (D) OTHER INFORMATION: - #/product= "VIP2A(a)" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 2475..5126 (D) OTHER INFORMATION: - #/note= "Coding sequence for the 100 kd VIP1A( - #a) protein. This coding sequence isrepeated in SEQ - #ID NO:4 and translated separately." - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: - - ATCGATACAA TGTTGTTTTA CTTAGACCGG TAGTCTCTGT AATTTGTTTA AT -#GCTATATT 60 - - CTTTACTTTG ATACATTTTA ATAGCCATTT CAACCTTATC AGTATGTTTT TG -#TGGTCTTC 120 - - CTCCTTTTTT TCCACGAGCT CTAGCTGCGT TTAATCCTGT TTTGGTACGT TC -#GCTAATAA 180 - - TATCTCTTTC TAATTCTGCA ATACTTGCCA TCATTCGAAA GAAGAATTTC CC -#CATAGCAT 240 - - TAGAGGTATC AATGTTGTCA TGAATAGAAA TAAAATCTAC ACCTAGCTCT TT -#GAATTTTT 300 - - CACTTAACTC AATTAGGTGT TTTGTAGAGC GAGAAATTCG ATCAAGTTTG TA -#AACAACTA 360 - - TCTTATCGCC TTTACGTAAT ACTTTTAGCA ACTCTTCGAG TTGAGGGCGC TC -#TTTTTTTA 420 - - TTCCTGTTAT TTTCTCCTGA TATAGCCTTT CTACACCATA TTGTTGCAAA GC -#ATCTATTT 480 - - GCATATCGAG ATTTTGTTCT TCTGTGCTGA CACGAGCATA ACCAAAAATC AA -#ATTGGTTT 540 - - CACTTCCTAT CTAAATATAT CTATTAAAAT AGCACCAAAA ACCTTATTAA AT -#TAAAATAA 600 - - GGAACTTTGT TTTTGGATAT GGATTTTGGT ACTCAATATG GATGAGTTTT TA -#ACGCTTTT 660 - - GTTAAAAAAC AAACAAGTGC CATAAACGGT CGTTTTTGGG ATGACATAAT AA -#ATAATCTG 720 - - TTTGATTAAC CTAACCTTGT ATCCTTACAG CCCAGTTTTA TTTGTACTTC AA -#CTGACTGA 780 - - ATATGAAAAC AACATGAAGG TTTCATAAAA TTTATATATT TTCCATAACG GA -#TGCTCTAT 840 - - CTTTAGGTTA TAGTTAAATT ATAAGAAAAA AACAAACGGA GGGAGTGAAA AA -#AAGCATCT 900 - - TCTCTATAAT TTTACAGGCT CTTTAATAAG AAGGGGGGAG ATTAGATAAT AA -#ATATGAAT 960 - - ATCTATCTAT AATTGTTTGC TTCTACAATA ACTTATCTAA CTTTCATATA CA -#ACAACAAA 1020 - - ACAGACTAAA TCCAGATTGT ATATTCATTT TCAGTTGTTC CTTTATAAAA TA -#ATTTCATA 1080 - - A ATG AAA AGA ATG GAG GGA AAG TTG TTT ATG - #GTG TCA AAA AAA TTA 1126 Met Lys Arg Met Glu Gly Lys Leu Phe M - #et Val Ser Lys Lys Leu 1 - # 5 - # 10 - # 15 - - CAA GTA GTT ACT AAA ACT GTA TTG CTT AGT AC - #A GTT TTC TCT ATA TCT 1174 Gln Val Val Thr Lys Thr Val Leu Leu Ser Th - #r Val Phe Ser Ile Ser 20 - # 25 - # 30 - - TTA TTA AAT AAT GAA GTG ATA AAA GCT GAA CA - #A TTA AAT ATA AAT TCT 1222 Leu Leu Asn Asn Glu Val Ile Lys Ala Glu Gl - #n Leu Asn Ile Asn Ser 35 - # 40 - # 45 - - CAA AGT AAA TAT ACT AAC TTG CAA AAT CTA AA - #A ATC ACT GAC AAG GTA 1270 Gln Ser Lys Tyr Thr Asn Leu Gln Asn Leu Ly - #s Ile Thr Asp Lys Val 50 - # 55 - # 60 - - GAG GAT TTT AAA GAA GAT AAG GAA AAA GCG AA - #A GAA TGG GGG AAA GAA 1318 Glu Asp Phe Lys Glu Asp Lys Glu Lys Ala Ly - #s Glu Trp Gly Lys Glu 65 - # 70 - # 75 - - AAA GAA AAA GAG TGG AAA CTA ACT GCT ACT GA - #A AAA GGA AAA ATG AAT 1366 Lys Glu Lys Glu Trp Lys Leu Thr Ala Thr Gl - #u Lys Gly Lys Met Asn 80 - # 85 - # 90 - # 95 - - AAT TTT TTA GAT AAT AAA AAT GAT ATA AAG AC - #A AAT TAT AAA GAA ATT 1414 Asn Phe Leu Asp Asn Lys Asn Asp Ile Lys Th - #r Asn Tyr Lys Glu Ile 100 - # 105 - # 110 - - ACT TTT TCT ATG GCA GGC TCA TTT GAA GAT GA - #A ATA AAA GAT TTA AAA 1462 Thr Phe Ser Met Ala Gly Ser Phe Glu Asp Gl - #u Ile Lys Asp Leu Lys 115 - # 120 - # 125 - - GAA ATT GAT AAG ATG TTT GAT AAA ACC AAT CT - #A TCA AAT TCT ATT ATC 1510 Glu Ile Asp Lys Met Phe Asp Lys Thr Asn Le - #u Ser Asn Ser Ile Ile 130 - # 135 - # 140 - - ACC TAT AAA AAT GTG GAA CCG ACA ACA ATT GG - #A TTT AAT AAA TCT TTA 1558 Thr Tyr Lys Asn Val Glu Pro Thr Thr Ile Gl - #y Phe Asn Lys Ser Leu 145 - # 150 - # 155 - - ACA GAA GGT AAT ACG ATT AAT TCT GAT GCA AT - #G GCA CAG TTT AAA GAA 1606 Thr Glu Gly Asn Thr Ile Asn Ser Asp Ala Me - #t Ala Gln Phe Lys Glu 160 1 - #65 1 - #70 1 -#75 - - CAA TTT TTA GAT AGG GAT ATT AAG TTT GAT AG - #T TAT CTA GAT ACGCAT 1654 Gln Phe Leu Asp Arg Asp Ile Lys Phe Asp Se - #r Tyr Leu Asp Thr His 180 - # 185 - # 190 - - TTA ACT GCT CAA CAA GTT TCC AGT AAA GAA AG - #A GTT ATT TTG AAG GTT 1702 Leu Thr Ala Gln Gln Val Ser Ser Lys Glu Ar - #g Val Ile Leu Lys Val 195 - # 200 - # 205 - - ACG GTT CCG AGT GGG AAA GGT TCT ACT ACT CC - #A ACA AAA GCA GGT GTC 1750 Thr Val Pro Ser Gly Lys Gly Ser Thr Thr Pr - #o Thr Lys Ala Gly Val 210 - # 215 - # 220 - - ATT TTA AAT AAT AGT GAA TAC AAA ATG CTC AT - #T GAT AAT GGG TAT ATG 1798 Ile Leu Asn Asn Ser Glu Tyr Lys Met Leu Il - #e Asp Asn Gly Tyr Met 225 - # 230 - # 235 - - GTC CAT GTA GAT AAG GTA TCA AAA GTG GTG AA - #A AAA GGG GTG GAG TGC 1846 Val His Val Asp Lys Val Ser Lys Val Val Ly - #s Lys Gly Val Glu Cys 240 2 - #45 2 - #50 2 -#55 - - TTA CAA ATT GAA GGG ACT TTA AAA AAG AGT CT - #T GAC TTT AAA AATGAT 1894 Leu Gln Ile Glu Gly Thr Leu Lys Lys Ser Le - #u Asp Phe Lys Asn Asp 260 - # 265 - # 270 - - ATA AAT GCT GAA GCG CAT AGC TGG GGT ATG AA - #G AAT TAT GAA GAG TGG 1942 Ile Asn Ala Glu Ala His Ser Trp Gly Met Ly - #s Asn Tyr Glu Glu Trp 275 - # 280 - # 285 - - GCT AAA GAT TTA ACC GAT TCG CAA AGG GAA GC - #T TTA GAT GGG TAT GCT 1990 Ala Lys Asp Leu Thr Asp Ser Gln Arg Glu Al - #a Leu Asp Gly Tyr Ala 290 - # 295 - # 300 - - AGG CAA GAT TAT AAA GAA ATC AAT AAT TAT TT - #A AGA AAT CAA GGC GGA 2038 Arg Gln Asp Tyr Lys Glu Ile Asn Asn Tyr Le - #u Arg Asn Gln Gly Gly 305 - # 310 - # 315 - - AGT GGA AAT GAA AAA CTA GAT GCT CAA ATA AA - #A AAT ATT TCT GAT GCT 2086 Ser Gly Asn Glu Lys Leu Asp Ala Gln Ile Ly - #s Asn Ile Ser Asp Ala 320 3 - #25 3 - #30 3 -#35 - - TTA GGG AAG AAA CCA ATA CCG GAA AAT ATT AC - #T GTG TAT AGA TGGTGT 2134 Leu Gly Lys Lys Pro Ile Pro Glu Asn Ile Th - #r Val Tyr Arg Trp Cys 340 - # 345 - # 350 - - GGC ATG CCG GAA TTT GGT TAT CAA ATT AGT GA - #T CCG TTA CCT TCT TTA 2182 Gly Met Pro Glu Phe Gly Tyr Gln Ile Ser As - #p Pro Leu Pro Ser Leu 355 - # 360 - # 365 - - AAA GAT TTT GAA GAA CAA TTT TTA AAT ACA AT - #C AAA GAA GAC AAA GGA 2230 Lys Asp Phe Glu Glu Gln Phe Leu Asn Thr Il - #e Lys Glu Asp Lys Gly 370 - # 375 - # 380 - - TAT ATG AGT ACA AGC TTA TCG AGT GAA CGT CT - #T GCA GCT TTT GGA TCT 2278 Tyr Met Ser Thr Ser Leu Ser Ser Glu Arg Le - #u Ala Ala Phe Gly Ser 385 - # 390 - # 395 - - AGA AAA ATT ATA TTA CGA TTA CAA GTT CCG AA - #A GGA AGT ACG GGT GCG 2326 Arg Lys Ile Ile Leu Arg Leu Gln Val Pro Ly - #s Gly Ser Thr Gly Ala 400 4 - #05 4 - #10 4 -#15 - - TAT TTA AGT GCC ATT GGT GGA TTT GCA AGT GA - #A AAA GAG ATC CTACTT 2374 Tyr Leu Ser Ala Ile Gly Gly Phe Ala Ser Gl - #u Lys Glu Ile Leu Leu 420 - # 425 - # 430 - - GAT AAA GAT AGT AAA TAT CAT ATT GAT AAA GT - #A ACA GAG GTA ATT ATT 2422 Asp Lys Asp Ser Lys Tyr His Ile Asp Lys Va - #l Thr Glu Val Ile Ile 435 - # 440 - # 445 - - AAA GGT GTT AAG CGA TAT GTA GTG GAT GCA AC - #A TTA TTA ACA AAT 2467 Lys Gly Val Lys Arg Tyr Val Val Asp Ala Th - #r Leu Leu Thr Asn 450 - # 455 - # 460 - - TAAGGAGATG AAAAATATGA AGAAAAAGTT AGCAAGTGTT GTAACGTGTA CG -#TTATTAGC 2527 - - TCCTATGTTT TTGAATGGAA ATGTGAATGC TGTTTACGCA GACAGCAAAA CA -#AATCAAAT 2587 - - TTCTACAACA CAGAAAAATC AACAGAAAGA GATGGACCGA AAAGGATTAC TT -#GGGTATTA 2647 - - TTTCAAAGGA AAAGATTTTA GTAATCTTAC TATGTTTGCA CCGACACGTG AT -#AGTACTCT 2707 - - TATTTATGAT CAACAAACAG CAAATAAACT ATTAGATAAA AAACAACAAG AA -#TATCAGTC 2767 - - TATTCGTTGG ATTGGTTTGA TTCAGAGTAA AGAAACGGGA GATTTCACAT TT -#AACTTATC 2827 - - TGAGGATGAA CAGGCAATTA TAGAAATCAA TGGGAAAATT ATTTCTAATA AA -#GGGAAAGA 2887 - - AAAGCAAGTT GTCCATTTAG AAAAAGGAAA ATTAGTTCCA ATCAAAATAG AG -#TATCAATC 2947 - - AGATACAAAA TTTAATATTG ACAGTAAAAC ATTTAAAGAA CTTAAATTAT TT -#AAAATAGA 3007 - - TAGTCAAAAC CAACCCCAGC AAGTCCAGCA AGATGAACTG AGAAATCCTG AA -#TTTAACAA 3067 - - GAAAGAATCA CAGGAATTCT TAGCGAAACC ATCGAAAATA AATCTTTTCA CT -#CAAAAAAT 3127 - - GAAAAGGGAA ATTGATGAAG ACACGGATAC GGATGGGGAC TCTATTCCTG AC -#CTTTGGGA 3187 - - AGAAAATGGG TATACGATTC ACAATAGAAT CGCTGTAAAG TGGGACGATT CT -#CTAGCAAG 3247 - - TAAAGGGTAT ACGAAATTTG TTTCAAATCC ACTAGAAAGT CACACAGTTG GT -#GATCCTTA 3307 - - TACAGATTAT GAAAAGGCAG CAAGAGATCT AGATTTGTCA AATGCAAAGG AA -#ACGTTTAA 3367 - - CCCATTGGTA GCTGCTTTTC CAAGTGTGAA TGTTAGTATG GAAAAGGTGA TA -#TTATCACC 3427 - - AAATGAAAAT TTATCCAATA GTGTAGAGTC TCATTCATCC ACGAATTGGT CT -#TATACAAA 3487 - - TACAGAAGGT GCTTCTGTTG AAGCGGGGAT TGGACCAAAA GGTATTTCGT TC -#GGAGTTAG 3547 - - CGTAAACTAT CAACACTCTG AAACAGTTGC ACAAGAATGG GGAACATCTA CA -#GGAAATAC 3607 - - TTCGCAATTC AATACGGCTT CAGCGGGATA TTTAAATGCA AATGTTCGAT AT -#AACAATGT 3667 - - AGGAACTGGT GCCATCTACG ATGTAAAACC TACAACAAGT TTTGTATTAA AT -#AACGATAC 3727 - - TATCGCAACT ATTACGGCGA AATCTAATTC TACAGCCTTA AATATATCTC CT -#GGAGAAAG 3787 - - TTACCCGAAA AAAGGACAAA ATGGAATCGC AATAACATCA ATGGATGATT TT -#AATTCCCA 3847 - - TCCGATTACA TTAAATAAAA AACAAGTAGA TAATCTGCTA AATAATAAAC CT -#ATGATGTT 3907 - - GGAAACAAAC CAAACAGATG GTGTTTATAA GATAAAAGAT ACACATGGAA AT -#ATAGTAAC 3967 - - TGGCGGAGAA TGGAATGGTG TCATACAACA AATCAAGGCT AAAACAGCGT CT -#ATTATTGT 4027 - - GGATGATGGG GAACGTGTAG CAGAAAAACG TGTAGCGGCA AAAGATTATG AA -#AATCCAGA 4087 - - AGATAAAACA CCGTCTTTAA CTTTAAAAGA TGCCCTGAAG CTTTCATATC CA -#GATGAAAT 4147 - - AAAAGAAATA GAGGGATTAT TATATTATAA AAACAAACCG ATATACGAAT CG -#AGCGTTAT 4207 - - GACTTACTTA GATGAAAATA CAGCAAAAGA AGTGACCAAA CAATTAAATG AT -#ACCACTGG 4267 - - GAAATTTAAA GATGTAAGTC ATTTATATGA TGTAAAACTG ACTCCAAAAA TG -#AATGTTAC 4327 - - AATCAAATTG TCTATACTTT ATGATAATGC TGAGTCTAAT GATAACTCAA TT -#GGTAAATG 4387 - - GACAAACACA AATATTGTTT CAGGTGGAAA TAACGGAAAA AAACAATATT CT -#TCTAATAA 4447 - - TCCGGATGCT AATTTGACAT TAAATACAGA TGCTCAAGAA AAATTAAATA AA -#AATCGTGA 4507 - - CTATTATATA AGTTTATATA TGAAGTCAGA AAAAAACACA CAATGTGAGA TT -#ACTATAGA 4567 - - TGGGGAGATT TATCCGATCA CTACAAAAAC AGTGAATGTG AATAAAGACA AT -#TACAAAAG 4627 - - ATTAGATATT ATAGCTCATA ATATAAAAAG TAATCCAATT TCTTCACTTC AT -#ATTAAAAC 4687 - - GAATGATGAA ATAACTTTAT TTTGGGATGA TATTTCTATA ACAGATGTAG CA -#TCAATAAA 4747 - - ACCGGAAAAT TTAACAGATT CAGAAATTAA ACAGATTTAT AGTAGGTATG GT -#ATTAAGTT 4807 - - AGAAGATGGA ATCCTTATTG ATAAAAAAGG TGGGATTCAT TATGGTGAAT TT -#ATTAATGA 4867 - - AGCTAGTTTT AATATTGAAC CATTGCAAAA TTATGTGACC AAATATGAAG TT -#ACTTATAG 4927 - - TAGTGAGTTA GGACCAAACG TGAGTGACAC ACTTGAAAGT GATAAAATTT AC -#AAGGATGG 4987 - - GACAATTAAA TTTGATTTTA CCAAATATAG TAAAAATGAA CAAGGATTAT TT -#TATGACAG 5047 - - TGGATTAAAT TGGGACTTTA AAATTAATGC TATTACTTAT GATGGTAAAG AG -#ATGAATGT 5107 - - TTTTCATAGA TATAATAAAT AGTTATTATA TCTATGAAGC TGGTGCTAAA GA -#TAGTGTAA 5167 - - AAGTTAATAT ACTGTAGGAT TGTAATAAAA GTAATGGAAT TGATATCGTA CT -#TTGGAGTG 5227 - - GGGGATACTT TGTAAATAGT TCTATCAGAA ACATTAGACT AAGAAAAGTT AC -#TACCCCCA 5287 - - CTTGAAAATG AAGATTCAAC TGATTACAAA CAACCTGTTA AATATTATAA GG -#TTTTAACA 5347 - - AAATATTAAA CTCTTTATGT TAATACTGTA ATATAAAGAG TTTAATTGTA TT -#CAAATGAA 5407 - - GCTTTCCCAC AAAATTAGAC TGATTATCTA ATGAAATAAT CAGTCTAATT TT -#GTAGAACA 5467 - - GGTCTGGTAT TATTGTACGT GGTCACTAAA AGATATCTAA TATTATTGGG CA -#AGGCGTTC 5527 - - CATGATTGAA TCCTCGAATG TCTTGCCCTT TTCATTTATT TAAGAAGGAT TG -#TGGAGAAA 5587 - - TTATGGTTTA GATAATGAAG AAAGACTTCA CTTCTAATTT TTGATGTTAA AT -#AAATCAAA 5647 - - ATTTGGCGAT TCACATTGTT TAATCCACTG ATAAAACATA CTGGAGTGTT CT -#TAAAAAAT 5707 - - CAGCTTTTTT CTTTATAAAA TTTTGCTTAG CGTACGAAAT TCGTGTTTTG TT -#GGTGGGAC 5767 - - CCCATGCCCA TCAACTTAAG AGTAAATTAG TAATGAACTT TCGTTCATCT GG -#ATTAAAAT 5827 - - AACCTCAAAT TAGGACATGT TTTTAAAAAT AAGCAGACCA AATAAGCCTA GA -#ATAGGTAT 5887 - - CATTTTTAAA AATTATGCTG CTTTCTTTTG TTTTCCAAAT CCATTATACT CA -#TAAGCAAC 5947 - - ACCCATAATG TCAAAGACTG TTTTTGTCTC ATATCGATAA GCTTGATATC GA -#ATTCCTGC 6007 - - AGCCCGGGGG ATCCACTAGT TCTAGAGCGG CCGCCACCGC GG - # - #6049 - - - - (2) INFORMATION FOR SEQ ID NO:2: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 462 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: - - Met Lys Arg Met Glu Gly Lys Leu Phe Met Va - #l Ser Lys Lys Leu Gln 1 5 - # 10 - # 15 - - Val Val Thr Lys Thr Val Leu Leu Ser Thr Va - #l Phe Ser Ile Ser Leu 20 - # 25 - # 30 - - Leu Asn Asn Glu Val Ile Lys Ala Glu Gln Le - #u Asn Ile Asn Ser Gln 35 - # 40 - # 45 - - Ser Lys Tyr Thr Asn Leu Gln Asn Leu Lys Il - #e Thr Asp Lys Val Glu 50 - # 55 - # 60 - - Asp Phe Lys Glu Asp Lys Glu Lys Ala Lys Gl - #u Trp Gly Lys Glu Lys 65 - # 70 - # 75 - # 80 - - Glu Lys Glu Trp Lys Leu Thr Ala Thr Glu Ly - #s Gly Lys Met Asn Asn 85 - # 90 - # 95 - - Phe Leu Asp Asn Lys Asn Asp Ile Lys Thr As - #n Tyr Lys Glu Ile Thr 100 - # 105 - # 110 - - Phe Ser Met Ala Gly Ser Phe Glu Asp Glu Il - #e Lys Asp Leu Lys Glu 115 - # 120 - # 125 - - Ile Asp Lys Met Phe Asp Lys Thr Asn Leu Se - #r Asn Ser Ile Ile Thr 130 - # 135 - # 140 - - Tyr Lys Asn Val Glu Pro Thr Thr Ile Gly Ph - #e Asn Lys Ser Leu Thr 145 1 - #50 1 - #55 1 -#60 - - Glu Gly Asn Thr Ile Asn Ser Asp Ala Met Al - #a Gln Phe Lys GluGln 165 - # 170 - # 175 - - Phe Leu Asp Arg Asp Ile Lys Phe Asp Ser Ty - #r Leu Asp Thr His Leu 180 - # 185 - # 190 - - Thr Ala Gln Gln Val Ser Ser Lys Glu Arg Va - #l Ile Leu Lys Val Thr 195 - # 200 - # 205 - - Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Th - #r Lys Ala Gly Val Ile 210 - # 215 - # 220 - - Leu Asn Asn Ser Glu Tyr Lys Met Leu Ile As - #p Asn Gly Tyr Met Val 225 2 - #30 2 - #35 2 -#40 - - His Val Asp Lys Val Ser Lys Val Val Lys Ly - #s Gly Val Glu CysLeu 245 - # 250 - # 255 - - Gln Ile Glu Gly Thr Leu Lys Lys Ser Leu As - #p Phe Lys Asn Asp Ile 260 - # 265 - # 270 - - Asn Ala Glu Ala His Ser Trp Gly Met Lys As - #n Tyr Glu Glu Trp Ala 275 - # 280 - # 285 - - Lys Asp Leu Thr Asp Ser Gln Arg Glu Ala Le - #u Asp Gly Tyr Ala Arg 290 - # 295 - # 300 - - Gln Asp Tyr Lys Glu Ile Asn Asn Tyr Leu Ar - #g Asn Gln Gly Gly Ser 305 3 - #10 3 - #15 3 -#20 - - Gly Asn Glu Lys Leu Asp Ala Gln Ile Lys As - #n Ile Ser Asp AlaLeu 325 - # 330 - # 335 - - Gly Lys Lys Pro Ile Pro Glu Asn Ile Thr Va - #l Tyr Arg Trp Cys Gly 340 - # 345 - # 350 - - Met Pro Glu Phe Gly Tyr Gln Ile Ser Asp Pr - #o Leu Pro Ser Leu Lys 355 - # 360 - # 365 - - Asp Phe Glu Glu Gln Phe Leu Asn Thr Ile Ly - #s Glu Asp Lys Gly Tyr 370 - # 375 - # 380 - - Met Ser Thr Ser Leu Ser Ser Glu Arg Leu Al - #a Ala Phe Gly Ser Arg 385 3 - #90 3 - #95 4 -#00 - - Lys Ile Ile Leu Arg Leu Gln Val Pro Lys Gl - #y Ser Thr Gly AlaTyr 405 - # 410 - # 415 - - Leu Ser Ala Ile Gly Gly Phe Ala Ser Glu Ly - #s Glu Ile Leu Leu Asp 420 - # 425 - # 430 - - Lys Asp Ser Lys Tyr His Ile Asp Lys Val Th - #r Glu Val Ile Ile Lys 435 - # 440 - # 445 - - Gly Val Lys Arg Tyr Val Val Asp Ala Thr Le - #u Leu Thr Asn 450 - # 455 - # 460 - - - - (2) INFORMATION FOR SEQ ID NO:3: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: peptide - - (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..20 (D) OTHER INFORMATION: - #/note= "Signal peptide for vacuolar targetting" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: - - Ser Ser Ser Ser Phe Ala Asp Ser Asn Pro Il - #e Arg Val Thr Asp Arg 1 5 - # 10 - # 15 - - Ala Ala Ser Thr 20 - - - - (2) INFORMATION FOR SEQ ID NO:4: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2655 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus - #cereus (B) STRAIN: AB78 (C) INDIVIDUAL ISOLATE: - #NRRL B-21058 - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..2652 (D) OTHER INFORMATION: - #/product= "100 kDa protein VIP1A(a)" /note= - #"This sequence is identical to the portion of SEQ ID - #NO:1 between and including nucleotide 2475 to 5126." - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: - - ATG AAA AAT ATG AAG AAA AAG TTA GCA AGT GT - #T GTA ACG TGT ACG TTA 48 Met Lys Asn Met Lys Lys Lys Leu Ala Ser Va - #l Val Thr Cys Thr Leu 465 - # 470 - # 475 - - TTA GCT CCT ATG TTT TTG AAT GGA AAT GTG AA - #T GCT GTT TAC GCA GAC 96 Leu Ala Pro Met Phe Leu Asn Gly Asn Val As - #n Ala Val Tyr Ala Asp 480 - # 485 - # 490 - - AGC AAA ACA AAT CAA ATT TCT ACA ACA CAG AA - #A AAT CAA CAG AAA GAG 144 Ser Lys Thr Asn Gln Ile Ser Thr Thr Gln Ly - #s Asn Gln Gln Lys Glu 495 5 - #00 5 - #05 5 -#10 - - ATG GAC CGA AAA GGA TTA CTT GGG TAT TAT TT - #C AAA GGA AAA GATTTT 192 Met Asp Arg Lys Gly Leu Leu Gly Tyr Tyr Ph - #e Lys Gly Lys Asp Phe 515 - # 520 - # 525 - - AGT AAT CTT ACT ATG TTT GCA CCG ACA CGT GA - #T AGT ACT CTT ATT TAT 240 Ser Asn Leu Thr Met Phe Ala Pro Thr Arg As - #p Ser Thr Leu Ile Tyr 530 - # 535 - # 540 - - GAT CAA CAA ACA GCA AAT AAA CTA TTA GAT AA - #A AAA CAA CAA GAA TAT 288 Asp Gln Gln Thr Ala Asn Lys Leu Leu Asp Ly - #s Lys Gln Gln Glu Tyr 545 - # 550 - # 555 - - CAG TCT ATT CGT TGG ATT GGT TTG ATT CAG AG - #T AAA GAA ACG GGA GAT 336 Gln Ser Ile Arg Trp Ile Gly Leu Ile Gln Se - #r Lys Glu Thr Gly Asp 560 - # 565 - # 570 - - TTC ACA TTT AAC TTA TCT GAG GAT GAA CAG GC - #A ATT ATA GAA ATC AAT 384 Phe Thr Phe Asn Leu Ser Glu Asp Glu Gln Al - #a Ile Ile Glu Ile Asn 575 5 - #80 5 - #85 5 -#90 - - GGG AAA ATT ATT TCT AAT AAA GGG AAA GAA AA - #G CAA GTT GTC CATTTA 432 Gly Lys Ile Ile Ser Asn Lys Gly Lys Glu Ly - #s Gln Val Val His Leu 595 - # 600 - # 605 - - GAA AAA GGA AAA TTA GTT CCA ATC AAA ATA GA - #G TAT CAA TCA GAT ACA 480 Glu Lys Gly Lys Leu Val Pro Ile Lys Ile Gl - #u Tyr Gln Ser Asp Thr 610 - # 615 - # 620 - - AAA TTT AAT ATT GAC AGT AAA ACA TTT AAA GA - #A CTT AAA TTA TTT AAA 528 Lys Phe Asn Ile Asp Ser Lys Thr Phe Lys Gl - #u Leu Lys Leu Phe Lys 625 - # 630 - # 635 - - ATA GAT AGT CAA AAC CAA CCC CAG CAA GTC CA - #G CAA GAT GAA CTG AGA 576 Ile Asp Ser Gln Asn Gln Pro Gln Gln Val Gl - #n Gln Asp Glu Leu Arg 640 - # 645 - # 650 - - AAT CCT GAA TTT AAC AAG AAA GAA TCA CAG GA - #A TTC TTA GCG AAA CCA 624 Asn Pro Glu Phe Asn Lys Lys Glu Ser Gln Gl - #u Phe Leu Ala Lys Pro 655 6 - #60 6 - #65 6 -#70 - - TCG AAA ATA AAT CTT TTC ACT CAA AAA ATG AA - #A AGG GAA ATT GATGAA 672 Ser Lys Ile Asn Leu Phe Thr Gln Lys Met Ly - #s Arg Glu Ile Asp Glu 675 - # 680 - # 685 - - GAC ACG GAT ACG GAT GGG GAC TCT ATT CCT GA - #C CTT TGG GAA GAA AAT 720 Asp Thr Asp Thr Asp Gly Asp Ser Ile Pro As - #p Leu Trp Glu Glu Asn 690 - # 695 - # 700 - - GGG TAT ACG ATT CAA AAT AGA ATC GCT GTA AA - #G TGG GAC GAT TCT CTA 768 Gly Tyr Thr Ile Gln Asn Arg Ile Ala Val Ly - #s Trp Asp Asp Ser Leu 705 - # 710 - # 715 - - GCA AGT AAA GGG TAT ACG AAA TTT GTT TCA AA - #T CCA CTA GAA AGT CAC 816 Ala Ser Lys Gly Tyr Thr Lys Phe Val Ser As - #n Pro Leu Glu Ser His 720 - # 725 - # 730 - - ACA GTT GGT GAT CCT TAT ACA GAT TAT GAA AA - #G GCA GCA AGA GAT CTA 864 Thr Val Gly Asp Pro Tyr Thr Asp Tyr Glu Ly - #s Ala Ala Arg Asp Leu 735 7 - #40 7 - #45 7 -#50 - - GAT TTG TCA AAT GCA AAG GAA ACG TTT AAC CC - #A TTG GTA GCT GCTTTT 912 Asp Leu Ser Asn Ala Lys Glu Thr Phe Asn Pr - #o Leu Val Ala Ala Phe 755 - # 760 - # 765 - - CCA AGT GTG AAT GTT AGT ATG GAA AAG GTG AT - #A TTA TCA CCA AAT GAA 960 Pro Ser Val Asn Val Ser Met Glu Lys Val Il - #e Leu Ser Pro Asn Glu 770 - # 775 - # 780 - - AAT TTA TCC AAT AGT GTA GAG TCT CAT TCA TC - #C ACG AAT TGG TCT TAT 1008 Asn Leu Ser Asn Ser Val Glu Ser His Ser Se - #r Thr Asn Trp Ser Tyr 785 - # 790 - # 795 - - ACA AAT ACA GAA GGT GCT TCT GTT GAA GCG GG - #G ATT GGA CCA AAA GGT 1056 Thr Asn Thr Glu Gly Ala Ser Val Glu Ala Gl - #y Ile Gly Pro Lys Gly 800 - # 805 - # 810 - - ATT TCG TTC GGA GTT AGC GTA AAC TAT CAA CA - #C TCT GAA ACA GTT GCA 1104 Ile Ser Phe Gly Val Ser Val Asn Tyr Gln Hi - #s Ser Glu Thr Val Ala 815 8 - #20 8 - #25 8 -#30 - - CAA GAA TGG GGA ACA TCT ACA GGA AAT ACT TC - #G CAA TTC AAT ACGGCT 1152 Gln Glu Trp Gly Thr Ser Thr Gly Asn Thr Se - #r Gln Phe Asn Thr Ala 835 - # 840 - # 845 - - TCA GCG GGA TAT TTA AAT GCA AAT GTT CGA TA - #T AAC AAT GTA GGA ACT 1200 Ser Ala Gly Tyr Leu Asn Ala Asn Val Arg Ty - #r Asn Asn Val Gly Thr 850 - # 855 - # 860 - - GGT GCC ATC TAC GAT GTA AAA CCT ACA ACA AG - #T TTT GTA TTA AAT AAC 1248 Gly Ala Ile Tyr Asp Val Lys Pro Thr Thr Se - #r Phe Val Leu Asn Asn 865 - # 870 - # 875 - - GAT ACT ATC GCA ACT ATT ACG GCG AAA TCT AA - #T TCT ACA GCC TTA AAT 1296 Asp Thr Ile Ala Thr Ile Thr Ala Lys Ser As - #n Ser Thr Ala Leu Asn 880 - # 885 - # 890 - - ATA TCT CCT GGA GAA AGT TAC CCG AAA AAA GG - #A CAA AAT GGA ATC GCA 1344 Ile Ser Pro Gly Glu Ser Tyr Pro Lys Lys Gl - #y Gln Asn Gly Ile Ala 895 9 - #00 9 - #05 9 -#10 - - ATA ACA TCA ATG GAT GAT TTT AAT TCC CAT CC - #G ATT ACA TTA AATAAA 1392 Ile Thr Ser Met Asp Asp Phe Asn Ser His Pr - #o Ile Thr Leu Asn Lys 915 - # 920 - # 925 - - AAA CAA GTA GAT AAT CTG CTA AAT AAT AAA CC - #T ATG ATG TTG GAA ACA 1440 Lys Gln Val Asp Asn Leu Leu Asn Asn Lys Pr - #o Met Met Leu Glu Thr 930 - # 935 - # 940 - - AAC CAA ACA GAT GGT GTT TAT AAG ATA AAA GA - #T ACA CAT GGA AAT ATA 1488 Asn Gln Thr Asp Gly Val Tyr Lys Ile Lys As - #p Thr His Gly Asn Ile 945 - # 950 - # 955 - - GTA ACT GGC GGA GAA TGG AAT GGT GTC ATA CA - #A CAA ATC AAG GCT AAA 1536 Val Thr Gly Gly Glu Trp Asn Gly Val Ile Gl - #n Gln Ile Lys Ala Lys 960 - # 965 - # 970 - - ACA GCG TCT ATT ATT GTG GAT GAT GGG GAA CG - #T GTA GCA GAA AAA CGT 1584 Thr Ala Ser Ile Ile Val Asp Asp Gly Glu Ar - #g Val Ala Glu Lys Arg 975 9 - #80 9 - #85 9 -#90 - - GTA GCG GCA AAA GAT TAT GAA AAT CCA GAA GA - #T AAA ACA CCG TCTTTA 1632 Val Ala Ala Lys Asp Tyr Glu Asn Pro Glu As - #p Lys Thr Pro Ser Leu 995 - # 1000 - # 1005 - - ACT TTA AAA GAT GCC CTG AAG CTT TCA TAT CC - #A GAT GAA ATA AAA GAA 1680 Thr Leu Lys Asp Ala Leu Lys Leu Ser Tyr Pr - #o Asp Glu Ile Lys Glu 1010 - # 1015 - # 1020 - - ATA GAG GGA TTA TTA TAT TAT AAA AAC AAA CC - #G ATA TAC GAA TCG AGC 1728 Ile Glu Gly Leu Leu Tyr Tyr Lys Asn Lys Pr - #o Ile Tyr Glu Ser Ser 1025 - # 1030 - # 1035 - - GTT ATG ACT TAC TTA GAT GAA AAT ACA GCA AA - #A GAA GTG ACC AAA CAA 1776 Val Met Thr Tyr Leu Asp Glu Asn Thr Ala Ly - #s Glu Val Thr Lys Gln 1040 - # 1045 - # 1050 - - TTA AAT GAT ACC ACT GGG AAA TTT AAA GAT GT - #A AGT CAT TTA TAT GAT 1824 Leu Asn Asp Thr Thr Gly Lys Phe Lys Asp Va - #l Ser His Leu Tyr Asp 1055 1060 - # 1065 - # 1070 - - GTA AAA CTG ACT CCA AAA ATG AAT GTT ACA AT - #C AAA TTG TCT ATA CTT 1872 Val Lys Leu Thr Pro Lys Met Asn Val Thr Il - #e Lys Leu Ser Ile Leu 1075 - # 1080 - # 1085 - - TAT GAT AAT GCT GAG TCT AAT GAT AAC TCA AT - #T GGT AAA TGG ACA AAC 1920 Tyr Asp Asn Ala Glu Ser Asn Asp Asn Ser Il - #e Gly Lys Trp Thr Asn 1090 - # 1095 - # 1100 - - ACA AAT ATT GTT TCA GGT GGA AAT AAC GGA AA - #A AAA CAA TAT TCT TCT 1968 Thr Asn Ile Val Ser Gly Gly Asn Asn Gly Ly - #s Lys Gln Tyr Ser Ser 1105 - # 1110 - # 1115 - - AAT AAT CCG GAT GCT AAT TTG ACA TTA AAT AC - #A GAT GCT CAA GAA AAA 2016 Asn Asn Pro Asp Ala Asn Leu Thr Leu Asn Th - #r Asp Ala Gln Glu Lys 1120 - # 1125 - # 1130 - - TTA AAT AAA AAT CGT GAC TAT TAT ATA AGT TT - #A TAT ATG AAG TCA GAA 2064 Leu Asn Lys Asn Arg Asp Tyr Tyr Ile Ser Le - #u Tyr Met Lys Ser Glu 1135 1140 - # 1145 - # 1150 - - AAA AAC ACA CAA TGT GAG ATT ACT ATA GAT GG - #G GAG ATT TAT CCG ATC 2112 Lys Asn Thr Gln Cys Glu Ile Thr Ile Asp Gl - #y Glu Ile Tyr Pro Ile 1155 - # 1160 - # 1165 - - ACT ACA AAA ACA GTG AAT GTG AAT AAA GAC AA - #T TAC AAA AGA TTA GAT 2160 Thr Thr Lys Thr Val Asn Val Asn Lys Asp As - #n Tyr Lys Arg Leu Asp 1170 - # 1175 - # 1180 - - ATT ATA GCT CAT AAT ATA AAA AGT AAT CCA AT - #T TCT TCA CTT CAT ATT 2208 Ile Ile Ala His Asn Ile Lys Ser Asn Pro Il - #e Ser Ser Leu His Ile 1185 - # 1190 - # 1195 - - AAA ACG AAT GAT GAA ATA ACT TTA TTT TGG GA - #T GAT ATT TCT ATA ACA 2256 Lys Thr Asn Asp Glu Ile Thr Leu Phe Trp As - #p Asp Ile Ser Ile Thr 1200 - # 1205 - # 1210 - - GAT GTA GCA TCA ATA AAA CCG GAA AAT TTA AC - #A GAT TCA GAA ATT AAA 2304 Asp Val Ala Ser Ile Lys Pro Glu Asn Leu Th - #r Asp Ser Glu Ile Lys 1215 1220 - # 1225 - # 1230 - - CAG ATT TAT AGT AGG TAT GGT ATT AAG TTA GA - #A GAT GGA ATC CTT ATT 2352 Gln Ile Tyr Ser Arg Tyr Gly Ile Lys Leu Gl - #u Asp Gly Ile Leu Ile 1235 - # 1240 - # 1245 - - GAT AAA AAA GGT GGG ATT CAT TAT GGT GAA TT - #T ATT AAT GAA GCT AGT 2400 Asp Lys Lys Gly Gly Ile His Tyr Gly Glu Ph - #e Ile Asn Glu Ala Ser 1250 - # 1255 - # 1260 - - TTT AAT ATT GAA CCA TTG CAA AAT TAT GTG AC - #C AAA TAT GAA GTT ACT 2448 Phe Asn Ile Glu Pro Leu Gln Asn Tyr Val Th - #r Lys Tyr Glu Val Thr 1265 - # 1270 - # 1275 - - TAT AGT AGT GAG TTA GGA CCA AAC GTG AGT GA - #C ACA CTT GAA AGT GAT 2496 Tyr Ser Ser Glu Leu Gly Pro Asn Val Ser As - #p Thr Leu Glu Ser Asp 1280 - # 1285 - # 1290 - - AAA ATT TAC AAG GAT GGG ACA ATT AAA TTT GA - #T TTT ACC AAA TAT AGT 2544 Lys Ile Tyr Lys Asp Gly Thr Ile Lys Phe As - #p Phe Thr Lys Tyr Ser 1295 1300 - # 1305 - # 1310 - - AAA AAT GAA CAA GGA TTA TTT TAT GAC AGT GG - #A TTA AAT TGG GAC TTT 2592 Lys Asn Glu Gln Gly Leu Phe Tyr Asp Ser Gl - #y Leu Asn Trp Asp Phe 1315 - # 1320 - # 1325 - - AAA ATT AAT GCT ATT ACT TAT GAT GGT AAA GA - #G ATG AAT GTT TTT CAT 2640 Lys Ile Asn Ala Ile Thr Tyr Asp Gly Lys Gl - #u Met Asn Val Phe His 1330 - # 1335 - # 1340 - - AGA TAT AAT AAA TAG - # - # - # 2655 Arg Tyr Asn Lys 1345 - - - - (2) INFORMATION FOR SEQ ID NO:5: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 884 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: - - Met Lys Asn Met Lys Lys Lys Leu Ala Ser Va - #l Val Thr Cys Thr Leu 1 5 - # 10 - # 15 - - Leu Ala Pro Met Phe Leu Asn Gly Asn Val As - #n Ala Val Tyr Ala Asp 20 - # 25 - # 30 - - Ser Lys Thr Asn Gln Ile Ser Thr Thr Gln Ly - #s Asn Gln Gln Lys Glu 35 - # 40 - # 45 - - Met Asp Arg Lys Gly Leu Leu Gly Tyr Tyr Ph - #e Lys Gly Lys Asp Phe 50 - # 55 - # 60 - - Ser Asn Leu Thr Met Phe Ala Pro Thr Arg As - #p Ser Thr Leu Ile Tyr 65 - # 70 - # 75 - # 80 - - Asp Gln Gln Thr Ala Asn Lys Leu Leu Asp Ly - #s Lys Gln Gln Glu Tyr 85 - # 90 - # 95 - - Gln Ser Ile Arg Trp Ile Gly Leu Ile Gln Se - #r Lys Glu Thr Gly Asp 100 - # 105 - # 110 - - Phe Thr Phe Asn Leu Ser Glu Asp Glu Gln Al - #a Ile Ile Glu Ile Asn 115 - # 120 - # 125 - - Gly Lys Ile Ile Ser Asn Lys Gly Lys Glu Ly - #s Gln Val Val His Leu 130 - # 135 - # 140 - - Glu Lys Gly Lys Leu Val Pro Ile Lys Ile Gl - #u Tyr Gln Ser Asp Thr 145 1 - #50 1 - #55 1 -#60 - - Lys Phe Asn Ile Asp Ser Lys Thr Phe Lys Gl - #u Leu Lys Leu PheLys 165 - # 170 - # 175 - - Ile Asp Ser Gln Asn Gln Pro Gln Gln Val Gl - #n Gln Asp Glu Leu Arg 180 - # 185 - # 190 - - Asn Pro Glu Phe Asn Lys Lys Glu Ser Gln Gl - #u Phe Leu Ala Lys Pro 195 - # 200 - # 205 - - Ser Lys Ile Asn Leu Phe Thr Gln Lys Met Ly - #s Arg Glu Ile Asp Glu 210 - # 215 - # 220 - - Asp Thr Asp Thr Asp Gly Asp Ser Ile Pro As - #p Leu Trp Glu Glu Asn 225 2 - #30 2 - #35 2 -#40 - - Gly Tyr Thr Ile Gln Asn Arg Ile Ala Val Ly - #s Trp Asp Asp SerLeu 245 - # 250 - # 255 - - Ala Ser Lys Gly Tyr Thr Lys Phe Val Ser As - #n Pro Leu Glu Ser His 260 - # 265 - # 270 - - Thr Val Gly Asp Pro Tyr Thr Asp Tyr Glu Ly - #s Ala Ala Arg Asp Leu 275 - # 280 - # 285 - - Asp Leu Ser Asn Ala Lys Glu Thr Phe Asn Pr - #o Leu Val Ala Ala Phe 290 - # 295 - # 300 - - Pro Ser Val Asn Val Ser Met Glu Lys Val Il - #e Leu Ser Pro Asn Glu 305 3 - #10 3 - #15 3 -#20 - - Asn Leu Ser Asn Ser Val Glu Ser His Ser Se - #r Thr Asn Trp SerTyr 325 - # 330 - # 335 - - Thr Asn Thr Glu Gly Ala Ser Val Glu Ala Gl - #y Ile Gly Pro Lys Gly 340 - # 345 - # 350 - - Ile Ser Phe Gly Val Ser Val Asn Tyr Gln Hi - #s Ser Glu Thr Val Ala 355 - # 360 - # 365 - - Gln Glu Trp Gly Thr Ser Thr Gly Asn Thr Se - #r Gln Phe Asn Thr Ala 370 - # 375 - # 380 - - Ser Ala Gly Tyr Leu Asn Ala Asn Val Arg Ty - #r Asn Asn Val Gly Thr 385 3 - #90 3 - #95 4 -#00 - - Gly Ala Ile Tyr Asp Val Lys Pro Thr Thr Se - #r Phe Val Leu AsnAsn 405 - # 410 - # 415 - - Asp Thr Ile Ala Thr Ile Thr Ala Lys Ser As - #n Ser Thr Ala Leu Asn 420 - # 425 - # 430 - - Ile Ser Pro Gly Glu Ser Tyr Pro Lys Lys Gl - #y Gln Asn Gly Ile Ala 435 - # 440 - # 445 - - Ile Thr Ser Met Asp Asp Phe Asn Ser His Pr - #o Ile Thr Leu Asn Lys 450 - # 455 - # 460 - - Lys Gln Val Asp Asn Leu Leu Asn Asn Lys Pr - #o Met Met Leu Glu Thr 465 4 - #70 4 - #75 4 -#80 - - Asn Gln Thr Asp Gly Val Tyr Lys Ile Lys As - #p Thr His Gly AsnIle 485 - # 490 - # 495 - - Val Thr Gly Gly Glu Trp Asn Gly Val Ile Gl - #n Gln Ile Lys Ala Lys 500 - # 505 - # 510 - - Thr Ala Ser Ile Ile Val Asp Asp Gly Glu Ar - #g Val Ala Glu Lys Arg 515 - # 520 - # 525 - - Val Ala Ala Lys Asp Tyr Glu Asn Pro Glu As - #p Lys Thr Pro Ser Leu 530 - # 535 - # 540 - - Thr Leu Lys Asp Ala Leu Lys Leu Ser Tyr Pr - #o Asp Glu Ile Lys Glu 545 5 - #50 5 - #55 5 -#60 - - Ile Glu Gly Leu Leu Tyr Tyr Lys Asn Lys Pr - #o Ile Tyr Glu SerSer 565 - # 570 - # 575 - - Val Met Thr Tyr Leu Asp Glu Asn Thr Ala Ly - #s Glu Val Thr Lys Gln 580 - # 585 - # 590 - - Leu Asn Asp Thr Thr Gly Lys Phe Lys Asp Va - #l Ser His Leu Tyr Asp 595 - # 600 - # 605 - - Val Lys Leu Thr Pro Lys Met Asn Val Thr Il - #e Lys Leu Ser Ile Leu 610 - # 615 - # 620 - - Tyr Asp Asn Ala Glu Ser Asn Asp Asn Ser Il - #e Gly Lys Trp Thr Asn 625 6 - #30 6 - #35 6 -#40 - - Thr Asn Ile Val Ser Gly Gly Asn Asn Gly Ly - #s Lys Gln Tyr SerSer 645 - # 650 - # 655 - - Asn Asn Pro Asp Ala Asn Leu Thr Leu Asn Th - #r Asp Ala Gln Glu Lys 660 - # 665 - # 670 - - Leu Asn Lys Asn Arg Asp Tyr Tyr Ile Ser Le - #u Tyr Met Lys Ser Glu 675 - # 680 - # 685 - - Lys Asn Thr Gln Cys Glu Ile Thr Ile Asp Gl - #y Glu Ile Tyr Pro Ile 690 - # 695 - # 700 - - Thr Thr Lys Thr Val Asn Val Asn Lys Asp As - #n Tyr Lys Arg Leu Asp 705 7 - #10 7 - #15 7 -#20 - - Ile Ile Ala His Asn Ile Lys Ser Asn Pro Il - #e Ser Ser Leu HisIle 725 - # 730 - # 735 - - Lys Thr Asn Asp Glu Ile Thr Leu Phe Trp As - #p Asp Ile Ser Ile Thr 740 - # 745 - # 750 - - Asp Val Ala Ser Ile Lys Pro Glu Asn Leu Th - #r Asp Ser Glu Ile Lys 755 - # 760 - # 765 - - Gln Ile Tyr Ser Arg Tyr Gly Ile Lys Leu Gl - #u Asp Gly Ile Leu Ile 770 - # 775 - # 780 - - Asp Lys Lys Gly Gly Ile His Tyr Gly Glu Ph - #e Ile Asn Glu Ala Ser 785 7 - #90 7 - #95 8 -#00 - - Phe Asn Ile Glu Pro Leu Gln Asn Tyr Val Th - #r Lys Tyr Glu ValThr 805 - # 810 - # 815 - - Tyr Ser Ser Glu Leu Gly Pro Asn Val Ser As - #p Thr Leu Glu Ser Asp 820 - # 825 - # 830 - - Lys Ile Tyr Lys Asp Gly Thr Ile Lys Phe As - #p Phe Thr Lys Tyr Ser 835 - # 840 - # 845 - - Lys Asn Glu Gln Gly Leu Phe Tyr Asp Ser Gl - #y Leu Asn Trp Asp Phe 850 - # 855 - # 860 - - Lys Ile Asn Ala Ile Thr Tyr Asp Gly Lys Gl - #u Met Asn Val Phe His 865 8 - #70 8 - #75 8 -#80 - - Arg Tyr Asn Lys - - - - (2) INFORMATION FOR SEQ ID NO:6: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2004 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus - #cereus (B) STRAIN: AB78 (C) INDIVIDUAL ISOLATE: - #NRRL B-21058 - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..2001 (D) OTHER INFORMATION: - #/product= "80 kDa protein VIP1A(a)" /note= - #"This sequence is identical to that found in SEQ ID - #NO:1 between and including nucleotide positions 3126 and - #5126" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: - - ATG AAA AGG GAA ATT GAT GAA GAC ACG GAT AC - #G GAT GGG GAC TCT ATT 48 Met Lys Arg Glu Ile Asp Glu Asp Thr Asp Th - #r Asp Gly Asp Ser Ile 885 8 - #90 8 - #95 9 -#00 - - CCT GAC CTT TGG GAA GAA AAT GGG TAT ACG AT - #T CAA AAT AGA ATCGCT 96 Pro Asp Leu Trp Glu Glu Asn Gly Tyr Thr Il - #e Gln Asn Arg Ile Ala 905 - # 910 - # 915 - - GTA AAG TGG GAC GAT TCT CTA GCA AGT AAA GG - #G TAT ACG AAA TTT GTT 144 Val Lys Trp Asp Asp Ser Leu Ala Ser Lys Gl - #y Tyr Thr Lys Phe Val 920 - # 925 - # 930 - - TCA AAT CCA CTA GAA AGT CAC ACA GTT GGT GA - #T CCT TAT ACA GAT TAT 192 Ser Asn Pro Leu Glu Ser His Thr Val Gly As - #p Pro Tyr Thr Asp Tyr 935 - # 940 - # 945 - - GAA AAG GCA GCA AGA GAT CTA GAT TTG TCA AA - #T GCA AAG GAA ACG TTT 240 Glu Lys Ala Ala Arg Asp Leu Asp Leu Ser As - #n Ala Lys Glu Thr Phe 950 - # 955 - # 960 - - AAC CCA TTG GTA GCT GCT TTT CCA AGT GTG AA - #T GTT AGT ATG GAA AAG 288 Asn Pro Leu Val Ala Ala Phe Pro Ser Val As - #n Val Ser Met Glu Lys 965 9 - #70 9 - #75 9 -#80 - - GTG ATA TTA TCA CCA AAT GAA AAT TTA TCC AA - #T AGT GTA GAG TCTCAT 336 Val Ile Leu Ser Pro Asn Glu Asn Leu Ser As - #n Ser Val Glu Ser His 985 - # 990 - # 995 - - TCA TCC ACG AAT TGG TCT TAT ACA AAT ACA GA - #A GGT GCT TCT GTT GAA 384 Ser Ser Thr Asn Trp Ser Tyr Thr Asn Thr Gl - #u Gly Ala Ser Val Glu 1000 - # 1005 - # 1010 - - GCG GGG ATT GGA CCA AAA GGT ATT TCG TTC GG - #A GTT AGC GTA AAC TAT 432 Ala Gly Ile Gly Pro Lys Gly Ile Ser Phe Gl - #y Val Ser Val Asn Tyr 1015 - # 1020 - # 1025 - - CAA CAC TCT GAA ACA GTT GCA CAA GAA TGG GG - #A ACA TCT ACA GGA AAT 480 Gln His Ser Glu Thr Val Ala Gln Glu Trp Gl - #y Thr Ser Thr Gly Asn 1030 - # 1035 - # 1040 - - ACT TCG CAA TTC AAT ACG GCT TCA GCG GGA TA - #T TTA AAT GCA AAT GTT 528 Thr Ser Gln Phe Asn Thr Ala Ser Ala Gly Ty - #r Leu Asn Ala Asn Val 1045 1050 - # 1055 - # 1060 - - CGA TAT AAC AAT GTA GGA ACT GGT GCC ATC TA - #C GAT GTA AAA CCT ACA 576 Arg Tyr Asn Asn Val Gly Thr Gly Ala Ile Ty - #r Asp Val Lys Pro Thr 1065 - # 1070 - # 1075 - - ACA AGT TTT GTA TTA AAT AAC GAT ACT ATC GC - #A ACT ATT ACG GCG AAA 624 Thr Ser Phe Val Leu Asn Asn Asp Thr Ile Al - #a Thr Ile Thr Ala Lys 1080 - # 1085 - # 1090 - - TCT AAT TCT ACA GCC TTA AAT ATA TCT CCT GG - #A GAA AGT TAC CCG AAA 672 Ser Asn Ser Thr Ala Leu Asn Ile Ser Pro Gl - #y Glu Ser Tyr Pro Lys 1095 - # 1100 - # 1105 - - AAA GGA CAA AAT GGA ATC GCA ATA ACA TCA AT - #G GAT GAT TTT AAT TCC 720 Lys Gly Gln Asn Gly Ile Ala Ile Thr Ser Me - #t Asp Asp Phe Asn Ser 1110 - # 1115 - # 1120 - - CAT CCG ATT ACA TTA AAT AAA AAA CAA GTA GA - #T AAT CTG CTA AAT AAT 768 His Pro Ile Thr Leu Asn Lys Lys Gln Val As - #p Asn Leu Leu Asn Asn 1125 1130 - # 1135 - # 1140 - - AAA CCT ATG ATG TTG GAA ACA AAC CAA ACA GA - #T GGT GTT TAT AAG ATA 816 Lys Pro Met Met Leu Glu Thr Asn Gln Thr As - #p Gly Val Tyr Lys Ile 1145 - # 1150 - # 1155 - - AAA GAT ACA CAT GGA AAT ATA GTA ACT GGC GG - #A GAA TGG AAT GGT GTC 864 Lys Asp Thr His Gly Asn Ile Val Thr Gly Gl - #y Glu Trp Asn Gly Val 1160 - # 1165 - # 1170 - - ATA CAA CAA ATC AAG GCT AAA ACA GCG TCT AT - #T ATT GTG GAT GAT GGG 912 Ile Gln Gln Ile Lys Ala Lys Thr Ala Ser Il - #e Ile Val Asp Asp Gly 1175 - # 1180 - # 1185 - - GAA CGT GTA GCA GAA AAA CGT GTA GCG GCA AA - #A GAT TAT GAA AAT CCA 960 Glu Arg Val Ala Glu Lys Arg Val Ala Ala Ly - #s Asp Tyr Glu Asn Pro 1190 - # 1195 - # 1200 - - GAA GAT AAA ACA CCG TCT TTA ACT TTA AAA GA - #T GCC CTG AAG CTT TCA 1008 Glu Asp Lys Thr Pro Ser Leu Thr Leu Lys As - #p Ala Leu Lys Leu Ser 1205 1210 - # 1215 - # 1220 - - TAT CCA GAT GAA ATA AAA GAA ATA GAG GGA TT - #A TTA TAT TAT AAA AAC 1056 Tyr Pro Asp Glu Ile Lys Glu Ile Glu Gly Le - #u Leu Tyr Tyr Lys Asn 1225 - # 1230 - # 1235 - - AAA CCG ATA TAC GAA TCG AGC GTT ATG ACT TA - #C TTA GAT GAA AAT ACA 1104 Lys Pro Ile Tyr Glu Ser Ser Val Met Thr Ty - #r Leu Asp Glu Asn Thr 1240 - # 1245 - # 1250 - - GCA AAA GAA GTG ACC AAA CAA TTA AAT GAT AC - #C ACT GGG AAA TTT AAA 1152 Ala Lys Glu Val Thr Lys Gln Leu Asn Asp Th - #r Thr Gly Lys Phe Lys 1255 - # 1260 - # 1265 - - GAT GTA AGT CAT TTA TAT GAT GTA AAA CTG AC - #T CCA AAA ATG AAT GTT 1200 Asp Val Ser His Leu Tyr Asp Val Lys Leu Th - #r Pro Lys Met Asn Val 1270 - # 1275 - # 1280 - - ACA ATC AAA TTG TCT ATA CTT TAT GAT AAT GC - #T GAG TCT AAT GAT AAC 1248 Thr Ile Lys Leu Ser Ile Leu Tyr Asp Asn Al - #a Glu Ser Asn Asp Asn 1285 1290 - # 1295 - # 1300 - - TCA ATT GGT AAA TGG ACA AAC ACA AAT ATT GT - #T TCA GGT GGA AAT AAC 1296 Ser Ile Gly Lys Trp Thr Asn Thr Asn Ile Va - #l Ser Gly Gly Asn Asn 1305 - # 1310 - # 1315 - - GGA AAA AAA CAA TAT TCT TCT AAT AAT CCG GA - #T GCT AAT TTG ACA TTA 1344 Gly Lys Lys Gln Tyr Ser Ser Asn Asn Pro As - #p Ala Asn Leu Thr Leu 1320 - # 1325 - # 1330 - - AAT ACA GAT GCT CAA GAA AAA TTA AAT AAA AA - #T CGT GAC TAT TAT ATA 1392 Asn Thr Asp Ala Gln Glu Lys Leu Asn Lys As - #n Arg Asp Tyr Tyr Ile 1335 - # 1340 - # 1345 - - AGT TTA TAT ATG AAG TCA GAA AAA AAC ACA CA - #A TGT GAG ATT ACT ATA 1440 Ser Leu Tyr Met Lys Ser Glu Lys Asn Thr Gl - #n Cys Glu Ile Thr Ile 1350 - # 1355 - # 1360 - - GAT GGG GAG ATT TAT CCG ATC ACT ACA AAA AC - #A GTG AAT GTG AAT AAA 1488 Asp Gly Glu Ile Tyr Pro Ile Thr Thr Lys Th - #r Val Asn Val Asn Lys 1365 1370 - # 1375 - # 1380 - - GAC AAT TAC AAA AGA TTA GAT ATT ATA GCT CA - #T AAT ATA AAA AGT AAT 1536 Asp Asn Tyr Lys Arg Leu Asp Ile Ile Ala Hi - #s Asn Ile Lys Ser Asn 1385 - # 1390 - # 1395 - - CCA ATT TCT TCA CTT CAT ATT AAA ACG AAT GA - #T GAA ATA ACT TTA TTT 1584 Pro Ile Ser Ser Leu His Ile Lys Thr Asn As - #p Glu Ile Thr Leu Phe 1400 - # 1405 - # 1410 - - TGG GAT GAT ATT TCT ATA ACA GAT GTA GCA TC - #A ATA AAA CCG GAA AAT 1632 Trp Asp Asp Ile Ser Ile Thr Asp Val Ala Se - #r Ile Lys Pro Glu Asn 1415 - # 1420 - # 1425 - - TTA ACA GAT TCA GAA ATT AAA CAG ATT TAT AG - #T AGG TAT GGT ATT AAG 1680 Leu Thr Asp Ser Glu Ile Lys Gln Ile Tyr Se - #r Arg Tyr Gly Ile Lys 1430 - # 1435 - # 1440 - - TTA GAA GAT GGA ATC CTT ATT GAT AAA AAA GG - #T GGG ATT CAT TAT GGT 1728 Leu Glu Asp Gly Ile Leu Ile Asp Lys Lys Gl - #y Gly Ile His Tyr Gly 1445 1450 - # 1455 - # 1460 - - GAA TTT ATT AAT GAA GCT AGT TTT AAT ATT GA - #A CCA TTG CCA AAT TAT 1776 Glu Phe Ile Asn Glu Ala Ser Phe Asn Ile Gl - #u Pro Leu Pro Asn Tyr 1465 - # 1470 - # 1475 - - GTG ACC AAA TAT GAA GTT ACT TAT AGT AGT GA - #G TTA GGA CCA AAC GTG 1824 Val Thr Lys Tyr Glu Val Thr Tyr Ser Ser Gl - #u Leu Gly Pro Asn Val 1480 - # 1485 - # 1490 - - AGT GAC ACA CTT GAA AGT GAT AAA ATT TAC AA - #G GAT GGG ACA ATT AAA 1872 Ser Asp Thr Leu Glu Ser Asp Lys Ile Tyr Ly - #s Asp Gly Thr Ile Lys 1495 - # 1500 - # 1505 - - TTT GAT TTT ACC AAA TAT AGT AAA AAT GAA CA - #A GGA TTA TTT TAT GAC 1920 Phe Asp Phe Thr Lys Tyr Ser Lys Asn Glu Gl - #n Gly Leu Phe Tyr Asp 1510 - # 1515 - # 1520 - - AGT GGA TTA AAT TGG GAC TTT AAA ATT AAT GC - #T ATT ACT TAT GAT GGT 1968 Ser Gly Leu Asn Trp Asp Phe Lys Ile Asn Al - #a Ile Thr Tyr Asp Gly 1525 1530 - # 1535 - # 1540 - - AAA GAG ATG AAT GTT TTT CAT AGA TAT AAT AA - #A TAG- # 2004 Lys Glu Met Asn Val Phe His Arg Tyr Asn Ly - #s 1545 - # 1550 - - - - (2) INFORMATION FOR SEQ ID NO:7: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 667 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: - - Met Lys Arg Glu Ile Asp Glu Asp Thr Asp Th - #r Asp Gly Asp SerIle 1 5 - # 10 - # 15 - - Pro Asp Leu Trp Glu Glu Asn Gly Tyr Thr Il - #e Gln Asn Arg Ile Ala 20 - # 25 - # 30 - - Val Lys Trp Asp Asp Ser Leu Ala Ser Lys Gl - #y Tyr Thr Lys Phe Val 35 - # 40 - # 45 - - Ser Asn Pro Leu Glu Ser His Thr Val Gly As - #p Pro Tyr Thr Asp Tyr 50 - # 55 - # 60 - - Glu Lys Ala Ala Arg Asp Leu Asp Leu Ser As - #n Ala Lys Glu Thr Phe 65 - # 70 - # 75 - # 80 - - Asn Pro Leu Val Ala Ala Phe Pro Ser Val As - #n Val Ser Met Glu Lys 85 - # 90 - # 95 - - Val Ile Leu Ser Pro Asn Glu Asn Leu Ser As - #n Ser Val Glu Ser His 100 - # 105 - # 110 - - Ser Ser Thr Asn Trp Ser Tyr Thr Asn Thr Gl - #u Gly Ala Ser Val Glu 115 - # 120 - # 125 - - Ala Gly Ile Gly Pro Lys Gly Ile Ser Phe Gl - #y Val Ser Val Asn Tyr 130 - # 135 - # 140 - - Gln His Ser Glu Thr Val Ala Gln Glu Trp Gl - #y Thr Ser Thr Gly Asn 145 1 - #50 1 - #55 1 -#60 - - Thr Ser Gln Phe Asn Thr Ala Ser Ala Gly Ty - #r Leu Asn Ala AsnVal 165 - # 170 - # 175 - - Arg Tyr Asn Asn Val Gly Thr Gly Ala Ile Ty - #r Asp Val Lys Pro Thr 180 - # 185 - # 190 - - Thr Ser Phe Val Leu Asn Asn Asp Thr Ile Al - #a Thr Ile Thr Ala Lys 195 - # 200 - # 205 - - Ser Asn Ser Thr Ala Leu Asn Ile Ser Pro Gl - #y Glu Ser Tyr Pro Lys 210 - # 215 - # 220 - - Lys Gly Gln Asn Gly Ile Ala Ile Thr Ser Me - #t Asp Asp Phe Asn Ser 225 2 - #30 2 - #35 2 -#40 - - His Pro Ile Thr Leu Asn Lys Lys Gln Val As - #p Asn Leu Leu AsnAsn 245 - # 250 - # 255 - - Lys Pro Met Met Leu Glu Thr Asn Gln Thr As - #p Gly Val Tyr Lys Ile 260 - # 265 - # 270 - - Lys Asp Thr His Gly Asn Ile Val Thr Gly Gl - #y Glu Trp Asn Gly Val 275 - # 280 - # 285 - - Ile Gln Gln Ile Lys Ala Lys Thr Ala Ser Il - #e Ile Val Asp Asp Gly 290 - # 295 - # 300 - - Glu Arg Val Ala Glu Lys Arg Val Ala Ala Ly - #s Asp Tyr Glu Asn Pro 305 3 - #10 3 - #15 3 -#20 - - Glu Asp Lys Thr Pro Ser Leu Thr Leu Lys As - #p Ala Leu Lys LeuSer 325 - # 330 - # 335 - - Tyr Pro Asp Glu Ile Lys Glu Ile Glu Gly Le - #u Leu Tyr Tyr Lys Asn 340 - # 345 - # 350 - - Lys Pro Ile Tyr Glu Ser Ser Val Met Thr Ty - #r Leu Asp Glu Asn Thr 355 - # 360 - # 365 - - Ala Lys Glu Val Thr Lys Gln Leu Asn Asp Th - #r Thr Gly Lys Phe Lys 370 - # 375 - # 380 - - Asp Val Ser His Leu Tyr Asp Val Lys Leu Th - #r Pro Lys Met Asn Val 385 3 - #90 3 - #95 4 -#00 - - Thr Ile Lys Leu Ser Ile Leu Tyr Asp Asn Al - #a Glu Ser Asn AspAsn 405 - # 410 - # 415 - - Ser Ile Gly Lys Trp Thr Asn Thr Asn Ile Va - #l Ser Gly Gly Asn Asn 420 - # 425 - # 430 - - Gly Lys Lys Gln Tyr Ser Ser Asn Asn Pro As - #p Ala Asn Leu Thr Leu 435 - # 440 - # 445 - - Asn Thr Asp Ala Gln Glu Lys Leu Asn Lys As - #n Arg Asp Tyr Tyr Ile 450 - # 455 - # 460 - - Ser Leu Tyr Met Lys Ser Glu Lys Asn Thr Gl - #n Cys Glu Ile Thr Ile 465 4 - #70 4 - #75 4 -#80 - - Asp Gly Glu Ile Tyr Pro Ile Thr Thr Lys Th - #r Val Asn Val AsnLys 485 - # 490 - # 495 - - Asp Asn Tyr Lys Arg Leu Asp Ile Ile Ala Hi - #s Asn Ile Lys Ser Asn 500 - # 505 - # 510 - - Pro Ile Ser Ser Leu His Ile Lys Thr Asn As - #p Glu Ile Thr Leu Phe 515 - # 520 - # 525 - - Trp Asp Asp Ile Ser Ile Thr Asp Val Ala Se - #r Ile Lys Pro Glu Asn 530 - # 535 - # 540 - - Leu Thr Asp Ser Glu Ile Lys Gln Ile Tyr Se - #r Arg Tyr Gly Ile Lys 545 5 - #50 5 - #55 5 -#60 - - Leu Glu Asp Gly Ile Leu Ile Asp Lys Lys Gl - #y Gly Ile His TyrGly 565 - # 570 - # 575 - - Glu Phe Ile Asn Glu Ala Ser Phe Asn Ile Gl - #u Pro Leu Pro Asn Tyr 580 - # 585 - # 590 - - Val Thr Lys Tyr Glu Val Thr Tyr Ser Ser Gl - #u Leu Gly Pro Asn Val 595 - # 600 - # 605 - - Ser Asp Thr Leu Glu Ser Asp Lys Ile Tyr Ly - #s Asp Gly Thr Ile Lys 610 - # 615 - # 620 - - Phe Asp Phe Thr Lys Tyr Ser Lys Asn Glu Gl - #n Gly Leu Phe Tyr Asp 625 6 - #30 6 - #35 6 -#40 - - Ser Gly Leu Asn Trp Asp Phe Lys Ile Asn Al - #a Ile Thr Tyr AspGly 645 - # 650 - # 655 - - Lys Glu Met Asn Val Phe His Arg Tyr Asn Ly - #s 660 - # 665 - - - - (2) INFORMATION FOR SEQ ID NO:8: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: peptide - - (iii) HYPOTHETICAL: NO - - (v) FRAGMENT TYPE: N-terminal - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus - #cereus (B) STRAIN: AB78 (C) INDIVIDUAL ISOLATE: - #NRRL B-21058 - - (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..16 (D) OTHER INFORMATION: - #/note= "N-terminal sequence of protein p - #urified from strain AB78" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: - - Lys Arg Glu Ile Asp Glu Asp Thr Asp Thr As - #x Gly Asp Ser Ile Pro 1 5 - # 10 - # 15 - - - - (2) INFORMATION FOR SEQ ID NO:9: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1..21 (D) OTHER INFORMATION: - #/note= "Oligonucleotide probe based on amino - #acids 3 to 9 of SEQ ID NO:8, using codon usage of Bacillus - #thuringiensis" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: - - GAAATTGATC AAGATACNGA T - # - # - #21 - - - - (2) INFORMATION FOR SEQ ID NO:10: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: peptide - - (iii) HYPOTHETICAL: NO - - (v) FRAGMENT TYPE: N-terminal - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus - #thuringiensis (B) STRAIN: AB88 - - (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..14 (D) OTHER INFORMATION: - #/note= "N-terminal amino acid sequence - #of protein known as anion exchange fraction23 (smaller)" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: - - Xaa Glu Pro Phe Val Ser Ala Xaa Xaa Xaa Gl - #n Xaa Xaa Xaa 1 5 - # 10 - - - - (2) INFORMATION FOR SEQ ID NO:11: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus - #thuringiensis - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: - - Xaa Glu Tyr Glu Asn Val Glu Pro Phe Val Se - #r Ala Xaa 1 5 - # 10 - - - - (2) INFORMATION FOR SEQ ID NO:12: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus - #thurigiensis - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: - - Met Asn Lys Asn Asn Thr Lys Leu Pro Thr Ar - #g Ala Leu Pro 1 5 - # 10 - - - - (2) INFORMATION FOR SEQ ID NO:13: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: peptide - - (iii) HYPOTHETICAL: NO - - (v) FRAGMENT TYPE: N-terminal - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus - #thuringiensis (B) STRAIN: AB88 - - (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..15 (D) OTHER INFORMATION: - #/note= "N-terminal amino acid sequence - #of 35 kDa VIP active against Agrotis ipsilon" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: - - Ala Leu Ser Glu Asn Thr Gly Lys Asp Gly Gl - #y Tyr Ile Val Pro 1 5 - # 10 - # 15 - - - - (2) INFORMATION FOR SEQ ID NO:14: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus - #thuringiensis - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: - - Met Asp Asn Asn Pro Asn Ile Asn Glu 1 5 - - - - (2) INFORMATION FOR SEQ ID NO:15: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: peptide - - (iii) HYPOTHETICAL: NO - - (v) FRAGMENT TYPE: N-terminal - - (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..9 (D) OTHER INFORMATION: - #/note= "N-terminal sequence of 80 kDa delta - #-endotoxin" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: - - Met Asp Asn Asn Pro Asn Ile Asn Glu 1 5 - - - - (2) INFORMATION FOR SEQ ID NO:16: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: peptide - - (iii) HYPOTHETICAL: NO - - (v) FRAGMENT TYPE: N-terminal - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus - #thuringiensis - - (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..11 (D) OTHER INFORMATION: - #/note= "N-terminal sequence from 60 kDa delta - #-endotoxin" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: - - Met Asn Val Leu Asn Ser Gly Arg Thr Thr Il - #e 1 5 - # 10 - - - - (2) INFORMATION FOR SEQ ID NO:17: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2655 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1..2652 (D) OTHER INFORMATION: - #/note= "Maize optimized DNA sequence - #for 100 kd VIP1A(a) protein from AB78" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: - - ATGAAGAACA TGAAGAAGAA GCTGGCCAGC GTGGTGACCT GCACCCTGCT GG -#CCCCCATG 60 - - TTCCTGAACG GCAACGTGAA CGCCGTGTAC GCCGACAGCA AGACCAACCA GA -#TCAGCACC 120 - - ACCCAGAAGA ACCAGCAGAA GGAGATGGAC CGCAAGGGCC TGCTGGGCTA CT -#ACTTCAAG 180 - - GGCAAGGACT TCAGCAACCT GACCATGTTC GCCCCCACGC GTGACAGCAC CC -#TGATCTAC 240 - - GACCAGCAGA CCGCCAACAA GCTGCTGGAC AAGAAGCAGC AGGAGTACCA GA -#GCATCCGC 300 - - TGGATCGGCC TGATCCAGAG CAAGGAGACC GGCGACTTCA CCTTCAACCT GA -#GCGAGGAC 360 - - GAGCAGGCCA TCATCGAGAT CAACGGCAAG ATCATCAGCA ACAAGGGCAA GG -#AGAAGCAG 420 - - GTGGTGCACC TGGAGAAGGG CAAGCTGGTG CCCATCAAGA TCGAGTACCA GA -#GCGACACC 480 - - AAGTTCAACA TCGACAGCAA GACCTTCAAG GAGCTGAAGC TTTTCAAGAT CG -#ACAGCCAG 540 - - AACCAGCCCC AGCAGGTGCA GCAGGACGAG CTGCGCAACC CCGAGTTCAA CA -#AGAAGGAG 600 - - AGCCAGGAGT TCCTGGCCAA GCCCAGCAAG ATCAACCTGT TCACCCAGCA GA -#TGAAGCGC 660 - - GAGATCGACG AGGACACCGA CACCGACGGC GACAGCATCC CCGACCTGTG GG -#AGGAGAAC 720 - - GGCTACACCA TCCAGAACCG CATCGCCGTG AAGTGGGACG ACAGCCTGGC TA -#GCAAGGGC 780 - - TACACCAAGT TCGTGAGCAA CCCCCTGGAG AGCCACACCG TGGGCGACCC CT -#ACACCGAC 840 - - TACGAGAAGG CCGCCCGCGA CCTGGACCTG AGCAACGCCA AGGAGACCTT CA -#ACCCCCTG 900 - - GTGGCCGCCT TCCCCAGCGT GAACGTGAGC ATGGAGAAGG TGATCCTGAG CC -#CCAACGAG 960 - - AACCTGAGCA ACAGCGTGGA GAGCCACTCG AGCACCAACT GGAGCTACAC CA -#ACACCGAG 1020 - - GGCGCCAGCG TGGAGGCCGG CATCGGTCCC AAGGGCATCA GCTTCGGCGT GA -#GCGTGAAC 1080 - - TACCAGCACA GCGAGACCGT GGCCCAGGAG TGGGGCACCA GCACCGGCAA CA -#CCAGCCAG 1140 - - TTCAACACCG CCAGCGCCGG CTACCTGAAC GCCAACGTGC GCTACAACAA CG -#TGGGCACC 1200 - - GGCGCCATCT ACGACGTGAA GCCCACCACC AGCTTCGTGC TGAACAACGA CA -#CCATCGCC 1260 - - ACCATCACCG CCAAGTCGAA TTCCACCGCC CTGAACATCA GCCCCGGCGA GA -#GCTACCCC 1320 - - AAGAAGGGCC AGAACGGCAT CGCCATCACC AGCATGGACG ACTTCAACAG CC -#ACCCCATC 1380 - - ACCCTGAACA AGAAGCAGGT GGACAACCTG CTGAACAACA AGCCCATGAT GC -#TGGAGACC 1440 - - AACCAGACCG ACGGCGTCTA CAAGATCAAG GACACCCACG GCAACATCGT GA -#CCGGCGGC 1500 - - GAGTGGAACG GCGTGATCCA GCAGATCAAG GCCAAGACCG CCAGCATCAT CG -#TCGACGAC 1560 - - GGCGAGCGCG TGGCCGAGAA GCGCGTGGCC GCCAAGGACT ACGAGAACCC CG -#AGGACAAG 1620 - - ACCCCCAGCC TGACCCTGAA GGACGCCCTG AAGCTGAGCT ACCCCGACGA GA -#TCAAGGAG 1680 - - ATCGAGGGCC TGCTGTACTA CAAGAACAAG CCCATCTACG AGAGCAGCGT GA -#TGACCTAT 1740 - - CTAGACGAGA ACACCGCCAA GGAGGTGACC AAGCAGCTGA ACGACACCAC CG -#GCAAGTTC 1800 - - AAGGACGTGA GCCACCTGTA CGACGTGAAG CTGACCCCCA AGATGAACGT GA -#CCATCAAG 1860 - - CTGAGCATCC TGTACGACAA CGCCGAGAGC AACGACAACA GCATCGGCAA GT -#GGACCAAC 1920 - - ACCAACATCG TGAGCGGCGG CAACAACGGC AAGAAGCAGT ACAGCAGCAA CA -#ACCCCGAC 1980 - - GCCAACCTGA CCCTGAACAC CGACGCCCAG GAGAAGCTGA ACAAGAACCG CG -#ACTACTAC 2040 - - ATCAGCCTGT ACATGAAGAG CGAGAAGAAC ACCCAGTGCG AGATCACCAT CG -#ACGGCGAG 2100 - - ATATACCCCA TCACCACCAA GACCGTGAAC GTGAACAAGG ACAACTACAA GC -#GCCTGGAC 2160 - - ATCATCGCCC ACAACATCAA GAGCAACCCC ATCAGCAGCC TGCACATCAA GA -#CCAACGAC 2220 - - GAGATCACCC TGTTCTGGGA CGACATATCG ATTACCGACG TCGCCAGCAT CA -#AGCCCGAG 2280 - - AACCTGACCG ACAGCGAGAT CAAGCAGATA TACAGTCGCT ACGGCATCAA GC -#TGGAGGAC 2340 - - GGCATCCTGA TCGACAAGAA GGGCGGCATC CACTACGGCG AGTTCATCAA CG -#AGGCCAGC 2400 - - TTCAACATCG AGCCCCTGCA GAACTACGTG ACCAAGTACG AGGTGACCTA CA -#GCAGCGAG 2460 - - CTGGGCCCCA ACGTGAGCGA CACCCTGGAG AGCGACAAGA TTTACAAGGA CG -#GCACCATC 2520 - - AAGTTCGACT TCACCAAGTA CAGCAAGAAC GAGCAGGGCC TGTTCTACGA CA -#GCGGCCTG 2580 - - AACTGGGACT TCAAGATCAA CGCCATCACC TACGACGGCA AGGAGATGAA CG -#TGTTCCAC 2640 - - CGCTACAACA AGTAG - # - # - # 2655 - - - - (2) INFORMATION FOR SEQ ID NO:18: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2004 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1..2004 (D) OTHER INFORMATION: - #/note= "Maize optimized DNA sequence - #for VIP1A(a) 80 kd protein from AB78" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: - - ATGAAGCGCG AGATCGACGA GGACACCGAC ACCGACGGCG ACAGCATCCC CG -#ACCTGTGG 60 - - GAGGAGAACG GCTACACCAT CCAGAACCGC ATCGCCGTGA AGTGGGACGA CA -#GCCTGGCT 120 - - AGCAAGGGCT ACACCAAGTT CGTGAGCAAC CCCCTGGAGA GCCACACCGT GG -#GCGACCCC 180 - - TACACCGACT ACGAGAAGGC CGCCCGCGAC CTGGACCTGA GCAACGCCAA GG -#AGACCTTC 240 - - AACCCCCTGG TGGCCGCCTT CCCCAGCGTG AACGTGAGCA TGGAGAAGGT GA -#TCCTGAGC 300 - - CCCAACGAGA ACCTGAGCAA CAGCGTGGAG AGCCACTCGA GCACCAACTG GA -#GCTACACC 360 - - AACACCGAGG GCGCCAGCGT GGAGGCCGGC ATCGGTCCCA AGGGCATCAG CT -#TCGGCGTG 420 - - AGCGTGAACT ACCAGCACAG CGAGACCGTG GCCCAGGAGT GGGGCACCAG CA -#CCGGCAAC 480 - - ACCAGCCAGT TCAACACCGC CAGCGCCGGC TACCTGAACG CCAACGTGCG CT -#ACAACAAC 540 - - GTGGGCACCG GCGCCATCTA CGACGTGAAG CCCACCACCA GCTTCGTGCT GA -#ACAACGAC 600 - - ACCATCGCCA CCATCACCGC CAAGTCGAAT TCCACCGCCC TGAACATCAG CC -#CCGGCGAG 660 - - AGCTACCCCA AGAAGGGCCA GAACGGCATC GCCATCACCA GCATGGACGA CT -#TCAACAGC 720 - - CACCCCATCA CCCTGAACAA GAAGCAGGTG GACAACCTGC TGAACAACAA GC -#CCATGATG 780 - - CTGGAGACCA ACCAGACCGA CGGCGTCTAC AAGATCAAGG ACACCCACGG CA -#ACATCGTG 840 - - ACCGGCGGCG AGTGGAACGG CGTGATCCAG CAGATCAAGG CCAAGACCGC CA -#GCATCATC 900 - - GTCGACGACG GCGAGCGCGT GGCCGAGAAG CGCGTGGCCG CCAAGGACTA CG -#AGAACCCC 960 - - GAGGACAAGA CCCCCAGCCT GACCCTGAAG GACGCCCTGA AGCTGAGCTA CC -#CCGACGAG 1020 - - ATCAAGGAGA TCGAGGGCCT GCTGTACTAC AAGAACAAGC CCATCTACGA GA -#GCAGCGTG 1080 - - ATGACCTATC TAGACGAGAA CACCGCCAAG GAGGTGACCA AGCAGCTGAA CG -#ACACCACC 1140 - - GGCAAGTTCA AGGACGTGAG CCACCTGTAC GACGTGAAGC TGACCCCCAA GA -#TGAACGTG 1200 - - ACCATCAAGC TGAGCATCCT GTACGACAAC GCCGAGAGCA ACGACAACAG CA -#TCGGCAAG 1260 - - TGGACCAACA CCAACATCGT GAGCGGCGGC AACAACGGCA AGAAGCAGTA CA -#GCAGCAAC 1320 - - AACCCCGACG CCAACCTGAC CCTGAACACC GACGCCCAGG AGAAGCTGAA CA -#AGAACCGC 1380 - - GACTACTACA TCAGCCTGTA CATGAAGAGC GAGAAGAACA CCCAGTGCGA GA -#TCACCATC 1440 - - GACGGCGAGA TATACCCCAT CACCACCAAG ACCGTGAACG TGAACAAGGA CA -#ACTACAAG 1500 - - CGCCTGGACA TCATCGCCCA CAACATCAAG AGCAACCCCA TCAGCAGCCT GC -#ACATCAAG 1560 - - ACCAACGACG AGATCACCCT GTTCTGGGAC GACATATCGA TTACCGACGT CG -#CCAGCATC 1620 - - AAGCCCGAGA ACCTGACCGA CAGCGAGATC AAGCAGATAT ACAGTCGCTA CG -#GCATCAAG 1680 - - CTGGAGGACG GCATCCTGAT CGACAAGAAG GGCGGCATCC ACTACGGCGA GT -#TCATCAAC 1740 - - GAGGCCAGCT TCAACATCGA GCCCCTGCAG AACTACGTGA CCAAGTACGA GG -#TGACCTAC 1800 - - AGCAGCGAGC TGGGCCCCAA CGTGAGCGAC ACCCTGGAGA GCGACAAGAT TT -#ACAAGGAC 1860 - - GGCACCATCA AGTTCGACTT CACCAAGTAC AGCAAGAACG AGCAGGGCCT GT -#TCTACGAC 1920 - - AGCGGCCTGA ACTGGGACTT CAAGATCAAC GCCATCACCT ACGACGGCAA GG -#AGATGAAC 1980 - - GTGTTCCACC GCTACAACAA GTAG - # - # 2004 - - - - (2) INFORMATION FOR SEQ ID NO:19: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4074 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1386 (D) OTHER INFORMATION: - #/product= "VIP2A(b) from Btt" - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1394..3895 (D) OTHER INFORMATION: - #/product= "VIP1A(b) from Btt" - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1..4074 (D) OTHER INFORMATION: - #/note= "Cloned DNA sequence from Btt which - # contains the genes for both VIP1A(b) and VIP2A(b)" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: - - ATG CAA AGA ATG GAG GGA AAG TTG TTT GTG GT - #G TCA AAA ACA TTA CAA 48 Met Gln Arg Met Glu Gly Lys Leu Phe Val Va - #l Ser Lys Thr Leu Gln 670 - # 675 - # 680 - - GTA GTT ACT AGA ACT GTA TTG CTT AGT ACA GT - #T TAC TCT ATA ACT TTA 96 Val Val Thr Arg Thr Val Leu Leu Ser Thr Va - #l Tyr Ser Ile Thr Leu 685 - # 690 - # 695 - - TTA AAT AAT GTA GTG ATA AAA GCT GAC CAA TT - #A AAT ATA AAT TCT CAA 144 Leu Asn Asn Val Val Ile Lys Ala Asp Gln Le - #u Asn Ile Asn Ser Gln 700 7 - #05 7 - #10 7 -#15 - - AGT AAA TAT ACT AAC TTG CAA AAT CTA AAA AT - #C CCT GAT AAT GCAGAG 192 Ser Lys Tyr Thr Asn Leu Gln Asn Leu Lys Il - #e Pro Asp Asn Ala Glu 720 - # 725 - # 730 - - GAT TTT AAA GAA GAT AAG GGG AAA GCG AAA GA - #A TGG GGG AAA GAG AAA 240 Asp Phe Lys Glu Asp Lys Gly Lys Ala Lys Gl - #u Trp Gly Lys Glu Lys 735 - # 740 - # 745 - - GGG GAA GAG TGG AGG CCT CCT GCT ACT GAG AA - #A GGA GAA ATG AAT AAT 288 Gly Glu Glu Trp Arg Pro Pro Ala Thr Glu Ly - #s Gly Glu Met Asn Asn 750 - # 755 - # 760 - - TTT TTA GAT AAT AAA AAT GAT ATA AAG ACC AA - #T TAT AAA GAA ATT ACT 336 Phe Leu Asp Asn Lys Asn Asp Ile Lys Thr As - #n Tyr Lys Glu Ile Thr 765 - # 770 - # 775 - - TTT TCT ATG GCA GGT TCA TGT GAA GAT GAA AT - #A AAA GAT TTA GAA GAA 384 Phe Ser Met Ala Gly Ser Cys Glu Asp Glu Il - #e Lys Asp Leu Glu Glu 780 7 - #85 7 - #90 7 -#95 - - ATT GAT AAG ATC TTT GAT AAA GCC AAT CTC TC - #G AGT TCT ATT ATCACC 432 Ile Asp Lys Ile Phe Asp Lys Ala Asn Leu Se - #r Ser Ser Ile Ile Thr 800 - # 805 - # 810 - - TAT AAA AAT GTG GAA CCA GCA ACA ATT GGA TT - #T AAT AAA TCT TTA ACA 480 Tyr Lys Asn Val Glu Pro Ala Thr Ile Gly Ph - #e Asn Lys Ser Leu Thr 815 - # 820 - # 825 - - GAA GGT AAT ACG ATT AAT TCT GAT GCA ATG GC - #A CAG TTT AAA GAA CAA 528 Glu Gly Asn Thr Ile Asn Ser Asp Ala Met Al - #a Gln Phe Lys Glu Gln 830 - # 835 - # 840 - - TTT TTA GGT AAG GAT ATG AAG TTT GAT AGT TA - #T CTA GAT ACT CAT TTA 576 Phe Leu Gly Lys Asp Met Lys Phe Asp Ser Ty - #r Leu Asp Thr His Leu 845 - # 850 - # 855 - - ACT GCT CAA CAA GTT TCC AGT AAA AAA AGA GT - #T ATT TTG AAG GTT ACG 624 Thr Ala Gln Gln Val Ser Ser Lys Lys Arg Va - #l Ile Leu Lys Val Thr 860 8 - #65 8 - #70 8 -#75 - - GTT CCG AGT GGG AAA GGT TCT ACT ACT CCA AC - #A AAA GCA GGT GTCATT 672 Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Th - #r Lys Ala Gly Val Ile 880 - # 885 - # 890 - - TTA AAC AAT AAT GAA TAC AAA ATG CTC ATT GA - #T AAT GGG TAT GTG CTC 720 Leu Asn Asn Asn Glu Tyr Lys Met Leu Ile As - #p Asn Gly Tyr Val Leu 895 - # 900 - # 905 - - CAT GTA GAT AAG GTA TCA AAA GTA GTA AAA AA - #A GGG ATG GAG TGC TTA 768 His Val Asp Lys Val Ser Lys Val Val Lys Ly - #s Gly Met Glu Cys Leu 910 - # 915 - # 920 - - CAA GTT GAA GGG ACT TTA AAA AAG AGT CTC GA - #C TTT AAA AAT GAT ATA 816 Gln Val Glu Gly Thr Leu Lys Lys Ser Leu As - #p Phe Lys Asn Asp Ile 925 - # 930 - # 935 - - AAT GCT GAA GCG CAT AGC TGG GGG ATG AAA AT - #T TAT GAA GAC TGG GCT 864 Asn Ala Glu Ala His Ser Trp Gly Met Lys Il - #e Tyr Glu Asp Trp Ala 940 9 - #45 9 - #50 9 -#55 - - AAA AAT TTA ACC GCT TCG CAA AGG GAA GCT TT - #A GAT GGG TAT GCTAGG 912 Lys Asn Leu Thr Ala Ser Gln Arg Glu Ala Le - #u Asp Gly Tyr Ala Arg 960 - # 965 - # 970 - - CAA GAT TAT AAA GAA ATC AAT AAT TAT TTG CG - #C AAT CAA GGC GGG AGT 960 Gln Asp Tyr Lys Glu Ile Asn Asn Tyr Leu Ar - #g Asn Gln Gly Gly Ser 975 - # 980 - # 985 - - GGA AAT GAA AAG CTG GAT GCC CAA TTA AAA AA - #T ATT TCT GAT GCT TTA 1008 Gly Asn Glu Lys Leu Asp Ala Gln Leu Lys As - #n Ile Ser Asp Ala Leu 990 - # 995 - # 1000 - - GGG AAG AAA CCC ATA CCA GAA AAT ATT ACC GT - #G TAT AGA TGG TGT GGC 1056 Gly Lys Lys Pro Ile Pro Glu Asn Ile Thr Va - #l Tyr Arg Trp Cys Gly 1005 - # 1010 - # 1015 - - ATG CCG GAA TTT GGT TAT CAA ATT AGT GAT CC - #G TTA CCT TCT TTA AAA 1104 Met Pro Glu Phe Gly Tyr Gln Ile Ser Asp Pr - #o Leu Pro Ser Leu Lys 1020 1025 - # 1030 - # 1035 - - GAT TTT GAA GAA CAA TTT TTA AAT ACA ATT AA - #A GAA GAC AAA GGG TAT 1152 Asp Phe Glu Glu Gln Phe Leu Asn Thr Ile Ly - #s Glu Asp Lys Gly Tyr 1040 - # 1045 - # 1050 - - ATG AGT ACA AGC TTA TCG AGT GAA CGT CTT GC - #A GCT TTT GGA TCT AGA 1200 Met Ser Thr Ser Leu Ser Ser Glu Arg Leu Al - #a Ala Phe Gly Ser Arg 1055 - # 1060 - # 1065 - - AAA ATT ATA TTA CGC TTA CAA GTT CCG AAA GG - #A AGT ACG GGG GCG TAT 1248 Lys Ile Ile Leu Arg Leu Gln Val Pro Lys Gl - #y Ser Thr Gly Ala Tyr 1070 - # 1075 - # 1080 - - TTA AGT GCC ATT GGT GGA TTT GCA AGT GAA AA - #A GAG ATC CTA CTT GAT 1296 Leu Ser Ala Ile Gly Gly Phe Ala Ser Glu Ly - #s Glu Ile Leu Leu Asp 1085 - # 1090 - # 1095 - - AAA GAT AGT AAA TAT CAT ATT GAT AAA GCA AC - #A GAG GTA ATC ATT AAA 1344 Lys Asp Ser Lys Tyr His Ile Asp Lys Ala Th - #r Glu Val Ile Ile Lys 1100 1105 - # 1110 - # 1115 - - GGT GTT AAG CGA TAT GTA GTG GAT GCA ACA TT - #A TTA ACA AAT - #1386 Gly Val Lys Arg Tyr Val Val Asp Ala Thr Le - #u Leu Thr Asn 1120 - # 1125 - - TAAGGAG ATG AAA AAT ATG AAG AAA AAG TTA GCA - #AGT GTT GTA ACC TGT 1435 Met Lys Asn Met Lys Lys - #Lys Leu Ala Ser Val Val Thr Cys 1 - # 5 - # 10 - - ATG TTA TTA GCT CCT ATG TTT TTG AAT GGA AA - #T GTG AAT GCT GTT AAC 1483 Met Leu Leu Ala Pro Met Phe Leu Asn Gly As - #n Val Asn Ala Val Asn 15 - # 20 - # 25 - # 30 - - GCG GAT AGT AAA ATA AAT CAG ATT TCT ACA AC - #G CAG GAA AAC CAA CAG 1531 Ala Asp Ser Lys Ile Asn Gln Ile Ser Thr Th - #r Gln Glu Asn Gln Gln 35 - # 40 - # 45 - - AAA GAG ATG GAC CGA AAG GGA TTA TTG GGA TA - #T TAT TTC AAA GGA AAA 1579 Lys Glu Met Asp Arg Lys Gly Leu Leu Gly Ty - #r Tyr Phe Lys Gly Lys 50 - # 55 - # 60 - - GAT TTT AAT AAT CTT ACT ATG TTT GCA CCG AC - #A CGT GAT AAT ACC CTT 1627 Asp Phe Asn Asn Leu Thr Met Phe Ala Pro Th - #r Arg Asp Asn Thr Leu 65 - # 70 - # 75 - - ATG TAT GAC CAA CAA ACA GCG AAT GCA TTA TT - #A GAT AAA AAA CAA CAA 1675 Met Tyr Asp Gln Gln Thr Ala Asn Ala Leu Le - #u Asp Lys Lys Gln Gln 80 - # 85 - # 90 - - GAA TAT CAG TCC ATT CGT TGG ATT GGT TTG AT - #T CAG CGT AAA GAA ACG 1723 Glu Tyr Gln Ser Ile Arg Trp Ile Gly Leu Il - #e Gln Arg Lys Glu Thr 95 - #100 - #105 - #110 - - GGC GAT TTC ACA TTT AAC TTA TCA AAG GAT GA - #A CAG GCA ATT ATA GAA 1771 Gly Asp Phe Thr Phe Asn Leu Ser Lys Asp Gl - #u Gln Ala Ile Ile Glu 115 - # 120 - # 125 - - ATC GAT GGG AAA ATC ATT TCT AAT AAA GGG AA - #A GAA AAG CAA GTT GTC 1819 Ile Asp Gly Lys Ile Ile Ser Asn Lys Gly Ly - #s Glu Lys Gln Val Val 130 - # 135 - # 140 - - CAT TTA GAA AAA GAA AAA TTA GTT CCA ATC AA - #A ATA GAG TAT CAA TCA 1867 His Leu Glu Lys Glu Lys Leu Val Pro Ile Ly - #s Ile Glu Tyr Gln Ser 145 - # 150 - # 155 - - GAT ACG AAA TTT AAT ATT GAT AGT AAA ACA TT - #T AAA GAA CTT AAA TTA 1915 Asp Thr Lys Phe Asn Ile Asp Ser Lys Thr Ph - #e Lys Glu Leu Lys Leu 160 - # 165 - # 170 - - TTT AAA ATA GAT AGT CAA AAC CAA TCT CAA CA - #A GTT CAA CTG AGA AAC 1963 Phe Lys Ile Asp Ser Gln Asn Gln Ser Gln Gl - #n Val Gln Leu Arg Asn 175 1 - #80 1 - #85 1 -#90 - - CCT GAA TTT AAC AAA AAA GAA TCA CAG GAA TT - #T TTA GCA AAA GCATCA 2011 Pro Glu Phe Asn Lys Lys Glu Ser Gln Glu Ph - #e Leu Ala Lys Ala Ser 195 - # 200 - # 205 - - AAA ACA AAC CTT TTT AAG CAA AAA ATG AAA AG - #A GAT ATT GAT GAA GAT 2059 Lys Thr Asn Leu Phe Lys Gln Lys Met Lys Ar - #g Asp Ile Asp Glu Asp 210 - # 215 - # 220 - - ACG GAT ACA GAT GGA GAC TCC ATT CCT GAT CT - #T TGG GAA GAA AAT GGG 2107 Thr Asp Thr Asp Gly Asp Ser Ile Pro Asp Le - #u Trp Glu Glu Asn Gly 225 - # 230 - # 235 - - TAC ACG ATT CAA AAT AAA GTT GCT GTC AAA TG - #G GAT GAT TCG CTA GCA 2155 Tyr Thr Ile Gln Asn Lys Val Ala Val Lys Tr - #p Asp Asp Ser Leu Ala 240 - # 245 - # 250 - - AGT AAG GGA TAT ACA AAA TTT GTT TCG AAT CC - #A TTA GAC AGC CAC ACA 2203 Ser Lys Gly Tyr Thr Lys Phe Val Ser Asn Pr - #o Leu Asp Ser His Thr 255 2 - #60 2 - #65 2 -#70 - - GTT GGC GAT CCC TAT ACT GAT TAT GAA AAG GC - #C GCA AGG GAT TTAGAT 2251 Val Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Al - #a Ala Arg Asp Leu Asp 275 - # 280 - # 285 - - TTA TCA AAT GCA AAG GAA ACG TTC AAC CCA TT - #G GTA GCT GCT TTT CCA 2299 Leu Ser Asn Ala Lys Glu Thr Phe Asn Pro Le - #u Val Ala Ala Phe Pro 290 - # 295 - # 300 - - AGT GTG AAT GTT AGT ATG GAA AAG GTG ATA TT - #A TCA CCA AAT GAA AAT 2347 Ser Val Asn Val Ser Met Glu Lys Val Ile Le - #u Ser Pro Asn Glu Asn 305 - # 310 - # 315 - - TTA TCC AAT AGT GTA GAG TCT CAT TCA TCC AC - #G AAT TGG TCT TAT ACG 2395 Leu Ser Asn Ser Val Glu Ser His Ser Ser Th - #r Asn Trp Ser Tyr Thr 320 - # 325 - # 330 - - AAT ACA GAA GGA GCT TCC ATT GAA GCT GGT GG - #C GGT CCA TTA GGC CTT 2443 Asn Thr Glu Gly Ala Ser Ile Glu Ala Gly Gl - #y Gly Pro Leu Gly Leu 335 3 - #40 3 - #45 3 -#50 - - TCT TTT GGC GTG AGT GTT ACT TAT CAA CAC TC - #T GAA ACA GTT GCACAA 2491 Ser Phe Gly Val Ser Val Thr Tyr Gln His Se - #r Glu Thr Val Ala Gln 355 - # 360 - # 365 - - GAA TGG GGA ACA TCT ACA GGA AAT ACT TCA CA - #A TTC AAT ACG GCT TCA 2539 Glu Trp Gly Thr Ser Thr Gly Asn Thr Ser Gl - #n Phe Asn Thr Ala Ser 370 - # 375 - # 380 - - GCG GGA TAT TTA AAT GCA AAT GTT CGG TAT AA - #C AAT GTA GGG ACT GGT 2587 Ala Gly Tyr Leu Asn Ala Asn Val Arg Tyr As - #n Asn Val Gly Thr Gly 385 - # 390 - # 395 - - GCC ATC TAT GAT GTA AAA CCT ACA ACA AGT TT - #T GTA TTA AAT AAC AAT 2635 Ala Ile Tyr Asp Val Lys Pro Thr Thr Ser Ph - #e Val Leu Asn Asn Asn 400 - # 405 - # 410 - - ACC ATC GCA ACG ATT ACA GCA AAA TCA AAT TC - #A ACA GCT TTA CGT ATA 2683 Thr Ile Ala Thr Ile Thr Ala Lys Ser Asn Se - #r Thr Ala Leu Arg Ile 415 4 - #20 4 - #25 4 -#30 - - TCT CCG GGG GAT AGT TAT CCA GAA ATA GGA GA - #A AAC GCT ATT GCGATT 2731 Ser Pro Gly Asp Ser Tyr Pro Glu Ile Gly Gl - #u Asn Ala Ile Ala Ile 435 - # 440 - # 445 - - ACA TCT ATG GAT GAT TTT AAT TCT CAT CCA AT - #T ACA TTA AAT AAA CAA 2779 Thr Ser Met Asp Asp Phe Asn Ser His Pro Il - #e Thr Leu Asn Lys Gln 450 - # 455 - # 460 - - CAG GTA AAT CAA TTG ATA AAT AAT AAG CCA AT - #T ATG CTA GAG ACA GAC 2827 Gln Val Asn Gln Leu Ile Asn Asn Lys Pro Il - #e Met Leu Glu Thr Asp 465 - # 470 - # 475 - - CAA ACA GAT GGT GTT TAT AAA ATA AGA GAT AC - #A CAT GGA AAT ATT GTA 2875 Gln Thr Asp Gly Val Tyr Lys Ile Arg Asp Th - #r His Gly Asn Ile Val 480 - # 485 - # 490 - - ACT GGT GGA GAA TGG AAT GGT GTA ACA CAA CA - #A ATT AAA GCA AAA ACA 2923 Thr Gly Gly Glu Trp Asn Gly Val Thr Gln Gl - #n Ile Lys Ala Lys Thr 495 5 - #00 5 - #05 5 -#10 - - GCG TCT ATT ATT GTG GAT GAC GGG AAA CAG GT - #A GCA GAA AAA CGTGTG 2971 Ala Ser Ile Ile Val Asp Asp Gly Lys Gln Va - #l Ala Glu Lys Arg Val 515 - # 520 - # 525 - - GCG GCA AAA GAT TAT GGT CAT CCA GAA GAT AA - #A ACA CCA CCT TTA ACT 3019 Ala Ala Lys Asp Tyr Gly His Pro Glu Asp Ly - #s Thr Pro Pro Leu Thr 530 - # 535 - # 540 - - TTA AAA GAT ACC CTG AAG CTT TCA TAC CCA GA - #T GAA ATA AAA GAA ACT 3067 Leu Lys Asp Thr Leu Lys Leu Ser Tyr Pro As - #p Glu Ile Lys Glu Thr 545 - # 550 - # 555 - - AAT GGA TTG TTG TAC TAT GAT GAC AAA CCA AT - #C TAT GAA TCG AGT GTC 3115 Asn Gly Leu Leu Tyr Tyr Asp Asp Lys Pro Il - #e Tyr Glu Ser Ser Val 560 - # 565 - # 570 - - ATG ACT TAT CTG GAT GAA AAT ACG GCA AAA GA - #A GTC AAA AAA CAA ATA 3163 Met Thr Tyr Leu Asp Glu Asn Thr Ala Lys Gl - #u Val Lys Lys Gln Ile 575 5 - #80 5 - #85 5 -#90 - - AAT GAT ACA ACC GGA AAA TTT AAG GAT GTA AA - #T CAC TTA TAT GATGTA 3211 Asn Asp Thr Thr Gly Lys Phe Lys Asp Val As - #n His Leu Tyr Asp Val 595 - # 600 - # 605 - - AAA CTG ACT CCA AAA ATG AAT TTT ACG ATT AA - #A ATG GCT TCC TTG TAT 3259 Lys Leu Thr Pro Lys Met Asn Phe Thr Ile Ly - #s Met Ala Ser Leu Tyr 610 - # 615 - # 620 - - GAT GGG GCT GAA AAT AAT CAT AAC TCT TTA GG - #A ACC TGG TAT TTA ACA 3307 Asp Gly Ala Glu Asn Asn His Asn Ser Leu Gl - #y Thr Trp Tyr Leu Thr 625 - # 630 - # 635 - - TAT AAT GTT GCT GGT GGA AAT ACT GGG AAG AG - #A CAA TAT CGT TCA GCT 3355 Tyr Asn Val Ala Gly Gly Asn Thr Gly Lys Ar - #g Gln Tyr Arg Ser Ala 640 - # 645 - # 650 - - CAT TCT TGT GCA CAT GTA GCT CTA TCT TCA GA - #A GCG AAA AAG AAA CTA 3403 His Ser Cys Ala His Val Ala Leu Ser Ser Gl - #u Ala Lys Lys Lys Leu 655 6 - #60 6 - #65 6 -#70 - - AAT CAA AAT GCG AAT TAC TAT CTT AGC ATG TA - #T ATG AAG GCT GATTCT 3451 Asn Gln Asn Ala Asn Tyr Tyr Leu Ser Met Ty - #r Met Lys Ala Asp Ser 675 - # 680 - # 685 - - ACT ACG GAA CCT ACA ATA GAA GTA GCT GGG GA - #A AAA TCT GCA ATA ACA 3499 Thr Thr Glu Pro Thr Ile Glu Val Ala Gly Gl - #u Lys Ser Ala Ile Thr 690 - # 695 - # 700 - - AGT AAA AAA GTA AAA TTA AAT AAT CAA AAT TA - #T CAA AGA GTT GAT ATT 3547 Ser Lys Lys Val Lys Leu Asn Asn Gln Asn Ty - #r Gln Arg Val Asp Ile 705 - # 710 - # 715 - - TTA GTG AAA AAT TCT GAA AGA AAT CCA ATG GA - #T AAA ATA TAT ATA AGA 3595 Leu Val Lys Asn Ser Glu Arg Asn Pro Met As - #p Lys Ile Tyr Ile Arg 720 - # 725 - # 730 - - GGA AAT GGC ACG ACA AAT GTT TAT GGG GAT GA - #T GTT ACT ATC CCA GAG 3643 Gly Asn Gly Thr Thr Asn Val Tyr Gly Asp As - #p Val Thr Ile Pro Glu 735 7 - #40 7 - #45 7 -#50 - - GTA TCA GCT ATA AAT CCG GCT AGT CTA TCA GA - #T GAA GAA ATT CAAGAA 3691 Val Ser Ala Ile Asn Pro Ala Ser Leu Ser As - #p Glu Glu Ile Gln Glu 755 - # 760 - # 765 - - ATA TTT AAA GAC TCA ACT ATT GAA TAT GGA AA - #T CCT AGT TTC GTT GCT 3739 Ile Phe Lys Asp Ser Thr Ile Glu Tyr Gly As - #n Pro Ser Phe Val Ala 770 - # 775 - # 780 - - GAT GCC GTA ACA TTT AAA AAT ATA AAA CCT TT - #A CAA AAT TAT GTA AAG 3787 Asp Ala Val Thr Phe Lys Asn Ile Lys Pro Le - #u Gln Asn Tyr Val Lys 785 - # 790 - # 795 - - GAA TAT GAA ATA TAT CAT AAA TCT CAT CGA TA - #T GAA AAG AAA ACG GTC 3835 Glu Tyr Glu Ile Tyr His Lys Ser His Arg Ty - #r Glu Lys Lys Thr Val 800 - # 805 - # 810 - - TTT GAT ATC ATG GGT GTT CAT TAT GAG TAT AG - #T ATA GCT AGG GAA CAA 3883 Phe Asp Ile Met Gly Val His Tyr Glu Tyr Se - #r Ile Ala Arg Glu Gln 815 8 - #20 8 - #25 8 -#30 - - AAG AAA GCC GCA TAATTTTAAA AATAAAACTC GTTAGAGTTT AT - #TTAGCATG 3935 Lys Lys Ala Ala - - GTATTTTTAA GAATAATCAA TATGTTGAAC CGTTTGTAGC TGTTTTGGAA GG -#GAATTTCA 3995 - - TTTTATTTGG TCTCTTAAGT TGATGGGCAT GGGATATGTT CAGCATCCAA GC -#GTTTNGGG 4055 - - GGTTANAAAA TCCAATTTT - # - # 407 - #4 - - - - (2) INFORMATION FOR SEQ ID NO:20: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 462 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: - - Met Gln Arg Met Glu Gly Lys Leu Phe Val Va - #l Ser Lys Thr Leu Gln 1 5 - # 10 - # 15 - - Val Val Thr Arg Thr Val Leu Leu Ser Thr Va - #l Tyr Ser Ile Thr Leu 20 - # 25 - # 30 - - Leu Asn Asn Val Val Ile Lys Ala Asp Gln Le - #u Asn Ile Asn Ser Gln 35 - # 40 - # 45 - - Ser Lys Tyr Thr Asn Leu Gln Asn Leu Lys Il - #e Pro Asp Asn Ala Glu 50 - # 55 - # 60 - - Asp Phe Lys Glu Asp Lys Gly Lys Ala Lys Gl - #u Trp Gly Lys Glu Lys 65 - # 70 - # 75 - # 80 - - Gly Glu Glu Trp Arg Pro Pro Ala Thr Glu Ly - #s Gly Glu Met Asn Asn 85 - # 90 - # 95 - - Phe Leu Asp Asn Lys Asn Asp Ile Lys Thr As - #n Tyr Lys Glu Ile Thr 100 - # 105 - # 110 - - Phe Ser Met Ala Gly Ser Cys Glu Asp Glu Il - #e Lys Asp Leu Glu Glu 115 - # 120 - # 125 - - Ile Asp Lys Ile Phe Asp Lys Ala Asn Leu Se - #r Ser Ser Ile Ile Thr 130 - # 135 - # 140 - - Tyr Lys Asn Val Glu Pro Ala Thr Ile Gly Ph - #e Asn Lys Ser Leu Thr 145 1 - #50 1 - #55 1 -#60 - - Glu Gly Asn Thr Ile Asn Ser Asp Ala Met Al - #a Gln Phe Lys GluGln 165 - # 170 - # 175 - - Phe Leu Gly Lys Asp Met Lys Phe Asp Ser Ty - #r Leu Asp Thr His Leu 180 - # 185 - # 190 - - Thr Ala Gln Gln Val Ser Ser Lys Lys Arg Va - #l Ile Leu Lys Val Thr 195 - # 200 - # 205 - - Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Th - #r Lys Ala Gly Val Ile 210 - # 215 - # 220 - - Leu Asn Asn Asn Glu Tyr Lys Met Leu Ile As - #p Asn Gly Tyr Val Leu 225 2 - #30 2 - #35 2 -#40 - - His Val Asp Lys Val Ser Lys Val Val Lys Ly - #s Gly Met Glu CysLeu 245 - # 250 - # 255 - - Gln Val Glu Gly Thr Leu Lys Lys Ser Leu As - #p Phe Lys Asn Asp Ile 260 - # 265 - # 270 - - Asn Ala Glu Ala His Ser Trp Gly Met Lys Il - #e Tyr Glu Asp Trp Ala 275 - # 280 - # 285 - - Lys Asn Leu Thr Ala Ser Gln Arg Glu Ala Le - #u Asp Gly Tyr Ala Arg 290 - # 295 - # 300 - - Gln Asp Tyr Lys Glu Ile Asn Asn Tyr Leu Ar - #g Asn Gln Gly Gly Ser 305 3 - #10 3 - #15 3 -#20 - - Gly Asn Glu Lys Leu Asp Ala Gln Leu Lys As - #n Ile Ser Asp AlaLeu 325 - # 330 - # 335 - - Gly Lys Lys Pro Ile Pro Glu Asn Ile Thr Va - #l Tyr Arg Trp Cys Gly 340 - # 345 - # 350 - - Met Pro Glu Phe Gly Tyr Gln Ile Ser Asp Pr - #o Leu Pro Ser Leu Lys 355 - # 360 - # 365 - - Asp Phe Glu Glu Gln Phe Leu Asn Thr Ile Ly - #s Glu Asp Lys Gly Tyr 370 - # 375 - # 380 - - Met Ser Thr Ser Leu Ser Ser Glu Arg Leu Al - #a Ala Phe Gly Ser Arg 385 3 - #90 3 - #95 4 -#00 - - Lys Ile Ile Leu Arg Leu Gln Val Pro Lys Gl - #y Ser Thr Gly AlaTyr 405 - # 410 - # 415 - - Leu Ser Ala Ile Gly Gly Phe Ala Ser Glu Ly - #s Glu Ile Leu Leu Asp 420 - # 425 - # 430 - - Lys Asp Ser Lys Tyr His Ile Asp Lys Ala Th - #r Glu Val Ile Ile Lys 435 - # 440 - # 445 - - Gly Val Lys Arg Tyr Val Val Asp Ala Thr Le - #u Leu Thr Asn 450 - # 455 - # 460 - - - - (2) INFORMATION FOR SEQ ID NO:21: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 834 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: - - Met Lys Asn Met Lys Lys Lys Leu Ala Ser Va - #l Val Thr Cys Met Leu 1 5 - # 10 - # 15 - - Leu Ala Pro Met Phe Leu Asn Gly Asn Val As - #n Ala Val Asn Ala Asp 20 - # 25 - # 30 - - Ser Lys Ile Asn Gln Ile Ser Thr Thr Gln Gl - #u Asn Gln Gln Lys Glu 35 - # 40 - # 45 - - Met Asp Arg Lys Gly Leu Leu Gly Tyr Tyr Ph - #e Lys Gly Lys Asp Phe 50 - # 55 - # 60 - - Asn Asn Leu Thr Met Phe Ala Pro Thr Arg As - #p Asn Thr Leu Met Tyr 65 - # 70 - # 75 - # 80 - - Asp Gln Gln Thr Ala Asn Ala Leu Leu Asp Ly - #s Lys Gln Gln Glu Tyr 85 - # 90 - # 95 - - Gln Ser Ile Arg Trp Ile Gly Leu Ile Gln Ar - #g Lys Glu Thr Gly Asp 100 - # 105 - # 110 - - Phe Thr Phe Asn Leu Ser Lys Asp Glu Gln Al - #a Ile Ile Glu Ile Asp 115 - # 120 - # 125 - - Gly Lys Ile Ile Ser Asn Lys Gly Lys Glu Ly - #s Gln Val Val His Leu 130 - # 135 - # 140 - - Glu Lys Glu Lys Leu Val Pro Ile Lys Ile Gl - #u Tyr Gln Ser Asp Thr 145 1 - #50 1 - #55 1 -#60 - - Lys Phe Asn Ile Asp Ser Lys Thr Phe Lys Gl - #u Leu Lys Leu PheLys 165 - # 170 - # 175 - - Ile Asp Ser Gln Asn Gln Ser Gln Gln Val Gl - #n Leu Arg Asn Pro Glu 180 - # 185 - # 190 - - Phe Asn Lys Lys Glu Ser Gln Glu Phe Leu Al - #a Lys Ala Ser Lys Thr 195 - # 200 - # 205 - - Asn Leu Phe Lys Gln Lys Met Lys Arg Asp Il - #e Asp Glu Asp Thr Asp 210 - # 215 - # 220 - - Thr Asp Gly Asp Ser Ile Pro Asp Leu Trp Gl - #u Glu Asn Gly Tyr Thr 225 2 - #30 2 - #35 2 -#40 - - Ile Gln Asn Lys Val Ala Val Lys Trp Asp As - #p Ser Leu Ala SerLys 245 - # 250 - # 255 - - Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu As - #p Ser His Thr Val Gly 260 - # 265 - # 270 - - Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Ar - #g Asp Leu Asp Leu Ser 275 - # 280 - # 285 - - Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Al - #a Ala Phe Pro Ser Val 290 - # 295 - # 300 - - Asn Val Ser Met Glu Lys Val Ile Leu Ser Pr - #o Asn Glu Asn Leu Ser 305 3 - #10 3 - #15 3 -#20 - - Asn Ser Val Glu Ser His Ser Ser Thr Asn Tr - #p Ser Tyr Thr AsnThr 325 - # 330 - # 335 - - Glu Gly Ala Ser Ile Glu Ala Gly Gly Gly Pr - #o Leu Gly Leu Ser Phe 340 - # 345 - # 350 - - Gly Val Ser Val Thr Tyr Gln His Ser Glu Th - #r Val Ala Gln Glu Trp 355 - # 360 - # 365 - - Gly Thr Ser Thr Gly Asn Thr Ser Gln Phe As - #n Thr Ala Ser Ala Gly 370 - # 375 - # 380 - - Tyr Leu Asn Ala Asn Val Arg Tyr Asn Asn Va - #l Gly Thr Gly Ala Ile 385 3 - #90 3 - #95 4 -#00 - - Tyr Asp Val Lys Pro Thr Thr Ser Phe Val Le - #u Asn Asn Asn ThrIle 405 - # 410 - # 415 - - Ala Thr Ile Thr Ala Lys Ser Asn Ser Thr Al - #a Leu Arg Ile Ser Pro 420 - # 425 - # 430 - - Gly Asp Ser Tyr Pro Glu Ile Gly Glu Asn Al - #a Ile Ala Ile Thr Ser 435 - # 440 - # 445 - - Met Asp Asp Phe Asn Ser His Pro Ile Thr Le - #u Asn Lys Gln Gln Val 450 - # 455 - # 460 - - Asn Gln Leu Ile Asn Asn Lys Pro Ile Met Le - #u Glu Thr Asp Gln Thr 465 4 - #70 4 - #75 4 -#80 - - Asp Gly Val Tyr Lys Ile Arg Asp Thr His Gl - #y Asn Ile Val ThrGly 485 - # 490 - # 495 - - Gly Glu Trp Asn Gly Val Thr Gln Gln Ile Ly - #s Ala Lys Thr Ala Ser 500 - # 505 - # 510 - - Ile Ile Val Asp Asp Gly Lys Gln Val Ala Gl - #u Lys Arg Val Ala Ala 515 - # 520 - # 525 - - Lys Asp Tyr Gly His Pro Glu Asp Lys Thr Pr - #o Pro Leu Thr Leu Lys 530 - # 535 - # 540 - - Asp Thr Leu Lys Leu Ser Tyr Pro Asp Glu Il - #e Lys Glu Thr Asn Gly 545 5 - #50 5 - #55 5 -#60 - - Leu Leu Tyr Tyr Asp Asp Lys Pro Ile Tyr Gl - #u Ser Ser Val MetThr 565 - # 570 - # 575 - - Tyr Leu Asp Glu Asn Thr Ala Lys Glu Val Ly - #s Lys Gln Ile Asn Asp 580 - # 585 - # 590 - - Thr Thr Gly Lys Phe Lys Asp Val Asn His Le - #u Tyr Asp Val Lys Leu 595 - # 600 - # 605 - - Thr Pro Lys Met Asn Phe Thr Ile Lys Met Al - #a Ser Leu Tyr Asp Gly 610 - # 615 - # 620 - - Ala Glu Asn Asn His Asn Ser Leu Gly Thr Tr - #p Tyr Leu Thr Tyr Asn 625 6 - #30 6 - #35 6 -#40 - - Val Ala Gly Gly Asn Thr Gly Lys Arg Gln Ty - #r Arg Ser Ala HisSer 645 - # 650 - # 655 - - Cys Ala His Val Ala Leu Ser Ser Glu Ala Ly - #s Lys Lys Leu Asn Gln 660 - # 665 - # 670 - - Asn Ala Asn Tyr Tyr Leu Ser Met Tyr Met Ly - #s Ala Asp Ser Thr Thr 675 - # 680 - # 685 - - Glu Pro Thr Ile Glu Val Ala Gly Glu Lys Se - #r Ala Ile Thr Ser Lys 690 - # 695 - # 700 - - Lys Val Lys Leu Asn Asn Gln Asn Tyr Gln Ar - #g Val Asp Ile Leu Val 705 7 - #10 7 - #15 7 -#20 - - Lys Asn Ser Glu Arg Asn Pro Met Asp Lys Il - #e Tyr Ile Arg GlyAsn 725 - # 730 - # 735 - - Gly Thr Thr Asn Val Tyr Gly Asp Asp Val Th - #r Ile Pro Glu Val Ser 740 - # 745 - # 750 - - Ala Ile Asn Pro Ala Ser Leu Ser Asp Glu Gl - #u Ile Gln Glu Ile Phe 755 - # 760 - # 765 - - Lys Asp Ser Thr Ile Glu Tyr Gly Asn Pro Se - #r Phe Val Ala Asp Ala 770 - # 775 - # 780 - - Val Thr Phe Lys Asn Ile Lys Pro Leu Gln As - #n Tyr Val Lys Glu Tyr 785 7 - #90 7 - #95 8 -#00 - - Glu Ile Tyr His Lys Ser His Arg Tyr Glu Ly - #s Lys Thr Val PheAsp 805 - # 810 - # 815 - - Ile Met Gly Val His Tyr Glu Tyr Ser Ile Al - #a Arg Glu Gln Lys Lys 820 - # 825 - # 830 - - Ala Ala - - - - (2) INFORMATION FOR SEQ ID NO:22: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4041 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..4038 (D) OTHER INFORMATION: - #/product= "VIP1A(a)/VIP2A(a) fusion product" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: - - ATG AAA AGA ATG GAG GGA AAG TTG TTT ATG GT - #G TCA AAA AAA TTA CAA 48 Met Lys Arg Met Glu Gly Lys Leu Phe Met Va - #l Ser Lys Lys Leu Gln 835 8 - #40 8 - #45 8 -#50 - - GTA GTT ACT AAA ACT GTA TTG CTT AGT ACA GT - #T TTC TCT ATA TCTTTA 96 Val Val Thr Lys Thr Val Leu Leu Ser Thr Va - #l Phe Ser Ile Ser Leu 855 - # 860 - # 865 - - TTA AAT AAT GAA GTG ATA AAA GCT GAA CAA TT - #A AAT ATA AAT TCT CAA 144 Leu Asn Asn Glu Val Ile Lys Ala Glu Gln Le - #u Asn Ile Asn Ser Gln 870 - # 875 - # 880 - - AGT AAA TAT ACT AAC TTG CAA AAT CTA AAA AT - #C ACT GAC AAG GTA GAG 192 Ser Lys Tyr Thr Asn Leu Gln Asn Leu Lys Il - #e Thr Asp Lys Val Glu 885 - # 890 - # 895 - - GAT TTT AAA GAA GAT AAG GAA AAA GCG AAA GA - #A TGG GGG AAA GAA AAA 240 Asp Phe Lys Glu Asp Lys Glu Lys Ala Lys Gl - #u Trp Gly Lys Glu Lys 900 - # 905 - # 910 - - GAA AAA GAG TGG AAA CTA ACT GCT ACT GAA AA - #A GGA AAA ATG AAT AAT 288 Glu Lys Glu Trp Lys Leu Thr Ala Thr Glu Ly - #s Gly Lys Met Asn Asn 915 9 - #20 9 - #25 9 -#30 - - TTT TTA GAT AAT AAA AAT GAT ATA AAG ACA AA - #T TAT AAA GAA ATTACT 336 Phe Leu Asp Asn Lys Asn Asp Ile Lys Thr As - #n Tyr Lys Glu Ile Thr 935 - # 940 - # 945 - - TTT TCT ATG GCA GGC TCA TTT GAA GAT GAA AT - #A AAA GAT TTA AAA GAA 384 Phe Ser Met Ala Gly Ser Phe Glu Asp Glu Il - #e Lys Asp Leu Lys Glu 950 - # 955 - # 960 - - ATT GAT AAG ATG TTT GAT AAA ACC AAT CTA TC - #A AAT TCT ATT ATC ACC 432 Ile Asp Lys Met Phe Asp Lys Thr Asn Leu Se - #r Asn Ser Ile Ile Thr 965 - # 970 - # 975 - - TAT AAA AAT GTG GAA CCG ACA ACA ATT GGA TT - #T AAT AAA TCT TTA ACA 480 Tyr Lys Asn Val Glu Pro Thr Thr Ile Gly Ph - #e Asn Lys Ser Leu Thr 980 - # 985 - # 990 - - GAA GGT AAT ACG ATT AAT TCT GAT GCA ATG GC - #A CAG TTT AAA GAA CAA 528 Glu Gly Asn Thr Ile Asn Ser Asp Ala Met Al - #a Gln Phe Lys Glu Gln 995 1 - #000 1005 - # 1010 - - TTT TTA GAT AGG GAT ATT AAG TTT GAT AGT TA - #T CTA GAT ACG CAT TTA 576 Phe Leu Asp Arg Asp Ile Lys Phe Asp Ser Ty - #r Leu Asp Thr His Leu 1015 - # 1020 - # 1025 - - ACT GCT CAA CAA GTT TCC AGT AAA GAA AGA GT - #T ATT TTG AAG GTT ACG 624 Thr Ala Gln Gln Val Ser Ser Lys Glu Arg Va - #l Ile Leu Lys Val Thr 1030 - # 1035 - # 1040 - - GTT CCG AGT GGG AAA GGT TCT ACT ACT CCA AC - #A AAA GCA GGT GTC ATT 672 Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Th - #r Lys Ala Gly Val Ile 1045 - # 1050 - # 1055 - - TTA AAT AAT AGT GAA TAC AAA ATG CTC ATT GA - #T AAT GGG TAT ATG GTC 720 Leu Asn Asn Ser Glu Tyr Lys Met Leu Ile As - #p Asn Gly Tyr Met Val 1060 - # 1065 - # 1070 - - CAT GTA GAT AAG GTA TCA AAA GTG GTG AAA AA - #A GGG GTG GAG TGC TTA 768 His Val Asp Lys Val Ser Lys Val Val Lys Ly - #s Gly Val Glu Cys Leu 1075 1080 - # 1085 - # 1090 - - CAA ATT GAA GGG ACT TTA AAA AAG AGT CTT GA - #C TTT AAA AAT GAT ATA 816 Gln Ile Glu Gly Thr Leu Lys Lys Ser Leu As - #p Phe Lys Asn Asp Ile 1095 - # 1100 - # 1105 - - AAT GCT GAA GCG CAT AGC TGG GGT ATG AAG AA - #T TAT GAA GAG TGG GCT 864 Asn Ala Glu Ala His Ser Trp Gly Met Lys As - #n Tyr Glu Glu Trp Ala 1110 - # 1115 - # 1120 - - AAA GAT TTA ACC GAT TCG CAA AGG GAA GCT TT - #A GAT GGG TAT GCT AGG 912 Lys Asp Leu Thr Asp Ser Gln Arg Glu Ala Le - #u Asp Gly Tyr Ala Arg 1125 - # 1130 - # 1135 - - CAA GAT TAT AAA GAA ATC AAT AAT TAT TTA AG - #A AAT CAA GGC GGA AGT 960 Gln Asp Tyr Lys Glu Ile Asn Asn Tyr Leu Ar - #g Asn Gln Gly Gly Ser 1140 - # 1145 - # 1150 - - GGA AAT GAA AAA CTA GAT GCT CAA ATA AAA AA - #T ATT TCT GAT GCT TTA 1008 Gly Asn Glu Lys Leu Asp Ala Gln Ile Lys As - #n Ile Ser Asp Ala Leu 1155 1160 - # 1165 - # 1170 - - GGG AAG AAA CCA ATA CCG GAA AAT ATT ACT GT - #G TAT AGA TGG TGT GGC 1056 Gly Lys Lys Pro Ile Pro Glu Asn Ile Thr Va - #l Tyr Arg Trp Cys Gly 1175 - # 1180 - # 1185 - - ATG CCG GAA TTT GGT TAT CAA ATT AGT GAT CC - #G TTA CCT TCT TTA AAA 1104 Met Pro Glu Phe Gly Tyr Gln Ile Ser Asp Pr - #o Leu Pro Ser Leu Lys 1190 - # 1195 - # 1200 - - GAT TTT GAA GAA CAA TTT TTA AAT ACA ATC AA - #A GAA GAC AAA GGA TAT 1152 Asp Phe Glu Glu Gln Phe Leu Asn Thr Ile Ly - #s Glu Asp Lys Gly Tyr 1205 - # 1210 - # 1215 - - ATG AGT ACA AGC TTA TCG AGT GAA CGT CTT GC - #A GCT TTT GGA TCT AGA 1200 Met Ser Thr Ser Leu Ser Ser Glu Arg Leu Al - #a Ala Phe Gly Ser Arg 1220 - # 1225 - # 1230 - - AAA ATT ATA TTA CGA TTA CAA GTT CCG AAA GG - #A AGT ACG GGT GCG TAT 1248 Lys Ile Ile Leu Arg Leu Gln Val Pro Lys Gl - #y Ser Thr Gly Ala Tyr 1235 1240 - # 1245 - # 1250 - - TTA AGT GCC ATT GGT GGA TTT GCA AGT GAA AA - #A GAG ATC CTA CTT GAT 1296 Leu Ser Ala Ile Gly Gly Phe Ala Ser Glu Ly - #s Glu Ile Leu Leu Asp 1255 - # 1260 - # 1265 - - AAA GAT AGT AAA TAT CAT ATT GAT AAA GTA AC - #A GAG GTA ATT ATT AAA 1344 Lys Asp Ser Lys Tyr His Ile Asp Lys Val Th - #r Glu Val Ile Ile Lys 1270 - # 1275 - # 1280 - - GGT GTT AAG CGA TAT GTA GTG GAT GCA ACA TT - #A TTA ACA AAT ATG AAA 1392 Gly Val Lys Arg Tyr Val Val Asp Ala Thr Le - #u Leu Thr Asn Met Lys 1285 - # 1290 - # 1295 - - AAT ATG AAG AAA AAG TTA GCA AGT GTT GTA AC - #G TGT ACG TTA TTA GCT 1440 Asn Met Lys Lys Lys Leu Ala Ser Val Val Th - #r Cys Thr Leu Leu Ala 1300 - # 1305 - # 1310 - - CCT ATG TTT TTG AAT GGA AAT GTG AAT GCT GT - #T TAC GCA GAC AGC AAA 1488 Pro Met Phe Leu Asn Gly Asn Val Asn Ala Va - #l Tyr Ala Asp Ser Lys 1315 1320 - # 1325 - # 1330 - - ACA AAT CAA ATT TCT ACA ACA CAG AAA AAT CA - #A CAG AAA GAG ATG GAC 1536 Thr Asn Gln Ile Ser Thr Thr Gln Lys Asn Gl - #n Gln Lys Glu Met Asp 1335 - # 1340 - # 1345 - - CGA AAA GGA TTA CTT GGG TAT TAT TTC AAA GG - #A AAA GAT TTT AGT AAT 1584 Arg Lys Gly Leu Leu Gly Tyr Tyr Phe Lys Gl - #y Lys Asp Phe Ser Asn 1350 - # 1355 - # 1360 - - CTT ACT ATG TTT GCA CCG ACA CGT GAT AGT AC - #T CTT ATT TAT GAT CAA 1632 Leu Thr Met Phe Ala Pro Thr Arg Asp Ser Th - #r Leu Ile Tyr Asp Gln 1365 - # 1370 - # 1375 - - CAA ACA GCA AAT AAA CTA TTA GAT AAA AAA CA - #A CAA GAA TAT CAG TCT 1680 Gln Thr Ala Asn Lys Leu Leu Asp Lys Lys Gl - #n Gln Glu Tyr Gln Ser 1380 - # 1385 - # 1390 - - ATT CGT TGG ATT GGT TTG ATT CAG AGT AAA GA - #A ACG GGA GAT TTC ACA 1728 Ile Arg Trp Ile Gly Leu Ile Gln Ser Lys Gl - #u Thr Gly Asp Phe Thr 1395 1400 - # 1405 - # 1410 - - TTT AAC TTA TCT GAG GAT GAA CAG GCA ATT AT - #A GAA ATC AAT GGG AAA 1776 Phe Asn Leu Ser Glu Asp Glu Gln Ala Ile Il - #e Glu Ile Asn Gly Lys 1415 - # 1420 - # 1425 - - ATT ATT TCT AAT AAA GGG AAA GAA AAG CAA GT - #T GTC CAT TTA GAA AAA 1824 Ile Ile Ser Asn Lys Gly Lys Glu Lys Gln Va - #l Val His Leu Glu Lys 1430 - # 1435 - # 1440 - - GGA AAA TTA GTT CCA ATC AAA ATA GAG TAT CA - #A TCA GAT ACA AAA TTT 1872 Gly Lys Leu Val Pro Ile Lys Ile Glu Tyr Gl - #n Ser Asp Thr Lys Phe 1445 - # 1450 - # 1455 - - AAT ATT GAC AGT AAA ACA TTT AAA GAA CTT AA - #A TTA TTT AAA ATA GAT 1920 Asn Ile Asp Ser Lys Thr Phe Lys Glu Leu Ly - #s Leu Phe Lys Ile Asp 1460 - # 1465 - # 1470 - - AGT CAA AAC CAA CCC CAG CAA GTC CAG CAA GA - #T GAA CTG AGA AAT CCT 1968 Ser Gln Asn Gln Pro Gln Gln Val Gln Gln As - #p Glu Leu Arg Asn Pro 1475 1480 - # 1485 - # 1490 - - GAA TTT AAC AAG AAA GAA TCA CAG GAA TTC TT - #A GCG AAA CCA TCG AAA 2016 Glu Phe Asn Lys Lys Glu Ser Gln Glu Phe Le - #u Ala Lys Pro Ser Lys 1495 - # 1500 - # 1505 - - ATA AAT CTT TTC ACT CAA AAA ATG AAA AGG GA - #A ATT GAT GAA GAC ACG 2064 Ile Asn Leu Phe Thr Gln Lys Met Lys Arg Gl - #u Ile Asp Glu Asp Thr 1510 - # 1515 - # 1520 - - GAT ACG GAT GGG GAC TCT ATT CCT GAC CTT TG - #G GAA GAA AAT GGG TAT 2112 Asp Thr Asp Gly Asp Ser Ile Pro Asp Leu Tr - #p Glu Glu Asn Gly Tyr 1525 - # 1530 - # 1535 - - ACG ATT CAA AAT AGA ATC GCT GTA AAG TGG GA - #C GAT TCT CTA GCA AGT 2160 Thr Ile Gln Asn Arg Ile Ala Val Lys Trp As - #p Asp Ser Leu Ala Ser 1540 - # 1545 - # 1550 - - AAA GGG TAT ACG AAA TTT GTT TCA AAT CCA CT - #A GAA AGT CAC ACA GTT 2208 Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Le - #u Glu Ser His Thr Val 1555 1560 - # 1565 - # 1570 - - GGT GAT CCT TAT ACA GAT TAT GAA AAG GCA GC - #A AGA GAT CTA GAT TTG 2256 Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Al - #a Arg Asp Leu Asp Leu 1575 - # 1580 - # 1585 - - TCA AAT GCA AAG GAA ACG TTT AAC CCA TTG GT - #A GCT GCT TTT CCA AGT 2304 Ser Asn Ala Lys Glu Thr Phe Asn Pro Leu Va - #l Ala Ala Phe Pro Ser 1590 - # 1595 - # 1600 - - GTG AAT GTT AGT ATG GAA AAG GTG ATA TTA TC - #A CCA AAT GAA AAT TTA 2352 Val Asn Val Ser Met Glu Lys Val Ile Leu Se - #r Pro Asn Glu Asn Leu 1605 - # 1610 - # 1615 - - TCC AAT AGT GTA GAG TCT CAT TCA TCC ACG AA - #T TGG TCT TAT ACA AAT 2400 Ser Asn Ser Val Glu Ser His Ser Ser Thr As - #n Trp Ser Tyr Thr Asn 1620 - # 1625 - # 1630 - - ACA GAA GGT GCT TCT GTT GAA GCG GGG ATT GG - #A CCA AAA GGT ATT TCG 2448 Thr Glu Gly Ala Ser Val Glu Ala Gly Ile Gl - #y Pro Lys Gly Ile Ser 1635 1640 - # 1645 - # 1650 - - TTC GGA GTT AGC GTA AAC TAT CAA CAC TCT GA - #A ACA GTT GCA CAA GAA 2496 Phe Gly Val Ser Val Asn Tyr Gln His Ser Gl - #u Thr Val Ala Gln Glu 1655 - # 1660 - # 1665 - - TGG GGA ACA TCT ACA GGA AAT ACT TCG CAA TT - #C AAT ACG GCT TCA GCG 2544 Trp Gly Thr Ser Thr Gly Asn Thr Ser Gln Ph - #e Asn Thr Ala Ser Ala 1670 - # 1675 - # 1680 - - GGA TAT TTA AAT GCA AAT GTT CGA TAT AAC AA - #T GTA GGA ACT GGT GCC 2592 Gly Tyr Leu Asn Ala Asn Val Arg Tyr Asn As - #n Val Gly Thr Gly Ala 1685 - # 1690 - # 1695 - - ATC TAC GAT GTA AAA CCT ACA ACA AGT TTT GT - #A TTA AAT AAC GAT ACT 2640 Ile Tyr Asp Val Lys Pro Thr Thr Ser Phe Va - #l Leu Asn Asn Asp Thr 1700 - # 1705 - # 1710 - - ATC GCA ACT ATT ACG GCG AAA TCT AAT TCT AC - #A GCC TTA AAT ATA TCT 2688 Ile Ala Thr Ile Thr Ala Lys Ser Asn Ser Th - #r Ala Leu Asn Ile Ser 1715 1720 - # 1725 - # 1730 - - CCT GGA GAA AGT TAC CCG AAA AAA GGA CAA AA - #T GGA ATC GCA ATA ACA 2736 Pro Gly Glu Ser Tyr Pro Lys Lys Gly Gln As - #n Gly Ile Ala Ile Thr 1735 - # 1740 - # 1745 - - TCA ATG GAT GAT TTT AAT TCC CAT CCG ATT AC - #A TTA AAT AAA AAA CAA 2784 Ser Met Asp Asp Phe Asn Ser His Pro Ile Th - #r Leu Asn Lys Lys Gln 1750 - # 1755 - # 1760 - - GTA GAT AAT CTG CTA AAT AAT AAA CCT ATG AT - #G TTG GAA ACA AAC CAA 2832 Val Asp Asn Leu Leu Asn Asn Lys Pro Met Me - #t Leu Glu Thr Asn Gln 1765 - # 1770 - # 1775 - - ACA GAT GGT GTT TAT AAG ATA AAA GAT ACA CA - #T GGA AAT ATA GTA ACT 2880 Thr Asp Gly Val Tyr Lys Ile Lys Asp Thr Hi - #s Gly Asn Ile Val Thr 1780 - # 1785 - # 1790 - - GGC GGA GAA TGG AAT GGT GTC ATA CAA CAA AT - #C AAG GCT AAA ACA GCG 2928 Gly Gly Glu Trp Asn Gly Val Ile Gln Gln Il - #e Lys Ala Lys Thr Ala 1795 1800 - # 1805 - # 1810 - - TCT ATT ATT GTG GAT GAT GGG GAA CGT GTA GC - #A GAA AAA CGT GTA GCG 2976 Ser Ile Ile Val Asp Asp Gly Glu Arg Val Al - #a Glu Lys Arg Val Ala 1815 - # 1820 - # 1825 - - GCA AAA GAT TAT GAA AAT CCA GAA GAT AAA AC - #A CCG TCT TTA ACT TTA 3024 Ala Lys Asp Tyr Glu Asn Pro Glu Asp Lys Th - #r Pro Ser Leu Thr Leu 1830 - # 1835 - # 1840 - - AAA GAT GCC CTG AAG CTT TCA TAT CCA GAT GA - #A ATA AAA GAA ATA GAG 3072 Lys Asp Ala Leu Lys Leu Ser Tyr Pro Asp Gl - #u Ile Lys Glu Ile Glu 1845 - # 1850 - # 1855 - - GGA TTA TTA TAT TAT AAA AAC AAA CCG ATA TA - #C GAA TCG AGC GTT ATG 3120 Gly Leu Leu Tyr Tyr Lys Asn Lys Pro Ile Ty - #r Glu Ser Ser Val Met 1860 - # 1865 - # 1870 - - ACT TAC TTA GAT GAA AAT ACA GCA AAA GAA GT - #G ACC AAA CAA TTA AAT 3168 Thr Tyr Leu Asp Glu Asn Thr Ala Lys Glu Va - #l Thr Lys Gln Leu Asn 1875 1880 - # 1885 - # 1890 - - GAT ACC ACT GGG AAA TTT AAA GAT GTA AGT CA - #T TTA TAT GAT GTA AAA 3216 Asp Thr Thr Gly Lys Phe Lys Asp Val Ser Hi - #s Leu Tyr Asp Val Lys 1895 - # 1900 - # 1905 - - CTG ACT CCA AAA ATG AAT GTT ACA ATC AAA TT - #G TCT ATA CTT TAT GAT 3264 Leu Thr Pro Lys Met Asn Val Thr Ile Lys Le - #u Ser Ile Leu Tyr Asp 1910 - # 1915 - # 1920 - - AAT GCT GAG TCT AAT GAT AAC TCA ATT GGT AA - #A TGG ACA AAC ACA AAT 3312 Asn Ala Glu Ser Asn Asp Asn Ser Ile Gly Ly - #s Trp Thr Asn Thr Asn 1925 - # 1930 - # 1935 - - ATT GTT TCA GGT GGA AAT AAC GGA AAA AAA CA - #A TAT TCT TCT AAT AAT 3360 Ile Val Ser Gly Gly Asn Asn Gly Lys Lys Gl - #n Tyr Ser Ser Asn Asn 1940 - # 1945 - # 1950 - - CCG GAT GCT AAT TTG ACA TTA AAT ACA GAT GC - #T CAA GAA AAA TTA AAT 3408 Pro Asp Ala Asn Leu Thr Leu Asn Thr Asp Al - #a Gln Glu Lys Leu Asn 1955 1960 - # 1965 - # 1970 - - AAA AAT CGT GAC TAT TAT ATA AGT TTA TAT AT - #G AAG TCA GAA AAA AAC 3456 Lys Asn Arg Asp Tyr Tyr Ile Ser Leu Tyr Me - #t Lys Ser Glu Lys Asn 1975 - # 1980 - # 1985 - - ACA CAA TGT GAG ATT ACT ATA GAT GGG GAG AT - #T TAT CCG ATC ACT ACA 3504 Thr Gln Cys Glu Ile Thr Ile Asp Gly Glu Il - #e Tyr Pro Ile Thr Thr 1990 - # 1995 - # 2000 - - AAA ACA GTG AAT GTG AAT AAA GAC AAT TAC AA - #A AGA TTA GAT ATT ATA 3552 Lys Thr Val Asn Val Asn Lys Asp Asn Tyr Ly - #s Arg Leu Asp Ile Ile 2005 - # 2010 - # 2015 - - GCT CAT AAT ATA AAA AGT AAT CCA ATT TCT TC - #A CTT CAT ATT AAA ACG 3600 Ala His Asn Ile Lys Ser Asn Pro Ile Ser Se - #r Leu His Ile Lys Thr 2020 - # 2025 - # 2030 - - AAT GAT GAA ATA ACT TTA TTT TGG GAT GAT AT - #T TCT ATA ACA GAT GTA 3648 Asn Asp Glu Ile Thr Leu Phe Trp Asp Asp Il - #e Ser Ile Thr Asp Val 2035 2040 - # 2045 - # 2050 - - GCA TCA ATA AAA CCG GAA AAT TTA ACA GAT TC - #A GAA ATT AAA CAG ATT 3696 Ala Ser Ile Lys Pro Glu Asn Leu Thr Asp Se - #r Glu Ile Lys Gln Ile 2055 - # 2060 - # 2065 - - TAT AGT AGG TAT GGT ATT AAG TTA GAA GAT GG - #A ATC CTT ATT GAT AAA 3744 Tyr Ser Arg Tyr Gly Ile Lys Leu Glu Asp Gl - #y Ile Leu Ile Asp Lys 2070 - # 2075 - # 2080 - - AAA GGT GGG ATT CAT TAT GGT GAA TTT ATT AA - #T GAA GCT AGT TTT AAT 3792 Lys Gly Gly Ile His Tyr Gly Glu Phe Ile As - #n Glu Ala Ser Phe Asn 2085 - # 2090 - # 2095 - - ATT GAA CCA TTG CAA AAT TAT GTG ACC AAA TA - #T GAA GTT ACT TAT AGT 3840 Ile Glu Pro Leu Gln Asn Tyr Val Thr Lys Ty - #r Glu Val Thr Tyr Ser 2100 - # 2105 - # 2110 - - AGT GAG TTA GGA CCA AAC GTG AGT GAC ACA CT - #T GAA AGT GAT AAA ATT 3888 Ser Glu Leu Gly Pro Asn Val Ser Asp Thr Le - #u Glu Ser Asp Lys Ile 2115 2120 - # 2125 - # 2130 - - TAC AAG GAT GGG ACA ATT AAA TTT GAT TTT AC - #C AAA TAT AGT AAA AAT 3936 Tyr Lys Asp Gly Thr Ile Lys Phe Asp Phe Th - #r Lys Tyr Ser Lys Asn 2135 - # 2140 - # 2145 - - GAA CAA GGA TTA TTT TAT GAC AGT GGA TTA AA - #T TGG GAC TTT AAA ATT 3984 Glu Gln Gly Leu Phe Tyr Asp Ser Gly Leu As - #n Trp Asp Phe Lys Ile 2150 - # 2155 - # 2160 - - AAT GCT ATT ACT TAT GAT GGT AAA GAG ATG AA - #T GTT TTT CAT AGA TAT 4032 Asn Ala Ile Thr Tyr Asp Gly Lys Glu Met As - #n Val Phe His Arg Tyr 2165 - # 2170 - # 2175 - - AAT AAA TAG - # - #- # 4041 Asn Lys 2180 - - - - (2) INFORMATION FOR SEQ ID NO:23: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1346 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: - - Met Lys Arg Met Glu Gly Lys Leu Phe Met Va - #l Ser Lys Lys LeuGln 1 5 - # 10 - # 15 - - Val Val Thr Lys Thr Val Leu Leu Ser Thr Va - #l Phe Ser Ile Ser Leu 20 - # 25 - # 30 - - Leu Asn Asn Glu Val Ile Lys Ala Glu Gln Le - #u Asn Ile Asn Ser Gln 35 - # 40 - # 45 - - Ser Lys Tyr Thr Asn Leu Gln Asn Leu Lys Il - #e Thr Asp Lys Val Glu 50 - # 55 - # 60 - - Asp Phe Lys Glu Asp Lys Glu Lys Ala Lys Gl - #u Trp Gly Lys Glu Lys 65 - # 70 - # 75 - # 80 - - Glu Lys Glu Trp Lys Leu Thr Ala Thr Glu Ly - #s Gly Lys Met Asn Asn 85 - # 90 - # 95 - - Phe Leu Asp Asn Lys Asn Asp Ile Lys Thr As - #n Tyr Lys Glu Ile Thr 100 - # 105 - # 110 - - Phe Ser Met Ala Gly Ser Phe Glu Asp Glu Il - #e Lys Asp Leu Lys Glu 115 - # 120 - # 125 - - Ile Asp Lys Met Phe Asp Lys Thr Asn Leu Se - #r Asn Ser Ile Ile Thr 130 - # 135 - # 140 - - Tyr Lys Asn Val Glu Pro Thr Thr Ile Gly Ph - #e Asn Lys Ser Leu Thr 145 1 - #50 1 - #55 1 -#60 - - Glu Gly Asn Thr Ile Asn Ser Asp Ala Met Al - #a Gln Phe Lys GluGln 165 - # 170 - # 175 - - Phe Leu Asp Arg Asp Ile Lys Phe Asp Ser Ty - #r Leu Asp Thr His Leu 180 - # 185 - # 190 - - Thr Ala Gln Gln Val Ser Ser Lys Glu Arg Va - #l Ile Leu Lys Val Thr 195 - # 200 - # 205 - - Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Th - #r Lys Ala Gly Val Ile 210 - # 215 - # 220 - - Leu Asn Asn Ser Glu Tyr Lys Met Leu Ile As - #p Asn Gly Tyr Met Val 225 2 - #30 2 - #35 2 -#40 - - His Val Asp Lys Val Ser Lys Val Val Lys Ly - #s Gly Val Glu CysLeu 245 - # 250 - # 255 - - Gln Ile Glu Gly Thr Leu Lys Lys Ser Leu As - #p Phe Lys Asn Asp Ile 260 - # 265 - # 270 - - Asn Ala Glu Ala His Ser Trp Gly Met Lys As - #n Tyr Glu Glu Trp Ala 275 - # 280 - # 285 - - Lys Asp Leu Thr Asp Ser Gln Arg Glu Ala Le - #u Asp Gly Tyr Ala Arg 290 - # 295 - # 300 - - Gln Asp Tyr Lys Glu Ile Asn Asn Tyr Leu Ar - #g Asn Gln Gly Gly Ser 305 3 - #10 3 - #15 3 -#20 - - Gly Asn Glu Lys Leu Asp Ala Gln Ile Lys As - #n Ile Ser Asp AlaLeu 325 - # 330 - # 335 - - Gly Lys Lys Pro Ile Pro Glu Asn Ile Thr Va - #l Tyr Arg Trp Cys Gly 340 - # 345 - # 350 - - Met Pro Glu Phe Gly Tyr Gln Ile Ser Asp Pr - #o Leu Pro Ser Leu Lys 355 - # 360 - # 365 - - Asp Phe Glu Glu Gln Phe Leu Asn Thr Ile Ly - #s Glu Asp Lys Gly Tyr 370 - # 375 - # 380 - - Met Ser Thr Ser Leu Ser Ser Glu Arg Leu Al - #a Ala Phe Gly Ser Arg 385 3 - #90 3 - #95 4 -#00 - - Lys Ile Ile Leu Arg Leu Gln Val Pro Lys Gl - #y Ser Thr Gly AlaTyr 405 - # 410 - # 415 - - Leu Ser Ala Ile Gly Gly Phe Ala Ser Glu Ly - #s Glu Ile Leu Leu Asp 420 - # 425 - # 430 - - Lys Asp Ser Lys Tyr His Ile Asp Lys Val Th - #r Glu Val Ile Ile Lys 435 - # 440 - # 445 - - Gly Val Lys Arg Tyr Val Val Asp Ala Thr Le - #u Leu Thr Asn Met Lys 450 - # 455 - # 460 - - Asn Met Lys Lys Lys Leu Ala Ser Val Val Th - #r Cys Thr Leu Leu Ala 465 4 - #70 4 - #75 4 -#80 - - Pro Met Phe Leu Asn Gly Asn Val Asn Ala Va - #l Tyr Ala Asp SerLys 485 - # 490 - # 495 - - Thr Asn Gln Ile Ser Thr Thr Gln Lys Asn Gl - #n Gln Lys Glu Met Asp 500 - # 505 - # 510 - - Arg Lys Gly Leu Leu Gly Tyr Tyr Phe Lys Gl - #y Lys Asp Phe Ser Asn 515 - # 520 - # 525 - - Leu Thr Met Phe Ala Pro Thr Arg Asp Ser Th - #r Leu Ile Tyr Asp Gln 530 - # 535 - # 540 - - Gln Thr Ala Asn Lys Leu Leu Asp Lys Lys Gl - #n Gln Glu Tyr Gln Ser 545 5 - #50 5 - #55 5 -#60 - - Ile Arg Trp Ile Gly Leu Ile Gln Ser Lys Gl - #u Thr Gly Asp PheThr 565 - # 570 - # 575 - - Phe Asn Leu Ser Glu Asp Glu Gln Ala Ile Il - #e Glu Ile Asn Gly Lys 580 - # 585 - # 590 - - Ile Ile Ser Asn Lys Gly Lys Glu Lys Gln Va - #l Val His Leu Glu Lys 595 - # 600 - # 605 - - Gly Lys Leu Val Pro Ile Lys Ile Glu Tyr Gl - #n Ser Asp Thr Lys Phe 610 - # 615 - # 620 - - Asn Ile Asp Ser Lys Thr Phe Lys Glu Leu Ly - #s Leu Phe Lys Ile Asp 625 6 - #30 6 - #35 6 -#40 - - Ser Gln Asn Gln Pro Gln Gln Val Gln Gln As - #p Glu Leu Arg AsnPro 645 - # 650 - # 655 - - Glu Phe Asn Lys Lys Glu Ser Gln Glu Phe Le - #u Ala Lys Pro Ser Lys 660 - # 665 - # 670 - - Ile Asn Leu Phe Thr Gln Lys Met Lys Arg Gl - #u Ile Asp Glu Asp Thr 675 - # 680 - # 685 - - Asp Thr Asp Gly Asp Ser Ile Pro Asp Leu Tr - #p Glu Glu Asn Gly Tyr 690 - # 695 - # 700 - - Thr Ile Gln Asn Arg Ile Ala Val Lys Trp As - #p Asp Ser Leu Ala Ser 705 7 - #10 7 - #15 7 -#20 - - Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Le - #u Glu Ser His ThrVal 725 - # 730 - # 735 - - Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Al - #a Arg Asp Leu Asp Leu 740 - # 745 - # 750 - - Ser Asn Ala Lys Glu Thr Phe Asn Pro Leu Va - #l Ala Ala Phe Pro Ser 755 - # 760 - # 765 - - Val Asn Val Ser Met Glu Lys Val Ile Leu Se - #r Pro Asn Glu Asn Leu 770 - # 775 - # 780 - - Ser Asn Ser Val Glu Ser His Ser Ser Thr As - #n Trp Ser Tyr Thr Asn 785 7 - #90 7 - #95 8 -#00 - - Thr Glu Gly Ala Ser Val Glu Ala Gly Ile Gl - #y Pro Lys Gly IleSer 805 - # 810 - # 815 - - Phe Gly Val Ser Val Asn Tyr Gln His Ser Gl - #u Thr Val Ala Gln Glu 820 - # 825 - # 830 - - Trp Gly Thr Ser Thr Gly Asn Thr Ser Gln Ph - #e Asn Thr Ala Ser Ala 835 - # 840 - # 845 - - Gly Tyr Leu Asn Ala Asn Val Arg Tyr Asn As - #n Val Gly Thr Gly Ala 850 - # 855 - # 860 - - Ile Tyr Asp Val Lys Pro Thr Thr Ser Phe Va - #l Leu Asn Asn Asp Thr 865 8 - #70 8 - #75 8 -#80 - - Ile Ala Thr Ile Thr Ala Lys Ser Asn Ser Th - #r Ala Leu Asn IleSer 885 - # 890 - # 895 - - Pro Gly Glu Ser Tyr Pro Lys Lys Gly Gln As - #n Gly Ile Ala Ile Thr 900 - # 905 - # 910 - - Ser Met Asp Asp Phe Asn Ser His Pro Ile Th - #r Leu Asn Lys Lys Gln 915 - # 920 - # 925 - - Val Asp Asn Leu Leu Asn Asn Lys Pro Met Me - #t Leu Glu Thr Asn Gln 930 - # 935 - # 940 - - Thr Asp Gly Val Tyr Lys Ile Lys Asp Thr Hi - #s Gly Asn Ile Val Thr 945 9 - #50 9 - #55 9 -#60 - - Gly Gly Glu Trp Asn Gly Val Ile Gln Gln Il - #e Lys Ala Lys ThrAla 965 - # 970 - # 975 - - Ser Ile Ile Val Asp Asp Gly Glu Arg Val Al - #a Glu Lys Arg Val Ala 980 - # 985 - # 990 - - Ala Lys Asp Tyr Glu Asn Pro Glu Asp Lys Th - #r Pro Ser Leu Thr Leu 995 - # 1000 - # 1005 - - Lys Asp Ala Leu Lys Leu Ser Tyr Pro Asp Gl - #u Ile Lys Glu Ile Glu 1010 - # 1015 - # 1020 - - Gly Leu Leu Tyr Tyr Lys Asn Lys Pro Ile Ty - #r Glu Ser Ser Val Met 1025 1030 - # 1035 - # 1040 - - Thr Tyr Leu Asp Glu Asn Thr Ala Lys Glu Va - #l Thr Lys Gln Leu Asn 1045 - # 1050 - # 1055 - - Asp Thr Thr Gly Lys Phe Lys Asp Val Ser Hi - #s Leu Tyr Asp Val Lys 1060 - # 1065 - # 1070 - - Leu Thr Pro Lys Met Asn Val Thr Ile Lys Le - #u Ser Ile Leu Tyr Asp 1075 - # 1080 - # 1085 - - Asn Ala Glu Ser Asn Asp Asn Ser Ile Gly Ly - #s Trp Thr Asn Thr Asn 1090 - # 1095 - # 1100 - - Ile Val Ser Gly Gly Asn Asn Gly Lys Lys Gl - #n Tyr Ser Ser Asn Asn 1105 1110 - # 1115 - # 1120 - - Pro Asp Ala Asn Leu Thr Leu Asn Thr Asp Al - #a Gln Glu Lys Leu Asn 1125 - # 1130 - # 1135 - - Lys Asn Arg Asp Tyr Tyr Ile Ser Leu Tyr Me - #t Lys Ser Glu Lys Asn 1140 - # 1145 - # 1150 - - Thr Gln Cys Glu Ile Thr Ile Asp Gly Glu Il - #e Tyr Pro Ile Thr Thr 1155 - # 1160 - # 1165 - - Lys Thr Val Asn Val Asn Lys Asp Asn Tyr Ly - #s Arg Leu Asp Ile Ile 1170 - # 1175 - # 1180 - - Ala His Asn Ile Lys Ser Asn Pro Ile Ser Se - #r Leu His Ile Lys Thr 1185 1190 - # 1195 - # 1200 - - Asn Asp Glu Ile Thr Leu Phe Trp Asp Asp Il - #e Ser Ile Thr Asp Val 1205 - # 1210 - # 1215 - - Ala Ser Ile Lys Pro Glu Asn Leu Thr Asp Se - #r Glu Ile Lys Gln Ile 1220 - # 1225 - # 1230 - - Tyr Ser Arg Tyr Gly Ile Lys Leu Glu Asp Gl - #y Ile Leu Ile Asp Lys 1235 - # 1240 - # 1245 - - Lys Gly Gly Ile His Tyr Gly Glu Phe Ile As - #n Glu Ala Ser Phe Asn 1250 - # 1255 - # 1260 - - Ile Glu Pro Leu Gln Asn Tyr Val Thr Lys Ty - #r Glu Val Thr Tyr Ser 1265 1270 - # 1275 - # 1280 - - Ser Glu Leu Gly Pro Asn Val Ser Asp Thr Le - #u Glu Ser Asp Lys Ile 1285 - # 1290 - # 1295 - - Tyr Lys Asp Gly Thr Ile Lys Phe Asp Phe Th - #r Lys Tyr Ser Lys Asn 1300 - # 1305 - # 1310 - - Glu Gln Gly Leu Phe Tyr Asp Ser Gly Leu As - #n Trp Asp Phe Lys Ile 1315 - # 1320 - # 1325 - - Asn Ala Ile Thr Tyr Asp Gly Lys Glu Met As - #n Val Phe His Arg Tyr 1330 - # 1335 - # 1340 - - Asn Lys 1345 - - - - (2) INFORMATION FOR SEQ ID NO:24: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1399 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1..1386 (D) OTHER INFORMATION: - #/note= "Maize optimized DNA sequence - #for VIP2A(a) protein from AB78" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: - - ATGAAGCGCA TGGAGGGCAA GCTGTTCATG GTGAGCAAGA AGCTCCAGGT GG -#TGACCAAG 60 - - ACCGTGCTGC TGAGCACCGT GTTCAGCATC AGCCTGCTGA ACAACGAGGT GA -#TCAAGGCC 120 - - GAGCAGCTGA ACATCAACAG CCAGAGCAAG TACACCAACC TCCAGAACCT GA -#AGATCACC 180 - - GACAAGGTGG AGGACTTCAA GGAGGACAAG GAGAAGGCCA AGGAGTGGGG CA -#AGGAGAAG 240 - - GAGAAGGAGT GGAAGCTTAC CGCCACCGAG AAGGGCAAGA TGAACAACTT CC -#TGGACAAC 300 - - AAGAACGACA TCAAGACCAA CTACAAGGAG ATCACCTTCA GCATGGCCGG CA -#GCTTCGAG 360 - - GACGAGATCA AGGACCTGAA GGAGATCGAC AAGATGTTCG ACAAGACCAA CC -#TGAGCAAC 420 - - AGCATCATCA CCTACAAGAA CGTGGAGCCC ACCACCATCG GCTTCAACAA GA -#GCCTGACC 480 - - GAGGGCAACA CCATCAACAG CGACGCCATG GCCCAGTTCA AGGAGCAGTT CC -#TGGACCGC 540 - - GACATCAAGT TCGACAGCTA CCTGGACACC CACCTGACCG CCCAGCAGGT GA -#GCAGCAAG 600 - - GAGCGCGTGA TCCTGAAGGT GACCGTCCCC AGCGGCAAGG GCAGCACCAC CC -#CCACCAAG 660 - - GCCGGCGTGA TCCTGAACAA CAGCGAGTAC AAGATGCTGA TCGACAACGG CT -#ACATGGTG 720 - - CACGTGGACA AGGTGAGCAA GGTGGTGAAG AAGGGCGTGG AGTGCCTCCA GA -#TCGAGGGC 780 - - ACCCTGAAGA AGAGTCTAGA CTTCAAGAAC GACATCAACG CCGAGGCCCA CA -#GCTGGGGC 840 - - ATGAAGAACT ACGAGGAGTG GGCCAAGGAC CTGACCGACA GCCAGCGCGA GG -#CCCTGGAC 900 - - GGCTACGCCC GCCAGGACTA CAAGGAGATC AACAACTACC TGCGCAACCA GG -#GCGGCAGC 960 - - GGCAACGAGA AGCTGGACGC CCAGATCAAG AACATCAGCG ACGCCCTGGG CA -#AGAAGCCC 1020 - - ATCCCCGAGA ACATCACCGT GTACCGCTGG TGCGGCATGC CCGAGTTCGG CT -#ACCAGATC 1080 - - AGCGACCCCC TGCCCAGCCT GAAGGACTTC GAGGAGCAGT TCCTGAACAC CA -#TCAAGGAG 1140 - - GACAAGGGCT ACATGAGCAC CAGCCTGAGC AGCGAGCGCC TGGCCGCCTT CG -#GCAGCCGC 1200 - - AAGATCATCC TGCGCCTGCA GGTGCCCAAG GGCAGCACCG GCGCCTACCT GA -#GCGCCATC 1260 - - GGCGGCTTCG CCAGCGAGAA GGAGATCCTG CTGGACAAGG ACAGCAAGTA CC -#ACATCGAC 1320 - - AAGGTGACCG AGGTGATCAT CAAGGGCGTG AAGCGCTACG TGGTGGACGC CA -#CCCTGCTG 1380 - - ACCAACTAGA TCTGAGCTC - # - # 139 - #9 - - - - (2) INFORMATION FOR SEQ ID NO:25: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: peptide - - (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..19 (D) OTHER INFORMATION: - #/note= "Secretion signal peptide to secrete V - #IP2 out of a cell" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: - - Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Se - #r Gly Ala Ala Gly Val 1 5 - # 10 - # 15 - - His Cys Leu - - - - (2) INFORMATION FOR SEQ ID NO:26: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2655 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "Synthetic DNA" - - (iii) HYPOTHETICAL: NO - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1..2655 (D) OTHER INFORMATION: - #/note= "maize optimized DNA sequence - #encoding VIP1A(a)" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: - - ATGAAGAACA TGAAGAAGAA GCTGGCCAGC GTGGTGACCT GCACCCTGCT GG -#CCCCCATG 60 - - TTCCTGAACG GCAACGTGAA CGCCGTGTAC GCCGACAGCA AGACCAACCA GA -#TCAGCACC 120 - - ACCCAGAAGA ACCAGCAGAA GGAGATGGAC CGCAAGGGCC TGCTGGGCTA CT -#ACTTCAAG 180 - - GGCAAGGACT TCAGCAACCT GACCATGTTC GCCCCCACGC GTGACAGCAC CC -#TGATCTAC 240 - - GACCAGCAGA CCGCCAACAA GCTGCTGGAC AAGAAGCAGC AGGAGTACCA GA -#GCATCCGC 300 - - TGGATCGGCC TGATCCAGAG CAAGGAGACC GGCGACTTCA CCTTCAACCT GA -#GCGAGGAC 360 - - GAGCAGGCCA TCATCGAGAT CAACGGCAAG ATCATCAGCA ACAAGGGCAA GG -#AGAAGCAG 420 - - GTGGTGCACC TGGAGAAGGG CAAGCTGGTG CCCATCAAGA TCGAGTACCA GA -#GCGACACC 480 - - AAGTTCAACA TCGACAGCAA GACCTTCAAG GAGCTGAAGC TTTTCAAGAT CG -#ACAGCCAG 540 - - AACCAGCCCC AGCAGGTGCA GCAGGACGAG CTGCGCAACC CCGAGTTCAA CA -#AGAAGGAG 600 - - AGCCAGGAGT TCCTGGCCAA GCCCAGCAAG ATCAACCTGT TCACCCAGCA GA -#TGAAGCGC 660 - - GAGATCGACG AGGACACCGA CACCGACGGC GACAGCATCC CCGACCTGTG GG -#AGGAGAAC 720 - - GGCTACACCA TCCAGAACCG CATCGCCGTG AAGTGGGACG ACAGCCTGGC TA -#GCAAGGGC 780 - - TACACCAAGT TCGTGAGCAA CCCCCTGGAG AGCCACACCG TGGGCGACCC CT -#ACACCGAC 840 - - TACGAGAAGG CCGCCCGCGA CCTGGACCTG AGCAACGCCA AGGAGACCTT CA -#ACCCCCTG 900 - - GTGGCCGCCT TCCCCAGCGT GAACGTGAGC ATGGAGAAGG TGATCCTGAG CC -#CCAACGAG 960 - - AACCTGAGCA ACAGCGTGGA GAGCCACTCG AGCACCAACT GGAGCTACAC CA -#ACACCGAG 1020 - - GGCGCCAGCG TGGAGGCCGG CATCGGTCCC AAGGGCATCA GCTTCGGCGT GA -#GCGTGAAC 1080 - - TACCAGCACA GCGAGACCGT GGCCCAGGAG TGGGGCACCA GCACCGGCAA CA -#CCAGCCAG 1140 - - TTCAACACCG CCAGCGCCGG CTACCTGAAC GCCAACGTGC GCTACAACAA CG -#TGGGCACC 1200 - - GGCGCCATCT ACGACGTGAA GCCCACCACC AGCTTCGTGC TGAACAACGA CA -#CCATCGCC 1260 - - ACCATCACCG CCAAGTCGAA TTCCACCGCC CTGAACATCA GCCCCGGCGA GA -#GCTACCCC 1320 - - AAGAAGGGCC AGAACGGCAT CGCCATCACC AGCATGGACG ACTTCAACAG CC -#ACCCCATC 1380 - - ACCCTGAACA AGAAGCAGGT GGACAACCTG CTGAACAACA AGCCCATGAT GC -#TGGAGACC 1440 - - AACCAGACCG ACGGCGTCTA CAAGATCAAG GACACCCACG GCAACATCGT GA -#CGGGCGGC 1500 - - GAGTGGAACG GCGTGATCCA GCAGATCAAG GCCAAGACCG CCAGCATCAT CG -#TCGACGAC 1560 - - GGCGAGCGCG TGGCCGAGAA GCGCGTGGCC GCCAAGGACT ACGAGAACCC CG -#AGGACAAG 1620 - - ACCCCCAGCC TGACCCTGAA GGACGCCCTG AAGCTGAGCT ACCCCGACGA GA -#TCAAGGAG 1680 - - ATCGAGGGCT TGCTGTACTA CAAGAACAAG CCCATCTACG AGAGCAGCGT GA -#TGACCTAT 1740 - - CTAGACGAGA ACACCGCCAA GGAGGTGACC AAGCAGCTGA ACGACACCAC CG -#GCAAGTTC 1800 - - AAGGACGTGA GCCACCTGTA CGACGTGAAG CTGACCCCCA AGATGAACGT GA -#CCATCAAG 1860 - - CTGAGCATCC TGTACGACAA CGCCGAGAGC AACGACAACA GCATCGGCAA GT -#GGACCAAC 1920 - - ACCAACATCG TGAGCGGCGG CAACAACGGC AAGAAGCAGT ACAGCAGCAA CA -#ACCCCGAC 1980 - - GCCAACCTGA CCCTGAACAC CGACGCCCAG GAGAAGCTGA ACAAGAACCG CG -#ACTACTAC 2040 - - ATCAGCCTGT ACATGAAGAG CGAGAAGAAC ACCCAGTGCG AGATCACCAT CG -#ACGGCGAG 2100 - - ATATACCCCA TCACCACCAA GACCGTGAAC GTGAACAAGG ACAACTACAA GC -#GCCTGGAC 2160 - - ATCATCGCCC ACAACATCAA GAGCAACCCC ATCAGCAGCC TGCACATCAA GA -#CCAACGAC 2220 - - GAGATCACCC TGTTCTGGGA CGACATATCG ATTACCGACG TCGCCAGCAT CA -#AGCCCGAG 2280 - - AACCTGACCG ACAGCGAGAT CAAGCAGATA TACAGTCGCT ACGGCATCAA GC -#TGGAGGAC 2340 - - GGCATCCTGA TCGACAAGAA AGGCGGCATC CACTACGGCG AGTTCATCAA CG -#AGGCCAGC 2400 - - TTCAACATCG AGCCCCTGCA GAACTACGTG ACCAAGTACG AGGTGACCTA CA -#GCAGCGAG 2460 - - CTGGGCCCCA ACGTGAGCGA CACCCTGGAG AGCGACAAGA TTTACAAGGA CG -#GCACCATC 2520 - - AAGTTCGACT TCACCAAGTA CAGCAAGAAC GAGCAGGGCC TGTTCTACGA CA -#GCGGCCTG 2580 - - AACTGGGACT TCAAGATCAA CGCCATCACC TACGACGGCA AGGAGATGAA CG -#TGTTCCAC 2640 - - CGCTACAACA AGTAG - # - # - # 2655 - - - - (2) INFORMATION FOR SEQ ID NO:27: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1389 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "Synthetic DNA" - - (iii) HYPOTHETICAL: NO - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 1..1389 (D) OTHER INFORMATION: - #/note= "maize optimized DNA sequence - #encoding VIP2A(a)" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: - - ATGAAGCGCA TGGAGGGCAA GCTGTTCATG GTGAGCAAGA AGCTCCAGGT GG -#TGACCAAG 60 - - ACCGTGCTGC TGAGCACCGT GTTCAGCATC AGCCTGCTGA ACAACGAGGT GA -#TCAAGGCC 120 - - GAGCAGCTGA ACATCAACAG CCAGAGCAAG TACACCAACC TCCAGAACCT GA -#AGATCACC 180 - - GACAAGGTGG AGGACTTCAA GGAGGACAAG GAGAAGGCCA AGGAGTGGGG CA -#AGGAGAAG 240 - - GAGAAGGAGT GGAAGCTTAC CGCCACCGAG AAGGGCAAGA TGAACAACTT CC -#TGGACAAC 300 - - AAGAACGACA TCAAGACCAA CTACAAGGAG ATCACCTTCA GCATAGCCGG CA -#GCTTCGAG 360 - - GACGAGATCA AGGACCTGAA GGAGATCGAC AAGATGTTCG ACAAGACCAA CC -#TGAGCAAC 420 - - AGCATCATCA CCTACAAGAA CGTGGAGCCC ACCACCATCG GCTTCAACAA GA -#GCCTGACC 480 - - GAGGGCAACA CCATCAACAG CGACGCCATG GCCCAGTTCA AGGAGCAGTT CC -#TGGACCGC 540 - - GACATCAAGT TCGACAGCTA CCTGGACACC CACCTGACCG CCCAGCAGGT GA -#GCAGCAAG 600 - - GAGCGCGTGA TCCTGAAGGT GACCGTCCCC AGCGGCAAGG GCAGCACCAC CC -#CCACCAAG 660 - - GCCGGCGTGA TCCTGAACAA CAGCGAGTAC AAGATGCTGA TCGACAACGG CT -#ACATGGTG 720 - - CACGTGGACA AGGTGAGCAA GGTGGTGAAG AAGGGCGTGG AGTGCCTCCA GA -#TCGAGGGC 780 - - ACCCTGAAGA AGAGTCTAGA CTTCAAGAAC GACATCAACG CCGAGGCCCA CA -#GCTGGGGC 840 - - ATGAAGAACT ACGAGGAGTG GGCCAAGGAC CTGACCGACA GCCAGCGCGA GG -#CCCTGGAC 900 - - GGCTACGCCC GCCAGGACTA CAAGGAGATC AACAACTACC TGCGCAACCA GG -#GCGGCAGC 960 - - GGCAACGAGA AGCTGGACGC CCAGATCAAG AACATCAGCG ACGCCCTGGG CA -#AGAAGCCC 1020 - - ATCCCCGAGA ACATCACCGT GTACCGCTGG TGCGGCATGC CCGAGTTCGG CT -#ACCAGATC 1080 - - AGCGACCCCC TGCCCAGCCT GAAGGACTTC GAGGAGCAGT TCCTGAACAC CA -#TCAAGGAG 1140 - - GACAAGGGCT ACATGAGCAC CAGCCTGAGC AGCGAGCGCC TGGCCGCCTT CG -#GCAGCCGC 1200 - - AAGATCATCC TGCGCCTGCA GGTGCCCAAG GGCAGCACTG GTGCCTACCT GA -#GCGCCATC 1260 - - GGCGGCTTCG CCAGCGAGAA GGAGATCCTG CTGGATAAGG ACAGCAAGTA CC -#ACATCGAC 1320 - - AAGGTGACCG AGGTGATCAT CAAGGGCGTG AAGCGCTACG TGGTGGACGC CA -#CCCTGCTG 1380 - - ACCAACTAG - # - #- # 1389 - - - - (2) INFORMATION FOR SEQ ID NO:28: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2378 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (iii) HYPOTHETICAL: NO - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 9..2375 (D) OTHER INFORMATION: - #/note= "Native DNA sequence encoding - #VIP3A(a) protein from AB88 as contained in pCIB7104" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: - - AGATGAAC ATG AAC AAG AAT AAT ACT AAA TTA AGC - #ACA AGA GCC TTA CCA 50 Met Asn Lys Asn Asn T - #hr Lys Leu Ser Thr Arg Ala Leu Pro 1 - # 5 - # 10 - - AGT TTT ATT GAT TAT TTT AAT GGC ATT TAT GG - #A TTT GCC ACT GGT ATC 98 Ser Phe Ile Asp Tyr Phe Asn Gly Ile Tyr Gl - #y Phe Ala Thr Gly Ile 15 - # 20 - # 25 - # 30 - - AAA GAC ATT ATG AAC ATG ATT TTT AAA ACG GA - #T ACA GGT GGT GAT CTA 146 Lys Asp Ile Met Asn Met Ile Phe Lys Thr As - #p Thr Gly Gly Asp Leu 35 - # 40 - # 45 - - ACC CTA GAC GAA ATT TTA AAG AAT CAG CAG TT - #A CTA AAT GAT ATT TCT 194 Thr Leu Asp Glu Ile Leu Lys Asn Gln Gln Le - #u Leu Asn Asp Ile Ser 50 - # 55 - # 60 - - GGT AAA TTG GAT GGG GTG AAT GGA AGC TTA AA - #T GAT CTT ATC GCA CAG 242 Gly Lys Leu Asp Gly Val Asn Gly Ser Leu As - #n Asp Leu Ile Ala Gln 65 - # 70 - # 75 - - GGA AAC TTA AAT ACA GAA TTA TCT AAG GAA AT - #A TTA AAA ATT GCA AAT 290 Gly Asn Leu Asn Thr Glu Leu Ser Lys Glu Il - #e Leu Lys Ile Ala Asn 80 - # 85 - # 90 - - GAA CAA AAT CAA GTT TTA AAT GAT GTT AAT AA - #C AAA CTC GAT GCG ATA 338 Glu Gln Asn Gln Val Leu Asn Asp Val Asn As - #n Lys Leu Asp Ala Ile 95 - #100 - #105 - #110 - - AAT ACG ATG CTT CGG GTA TAT CTA CCT AAA AT - #T ACC TCT ATG TTG AGT 386 Asn Thr Met Leu Arg Val Tyr Leu Pro Lys Il - #e Thr Ser Met Leu Ser 115 - # 120 - # 125 - - GAT GTA ATG AAA CAA AAT TAT GCG CTA AGT CT - #G CAA ATA GAA TAC TTA 434 Asp Val Met Lys Gln Asn Tyr Ala Leu Ser Le - #u Gln Ile Glu Tyr Leu 130 - # 135 - # 140 - - AGT AAA CAA TTG CAA GAG ATT TCT GAT AAG TT - #G GAT ATT ATT AAT GTA 482 Ser Lys Gln Leu Gln Glu Ile Ser Asp Lys Le - #u Asp Ile Ile Asn Val 145 - # 150 - # 155 - - AAT GTA CTT ATT AAC TCT ACA CTT ACT GAA AT - #T ACA CCT GCG TAT CAA 530 Asn Val Leu Ile Asn Ser Thr Leu Thr Glu Il - #e Thr Pro Ala Tyr Gln 160 - # 165 - # 170 - - AGG ATT AAA TAT GTG AAC GAA AAA TTT GAG GA - #A TTA ACT TTT GCT ACA 578 Arg Ile Lys Tyr Val Asn Glu Lys Phe Glu Gl - #u Leu Thr Phe Ala Thr 175 1 - #80 1 - #85 1 -#90 - - GAA ACT AGT TCA AAA GTA AAA AAG GAT GGC TC - #T CCT GCA GAT ATTCTT 626 Glu Thr Ser Ser Lys Val Lys Lys Asp Gly Se - #r Pro Ala Asp Ile Leu 195 - # 200 - # 205 - - GAT GAG TTA ACT GAG TTA ACT GAA CTA GCG AA - #A AGT GTA ACA AAA AAT 674 Asp Glu Leu Thr Glu Leu Thr Glu Leu Ala Ly - #s Ser Val Thr Lys Asn 210 - # 215 - # 220 - - GAT GTG GAT GGT TTT GAA TTT TAC CTT AAT AC - #A TTC CAC GAT GTA ATG 722 Asp Val Asp Gly Phe Glu Phe Tyr Leu Asn Th - #r Phe His Asp Val Met 225 - # 230 - # 235 - - GTA GGA AAT AAT TTA TTC GGG CGT TCA GCT TT - #A AAA ACT GCA TCG GAA 770 Val Gly Asn Asn Leu Phe Gly Arg Ser Ala Le - #u Lys Thr Ala Ser Glu 240 - # 245 - # 250 - - TTA ATT ACT AAA GAA AAT GTG AAA ACA AGT GG - #C AGT GAG GTC GGA AAT 818 Leu Ile Thr Lys Glu Asn Val Lys Thr Ser Gl - #y Ser Glu Val Gly Asn 255 2 - #60 2 - #65 2 -#70 - - GTT TAT AAC TTC TTA ATT GTA TTA ACA GCT CT - #G CAA GCC CAA GCTTTT 866 Val Tyr Asn Phe Leu Ile Val Leu Thr Ala Le - #u Gln Ala Gln Ala Phe 275 - # 280 - # 285 - - CTT ACT TTA ACA ACA TGC CGA AAA TTA TTA GG - #C TTA GCA GAT ATT GAT 914 Leu Thr Leu Thr Thr Cys Arg Lys Leu Leu Gl - #y Leu Ala Asp Ile Asp 290 - # 295 - # 300 - - TAT ACT TCT ATT ATG AAT GAA CAT TTA AAT AA - #G GAA AAA GAG GAA TTT 962 Tyr Thr Ser Ile Met Asn Glu His Leu Asn Ly - #s Glu Lys Glu Glu Phe 305 - # 310 - # 315 - - AGA GTA AAC ATC CTC CCT ACA CTT TCT AAT AC - #T TTT TCT AAT CCT AAT 1010 Arg Val Asn Ile Leu Pro Thr Leu Ser Asn Th - #r Phe Ser Asn Pro Asn 320 - # 325 - # 330 - - TAT GCA AAA GTT AAA GGA AGT GAT GAA GAT GC - #A AAG ATG ATT GTG GAA 1058 Tyr Ala Lys Val Lys Gly Ser Asp Glu Asp Al - #a Lys Met Ile Val Glu 335 3 - #40 3 - #45 3 -#50 - - GCT AAA CCA GGA CAT GCA TTG ATT GGG TTT GA - #A ATT AGT AAT GATTCA 1106 Ala Lys Pro Gly His Ala Leu Ile Gly Phe Gl - #u Ile Ser Asn Asp Ser 355 - # 360 - # 365 - - ATT ACA GTA TTA AAA GTA TAT GAG GCT AAG CT - #A AAA CAA AAT TAT CAA 1154 Ile Thr Val Leu Lys Val Tyr Glu Ala Lys Le - #u Lys Gln Asn Tyr Gln 370 - # 375 - # 380 - - GTC GAT AAG GAT TCC TTA TCG GAA GTT ATT TA - #T GGT GAT ATG GAT AAA 1202 Val Asp Lys Asp Ser Leu Ser Glu Val Ile Ty - #r Gly Asp Met Asp Lys 385 - # 390 - # 395 - - TTA TTG TGC CCA GAT CAA TCT GAA CAA ATC TA - #T TAT ACA AAT AAC ATA 1250 Leu Leu Cys Pro Asp Gln Ser Glu Gln Ile Ty - #r Tyr Thr Asn Asn Ile 400 - # 405 - # 410 - - GTA TTT CCA AAT GAA TAT GTA ATT ACT AAA AT - #T GAT TTC ACT AAA AAA 1298 Val Phe Pro Asn Glu Tyr Val Ile Thr Lys Il - #e Asp Phe Thr Lys Lys 415 4 - #20 4 - #25 4 -#30 - - ATG AAA ACT TTA AGA TAT GAG GTA ACA GCG AA - #T TTT TAT GAT TCTTCT 1346 Met Lys Thr Leu Arg Tyr Glu Val Thr Ala As - #n Phe Tyr Asp Ser Ser 435 - # 440 - # 445 - - ACA GGA GAA ATT GAC TTA AAT AAG AAA AAA GT - #A GAA TCA AGT GAA GCG 1394 Thr Gly Glu Ile Asp Leu Asn Lys Lys Lys Va - #l Glu Ser Ser Glu Ala 450 - # 455 - # 460 - - GAG TAT AGA ACG TTA AGT GCT AAT GAT GAT GG - #G GTG TAT ATG CCG TTA 1442 Glu Tyr Arg Thr Leu Ser Ala Asn Asp Asp Gl - #y Val Tyr Met Pro Leu 465 - # 470 - # 475 - - GGT GTC ATC AGT GAA ACA TTT TTG ACT CCG AT - #T AAT GGG TTT GGC CTC 1490 Gly Val Ile Ser Glu Thr Phe Leu Thr Pro Il - #e Asn Gly Phe Gly Leu 480 - # 485 - # 490 - - CAA GCT GAT GAA AAT TCA AGA TTA ATT ACT TT - #A ACA TGT AAA TCA TAT 1538 Gln Ala Asp Glu Asn Ser Arg Leu Ile Thr Le - #u Thr Cys Lys Ser Tyr 495 5 - #00 5 - #05 5 -#10 - - TTA AGA GAA CTA CTG CTA GCA ACA GAC TTA AG - #C AAT AAA GAA ACTAAA 1586 Leu Arg Glu Leu Leu Leu Ala Thr Asp Leu Se - #r Asn Lys Glu Thr Lys 515 - # 520 - # 525 - - TTG ATC GTC CCG CCA AGT GGT TTT ATT AGC AA - #T ATT GTA GAG AAC GGG 1634 Leu Ile Val Pro Pro Ser Gly Phe Ile Ser As - #n Ile Val Glu Asn Gly 530 - # 535 - # 540 - - TCC ATA GAA GAG GAC AAT TTA GAG CCG TGG AA - #A GCA AAT AAT AAG AAT 1682 Ser Ile Glu Glu Asp Asn Leu Glu Pro Trp Ly - #s Ala Asn Asn Lys Asn 545 - # 550 - # 555 - - GCG TAT GTA GAT CAT ACA GGC GGA GTG AAT GG - #A ACT AAA GCT TTA TAT 1730 Ala Tyr Val Asp His Thr Gly Gly Val Asn Gl - #y Thr Lys Ala Leu Tyr 560 - # 565 - # 570 - - GTT CAT AAG GAC GGA GGA ATT TCA CAA TTT AT - #T GGA GAT AAG TTA AAA 1778 Val His Lys Asp Gly Gly Ile Ser Gln Phe Il - #e Gly Asp Lys Leu Lys 575 5 - #80 5 - #85 5 -#90 - - CCG AAA ACT GAG TAT GTA ATC CAA TAT ACT GT - #T AAA GGA AAA CCTTCT 1826 Pro Lys Thr Glu Tyr Val Ile Gln Tyr Thr Va - #l Lys Gly Lys Pro Ser 595 - # 600 - # 605 - - ATT CAT TTA AAA GAT GAA AAT ACT GGA TAT AT - #T CAT TAT GAA GAT ACA 1874 Ile His Leu Lys Asp Glu Asn Thr Gly Tyr Il - #e His Tyr Glu Asp Thr 610 - # 615 - # 620 - - AAT AAT AAT TTA GAA GAT TAT CAA ACT ATT AA - #T AAA CGT TTT ACT ACA 1922 Asn Asn Asn Leu Glu Asp Tyr Gln Thr Ile As - #n Lys Arg Phe Thr Thr 625 - # 630 - # 635 - - GGA ACT GAT TTA AAG GGA GTG TAT TTA ATT TT - #A AAA AGT CAA AAT GGA 1970 Gly Thr Asp Leu Lys Gly Val Tyr Leu Ile Le - #u Lys Ser Gln Asn Gly 640 - # 645 - # 650 - - GAT GAA GCT TGG GGA GAT AAC TTT ATT ATT TT - #G GAA ATT AGT CCT TCT 2018 Asp Glu Ala Trp Gly Asp Asn Phe Ile Ile Le - #u Glu Ile Ser Pro Ser 655 6 - #60 6 - #65 6 -#70 - - GAA AAG TTA TTA AGT CCA GAA TTA ATT AAT AC - #A AAT AAT TGG ACGAGT 2066 Glu Lys Leu Leu Ser Pro Glu Leu Ile Asn Th - #r Asn Asn Trp Thr Ser 675 - # 680 - # 685 - - ACG GGA TCA ACT AAT ATT AGC GGT AAT ACA CT - #C ACT CTT TAT CAG GGA 2114 Thr Gly Ser Thr Asn Ile Ser Gly Asn Thr Le - #u Thr Leu Tyr Gln Gly 690 - # 695 - # 700 - - GGA CGA GGG ATT CTA AAA CAA AAC CTT CAA TT - #A GAT AGT TTT TCA ACT 2162 Gly Arg Gly Ile Leu Lys Gln Asn Leu Gln Le - #u Asp Ser Phe Ser Thr 705 - # 710 - # 715 - - TAT AGA GTG TAT TTT TCT GTG TCC GGA GAT GC - #T AAT GTA AGG ATT AGA 2210 Tyr Arg Val Tyr Phe Ser Val Ser Gly Asp Al - #a Asn Val Arg Ile Arg 720 - # 725 - # 730 - - AAT TCT AGG GAA GTG TTA TTT GAA AAA AGA TA - #T ATG AGC GGT GCT AAA 2258 Asn Ser Arg Glu Val Leu Phe Glu Lys Arg Ty - #r Met Ser Gly Ala Lys 735 7 - #40 7 - #45 7 -#50 - - GAT GTT TCT GAA ATG TTC ACT ACA AAA TTT GA - #G AAA GAT AAC TTTTAT 2306 Asp Val Ser Glu Met Phe Thr Thr Lys Phe Gl - #u Lys Asp Asn Phe Tyr 755 - # 760 - # 765 - - ATA GAG CTT TCT CAA GGG AAT AAT TTA TAT GG - #T GGT CCT ATT GTA CAT 2354 Ile Glu Leu Ser Gln Gly Asn Asn Leu Tyr Gl - #y Gly Pro Ile Val His 770 - # 775 - # 780 - - TTT TAC GAT GTC TCT ATT AAG TAA - # - # 2378 Phe Tyr Asp Val Ser Ile Lys 785 - - - - (2) INFORMATION FOR SEQ ID NO:29: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 789 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: - - Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Ar - #g Ala Leu Pro Ser Phe 1 5 - # 10 - # 15 - - Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Al - #a Thr Gly Ile Lys Asp 20 - # 25 - # 30 - - Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gl - #y Gly Asp Leu Thr Leu 35 - # 40 - # 45 - - Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu As - #n Asp Ile Ser Gly Lys 50 - # 55 - # 60 - - Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Le - #u Ile Ala Gln Gly Asn 65 - # 70 - # 75 - # 80 - - Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Ly - #s Ile Ala Asn Glu Gln 85 - # 90 - # 95 - - Asn Gln Val Leu Asn Asp Val Asn Asn Lys Le - #u Asp Ala Ile Asn Thr 100 - # 105 - # 110 - - Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Se - #r Met Leu Ser Asp Val 115 - # 120 - # 125 - - Met Lys Gln Asn Tyr Ala Leu Ser Leu Gln Il - #e Glu Tyr Leu Ser Lys 130 - # 135 - # 140 - - Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Il - #e Ile Asn Val Asn Val 145 1 - #50 1 - #55 1 -#60 - - Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pr - #o Ala Tyr Gln ArgIle 165 - # 170 - # 175 - - Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Th - #r Phe Ala Thr Glu Thr 180 - # 185 - # 190 - - Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Al - #a Asp Ile Leu Asp Glu 195 - # 200 - # 205 - - Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Va - #l Thr Lys Asn Asp Val 210 - # 215 - # 220 - - Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe Hi - #s Asp Val Met Val Gly 225 2 - #30 2 - #35 2 -#40 - - Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Th - #r Ala Ser Glu LeuIle 245 - # 250 - # 255 - - Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Gl - #u Val Gly Asn Val Tyr 260 - # 265 - # 270 - - Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Al - #a Gln Ala Phe Leu Thr 275 - # 280 - # 285 - - Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Al - #a Asp Ile Asp Tyr Thr 290 - # 295 - # 300 - - Ser Ile Met Asn Glu His Leu Asn Lys Glu Ly - #s Glu Glu Phe Arg Val 305 3 - #10 3 - #15 3 -#20 - - Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Se - #r Asn Pro Asn TyrAla 325 - # 330 - # 335 - - Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Me - #t Ile Val Glu Ala Lys 340 - # 345 - # 350 - - Pro Gly His Ala Leu Ile Gly Phe Glu Ile Se - #r Asn Asp Ser Ile Thr 355 - # 360 - # 365 - - Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gl - #n Asn Tyr Gln Val Asp 370 - # 375 - # 380 - - Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly As - #p Met Asp Lys Leu Leu 385 3 - #90 3 - #95 4 -#00 - - Cys Pro Asp Gln Ser Glu Gln Ile Tyr Tyr Th - #r Asn Asn Ile ValPhe 405 - # 410 - # 415 - - Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Ph - #e Thr Lys Lys Met Lys 420 - # 425 - # 430 - - Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Ty - #r Asp Ser Ser Thr Gly 435 - # 440 - # 445 - - Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Se - #r Ser Glu Ala Glu Tyr 450 - # 455 - # 460 - - Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Ty - #r Met Pro Leu Gly Val 465 4 - #70 4 - #75 4 -#80 - - Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gl - #y Phe Gly Leu GlnAla 485 - # 490 - # 495 - - Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cy - #s Lys Ser Tyr Leu Arg 500 - # 505 - # 510 - - Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Ly - #s Glu Thr Lys Leu Ile 515 - # 520 - # 525 - - Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Va - #l Glu Asn Gly Ser Ile 530 - # 535 - # 540 - - Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala As - #n Asn Lys Asn Ala Tyr 545 5 - #50 5 - #55 5 -#60 - - Val Asp His Thr Gly Gly Val Asn Gly Thr Ly - #s Ala Leu Tyr ValHis 565 - # 570 - # 575 - - Lys Asp Gly Gly Ile Ser Gln Phe Ile Gly As - #p Lys Leu Lys Pro Lys 580 - # 585 - # 590 - - Thr Glu Tyr Val Ile Gln Tyr Thr Val Lys Gl - #y Lys Pro Ser Ile His 595 - # 600 - # 605 - - Leu Lys Asp Glu Asn Thr Gly Tyr Ile His Ty - #r Glu Asp Thr Asn Asn 610 - # 615 - # 620 - - Asn Leu Glu Asp Tyr Gln Thr Ile Asn Lys Ar - #g Phe Thr Thr Gly Thr 625 6 - #30 6 - #35 6 -#40 - - Asp Leu Lys Gly Val Tyr Leu Ile Leu Lys Se - #r Gln Asn Gly AspGlu 645 - # 650 - # 655 - - Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Il - #e Ser Pro Ser Glu Lys 660 - # 665 - # 670 - - Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn As - #n Trp Thr Ser Thr Gly 675 - # 680 - # 685 - - Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Le - #u Tyr Gln Gly Gly Arg 690 - # 695 - # 700 - - Gly Ile Leu Lys Gln Asn Leu Gln Leu Asp Se - #r Phe Ser Thr Tyr Arg 705 7 - #10 7 - #15 7 -#20 - - Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Va - #l Arg Ile Arg AsnSer 725 - # 730 - # 735 - - Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Se - #r Gly Ala Lys Asp Val 740 - # 745 - # 750 - - Ser Glu Met Phe Thr Thr Lys Phe Glu Lys As - #p Asn Phe Tyr Ile Glu 755 - # 760 - # 765 - - Leu Ser Gln Gly Asn Asn Leu Tyr Gly Gly Pr - #o Ile Val His Phe Tyr 770 - # 775 - # 780 - - Asp Val Ser Ile Lys 785 - - - - (2) INFORMATION FOR SEQ ID NO:30: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2403 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "Synthetic DNA" - - (iii) HYPOTHETICAL: NO - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- - #feature (B) LOCATION: 11..2389 (D) OTHER INFORMATION: - #/note= "maize optimized DNA sequence - #encoding VIP3A(a)" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: - - GGATCCACCA ATGAACATGA ACAAGAACAA CACCAAGCTG AGCACCCGCG CC -#CTGCCGAG 60 - - CTTCATCGAC TACTTCAACG GCATCTACGG CTTCGCCACC GGCATCAAGG AC -#ATCATGAA 120 - - CATGATCTTC AAGACCGACA CCGGCGGCGA CCTGACCCTG GACGAGATCC TG -#AAGAACCA 180 - - GCAGCTGCTG AACGACATCA GCGGCAAGCT GGACGGCGTG AACGGCAGCC TG -#AACGACCT 240 - - GATCGCCCAG GGCAACCTGA ACACCGAGCT GAGCAAGGAG ATCCTTAAGA TC -#GCCAACGA 300 - - GCAGAACCAG GTGCTGAACG ACGTGAACAA CAAGCTGGAC GCCATCAACA CC -#ATGCTGCG 360 - - CGTGTACCTG CCGAAGATCA CCAGCATGCT GAGCGACGTG ATGAAGCAGA AC -#TACGCCCT 420 - - GAGCCTGCAG ATCGAGTACC TGAGCAAGCA GCTGCAGGAG ATCAGCGACA AG -#CTGGACAT 480 - - CATCAACGTG AACGTCCTGA TCAACAGCAC CCTGACCGAG ATCACCCCGG CC -#TACCAGCG 540 - - CATCAAGTAC GTGAACGAGA AGTTCGAAGA GCTGACCTTC GCCACCGAGA CC -#AGCAGCAA 600 - - GGTGAAGAAG GACGGCAGCC CGGCCGACAT CCTGGACGAG CTGACCGAGC TG -#ACCGAGCT 660 - - GGCCAAGAGC GTGACCAAGA ACGACGTGGA CGGCTTCGAG TTCTACCTGA AC -#ACCTTCCA 720 - - CGACGTGATG GTGGGCAACA ACCTGTTCGG CCGCAGCGCC CTGAAGACCG CC -#AGCGAGCT 780 - - GATCACCAAG GAGAACGTGA AGACCAGCGG CAGCGAGGTG GGCAACGTGT AC -#AACTTCCT 840 - - GATCGTGCTG ACCGCCCTGC AGGCCCAGGC CTTCCTGACC CTGACCACCT GT -#CGCAAGCT 900 - - GCTGGGCCTG GCCGACATCG ACTACACCAG CATCATGAAC GAGCACTTGA AC -#AAGGAGAA 960 - - GGAGGAGTTC CGCGTGAACA TCCTGCCGAC CCTGAGCAAC ACCTTCAGCA AC -#CCGAACTA 1020 - - CGCCAAGGTG AAGGGCAGCG ACGAGGACGC CAAGATGATC GTGGAGGCTA AG -#CCGGGCCA 1080 - - CGCGTTGATC GGCTTCGAGA TCAGCAACGA CAGCATCACC GTGCTGAAGG TG -#TACGAGGC 1140 - - CAAGCTGAAG CAGAACTACC AGGTGGACAA GGACAGCTTG AGCGAGGTGA TC -#TACGGCGA 1200 - - CATGGACAAG CTGCTGTGTC CGGACCAGAG CGAGCAAATC TACTACACCA AC -#AACATCGT 1260 - - GTTCCCGAAC GAGTACGTGA TCACCAAGAT CGACTTCACC AAGAAGATGA AG -#ACCCTGCG 1320 - - CTACGAGGTG ACCGCCAACT TCTACGACAG CAGCACCGGC GAGATCGACC TG -#AACAAGAA 1380 - - GAAGGTGGAG AGCAGCGAGG CCGAGTACCG CACCCTGAGC GCGAACGACG AC -#GGCGTCTA 1440 - - CATGCCACTG GGCGTGATCA GCGAGACCTT CCTGACCCCG ATCAACGGCT TT -#GGCCTGCA 1500 - - GGCCGACGAG AACAGCCGCC TGATCACCCT GACCTGTAAG AGCTACCTGC GC -#GAGCTGCT 1560 - - GCTAGCCACC GACCTGAGCA ACAAGGAGAC CAAGCTGATC GTGCCACCGA GC -#GGCTTCAT 1620 - - CAGCAACATC GTGGAGAACG GCAGCATCGA GGAGGACAAC CTGGAGCCGT GG -#AAGGCCAA 1680 - - CAACAAGAAC GCCTACGTGG ACCACACCGG CGGCGTGAAC GGCACCAAGG CC -#CTGTACGT 1740 - - GCACAAGGAC GGCGGCATCA GCCAGTTCAT CGGCGACAAG CTGAAGCCGA AG -#ACCGAGTA 1800 - - CGTGATCCAG TACACCGTGA AGGGCAAGCC ATCGATTCAC CTGAAGGACG AG -#AACACCGG 1860 - - CTACATCCAC TACGAGGACA CCAACAACAA CCTGGAGGAC TACCAGACCA TC -#AACAAGCG 1920 - - CTTCACCACC GGCACCGACC TGAAGGGCGT GTACCTGATC CTGAAGAGCC AG -#AACGGCGA 1980 - - CGAGGCCTGG GGCGACAACT TCATCATCCT GGAGATCAGC CCGAGCGAGA AG -#CTGCTGAG 2040 - - CCCGGAGCTG ATCAACACCA ACAACTGGAC CAGCACCGGC AGCACCAACA TC -#AGCGGCAA 2100 - - CACCCTGACC CTGTACCAGG GCGGCCGCGG CATCCTGAAG CAGAACCTGC AG -#CTGGACAG 2160 - - CTTCAGCACC TACCGCGTGT ACTTCAGCGT GAGCGGCGAC GCCAACGTGC GC -#ATCCGCAA 2220 - - CAGCCGCGAG GTGCTGTTCG AGAAGAGGTA CATGAGCGGC GCCAAGGACG TG -#AGCGAGAT 2280 - - GTTCACCACC AAGTTCGAGA AGGACAACTT CTACATCGAG CTGAGCCAGG GC -#AACAACCT 2340 - - GTACGGCGGC CCGATCGTGC ACTTCTACGA CGTGAGCATC AAGTTAACGT AG -#AGCTCAGA 2400 - - TCT - # - # - # 2403 - - - - (2) INFORMATION FOR SEQ ID NO:31: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2612 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (iii) HYPOTHETICAL: NO - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 118..2484 (D) OTHER INFORMATION: - #/note= "Native DNA sequence encoding - #VIP3A(b) from AB424" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: - - ATTGAAATTG ATAAAAAGTT ATGAGTGTTT AATAATCAGT AATTACCAAT AA -#AGAATTAA 60 - - GAATACAAGT TTACAAGAAA TAAGTGTTAC AAAAAATAGC TGAAAAGGAA GA - #TGAAC 117 - - ATG AAC AAG AAT AAT ACT AAA TTA AGC ACA AG - #A GCC TTA CCA AGT TTT 165 Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Ar - #g Ala Leu Pro Ser Phe 790 7 - #95 8 - #00 8 -#05 - - ATT GAT TAT TTC AAT GGC ATT TAT GGA TTT GC - #C ACT GGT ATC AAAGAC 213 Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Al - #a Thr Gly Ile Lys Asp 810 - # 815 - # 820 - - ATT ATG AAC ATG ATT TTT AAA ACG GAT ACA GG - #T GGT GAT CTA ACC CTA 261 Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gl - #y Gly Asp Leu Thr Leu 825 - # 830 - # 835 - - GAC GAA ATT TTA AAG AAT CAG CAG CTA CTA AA - #T GAT ATT TCT GGT AAA 309 Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu As - #n Asp Ile Ser Gly Lys 840 - # 845 - # 850 - - TTG GAT GGG GTG AAT GGA AGC TTA AAT GAT CT - #T ATC GCA CAG GGA AAC 357 Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Le - #u Ile Ala Gln Gly Asn 855 - # 860 - # 865 - - TTA AAT ACA GAA TTA TCT AAG GAA ATA TTA AA - #A ATT GCA AAT GAA CAA 405 Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Ly - #s Ile Ala Asn Glu Gln 870 8 - #75 8 - #80 8 -#85 - - AAT CAA GTT TTA AAT GAT GTT AAT AAC AAA CT - #C GAT GCG ATA AATACG 453 Asn Gln Val Leu Asn Asp Val Asn Asn Lys Le - #u Asp Ala Ile Asn Thr 890 - # 895 - # 900 - - ATG CTT CGG GTA TAT CTA CCT AAA ATT ACC TC - #T ATG TTG AGT GAT GTA 501 Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Se - #r Met Leu Ser Asp Val 905 - # 910 - # 915 - - ATG AAA CAA AAT TAT GCG CTA AGT CTG CAA AT - #A GAA TAC TTA AGT AAA 549 Met Lys Gln Asn Tyr Ala Leu Ser Leu Gln Il - #e Glu Tyr Leu Ser Lys 920 - # 925 - # 930 - - CAA TTG CAA GAG ATT TCT GAT AAG TTG GAT AT - #T ATT AAT GTA AAT GTA 597 Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Il - #e Ile Asn Val Asn Val 935 - # 940 - # 945 - - CTT ATT AAC TCT ACA CTT ACT GAA ATT ACA CC - #T GCG TAT CAA AGG ATT 645 Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pr - #o Ala Tyr Gln Arg Ile 950 9 - #55 9 - #60 9 -#65 - - AAA TAT GTG AAC GAA AAA TTT GAG GAA TTA AC - #T TTT GCT ACA GAAACT 693 Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Th - #r Phe Ala Thr Glu Thr 970 - # 975 - # 980 - - AGT TCA AAA GTA AAA AAG GAT GGC TCT CCT GC - #A GAT ATT CGT GAT GAG 741 Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Al - #a Asp Ile Arg Asp Glu 985 - # 990 - # 995 - - TTA ACT GAG TTA ACT GAA CTA GCG AAA AGT GT - #A ACA AAA AAT GAT GTG 789 Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Va - #l Thr Lys Asn Asp Val 1000 - # 1005 - # 1010 - - GAT GGT TTT GAA TTT TAC CTT AAT ACA TTC CA - #C GAT GTA ATG GTA GGA 837 Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe Hi - #s Asp Val Met Val Gly 1015 - # 1020 - # 1025 - - AAT AAT TTA TTC GGG CGT TCA GCT TTA AAA AC - #T GCA TCG GAA TTA ATT 885 Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Th - #r Ala Ser Glu Leu Ile 1030 1035 - # 1040 - # 1045 - - ACT AAA GAA AAT GTG AAA ACA AGT GGC AGT GA - #G GTC GGA AAT GTT TAT 933 Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Gl - #u Val Gly Asn Val Tyr 1050 - # 1055 - # 1060 - - AAC TTC CTA ATT GTA TTA ACA GCT CTG CAA GC - #A AAA GCT TTT CTT ACT 981 Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Al - #a Lys Ala Phe Leu Thr 1065 - # 1070 - # 1075 - - TTA ACA CCA TGC CGA AAA TTA TTA GGC TTA GC - #A GAT ATT GAT TAT ACT 1029 Leu Thr Pro Cys Arg Lys Leu Leu Gly Leu Al - #a Asp Ile Asp Tyr Thr 1080 - # 1085 - # 1090 - - TCT ATT ATG AAT GAA CAT TTA AAT AAG GAA AA - #A GAG GAA TTT AGA GTA 1077 Ser Ile Met Asn Glu His Leu Asn Lys Glu Ly - #s Glu Glu Phe Arg Val 1095 - # 1100 - # 1105 - - AAC ATC CTC CCT ACA CTT TCT AAT ACT TTT TC - #T AAT CCT AAT TAT GCA 1125 Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Se - #r Asn Pro Asn Tyr Ala 1110 1115 - # 1120 - # 1125 - - AAA GTT AAA GGA AGT GAT GAA GAT GCA AAG AT - #G ATT GTG GAA GCT AAA 1173 Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Me - #t Ile Val Glu Ala Lys 1130 - # 1135 - # 1140 - - CCA GGA CAT GCA TTG ATT GGG TTT GAA ATT AG - #T AAT GAT TCA ATT ACA 1221 Pro Gly His Ala Leu Ile Gly Phe Glu Ile Se - #r Asn Asp Ser Ile Thr 1145 - # 1150 - # 1155 - - GTA TTA AAA GTA TAT GAG GCT AAG CTA AAA CA - #A AAT TAT CAA GTC GAT 1269 Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gl - #n Asn Tyr Gln Val Asp 1160 - # 1165 - # 1170 - - AAG GAT TCC TTA TCG GAA GTT ATT TAT GGC GA - #T ATG GAT AAA TTA TTG 1317 Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly As - #p Met Asp Lys Leu Leu 1175 - # 1180 - # 1185 - - TGC CCA GAT CAA TCT GGA CAA ATC TAT TAT AC - #A AAT AAC ATA GTA TTT 1365 Cys Pro Asp Gln Ser Gly Gln Ile Tyr Tyr Th - #r Asn Asn Ile Val Phe 1190 1195 - # 1200 - # 1205 - - CCA AAT GAA TAT GTA ATT ACT AAA ATT GAT TT - #C ACT AAA AAA ATG AAA 1413 Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Ph - #e Thr Lys Lys Met Lys 1210 - # 1215 - # 1220 - - ACT TTA AGA TAT GAG GTA ACA GCG AAT TTT TA - #T GAT TCT TCT ACA GGA 1461 Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Ty - #r Asp Ser Ser Thr Gly 1225 - # 1230 - # 1235 - - GAA ATT GAC TTA AAT AAG AAA AAA GTA GAA TC - #A AGT GAA GCG GAG TAT 1509 Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Se - #r Ser Glu Ala Glu Tyr 1240 - # 1245 - # 1250 - - AGA ACG TTA AGT GCT AAT GAT GAT GGG GTG TA - #T ATG CCG TTA GGT GTC 1557 Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Ty - #r Met Pro Leu Gly Val 1255 - # 1260 - # 1265 - - ATC AGT GAA ACA TTT TTG ACT CCG ATT AAT GG - #G TTT GGC CTC CAA GCT 1605 Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gl - #y Phe Gly Leu Gln Ala 1270 1275 - # 1280 - # 1285 - - GAT GAA AAT TCA AGA TTA ATT ACT TTA ACA TG - #T AAA TCA TAT TTA AGA 1653 Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cy - #s Lys Ser Tyr Leu Arg 1290 - # 1295 - # 1300 - - GAA CTA CTG CTA GCA ACA GAC TTA AGC AAT AA - #A GAA ACT AAA TTG ATC 1701 Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Ly - #s Glu Thr Lys Leu Ile 1305 - # 1310 - # 1315 - - GTC CCG CCA AGT GGT TTT ATT AGC AAT ATT GT - #A GAG AAC GGG TCC ATA 1749 Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Va - #l Glu Asn Gly Ser Ile 1320 - # 1325 - # 1330 - - GAA GAG GAC AAT TTA GAG CCG TGG AAA GCA AA - #T AAT AAG AAT GCG TAT 1797 Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala As - #n Asn Lys Asn Ala Tyr 1335 - # 1340 - # 1345 - - GTA GAT CAT ACA GGC GGA GTG AAT GGA ACT AA - #A GCT TTA TAT GTT CAT 1845 Val Asp His Thr Gly Gly Val Asn Gly Thr Ly - #s Ala Leu Tyr Val His 1350 1355 - # 1360 - # 1365 - - AAG GAC GGA GGA ATT TCA CAA TTT ATT GGA GA - #T AAG TTA AAA CCG AAA 1893 Lys Asp Gly Gly Ile Ser Gln Phe Ile Gly As - #p Lys Leu Lys Pro Lys 1370 - # 1375 - # 1380 - - ACT GAG TAT GTA ATC CAA TAT ACT GTT AAA GG - #A AAA CCT TCT ATT CAT 1941 Thr Glu Tyr Val Ile Gln Tyr Thr Val Lys Gl - #y Lys Pro Ser Ile His 1385 - # 1390 - # 1395 - - TTA AAA GAT GAA AAT ACT GGA TAT ATT CAT TA - #T GAA GAT ACA AAT AAT 1989 Leu Lys Asp Glu Asn Thr Gly Tyr Ile His Ty - #r Glu Asp Thr Asn Asn 1400 - # 1405 - # 1410 - - AAT TTA GAA GAT TAT CAA ACT ATT AAT AAA CG - #T TTT ACT ACA GGA ACT 2037 Asn Leu Glu Asp Tyr Gln Thr Ile Asn Lys Ar - #g Phe Thr Thr Gly Thr 1415 - # 1420 - # 1425 - - GAT TTA AAG GGA GTG TAT TTA ATT TTA AAA AG - #T CAA AAT GGA GAT GAA 2085 Asp Leu Lys Gly Val Tyr Leu Ile Leu Lys Se - #r Gln Asn Gly Asp Glu 1430 1435 - # 1440 - # 1445 - - GCT TGG GGA GAT AAC TTT ATT ATT TTG GAA AT - #T AGT CCT TCT GAA AAG 2133 Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Il - #e Ser Pro Ser Glu Lys 1450 - # 1455 - # 1460 - - TTA TTA AGT CCA GAA TTA ATT AAT ACA AAT AA - #T TGG ACG AGT ACG GGA 2181 Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn As - #n Trp Thr Ser Thr Gly 1465 - # 1470 - # 1475 - - TCA ACT AAT ATT AGC GGT AAT ACA CTC ACT CT - #T TAT CAG GGA GGA CGA 2229 Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Le - #u Tyr Gln Gly Gly Arg 1480 - # 1485 - # 1490 - - GGG ATT CTA AAA CAA AAC CTT CAA TTA GAT AG - #T TTT TCA ACT TAT AGA 2277 Gly Ile Leu Lys Gln Asn Leu Gln Leu Asp Se - #r Phe Ser Thr Tyr Arg 1495 - # 1500 - # 1505 - - GTG TAT TTC TCT GTG TCC GGA GAT GCT AAT GT - #A AGG ATT AGA AAT TCT 2325 Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Va - #l Arg Ile Arg Asn Ser 1510 1515 - # 1520 - # 1525 - - AGG GAA GTG TTA TTT GAA AAA AGA TAT ATG AG - #C GGT GCT AAA GAT GTT 2373 Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Se - #r Gly Ala Lys Asp Val 1530 - # 1535 - # 1540 - - TCT GAA ATG TTC ACT ACA AAA TTT GAG AAA GA - #T AAC TTC TAT ATA GAG 2421 Ser Glu Met Phe Thr Thr Lys Phe Glu Lys As - #p Asn Phe Tyr Ile Glu 1545 - # 1550 - # 1555 - - CTT TCT CAA GGG AAT AAT TTA TAT GGT GGT CC - #T ATT GTA CAT TTT TAC 2469 Leu Ser Gln Gly Asn Asn Leu Tyr Gly Gly Pr - #o Ile Val His Phe Tyr 1560 - # 1565 - # 1570 - - GAT GTC TCT ATT AAG TAAGATCGGG ATCTAATATT AACAGTTTT - #T AGAAGCTAAT 2524 Asp Val Ser Ile Lys 1575 - - TCTTGTATAA TGTCCTTGAT TATGGAAAAA CACAATTTTG TTTGCTAAGA TG -#TATATATA 2584 - - GCTCACTCAT TAAAAGGCAA TCAAGCTT - # - # 2612 - - - - (2) INFORMATION FOR SEQ ID NO:32: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 789 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: - - Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Ar - #g Ala Leu Pro Ser Phe 1 5 - # 10 - # 15 - - Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Al - #a Thr Gly Ile Lys Asp 20 - # 25 - # 30 - - Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gl - #y Gly Asp Leu Thr Leu 35 - # 40 - # 45 - - Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu As - #n Asp Ile Ser Gly Lys 50 - # 55 - # 60 - - Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Le - #u Ile Ala Gln Gly Asn 65 - # 70 - # 75 - # 80 - - Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Ly - #s Ile Ala Asn Glu Gln 85 - # 90 - # 95 - - Asn Gln Val Leu Asn Asp Val Asn Asn Lys Le - #u Asp Ala Ile Asn Thr 100 - # 105 - # 110 - - Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Se - #r Met Leu Ser Asp Val 115 - # 120 - # 125 - - Met Lys Gln Asn Tyr Ala Leu Ser Leu Gln Il - #e Glu Tyr Leu Ser Lys 130 - # 135 - # 140 - - Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Il - #e Ile Asn Val Asn Val 145 1 - #50 1 - #55 1 -#60 - - Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pr - #o Ala Tyr Gln ArgIle 165 - # 170 - # 175 - - Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Th - #r Phe Ala Thr Glu Thr 180 - # 185 - # 190 - - Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Al - #a Asp Ile Arg Asp Glu 195 - # 200 - # 205 - - Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Va - #l Thr Lys Asn Asp Val 210 - # 215 - # 220 - - Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe Hi - #s Asp Val Met Val Gly 225 2 - #30 2 - #35 2 -#40 - - Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Th - #r Ala Ser Glu LeuIle 245 - # 250 - # 255 - - Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Gl - #u Val Gly Asn Val Tyr 260 - # 265 - # 270 - - Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Al - #a Lys Ala Phe Leu Thr 275 - # 280 - # 285 - - Leu Thr Pro Cys Arg Lys Leu Leu Gly Leu Al - #a Asp Ile Asp Tyr Thr 290 - # 295 - # 300 - - Ser Ile Met Asn Glu His Leu Asn Lys Glu Ly - #s Glu Glu Phe Arg Val 305 3 - #10 3 - #15 3 -#20 - - Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Se - #r Asn Pro Asn TyrAla 325 - # 330 - # 335 - - Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Me - #t Ile Val Glu Ala Lys 340 - # 345 - # 350 - - Pro Gly His Ala Leu Ile Gly Phe Glu Ile Se - #r Asn Asp Ser Ile Thr 355 - # 360 - # 365 - - Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gl - #n Asn Tyr Gln Val Asp 370 - # 375 - # 380 - - Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly As - #p Met Asp Lys Leu Leu 385 3 - #90 3 - #95 4 -#00 - - Cys Pro Asp Gln Ser Gly Gln Ile Tyr Tyr Th - #r Asn Asn Ile ValPhe 405 - # 410 - # 415 - - Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Ph - #e Thr Lys Lys Met Lys 420 - # 425 - # 430 - - Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Ty - #r Asp Ser Ser Thr Gly 435 - # 440 - # 445 - - Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Se - #r Ser Glu Ala Glu Tyr 450 - # 455 - # 460 - - Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Ty - #r Met Pro Leu Gly Val 465 4 - #70 4 - #75 4 -#80 - - Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gl - #y Phe Gly Leu GlnAla 485 - # 490 - # 495 - - Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cy - #s Lys Ser Tyr Leu Arg 500 - # 505 - # 510 - - Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Ly - #s Glu Thr Lys Leu Ile 515 - # 520 - # 525 - - Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Va - #l Glu Asn Gly Ser Ile 530 - # 535 - # 540 - - Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala As - #n Asn Lys Asn Ala Tyr 545 5 - #50 5 - #55 5 -#60 - - Val Asp His Thr Gly Gly Val Asn Gly Thr Ly - #s Ala Leu Tyr ValHis 565 - # 570 - # 575 - - Lys Asp Gly Gly Ile Ser Gln Phe Ile Gly As - #p Lys Leu Lys Pro Lys 580 - # 585 - # 590 - - Thr Glu Tyr Val Ile Gln Tyr Thr Val Lys Gl - #y Lys Pro Ser Ile His 595 - # 600 - # 605 - - Leu Lys Asp Glu Asn Thr Gly Tyr Ile His Ty - #r Glu Asp Thr Asn Asn 610 - # 615 - # 620 - - Asn Leu Glu Asp Tyr Gln Thr Ile Asn Lys Ar - #g Phe Thr Thr Gly Thr 625 6 - #30 6 - #35 6 -#40 - - Asp Leu Lys Gly Val Tyr Leu Ile Leu Lys Se - #r Gln Asn Gly AspGlu 645 - # 650 - # 655 - - Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Il - #e Ser Pro Ser Glu Lys 660 - # 665 - # 670 - - Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn As - #n Trp Thr Ser Thr Gly 675 - # 680 - # 685 - - Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Le - #u Tyr Gln Gly Gly Arg 690 - # 695 - # 700 - - Gly Ile Leu Lys Gln Asn Leu Gln Leu Asp Se - #r Phe Ser Thr Tyr Arg 705 7 - #10 7 - #15 7 -#20 - - Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Va - #l Arg Ile Arg AsnSer 725 - # 730 - # 735 - - Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Se - #r Gly Ala Lys Asp Val 740 - # 745 - # 750 - - Ser Glu Met Phe Thr Thr Lys Phe Glu Lys As - #p Asn Phe Tyr Ile Glu 755 - # 760 - # 765 - - Leu Ser Gln Gly Asn Asn Leu Tyr Gly Gly Pr - #o Ile Val His Phe Tyr 770 - # 775 - # 780 - - Asp Val Ser Ile Lys 785 - - - - (2) INFORMATION FOR SEQ ID NO:33: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "forward primer used to make pCIB5526" - - (iii) HYPOTHETICAL: NO - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: - - GGATCCACCA TGAAGACCAA CCAGATCAGC - # - # 30 - - - - (2) INFORMATION FOR SEQ ID NO:34: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "reverse primer used to make pCIB5526" - - (iii) HYPOTHETICAL: NO - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: - - AAGCTTCAGC TCCTT - # - # - # 15 - - - - (2) INFORMATION FOR SEQ ID NO:35: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2576 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "Synthetic DNA" - - (iii) HYPOTHETICAL: NO - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 9..2564 (D) OTHER INFORMATION: - #/note= "Maize optimized sequence encoding - #VIP1A(a) with the Bacillus secretion signal removed a - #s contained in pCIB5526" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: - - GATCCACC ATG AAG ACC AAC CAG ATC AGC ACC ACC - #CAG AAG AAC CAG CAG 50 Met Lys Thr Asn Gln I - #le Ser Thr Thr Gln Lys Asn Gln Gln - # 825 - # 830 - #835 - - AAG GAG ATG GAC CGC AAG GGC CTG CTG GGC TA - #C TAC TTC AAG GGCAAG 98 Lys Glu Met Asp Arg Lys Gly Leu Leu Gly Ty - #r Tyr Phe Lys Gly Lys 840 - # 845 - # 850 - - GAC TTC AGC AAC CTG ACC ATG TTC GCC CCC AC - #G CGT GAC AGC ACC CTG 146 Asp Phe Ser Asn Leu Thr Met Phe Ala Pro Th - #r Arg Asp Ser Thr Leu 855 - # 860 - # 865 - - ATC TAC GAC CAG CAG ACC GCC AAC AAG CTG CT - #G GAC AAG AAG CAG CAG 194 Ile Tyr Asp Gln Gln Thr Ala Asn Lys Leu Le - #u Asp Lys Lys Gln Gln 870 - # 875 - # 880 - - GAG TAC CAG AGC ATC CGC TGG ATC GGC CTG AT - #C CAG AGC AAG GAG ACC 242 Glu Tyr Gln Ser Ile Arg Trp Ile Gly Leu Il - #e Gln Ser Lys Glu Thr 885 - # 890 - # 895 - - GGC GAC TTC ACC TTC AAC CTG AGC GAG GAC GA - #G CAG GCC ATC ATC GAG 290 Gly Asp Phe Thr Phe Asn Leu Ser Glu Asp Gl - #u Gln Ala Ile Ile Glu 900 9 - #05 9 - #10 9 -#15 - - ATC AAC GGC AAG ATC ATC AGC AAC AAG GGC AA - #G GAG AAG CAG GTGGTG 338 Ile Asn Gly Lys Ile Ile Ser Asn Lys Gly Ly - #s Glu Lys Gln Val Val 920 - # 925 - # 930 - - CAC CTG GAG AAG GGC AAG CTG GTG CCC ATC AA - #G ATC GAG TAC CAG AGC 386 His Leu Glu Lys Gly Lys Leu Val Pro Ile Ly - #s Ile Glu Tyr Gln Ser 935 - # 940 - # 945 - - GAC ACC AAG TTC AAC ATC GAC AGC AAG ACC TT - #C AAG GAG CTG AAG CTT 434 Asp Thr Lys Phe Asn Ile Asp Ser Lys Thr Ph - #e Lys Glu Leu Lys Leu 950 - # 955 - # 960 - - TTC AAG ATC GAC AGC CAG AAC CAG CCC CAG CA - #G GTG CAG CAG GAC GAG 482 Phe Lys Ile Asp Ser Gln Asn Gln Pro Gln Gl - #n Val Gln Gln Asp Glu 965 - # 970 - # 975 - - CTG CGC AAC CCC GAG TTC AAC AAG AAG GAG AG - #C CAG GAG TTC CTG GCC 530 Leu Arg Asn Pro Glu Phe Asn Lys Lys Glu Se - #r Gln Glu Phe Leu Ala 980 9 - #85 9 - #90 9 -#95 - - AAG CCC AGC AAG ATC AAC CTG TTC ACC CAG CA - #G ATG AAG CGC GAGATC 578 Lys Pro Ser Lys Ile Asn Leu Phe Thr Gln Gl - #n Met Lys Arg Glu Ile 1000 - # 1005 - # 1010 - - GAC GAG GAC ACC GAC ACC GAC GGC GAC AGC AT - #C CCC GAC CTG TGG GAG 626 Asp Glu Asp Thr Asp Thr Asp Gly Asp Ser Il - #e Pro Asp Leu Trp Glu 1015 - # 1020 - # 1025 - - GAG AAC GGC TAC ACC ATC CAG AAC CGC ATC GC - #C GTG AAG TGG GAC GAC 674 Glu Asn Gly Tyr Thr Ile Gln Asn Arg Ile Al - #a Val Lys Trp Asp Asp 1030 - # 1035 - # 1040 - - AGC CTG GCT AGC AAG GGC TAC ACC AAG TTC GT - #G AGC AAC CCC CTG GAG 722 Ser Leu Ala Ser Lys Gly Tyr Thr Lys Phe Va - #l Ser Asn Pro Leu Glu 1045 - # 1050 - # 1055 - - AGC CAC ACC GTG GGC GAC CCC TAC ACC GAC TA - #C GAG AAG GCC GCC CGC 770 Ser His Thr Val Gly Asp Pro Tyr Thr Asp Ty - #r Glu Lys Ala Ala Arg 1060 1065 - # 1070 - # 1075 - - GAC CTG GAC CTG AGC AAC GCC AAG GAG ACC TT - #C AAC CCC CTG GTG GCC 818 Asp Leu Asp Leu Ser Asn Ala Lys Glu Thr Ph - #e Asn Pro Leu Val Ala 1080 - # 1085 - # 1090 - - GCC TTC CCC AGC GTG AAC GTG AGC ATG GAG AA - #G GTG ATC CTG AGC CCC 866 Ala Phe Pro Ser Val Asn Val Ser Met Glu Ly - #s Val Ile Leu Ser Pro 1095 - # 1100 - # 1105 - - AAC GAG AAC CTG AGC AAC AGC GTG GAG AGC CA - #C TCG AGC ACC AAC TGG 914 Asn Glu Asn Leu Ser Asn Ser Val Glu Ser Hi - #s Ser Ser Thr Asn Trp 1110 - # 1115 - # 1120 - - AGC TAC ACC AAC ACC GAG GGC GCC AGC GTG GA - #G GCC GGC ATC GGT CCC 962 Ser Tyr Thr Asn Thr Glu Gly Ala Ser Val Gl - #u Ala Gly Ile Gly Pro 1125 - # 1130 - # 1135 - - AAG GGC ATC AGC TTC GGC GTG AGC GTG AAC TA - #C CAG CAC AGC GAG ACC 1010 Lys Gly Ile Ser Phe Gly Val Ser Val Asn Ty - #r Gln His Ser Glu Thr 1140 1145 - # 1150 - # 1155 - - GTG GCC CAG GAG TGG GGC ACC AGC ACC GGC AA - #C ACC AGC CAG TTC AAC 1058 Val Ala Gln Glu Trp Gly Thr Ser Thr Gly As - #n Thr Ser Gln Phe Asn 1160 - # 1165 - # 1170 - - ACC GCC AGC GCC GGC TAC CTG AAC GCC AAC GT - #G CGC TAC AAC AAC GTG 1106 Thr Ala Ser Ala Gly Tyr Leu Asn Ala Asn Va - #l Arg Tyr Asn Asn Val 1175 - # 1180 - # 1185 - - GGC ACC GGC GCC ATC TAC GAC GTG AAG CCC AC - #C ACC AGC TTC GTG CTG 1154 Gly Thr Gly Ala Ile Tyr Asp Val Lys Pro Th - #r Thr Ser Phe Val Leu 1190 - # 1195 - # 1200 - - AAC AAC GAC ACC ATC GCC ACC ATC ACC GCC AA - #G TCG AAT TCC ACC GCC 1202 Asn Asn Asp Thr Ile Ala Thr Ile Thr Ala Ly - #s Ser Asn Ser Thr Ala 1205 - # 1210 - # 1215 - - CTG AAC ATC AGC CCC GGC GAG AGC TAC CCC AA - #G AAG GGC CAG AAC GGC 1250 Leu Asn Ile Ser Pro Gly Glu Ser Tyr Pro Ly - #s Lys Gly Gln Asn Gly 1220 1225 - # 1230 - # 1235 - - ATC GCC ATC ACC AGC ATG GAC GAC TTC AAC AG - #C CAC CCC ATC ACC CTG 1298 Ile Ala Ile Thr Ser Met Asp Asp Phe Asn Se - #r His Pro Ile Thr Leu 1240 - # 1245 - # 1250 - - AAC AAG AAG CAG GTG GAC AAC CTG CTG AAC AA - #C AAG CCC ATG ATG CTG 1346 Asn Lys Lys Gln Val Asp Asn Leu Leu Asn As - #n Lys Pro Met Met Leu 1255 - # 1260 - # 1265 - - GAG ACC AAC CAG ACC GAC GGC GTC TAC AAG AT - #C AAG GAC ACC CAC GGC 1394 Glu Thr Asn Gln Thr Asp Gly Val Tyr Lys Il - #e Lys Asp Thr His Gly 1270 - # 1275 - # 1280 - - AAC ATC GTG ACG GGC GGC GAG TGG AAC GGC GT - #G ATC CAG CAG ATC AAG 1442 Asn Ile Val Thr Gly Gly Glu Trp Asn Gly Va - #l Ile Gln Gln Ile Lys 1285 - # 1290 - # 1295 - - GCC AAG ACC GCC AGC ATC ATC GTC GAC GAC GG - #C GAG CGC GTG GCC GAG 1490 Ala Lys Thr Ala Ser Ile Ile Val Asp Asp Gl - #y Glu Arg Val Ala Glu 1300 1305 - # 1310 - # 1315 - - AAG CGC GTG GCC GCC AAG GAC TAC GAG AAC CC - #C GAG GAC AAG ACC CCC 1538 Lys Arg Val Ala Ala Lys Asp Tyr Glu Asn Pr - #o Glu Asp Lys Thr Pro 1320 - # 1325 - # 1330 - - AGC CTG ACC CTG AAG GAC GCC CTG AAG CTG AG - #C TAC CCC GAC GAG ATC 1586 Ser Leu Thr Leu Lys Asp Ala Leu Lys Leu Se - #r Tyr Pro Asp Glu Ile 1335 - # 1340 - # 1345 - - AAG GAG ATC GAG GGC TTG CTG TAC TAC AAG AA - #C AAG CCC ATC TAC GAG 1634 Lys Glu Ile Glu Gly Leu Leu Tyr Tyr Lys As - #n Lys Pro Ile Tyr Glu 1350 - # 1355 - # 1360 - - AGC AGC GTG ATG ACC TAT CTA GAC GAG AAC AC - #C GCC AAG GAG GTG ACC 1682 Ser Ser Val Met Thr Tyr Leu Asp Glu Asn Th - #r Ala Lys Glu Val Thr 1365 - # 1370 - # 1375 - - AAG CAG CTG AAC GAC ACC ACC GGC AAG TTC AA - #G GAC GTG AGC CAC CTG 1730 Lys Gln Leu Asn Asp Thr Thr Gly Lys Phe Ly - #s Asp Val Ser His Leu 1380 1385 - # 1390 - # 1395 - - TAC GAC GTG AAG CTG ACC CCC AAG ATG AAC GT - #G ACC ATC AAG CTG AGC 1778 Tyr Asp Val Lys Leu Thr Pro Lys Met Asn Va - #l Thr Ile Lys Leu Ser 1400 - # 1405 - # 1410 - - ATC CTG TAC GAC AAC GCC GAG AGC AAC GAC AA - #C AGC ATC GGC AAG TGG 1826 Ile Leu Tyr Asp Asn Ala Glu Ser Asn Asp As - #n Ser Ile Gly Lys Trp 1415 - # 1420 - # 1425 - - ACC AAC ACC AAC ATC GTG AGC GGC GGC AAC AA - #C GGC AAG AAG CAG TAC 1874 Thr Asn Thr Asn Ile Val Ser Gly Gly Asn As - #n Gly Lys Lys Gln Tyr 1430 - # 1435 - # 1440 - - AGC AGC AAC AAC CCC GAC GCC AAC CTG ACC CT - #G AAC ACC GAC GCC CAG 1922 Ser Ser Asn Asn Pro Asp Ala Asn Leu Thr Le - #u Asn Thr Asp Ala Gln 1445 - # 1450 - # 1455 - - GAG AAG CTG AAC AAG AAC CGC GAC TAC TAC AT - #C AGC CTG TAC ATG AAG 1970 Glu Lys Leu Asn Lys Asn Arg Asp Tyr Tyr Il - #e Ser Leu Tyr Met Lys 1460 1465 - # 1470 - # 1475 - - AGC GAG AAG AAC ACC CAG TGC GAG ATC ACC AT - #C GAC GGC GAG ATA TAC 2018 Ser Glu Lys Asn Thr Gln Cys Glu Ile Thr Il - #e Asp Gly Glu Ile Tyr 1480 - # 1485 - # 1490 - - CCC ATC ACC ACC AAG ACC GTG AAC GTG AAC AA - #G GAC AAC TAC AAG CGC 2066 Pro Ile Thr Thr Lys Thr Val Asn Val Asn Ly - #s Asp Asn Tyr Lys Arg 1495 - # 1500 - # 1505 - - CTG GAC ATC ATC GCC CAC AAC ATC AAG AGC AA - #C CCC ATC AGC AGC CTG 2114 Leu Asp Ile Ile Ala His Asn Ile Lys Ser As - #n Pro Ile Ser Ser Leu 1510 - # 1515 - # 1520 - - CAC ATC AAG ACC AAC GAC GAG ATC ACC CTG TT - #C TGG GAC GAC ATA TCG 2162 His Ile Lys Thr Asn Asp Glu Ile Thr Leu Ph - #e Trp Asp Asp Ile Ser 1525 - # 1530 - # 1535 - - ATT ACC GAC GTC GCC AGC ATC AAG CCC GAG AA - #C CTG ACC GAC AGC GAG 2210 Ile Thr Asp Val Ala Ser Ile Lys Pro Glu As - #n Leu Thr Asp Ser Glu 1540 1545 - # 1550 - # 1555 - - ATC AAG CAG ATA TAC AGT CGC TAC GGC ATC AA - #G CTG GAG GAC GGC ATC 2258 Ile Lys Gln Ile Tyr Ser Arg Tyr Gly Ile Ly - #s Leu Glu Asp Gly Ile 1560 - # 1565 - # 1570 - - CTG ATC GAC AAG AAA GGC GGC ATC CAC TAC GG - #C GAG TTC ATC AAC GAG 2306 Leu Ile Asp Lys Lys Gly Gly Ile His Tyr Gl - #y Glu Phe Ile Asn Glu 1575 - # 1580 - # 1585 - - GCC AGC TTC AAC ATC GAG CCC CTG CAG AAC TA - #C GTG ACC AAG TAC GAG 2354 Ala Ser Phe Asn Ile Glu Pro Leu Gln Asn Ty - #r Val Thr Lys Tyr Glu 1590 - # 1595 - # 1600 - - GTG ACC TAC AGC AGC GAG CTG GGC CCC AAC GT - #G AGC GAC ACC CTG GAG 2402 Val Thr Tyr Ser Ser Glu Leu Gly Pro Asn Va - #l Ser Asp Thr Leu Glu 1605 - # 1610 - # 1615 - - AGC GAC AAG ATT TAC AAG GAC GGC ACC ATC AA - #G TTC GAC TTC ACC AAG 2450 Ser Asp Lys Ile Tyr Lys Asp Gly Thr Ile Ly - #s Phe Asp Phe Thr Lys 1620 1625 - # 1630 - # 1635 - - TAC AGC AAG AAC GAG CAG GGC CTG TTC TAC GA - #C AGC GGC CTG AAC TGG 2498 Tyr Ser Lys Asn Glu Gln Gly Leu Phe Tyr As - #p Ser Gly Leu Asn Trp 1640 - # 1645 - # 1650 - - GAC TTC AAG ATC AAC GCC ATC ACC TAC GAC GG - #C AAG GAG ATG AAC GTG 2546 Asp Phe Lys Ile Asn Ala Ile Thr Tyr Asp Gl - #y Lys Glu Met Asn Val 1655 - # 1660 - # 1665 - - TTC CAC CGC TAC AAC AAG TAGATCTGAG CT - # - # 2576 Phe His Arg Tyr Asn Lys 1670 - - - - (2) INFORMATION FOR SEQ ID NO:36: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 852 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: - - Met Lys Thr Asn Gln Ile Ser Thr Thr Gln Ly - #s Asn Gln Gln Lys Glu 1 5 - # 10 - # 15 - - Met Asp Arg Lys Gly Leu Leu Gly Tyr Tyr Ph - #e Lys Gly Lys Asp Phe 20 - # 25 - # 30 - - Ser Asn Leu Thr Met Phe Ala Pro Thr Arg As - #p Ser Thr Leu Ile Tyr 35 - # 40 - # 45 - - Asp Gln Gln Thr Ala Asn Lys Leu Leu Asp Ly - #s Lys Gln Gln Glu Tyr 50 - # 55 - # 60 - - Gln Ser Ile Arg Trp Ile Gly Leu Ile Gln Se - #r Lys Glu Thr Gly Asp 65 - # 70 - # 75 - # 80 - - Phe Thr Phe Asn Leu Ser Glu Asp Glu Gln Al - #a Ile Ile Glu Ile Asn 85 - # 90 - # 95 - - Gly Lys Ile Ile Ser Asn Lys Gly Lys Glu Ly - #s Gln Val Val His Leu 100 - # 105 - # 110 - - Glu Lys Gly Lys Leu Val Pro Ile Lys Ile Gl - #u Tyr Gln Ser Asp Thr 115 - # 120 - # 125 - - Lys Phe Asn Ile Asp Ser Lys Thr Phe Lys Gl - #u Leu Lys Leu Phe Lys 130 - # 135 - # 140 - - Ile Asp Ser Gln Asn Gln Pro Gln Gln Val Gl - #n Gln Asp Glu Leu Arg 145 1 - #50 1 - #55 1 -#60 - - Asn Pro Glu Phe Asn Lys Lys Glu Ser Gln Gl - #u Phe Leu Ala LysPro 165 - # 170 - # 175 - - Ser Lys Ile Asn Leu Phe Thr Gln Gln Met Ly - #s Arg Glu Ile Asp Glu 180 - # 185 - # 190 - - Asp Thr Asp Thr Asp Gly Asp Ser Ile Pro As - #p Leu Trp Glu Glu Asn 195 - # 200 - # 205 - - Gly Tyr Thr Ile Gln Asn Arg Ile Ala Val Ly - #s Trp Asp Asp Ser Leu 210 - # 215 - # 220 - - Ala Ser Lys Gly Tyr Thr Lys Phe Val Ser As - #n Pro Leu Glu Ser His 225 2 - #30 2 - #35 2 -#40 - - Thr Val Gly Asp Pro Tyr Thr Asp Tyr Glu Ly - #s Ala Ala Arg AspLeu 245 - # 250 - # 255 - - Asp Leu Ser Asn Ala Lys Glu Thr Phe Asn Pr - #o Leu Val Ala Ala Phe 260 - # 265 - # 270 - - Pro Ser Val Asn Val Ser Met Glu Lys Val Il - #e Leu Ser Pro Asn Glu 275 - # 280 - # 285 - - Asn Leu Ser Asn Ser Val Glu Ser His Ser Se - #r Thr Asn Trp Ser Tyr 290 - # 295 - # 300 - - Thr Asn Thr Glu Gly Ala Ser Val Glu Ala Gl - #y Ile Gly Pro Lys Gly 305 3 - #10 3 - #15 3 -#20 - - Ile Ser Phe Gly Val Ser Val Asn Tyr Gln Hi - #s Ser Glu Thr ValAla 325 - # 330 - # 335 - - Gln Glu Trp Gly Thr Ser Thr Gly Asn Thr Se - #r Gln Phe Asn Thr Ala 340 - # 345 - # 350 - - Ser Ala Gly Tyr Leu Asn Ala Asn Val Arg Ty - #r Asn Asn Val Gly Thr 355 - # 360 - # 365 - - Gly Ala Ile Tyr Asp Val Lys Pro Thr Thr Se - #r Phe Val Leu Asn Asn 370 - # 375 - # 380 - - Asp Thr Ile Ala Thr Ile Thr Ala Lys Ser As - #n Ser Thr Ala Leu Asn 385 3 - #90 3 - #95 4 -#00 - - Ile Ser Pro Gly Glu Ser Tyr Pro Lys Lys Gl - #y Gln Asn Gly IleAla 405 - # 410 - # 415 - - Ile Thr Ser Met Asp Asp Phe Asn Ser His Pr - #o Ile Thr Leu Asn Lys 420 - # 425 - # 430 - - Lys Gln Val Asp Asn Leu Leu Asn Asn Lys Pr - #o Met Met Leu Glu Thr 435 - # 440 - # 445 - - Asn Gln Thr Asp Gly Val Tyr Lys Ile Lys As - #p Thr His Gly Asn Ile 450 - # 455 - # 460 - - Val Thr Gly Gly Glu Trp Asn Gly Val Ile Gl - #n Gln Ile Lys Ala Lys 465 4 - #70 4 - #75 4 -#80 - - Thr Ala Ser Ile Ile Val Asp Asp Gly Glu Ar - #g Val Ala Glu LysArg 485 - # 490 - # 495 - - Val Ala Ala Lys Asp Tyr Glu Asn Pro Glu As - #p Lys Thr Pro Ser Leu 500 - # 505 - # 510 - - Thr Leu Lys Asp Ala Leu Lys Leu Ser Tyr Pr - #o Asp Glu Ile Lys Glu 515 - # 520 - # 525 - - Ile Glu Gly Leu Leu Tyr Tyr Lys Asn Lys Pr - #o Ile Tyr Glu Ser Ser 530 - # 535 - # 540 - - Val Met Thr Tyr Leu Asp Glu Asn Thr Ala Ly - #s Glu Val Thr Lys Gln 545 5 - #50 5 - #55 5 -#60 - - Leu Asn Asp Thr Thr Gly Lys Phe Lys Asp Va - #l Ser His Leu TyrAsp 565 - # 570 - # 575 - - Val Lys Leu Thr Pro Lys Met Asn Val Thr Il - #e Lys Leu Ser Ile Leu 580 - # 585 - # 590 - - Tyr Asp Asn Ala Glu Ser Asn Asp Asn Ser Il - #e Gly Lys Trp Thr Asn 595 - # 600 - # 605 - - Thr Asn Ile Val Ser Gly Gly Asn Asn Gly Ly - #s Lys Gln Tyr Ser Ser 610 - # 615 - # 620 - - Asn Asn Pro Asp Ala Asn Leu Thr Leu Asn Th - #r Asp Ala Gln Glu Lys 625 6 - #30 6 - #35 6 -#40 - - Leu Asn Lys Asn Arg Asp Tyr Tyr Ile Ser Le - #u Tyr Met Lys SerGlu 645 - # 650 - # 655 - - Lys Asn Thr Gln Cys Glu Ile Thr Ile Asp Gl - #y Glu Ile Tyr Pro Ile 660 - # 665 - # 670 - - Thr Thr Lys Thr Val Asn Val Asn Lys Asp As - #n Tyr Lys Arg Leu Asp 675 - # 680 - # 685 - - Ile Ile Ala His Asn Ile Lys Ser Asn Pro Il - #e Ser Ser Leu His Ile 690 - # 695 - # 700 - - Lys Thr Asn Asp Glu Ile Thr Leu Phe Trp As - #p Asp Ile Ser Ile Thr 705 7 - #10 7 - #15 7 -#20 - - Asp Val Ala Ser Ile Lys Pro Glu Asn Leu Th - #r Asp Ser Glu IleLys 725 - # 730 - # 735 - - Gln Ile Tyr Ser Arg Tyr Gly Ile Lys Leu Gl - #u Asp Gly Ile Leu Ile 740 - # 745 - # 750 - - Asp Lys Lys Gly Gly Ile His Tyr Gly Glu Ph - #e Ile Asn Glu Ala Ser 755 - # 760 - # 765 - - Phe Asn Ile Glu Pro Leu Gln Asn Tyr Val Th - #r Lys Tyr Glu Val Thr 770 - # 775 - # 780 - - Tyr Ser Ser Glu Leu Gly Pro Asn Val Ser As - #p Thr Leu Glu Ser Asp 785 7 - #90 7 - #95 8 -#00 - - Lys Ile Tyr Lys Asp Gly Thr Ile Lys Phe As - #p Phe Thr Lys TyrSer 805 - # 810 - # 815 - - Lys Asn Glu Gln Gly Leu Phe Tyr Asp Ser Gl - #y Leu Asn Trp Asp Phe 820 - # 825 - # 830 - - Lys Ile Asn Ala Ile Thr Tyr Asp Gly Lys Gl - #u Met Asn Val Phe His 835 - # 840 - # 845 - - Arg Tyr Asn Lys 850 - - - - (2) INFORMATION FOR SEQ ID NO:37: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "forward primer used to make pCIB5527" - - (iii) HYPOTHETICAL: NO - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: - - GGATCCACCA TGCTGCAGAA CCTGAAGATC AC - # - # 32 - - - - (2) INFORMATION FOR SEQ ID NO:38: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "reverse primer used to make pCIB5527" - - (iii) HYPOTHETICAL: NO - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: - - AAGCTTCCAC TCCTTCTC - # - # - # 18 - - - - (2) INFORMATION FOR SEQ ID NO:39: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1241 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "Synthetic DNA" - - (iii) HYPOTHETICAL: NO - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 9..1238 (D) OTHER INFORMATION: - #/note= "Maize optimized DNA sequence - #encoding VIP2A(a) with the Bacillus secretion signal re - #moved as contained in pCIB5527" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: - - GATCCACC ATG CTG CAG AAC CTG AAG ATC ACC GAC - #AAG GTG GAG GAC TTC 50 Met Leu Gln Asn Leu L - #ys Ile Thr Asp Lys Val Glu Asp Phe 855 - # 860 - # 865 - - AAG GAG GAC AAG GAG AAG GCC AAG GAG TGG GG - #C AAG GAG AAG GAG AAG 98 Lys Glu Asp Lys Glu Lys Ala Lys Glu Trp Gl - #y Lys Glu Lys Glu Lys 870 - # 875 - # 880 - - GAG TGG AAG CTT ACC GCC ACC GAG AAG GGC AA - #G ATG AAC AAC TTC CTG 146 Glu Trp Lys Leu Thr Ala Thr Glu Lys Gly Ly - #s Met Asn Asn Phe Leu 885 - # 890 - # 895 - - GAC AAC AAG AAC GAC ATC AAG ACC AAC TAC AA - #G GAG ATC ACC TTC AGC 194 Asp Asn Lys Asn Asp Ile Lys Thr Asn Tyr Ly - #s Glu Ile Thr Phe Ser 900 - # 905 - # 910 - - ATA GCC GGC AGC TTC GAG GAC GAG ATC AAG GA - #C CTG AAG GAG ATC GAC 242 Ile Ala Gly Ser Phe Glu Asp Glu Ile Lys As - #p Leu Lys Glu Ile Asp 915 9 - #20 9 - #25 9 -#30 - - AAG ATG TTC GAC AAG ACC AAC CTG AGC AAC AG - #C ATC ATC ACC TACAAG 290 Lys Met Phe Asp Lys Thr Asn Leu Ser Asn Se - #r Ile Ile Thr Tyr Lys 935 - # 940 - # 945 - - AAC GTG GAG CCC ACC ACC ATC GGC TTC AAC AA - #G AGC CTG ACC GAG GGC 338 Asn Val Glu Pro Thr Thr Ile Gly Phe Asn Ly - #s Ser Leu Thr Glu Gly 950 - # 955 - # 960 - - AAC ACC ATC AAC AGC GAC GCC ATG GCC CAG TT - #C AAG GAG CAG TTC CTG 386 Asn Thr Ile Asn Ser Asp Ala Met Ala Gln Ph - #e Lys Glu Gln Phe Leu 965 - # 970 - # 975 - - GAC CGC GAC ATC AAG TTC GAC AGC TAC CTG GA - #C ACC CAC CTG ACC GCC 434 Asp Arg Asp Ile Lys Phe Asp Ser Tyr Leu As - #p Thr His Leu Thr Ala 980 - # 985 - # 990 - - CAG CAG GTG AGC AGC AAG GAG CGC GTG ATC CT - #G AAG GTG ACC GTC CCC 482 Gln Gln Val Ser Ser Lys Glu Arg Val Ile Le - #u Lys Val Thr Val Pro 995 1 - #000 1005 - # 1010 - - AGC GGC AAG GGC AGC ACC ACC CCC ACC AAG GC - #C GGC GTG ATC CTG AAC 530 Ser Gly Lys Gly Ser Thr Thr Pro Thr Lys Al - #a Gly Val Ile Leu Asn 1015 - # 1020 - # 1025 - - AAC AGC GAG TAC AAG ATG CTG ATC GAC AAC GG - #C TAC ATG GTG CAC GTG 578 Asn Ser Glu Tyr Lys Met Leu Ile Asp Asn Gl - #y Tyr Met Val His Val 1030 - # 1035 - # 1040 - - GAC AAG GTG AGC AAG GTG GTG AAG AAG GGC GT - #G GAG TGC CTC CAG ATC 626 Asp Lys Val Ser Lys Val Val Lys Lys Gly Va - #l Glu Cys Leu Gln Ile 1045 - # 1050 - # 1055 - - GAG GGC ACC CTG AAG AAG AGT CTA GAC TTC AA - #G AAC GAC ATC AAC GCC 674 Glu Gly Thr Leu Lys Lys Ser Leu Asp Phe Ly - #s Asn Asp Ile Asn Ala 1060 - # 1065 - # 1070 - - GAG GCC CAC AGC TGG GGC ATG AAG AAC TAC GA - #G GAG TGG GCC AAG GAC 722 Glu Ala His Ser Trp Gly Met Lys Asn Tyr Gl - #u Glu Trp Ala Lys Asp 1075 1080 - # 1085 - # 1090 - - CTG ACC GAC AGC CAG CGC GAG GCC CTG GAC GG - #C TAC GCC CGC CAG GAC 770 Leu Thr Asp Ser Gln Arg Glu Ala Leu Asp Gl - #y Tyr Ala Arg Gln Asp 1095 - # 1100 - # 1105 - - TAC AAG GAG ATC AAC AAC TAC CTG CGC AAC CA - #G GGC GGC AGC GGC AAC 818 Tyr Lys Glu Ile Asn Asn Tyr Leu Arg Asn Gl - #n Gly Gly Ser Gly Asn 1110 - # 1115 - # 1120 - - GAG AAG CTG GAC GCC CAG ATC AAG AAC ATC AG - #C GAC GCC CTG GGC AAG 866 Glu Lys Leu Asp Ala Gln Ile Lys Asn Ile Se - #r Asp Ala Leu Gly Lys 1125 - # 1130 - # 1135 - - AAG CCC ATC CCC GAG AAC ATC ACC GTG TAC CG - #C TGG TGC GGC ATG CCC 914 Lys Pro Ile Pro Glu Asn Ile Thr Val Tyr Ar - #g Trp Cys Gly Met Pro 1140 - # 1145 - # 1150 - - GAG TTC GGC TAC CAG ATC AGC GAC CCC CTG CC - #C AGC CTG AAG GAC TTC 962 Glu Phe Gly Tyr Gln Ile Ser Asp Pro Leu Pr - #o Ser Leu Lys Asp Phe 1155 1160 - # 1165 - # 1170 - - GAG GAG CAG TTC CTG AAC ACC ATC AAG GAG GA - #C AAG GGC TAC ATG AGC 1010 Glu Glu Gln Phe Leu Asn Thr Ile Lys Glu As - #p Lys Gly Tyr Met Ser 1175 - # 1180 - # 1185 - - ACC AGC CTG AGC AGC GAG CGC CTG GCC GCC TT - #C GGC AGC CGC AAG ATC 1058 Thr Ser Leu Ser Ser Glu Arg Leu Ala Ala Ph - #e Gly Ser Arg Lys Ile 1190 - # 1195 - # 1200 - - ATC CTG CGC CTG CAG GTG CCC AAG GGC AGC AC - #T GGT GCC TAC CTG AGC 1106 Ile Leu Arg Leu Gln Val Pro Lys Gly Ser Th - #r Gly Ala Tyr Leu Ser 1205 - # 1210 - # 1215 - - GCC ATC GGC GGC TTC GCC AGC GAG AAG GAG AT - #C CTG CTG GAT AAG GAC 1154 Ala Ile Gly Gly Phe Ala Ser Glu Lys Glu Il - #e Leu Leu Asp Lys Asp 1220 - # 1225 - # 1230 - - AGC AAG TAC CAC ATC GAC AAG GTG ACC GAG GT - #G ATC ATC AAG GGC GTG 1202 Ser Lys Tyr His Ile Asp Lys Val Thr Glu Va - #l Ile Ile Lys Gly Val 1235 1240 - # 1245 - # 1250 - - AAG CGC TAC GTG GTG GAC GCC ACC CTG CTG AC - #C AAC TAG - # 1241 Lys Arg Tyr Val Val Asp Ala Thr Leu Leu Th - #r Asn 1255 - # 1260 - - - - (2) INFORMATION FOR SEQ ID NO:40: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 410 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: - - Met Leu Gln Asn Leu Lys Ile Thr Asp Lys Va - #l Glu Asp Phe Lys Glu 1 5 - # 10 - # 15 - - Asp Lys Glu Lys Ala Lys Glu Trp Gly Lys Gl - #u Lys Glu Lys Glu Trp 20 - # 25 - # 30 - - Lys Leu Thr Ala Thr Glu Lys Gly Lys Met As - #n Asn Phe Leu Asp Asn 35 - # 40 - # 45 - - Lys Asn Asp Ile Lys Thr Asn Tyr Lys Glu Il - #e Thr Phe Ser Ile Ala 50 - # 55 - # 60 - - Gly Ser Phe Glu Asp Glu Ile Lys Asp Leu Ly - #s Glu Ile Asp Lys Met 65 - # 70 - # 75 - # 80 - - Phe Asp Lys Thr Asn Leu Ser Asn Ser Ile Il - #e Thr Tyr Lys Asn Val 85 - # 90 - # 95 - - Glu Pro Thr Thr Ile Gly Phe Asn Lys Ser Le - #u Thr Glu Gly Asn Thr 100 - # 105 - # 110 - - Ile Asn Ser Asp Ala Met Ala Gln Phe Lys Gl - #u Gln Phe Leu Asp Arg 115 - # 120 - # 125 - - Asp Ile Lys Phe Asp Ser Tyr Leu Asp Thr Hi - #s Leu Thr Ala Gln Gln 130 - # 135 - # 140 - - Val Ser Ser Lys Glu Arg Val Ile Leu Lys Va - #l Thr Val Pro Ser Gly 145 1 - #50 1 - #55 1 -#60 - - Lys Gly Ser Thr Thr Pro Thr Lys Ala Gly Va - #l Ile Leu Asn AsnSer 165 - # 170 - # 175 - - Glu Tyr Lys Met Leu Ile Asp Asn Gly Tyr Me - #t Val His Val Asp Lys 180 - # 185 - # 190 - - Val Ser Lys Val Val Lys Lys Gly Val Glu Cy - #s Leu Gln Ile Glu Gly 195 - # 200 - # 205 - - Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn As - #p Ile Asn Ala Glu Ala 210 - # 215 - # 220 - - His Ser Trp Gly Met Lys Asn Tyr Glu Glu Tr - #p Ala Lys Asp Leu Thr 225 2 - #30 2 - #35 2 -#40 - - Asp Ser Gln Arg Glu Ala Leu Asp Gly Tyr Al - #a Arg Gln Asp TyrLys 245 - # 250 - # 255 - - Glu Ile Asn Asn Tyr Leu Arg Asn Gln Gly Gl - #y Ser Gly Asn Glu Lys 260 - # 265 - # 270 - - Leu Asp Ala Gln Ile Lys Asn Ile Ser Asp Al - #a Leu Gly Lys Lys Pro 275 - # 280 - # 285 - - Ile Pro Glu Asn Ile Thr Val Tyr Arg Trp Cy - #s Gly Met Pro Glu Phe 290 - # 295 - # 300 - - Gly Tyr Gln Ile Ser Asp Pro Leu Pro Ser Le - #u Lys Asp Phe Glu Glu 305 3 - #10 3 - #15 3 -#20 - - Gln Phe Leu Asn Thr Ile Lys Glu Asp Lys Gl - #y Tyr Met Ser ThrSer 325 - # 330 - # 335 - - Leu Ser Ser Glu Arg Leu Ala Ala Phe Gly Se - #r Arg Lys Ile Ile Leu 340 - # 345 - # 350 - - Arg Leu Gln Val Pro Lys Gly Ser Thr Gly Al - #a Tyr Leu Ser Ala Ile 355 - # 360 - # 365 - - Gly Gly Phe Ala Ser Glu Lys Glu Ile Leu Le - #u Asp Lys Asp Ser Lys 370 - # 375 - # 380 - - Tyr His Ile Asp Lys Val Thr Glu Val Ile Il - #e Lys Gly Val Lys Arg 385 3 - #90 3 - #95 4 -#00 - - Tyr Val Val Asp Ala Thr Leu Leu Thr Asn 405 - # 410 - - - - (2) INFORMATION FOR SEQ ID NO:41: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 72 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "oligonucleotide encoding eukaryotic - #secretion signal used to construct pCIB5527" - - (iii) HYPOTHETICAL: NO - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: - - GGATCCACCA TGGGCTGGAG CTGGATCTTC CTGTTCCTGC TGAGCGGCGC CG -#CGGGCGTG 60 - - CACTGCCTGC AG - # - # - # 72 - - - - (2) INFORMATION FOR SEQ ID NO:42: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1241 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "Synthetic DNA" - - (iii) HYPOTHETICAL: NO - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 9..1238 (D) OTHER INFORMATION: - #/note= "Maize optimized DNA sequence - #encoding VIP2A(a) with the Bacillus secretion signal re - #moved and the eukaryotic secretion signal inserted - #as contained in pCIB5528" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: - - GATCCACC ATG CTG CAG AAC CTG AAG ATC ACC GAC - #AAG GTG GAG GAC TTC 50 Met Leu Gln Asn Leu L - #ys Ile Thr Asp Lys Val Glu Asp Phe - # 415 - # 420 - - AAG GAG GAC AAG GAG AAG GCC AAG GAG TGG GG - #C AAG GAG AAG GAG AAG 98 Lys Glu Asp Lys Glu Lys Ala Lys Glu Trp Gl - #y Lys Glu Lys Glu Lys 425 4 - #30 4 - #35 4 -#40 - - GAG TGG AAG CTT ACC GCC ACC GAG AAG GGC AA - #G ATG AAC AAC TTCCTG 146 Glu Trp Lys Leu Thr Ala Thr Glu Lys Gly Ly - #s Met Asn Asn Phe Leu 445 - # 450 - # 455 - - GAC AAC AAG AAC GAC ATC AAG ACC AAC TAC AA - #G GAG ATC ACC TTC AGC 194 Asp Asn Lys Asn Asp Ile Lys Thr Asn Tyr Ly - #s Glu Ile Thr Phe Ser 460 - # 465 - # 470 - - ATA GCC GGC AGC TTC GAG GAC GAG ATC AAG GA - #C CTG AAG GAG ATC GAC 242 Ile Ala Gly Ser Phe Glu Asp Glu Ile Lys As - #p Leu Lys Glu Ile Asp 475 - # 480 - # 485 - - AAG ATG TTC GAC AAG ACC AAC CTG AGC AAC AG - #C ATC ATC ACC TAC AAG 290 Lys Met Phe Asp Lys Thr Asn Leu Ser Asn Se - #r Ile Ile Thr Tyr Lys 490 - # 495 - # 500 - - AAC GTG GAG CCC ACC ACC ATC GGC TTC AAC AA - #G AGC CTG ACC GAG GGC 338 Asn Val Glu Pro Thr Thr Ile Gly Phe Asn Ly - #s Ser Leu Thr Glu Gly 505 5 - #10 5 - #15 5 -#20 - - AAC ACC ATC AAC AGC GAC GCC ATG GCC CAG TT - #C AAG GAG CAG TTCCTG 386 Asn Thr Ile Asn Ser Asp Ala Met Ala Gln Ph - #e Lys Glu Gln Phe Leu 525 - # 530 - # 535 - - GAC CGC GAC ATC AAG TTC GAC AGC TAC CTG GA - #C ACC CAC CTG ACC GCC 434 Asp Arg Asp Ile Lys Phe Asp Ser Tyr Leu As - #p Thr His Leu Thr Ala 540 - # 545 - # 550 - - CAG CAG GTG AGC AGC AAG GAG CGC GTG ATC CT - #G AAG GTG ACC GTC CCC 482 Gln Gln Val Ser Ser Lys Glu Arg Val Ile Le - #u Lys Val Thr Val Pro 555 - # 560 - # 565 - - AGC GGC AAG GGC AGC ACC ACC CCC ACC AAG GC - #C GGC GTG ATC CTG AAC 530 Ser Gly Lys Gly Ser Thr Thr Pro Thr Lys Al - #a Gly Val Ile Leu Asn 570 - # 575 - # 580 - - AAC AGC GAG TAC AAG ATG CTG ATC GAC AAC GG - #C TAC ATG GTG CAC GTG 578 Asn Ser Glu Tyr Lys Met Leu Ile Asp Asn Gl - #y Tyr Met Val His Val 585 5 - #90 5 - #95 6 -#00 - - GAC AAG GTG AGC AAG GTG GTG AAG AAG GGC GT - #G GAG TGC CTC CAGATC 626 Asp Lys Val Ser Lys Val Val Lys Lys Gly Va - #l Glu Cys Leu Gln Ile 605 - # 610 - # 615 - - GAG GGC ACC CTG AAG AAG AGT CTA GAC TTC AA - #G AAC GAC ATC AAC GCC 674 Glu Gly Thr Leu Lys Lys Ser Leu Asp Phe Ly - #s Asn Asp Ile Asn Ala 620 - # 625 - # 630 - - GAG GCC CAC AGC TGG GGC ATG AAG AAC TAC GA - #G GAG TGG GCC AAG GAC 722 Glu Ala His Ser Trp Gly Met Lys Asn Tyr Gl - #u Glu Trp Ala Lys Asp 635 - # 640 - # 645 - - CTG ACC GAC AGC CAG CGC GAG GCC CTG GAC GG - #C TAC GCC CGC CAG GAC 770 Leu Thr Asp Ser Gln Arg Glu Ala Leu Asp Gl - #y Tyr Ala Arg Gln Asp 650 - # 655 - # 660 - - TAC AAG GAG ATC AAC AAC TAC CTG CGC AAC CA - #G GGC GGC AGC GGC AAC 818 Tyr Lys Glu Ile Asn Asn Tyr Leu Arg Asn Gl - #n Gly Gly Ser Gly Asn 665 6 - #70 6 - #75 6 -#80 - - GAG AAG CTG GAC GCC CAG ATC AAG AAC ATC AG - #C GAC GCC CTG GGCAAG 866 Glu Lys Leu Asp Ala Gln Ile Lys Asn Ile Se - #r Asp Ala Leu Gly Lys 685 - # 690 - # 695 - - AAG CCC ATC CCC GAG AAC ATC ACC GTG TAC CG - #C TGG TGC GGC ATG CCC 914 Lys Pro Ile Pro Glu Asn Ile Thr Val Tyr Ar - #g Trp Cys Gly Met Pro 700 - # 705 - # 710 - - GAG TTC GGC TAC CAG ATC AGC GAC CCC CTG CC - #C AGC CTG AAG GAC TTC 962 Glu Phe Gly Tyr Gln Ile Ser Asp Pro Leu Pr - #o Ser Leu Lys Asp Phe 715 - # 720 - # 725 - - GAG GAG CAG TTC CTG AAC ACC ATC AAG GAG GA - #C AAG GGC TAC ATG AGC 1010 Glu Glu Gln Phe Leu Asn Thr Ile Lys Glu As - #p Lys Gly Tyr Met Ser 730 - # 735 - # 740 - - ACC AGC CTG AGC AGC GAG CGC CTG GCC GCC TT - #C GGC AGC CGC AAG ATC 1058 Thr Ser Leu Ser Ser Glu Arg Leu Ala Ala Ph - #e Gly Ser Arg Lys Ile 745 7 - #50 7 - #55 7 -#60 - - ATC CTG CGC CTG CAG GTG CCC AAG GGC AGC AC - #T GGT GCC TAC CTGAGC 1106 Ile Leu Arg Leu Gln Val Pro Lys Gly Ser Th - #r Gly Ala Tyr Leu Ser 765 - # 770 - # 775 - - GCC ATC GGC GGC TTC GCC AGC GAG AAG GAG AT - #C CTG CTG GAT AAG GAC 1154 Ala Ile Gly Gly Phe Ala Ser Glu Lys Glu Il - #e Leu Leu Asp Lys Asp 780 - # 785 - # 790 - - AGC AAG TAC CAC ATC GAC AAG GTG ACC GAG GT - #G ATC ATC AAG GGC GTG 1202 Ser Lys Tyr His Ile Asp Lys Val Thr Glu Va - #l Ile Ile Lys Gly Val 795 - # 800 - # 805 - - AAG CGC TAC GTG GTG GAC GCC ACC CTG CTG AC - #C AAC TAG - # 1241 Lys Arg Tyr Val Val Asp Ala Thr Leu Leu Th - #r Asn 810 - # 815 - # 820 - - - - (2) INFORMATION FOR SEQ ID NO:43: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 410 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: - - Met Leu Gln Asn Leu Lys Ile Thr Asp Lys Va - #l Glu Asp Phe Lys Glu 1 5 - # 10 - # 15 - - Asp Lys Glu Lys Ala Lys Glu Trp Gly Lys Gl - #u Lys Glu Lys Glu Trp 20 - # 25 - # 30 - - Lys Leu Thr Ala Thr Glu Lys Gly Lys Met As - #n Asn Phe Leu Asp Asn 35 - # 40 - # 45 - - Lys Asn Asp Ile Lys Thr Asn Tyr Lys Glu Il - #e Thr Phe Ser Ile Ala 50 - # 55 - # 60 - - Gly Ser Phe Glu Asp Glu Ile Lys Asp Leu Ly - #s Glu Ile Asp Lys Met 65 - # 70 - # 75 - # 80 - - Phe Asp Lys Thr Asn Leu Ser Asn Ser Ile Il - #e Thr Tyr Lys Asn Val 85 - # 90 - # 95 - - Glu Pro Thr Thr Ile Gly Phe Asn Lys Ser Le - #u Thr Glu Gly Asn Thr 100 - # 105 - # 110 - - Ile Asn Ser Asp Ala Met Ala Gln Phe Lys Gl - #u Gln Phe Leu Asp Arg 115 - # 120 - # 125 - - Asp Ile Lys Phe Asp Ser Tyr Leu Asp Thr Hi - #s Leu Thr Ala Gln Gln 130 - # 135 - # 140 - - Val Ser Ser Lys Glu Arg Val Ile Leu Lys Va - #l Thr Val Pro Ser Gly 145 1 - #50 1 - #55 1 -#60 - - Lys Gly Ser Thr Thr Pro Thr Lys Ala Gly Va - #l Ile Leu Asn AsnSer 165 - # 170 - # 175 - - Glu Tyr Lys Met Leu Ile Asp Asn Gly Tyr Me - #t Val His Val Asp Lys 180 - # 185 - # 190 - - Val Ser Lys Val Val Lys Lys Gly Val Glu Cy - #s Leu Gln Ile Glu Gly 195 - # 200 - # 205 - - Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn As - #p Ile Asn Ala Glu Ala 210 - # 215 - # 220 - - His Ser Trp Gly Met Lys Asn Tyr Glu Glu Tr - #p Ala Lys Asp Leu Thr 225 2 - #30 2 - #35 2 -#40 - - Asp Ser Gln Arg Glu Ala Leu Asp Gly Tyr Al - #a Arg Gln Asp TyrLys 245 - # 250 - # 255 - - Glu Ile Asn Asn Tyr Leu Arg Asn Gln Gly Gl - #y Ser Gly Asn Glu Lys 260 - # 265 - # 270 - - Leu Asp Ala Gln Ile Lys Asn Ile Ser Asp Al - #a Leu Gly Lys Lys Pro 275 - # 280 - # 285 - - Ile Pro Glu Asn Ile Thr Val Tyr Arg Trp Cy - #s Gly Met Pro Glu Phe 290 - # 295 - # 300 - - Gly Tyr Gln Ile Ser Asp Pro Leu Pro Ser Le - #u Lys Asp Phe Glu Glu 305 3 - #10 3 - #15 3 -#20 - - Gln Phe Leu Asn Thr Ile Lys Glu Asp Lys Gl - #y Tyr Met Ser ThrSer 325 - # 330 - # 335 - - Leu Ser Ser Glu Arg Leu Ala Ala Phe Gly Se - #r Arg Lys Ile Ile Leu 340 - # 345 - # 350 - - Arg Leu Gln Val Pro Lys Gly Ser Thr Gly Al - #a Tyr Leu Ser Ala Ile 355 - # 360 - # 365 - - Gly Gly Phe Ala Ser Glu Lys Glu Ile Leu Le - #u Asp Lys Asp Ser Lys 370 - # 375 - # 380 - - Tyr His Ile Asp Lys Val Thr Glu Val Ile Il - #e Lys Gly Val Lys Arg 385 3 - #90 3 - #95 4 -#00 - - Tyr Val Val Asp Ala Thr Leu Leu Thr Asn 405 - # 410 - - - - (2) INFORMATION FOR SEQ ID NO:44: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 86 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "oligonucleotide encoding vacuolar - #targetting peptide used to construct pCIB5533" - - (iii) HYPOTHETICAL: NO - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: - - CCGCGGGCGT GCACTGCCTC AGCAGCAGCA GCTTCGCCGA CAGCAACCCC AT -#CCGCGTGA 60 - - CCGACCGCGC CGCCAGCACC CTGCAG - # - # 86 - - - - (2) INFORMATION FOR SEQ ID NO:45: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1358 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "Synthetic DNA" - - (iii) HYPOTHETICAL: NO - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 9..1355 (D) OTHER INFORMATION: - #/note= "Maize optimized VIP2A(a) with the - #Bacillus secretion signal removed and the vacuolar targetting - #signal inserted as contained in pCIB5533" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: - - GATCCACC ATG GGC TGG AGC TGG ATC TTC CTG TTC - #CTG CTG AGC GGC GCC 50 Met Gly Trp Ser Trp I - #le Phe Leu Phe Leu Leu Ser Gly Ala - # 415 - # 420 - - GCG GGC GTG CAC TGC CTC AGC AGC AGC AGC TT - #C GCC GAC AGC AAC CCC 98 Ala Gly Val His Cys Leu Ser Ser Ser Ser Ph - #e Ala Asp Ser Asn Pro 425 4 - #30 4 - #35 4 -#40 - - ATC CGC GTG ACC GAC CGC GCC GCC AGC ACC CT - #G CAG AAC CTG AAGATC 146 Ile Arg Val Thr Asp Arg Ala Ala Ser Thr Le - #u Gln Asn Leu Lys Ile 445 - # 450 - # 455 - - ACC GAC AAG GTG GAG GAC TTC AAG GAG GAC AA - #G GAG AAG GCC AAG GAG 194 Thr Asp Lys Val Glu Asp Phe Lys Glu Asp Ly - #s Glu Lys Ala Lys Glu 460 - # 465 - # 470 - - TGG GGC AAG GAG AAG GAG AAG GAG TGG AAG CT - #T ACC GCC ACC GAG AAG 242 Trp Gly Lys Glu Lys Glu Lys Glu Trp Lys Le - #u Thr Ala Thr Glu Lys 475 - # 480 - # 485 - - GGC AAG ATG AAC AAC TTC CTG GAC AAC AAG AA - #C GAC ATC AAG ACC AAC 290 Gly Lys Met Asn Asn Phe Leu Asp Asn Lys As - #n Asp Ile Lys Thr Asn 490 - # 495 - # 500 - - TAC AAG GAG ATC ACC TTC AGC ATA GCC GGC AG - #C TTC GAG GAC GAG ATC 338 Tyr Lys Glu Ile Thr Phe Ser Ile Ala Gly Se - #r Phe Glu Asp Glu Ile 505 5 - #10 5 - #15 5 -#20 - - AAG GAC CTG AAG GAG ATC GAC AAG ATG TTC GA - #C AAG ACC AAC CTGAGC 386 Lys Asp Leu Lys Glu Ile Asp Lys Met Phe As - #p Lys Thr Asn Leu Ser 525 - # 530 - # 535 - - AAC AGC ATC ATC ACC TAC AAG AAC GTG GAG CC - #C ACC ACC ATC GGC TTC 434 Asn Ser Ile Ile Thr Tyr Lys Asn Val Glu Pr - #o Thr Thr Ile Gly Phe 540 - # 545 - # 550 - - AAC AAG AGC CTG ACC GAG GGC AAC ACC ATC AA - #C AGC GAC GCC ATG GCC 482 Asn Lys Ser Leu Thr Glu Gly Asn Thr Ile As - #n Ser Asp Ala Met Ala 555 - # 560 - # 565 - - CAG TTC AAG GAG CAG TTC CTG GAC CGC GAC AT - #C AAG TTC GAC AGC TAC 530 Gln Phe Lys Glu Gln Phe Leu Asp Arg Asp Il - #e Lys Phe Asp Ser Tyr 570 - # 575 - # 580 - - CTG GAC ACC CAC CTG ACC GCC CAG CAG GTG AG - #C AGC AAG GAG CGC GTG 578 Leu Asp Thr His Leu Thr Ala Gln Gln Val Se - #r Ser Lys Glu Arg Val 585 5 - #90 5 - #95 6 -#00 - - ATC CTG AAG GTG ACC GTC CCC AGC GGC AAG GG - #C AGC ACC ACC CCCACC 626 Ile Leu Lys Val Thr Val Pro Ser Gly Lys Gl - #y Ser Thr Thr Pro Thr 605 - # 610 - # 615 - - AAG GCC GGC GTG ATC CTG AAC AAC AGC GAG TA - #C AAG ATG CTG ATC GAC 674 Lys Ala Gly Val Ile Leu Asn Asn Ser Glu Ty - #r Lys Met Leu Ile Asp 620 - # 625 - # 630 - - AAC GGC TAC ATG GTG CAC GTG GAC AAG GTG AG - #C AAG GTG GTG AAG AAG 722 Asn Gly Tyr Met Val His Val Asp Lys Val Se - #r Lys Val Val Lys Lys 635 - # 640 - # 645 - - GGC GTG GAG TGC CTC CAG ATC GAG GGC ACC CT - #G AAG AAG AGT CTA GAC 770 Gly Val Glu Cys Leu Gln Ile Glu Gly Thr Le - #u Lys Lys Ser Leu Asp 650 - # 655 - # 660 - - TTC AAG AAC GAC ATC AAC GCC GAG GCC CAC AG - #C TGG GGC ATG AAG AAC 818 Phe Lys Asn Asp Ile Asn Ala Glu Ala His Se - #r Trp Gly Met Lys Asn 665 6 - #70 6 - #75 6 -#80 - - TAC GAG GAG TGG GCC AAG GAC CTG ACC GAC AG - #C CAG CGC GAG GCCCTG 866 Tyr Glu Glu Trp Ala Lys Asp Leu Thr Asp Se - #r Gln Arg Glu Ala Leu 685 - # 690 - # 695 - - GAC GGC TAC GCC CGC CAG GAC TAC AAG GAG AT - #C AAC AAC TAC CTG CGC 914 Asp Gly Tyr Ala Arg Gln Asp Tyr Lys Glu Il - #e Asn Asn Tyr Leu Arg 700 - # 705 - # 710 - - AAC CAG GGC GGC AGC GGC AAC GAG AAG CTG GA - #C GCC CAG ATC AAG AAC 962 Asn Gln Gly Gly Ser Gly Asn Glu Lys Leu As - #p Ala Gln Ile Lys Asn 715 - # 720 - # 725 - - ATC AGC GAC GCC CTG GGC AAG AAG CCC ATC CC - #C GAG AAC ATC ACC GTG 1010 Ile Ser Asp Ala Leu Gly Lys Lys Pro Ile Pr - #o Glu Asn Ile Thr Val 730 - # 735 - # 740 - - TAC CGC TGG TGC GGC ATG CCC GAG TTC GGC TA - #C CAG ATC AGC GAC CCC 1058 Tyr Arg Trp Cys Gly Met Pro Glu Phe Gly Ty - #r Gln Ile Ser Asp Pro 745 7 - #50 7 - #55 7 -#60 - - CTG CCC AGC CTG AAG GAC TTC GAG GAG CAG TT - #C CTG AAC ACC ATCAAG 1106 Leu Pro Ser Leu Lys Asp Phe Glu Glu Gln Ph - #e Leu Asn Thr Ile Lys 765 - # 770 - # 775 - - GAG GAC AAG GGC TAC ATG AGC ACC AGC CTG AG - #C AGC GAG CGC CTG GCC 1154 Glu Asp Lys Gly Tyr Met Ser Thr Ser Leu Se - #r Ser Glu Arg Leu Ala 780 - # 785 - # 790 - - GCC TTC GGC AGC CGC AAG ATC ATC CTG CGC CT - #G CAG GTG CCC AAG GGC 1202 Ala Phe Gly Ser Arg Lys Ile Ile Leu Arg Le - #u Gln Val Pro Lys Gly 795 - # 800 - # 805 - - AGC ACT GGT GCC TAC CTG AGC GCC ATC GGC GG - #C TTC GCC AGC GAG AAG 1250 Ser Thr Gly Ala Tyr Leu Ser Ala Ile Gly Gl - #y Phe Ala Ser Glu Lys 810 - # 815 - # 820 - - GAG ATC CTG CTG GAT AAG GAC AGC AAG TAC CA - #C ATC GAC AAG GTG ACC 1298 Glu Ile Leu Leu Asp Lys Asp Ser Lys Tyr Hi - #s Ile Asp Lys Val Thr 825 8 - #30 8 - #35 8 -#40 - - GAG GTG ATC ATC AAG GGC GTG AAG CGC TAC GT - #G GTG GAC GCC ACCCTG 1346 Glu Val Ile Ile Lys Gly Val Lys Arg Tyr Va - #l Val Asp Ala Thr Leu 845 - # 850 - # 855 - - CTG ACC AAC TAG - # - # - # 1358 Leu Thr Asn - - - - (2) INFORMATION FOR SEQ ID NO:46: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 449 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: - - Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Le - #u Ser Gly Ala Ala Gly 1 5 - # 10 - # 15 - - Val His Cys Leu Ser Ser Ser Ser Phe Ala As - #p Ser Asn Pro Ile Arg 20 - # 25 - # 30 - - Val Thr Asp Arg Ala Ala Ser Thr Leu Gln As - #n Leu Lys Ile Thr Asp 35 - # 40 - # 45 - - Lys Val Glu Asp Phe Lys Glu Asp Lys Glu Ly - #s Ala Lys Glu Trp Gly 50 - # 55 - # 60 - - Lys Glu Lys Glu Lys Glu Trp Lys Leu Thr Al - #a Thr Glu Lys Gly Lys 65 - # 70 - # 75 - # 80 - - Met Asn Asn Phe Leu Asp Asn Lys Asn Asp Il - #e Lys Thr Asn Tyr Lys 85 - # 90 - # 95 - - Glu Ile Thr Phe Ser Ile Ala Gly Ser Phe Gl - #u Asp Glu Ile Lys Asp 100 - # 105 - # 110 - - Leu Lys Glu Ile Asp Lys Met Phe Asp Lys Th - #r Asn Leu Ser Asn Ser 115 - # 120 - # 125 - - Ile Ile Thr Tyr Lys Asn Val Glu Pro Thr Th - #r Ile Gly Phe Asn Lys 130 - # 135 - # 140 - - Ser Leu Thr Glu Gly Asn Thr Ile Asn Ser As - #p Ala Met Ala Gln Phe 145 1 - #50 1 - #55 1 -#60 - - Lys Glu Gln Phe Leu Asp Arg Asp Ile Lys Ph - #e Asp Ser Tyr LeuAsp 165 - # 170 - # 175 - - Thr His Leu Thr Ala Gln Gln Val Ser Ser Ly - #s Glu Arg Val Ile Leu 180 - # 185 - # 190 - - Lys Val Thr Val Pro Ser Gly Lys Gly Ser Th - #r Thr Pro Thr Lys Ala 195 - # 200 - # 205 - - Gly Val Ile Leu Asn Asn Ser Glu Tyr Lys Me - #t Leu Ile Asp Asn Gly 210 - # 215 - # 220 - - Tyr Met Val His Val Asp Lys Val Ser Lys Va - #l Val Lys Lys Gly Val 225 2 - #30 2 - #35 2 -#40 - - Glu Cys Leu Gln Ile Glu Gly Thr Leu Lys Ly - #s Ser Leu Asp PheLys 245 - # 250 - # 255 - - Asn Asp Ile Asn Ala Glu Ala His Ser Trp Gl - #y Met Lys Asn Tyr Glu 260 - # 265 - # 270 - - Glu Trp Ala Lys Asp Leu Thr Asp Ser Gln Ar - #g Glu Ala Leu Asp Gly 275 - # 280 - # 285 - - Tyr Ala Arg Gln Asp Tyr Lys Glu Ile Asn As - #n Tyr Leu Arg Asn Gln 290 - # 295 - # 300 - - Gly Gly Ser Gly Asn Glu Lys Leu Asp Ala Gl - #n Ile Lys Asn Ile Ser 305 3 - #10 3 - #15 3 -#20 - - Asp Ala Leu Gly Lys Lys Pro Ile Pro Glu As - #n Ile Thr Val TyrArg 325 - # 330 - # 335 - - Trp Cys Gly Met Pro Glu Phe Gly Tyr Gln Il - #e Ser Asp Pro Leu Pro 340 - # 345 - # 350 - - Ser Leu Lys Asp Phe Glu Glu Gln Phe Leu As - #n Thr Ile Lys Glu Asp 355 - # 360 - # 365 - - Lys Gly Tyr Met Ser Thr Ser Leu Ser Ser Gl - #u Arg Leu Ala Ala Phe 370 - # 375 - # 380 - - Gly Ser Arg Lys Ile Ile Leu Arg Leu Gln Va - #l Pro Lys Gly Ser Thr 385 3 - #90 3 - #95 4 -#00 - - Gly Ala Tyr Leu Ser Ala Ile Gly Gly Phe Al - #a Ser Glu Lys GluIle 405 - # 410 - # 415 - - Leu Leu Asp Lys Asp Ser Lys Tyr His Ile As - #p Lys Val Thr Glu Val 420 - # 425 - # 430 - - Ile Ile Lys Gly Val Lys Arg Tyr Val Val As - #p Ala Thr Leu Leu Thr 435 - # 440 - # 445 - - Asn - - - - (2) INFORMATION FOR SEQ ID NO:47: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: peptide - - (iii) HYPOTHETICAL: NO - - (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..16 (D) OTHER INFORMATION: - #/note= "linker peptide for fusion of VIP1A( - #a) and VIP2A(a) used to construct pCIB5533" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: - - Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Th - #r Pro Pro Thr Pro Ser 1 5 - # 10 - # 15 - - - - (2) INFORMATION FOR SEQ ID NO:48: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "DNA encoding linker peptide used to - #construct pCIB5533" - - (iii) HYPOTHETICAL: NO - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: - - CCCGGGCCTT CTACTCCCCC AACTCCCTCT CCTAGCACGC CTCCGACACC TA -#GCGATATC 60 - - GGATCC - # - # -# 66 - - - - (2) INFORMATION FOR SEQ ID NO:49: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4031 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "Synthetic DNA" - - (iii) HYPOTHETICAL: NO - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 6..4019 (D) OTHER INFORMATION: - #/note= "Maize optimized DNA sequence - #encoding a VIP2A(a) - VIP1A(a) fusion protein as contai - #ned in pCIB5531" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: - - GATCC ATG AAG CGC ATG GAG GGC AAG CTG TTC - #ATG GTG AGC AAG AAG 47 Met Lys Arg Met Glu Gly Lys - #Leu Phe Met Val Ser Lys Lys 450 - # 455 - # 460 - - CTC CAG GTG GTG ACC AAG ACC GTG CTG CTG AG - #C ACC GTG TTC AGC ATC 95 Leu Gln Val Val Thr Lys Thr Val Leu Leu Se - #r Thr Val Phe Ser Ile 465 - # 470 - # 475 - - AGC CTG CTG AAC AAC GAG GTG ATC AAG GCC GA - #G CAG CTG AAC ATC AAC 143 Ser Leu Leu Asn Asn Glu Val Ile Lys Ala Gl - #u Gln Leu Asn Ile Asn 480 4 - #85 4 - #90 4 -#95 - - AGC CAG AGC AAG TAC ACC AAC CTC CAG AAC CT - #G AAG ATC ACC GACAAG 191 Ser Gln Ser Lys Tyr Thr Asn Leu Gln Asn Le - #u Lys Ile Thr Asp Lys 500 - # 505 - # 510 - - GTG GAG GAC TTC AAG GAG GAC AAG GAG AAG GC - #C AAG GAG TGG GGC AAG 239 Val Glu Asp Phe Lys Glu Asp Lys Glu Lys Al - #a Lys Glu Trp Gly Lys 515 - # 520 - # 525 - - GAG AAG GAG AAG GAG TGG AAG CTT ACC GCC AC - #C GAG AAG GGC AAG ATG 287 Glu Lys Glu Lys Glu Trp Lys Leu Thr Ala Th - #r Glu Lys Gly Lys Met 530 - # 535 - # 540 - - AAC AAC TTC CTG GAC AAC AAG AAC GAC ATC AA - #G ACC AAC TAC AAG GAG 335 Asn Asn Phe Leu Asp Asn Lys Asn Asp Ile Ly - #s Thr Asn Tyr Lys Glu 545 - # 550 - # 555 - - ATC ACC TTC AGC ATA GCC GGC AGC TTC GAG GA - #C GAG ATC AAG GAC CTG 383 Ile Thr Phe Ser Ile Ala Gly Ser Phe Glu As - #p Glu Ile Lys Asp Leu 560 5 - #65 5 - #70 5 -#75 - - AAG GAG ATC GAC AAG ATG TTC GAC AAG ACC AA - #C CTG AGC AAC AGCATC 431 Lys Glu Ile Asp Lys Met Phe Asp Lys Thr As - #n Leu Ser Asn Ser Ile 580 - # 585 - # 590 - - ATC ACC TAC AAG AAC GTG GAG CCC ACC ACC AT - #C GGC TTC AAC AAG AGC 479 Ile Thr Tyr Lys Asn Val Glu Pro Thr Thr Il - #e Gly Phe Asn Lys Ser 595 - # 600 - # 605 - - CTG ACC GAG GGC AAC ACC ATC AAC AGC GAC GC - #C ATG GCC CAG TTC AAG 527 Leu Thr Glu Gly Asn Thr Ile Asn Ser Asp Al - #a Met Ala Gln Phe Lys 610 - # 615 - # 620 - - GAG CAG TTC CTG GAC CGC GAC ATC AAG TTC GA - #C AGC TAC CTG GAC ACC 575 Glu Gln Phe Leu Asp Arg Asp Ile Lys Phe As - #p Ser Tyr Leu Asp Thr 625 - # 630 - # 635 - - CAC CTG ACC GCC CAG CAG GTG AGC AGC AAG GA - #G CGC GTG ATC CTG AAG 623 His Leu Thr Ala Gln Gln Val Ser Ser Lys Gl - #u Arg Val Ile Leu Lys 640 6 - #45 6 - #50 6 -#55 - - GTG ACC GTC CCC AGC GGC AAG GGC AGC ACC AC - #C CCC ACC AAG GCCGGC 671 Val Thr Val Pro Ser Gly Lys Gly Ser Thr Th - #r Pro Thr Lys Ala Gly 660 - # 665 - # 670 - - GTG ATC CTG AAC AAC AGC GAG TAC AAG ATG CT - #G ATC GAC AAC GGC TAC 719 Val Ile Leu Asn Asn Ser Glu Tyr Lys Met Le - #u Ile Asp Asn Gly Tyr 675 - # 680 - # 685 - - ATG GTG CAC GTG GAC AAG GTG AGC AAG GTG GT - #G AAG AAG GGC GTG GAG 767 Met Val His Val Asp Lys Val Ser Lys Val Va - #l Lys Lys Gly Val Glu 690 - # 695 - # 700 - - TGC CTC CAG ATC GAG GGC ACC CTG AAG AAG AG - #T CTA GAC TTC AAG AAC 815 Cys Leu Gln Ile Glu Gly Thr Leu Lys Lys Se - #r Leu Asp Phe Lys Asn 705 - # 710 - # 715 - - GAC ATC AAC GCC GAG GCC CAC AGC TGG GGC AT - #G AAG AAC TAC GAG GAG 863 Asp Ile Asn Ala Glu Ala His Ser Trp Gly Me - #t Lys Asn Tyr Glu Glu 720 7 - #25 7 - #30 7 -#35 - - TGG GCC AAG GAC CTG ACC GAC AGC CAG CGC GA - #G GCC CTG GAC GGCTAC 911 Trp Ala Lys Asp Leu Thr Asp Ser Gln Arg Gl - #u Ala Leu Asp Gly Tyr 740 - # 745 - # 750 - - GCC CGC CAG GAC TAC AAG GAG ATC AAC AAC TA - #C CTG CGC AAC CAG GGC 959 Ala Arg Gln Asp Tyr Lys Glu Ile Asn Asn Ty - #r Leu Arg Asn Gln Gly 755 - # 760 - # 765 - - GGC AGC GGC AAC GAG AAG CTG GAC GCC CAG AT - #C AAG AAC ATC AGC GAC 1007 Gly Ser Gly Asn Glu Lys Leu Asp Ala Gln Il - #e Lys Asn Ile Ser Asp 770 - # 775 - # 780 - - GCC CTG GGC AAG AAG CCC ATC CCC GAG AAC AT - #C ACC GTG TAC CGC TGG 1055 Ala Leu Gly Lys Lys Pro Ile Pro Glu Asn Il - #e Thr Val Tyr Arg Trp 785 - # 790 - # 795 - - TGC GGC ATG CCC GAG TTC GGC TAC CAG ATC AG - #C GAC CCC CTG CCC AGC 1103 Cys Gly Met Pro Glu Phe Gly Tyr Gln Ile Se - #r Asp Pro Leu Pro Ser 800 8 - #05 8 - #10 8 -#15 - - CTG AAG GAC TTC GAG GAG CAG TTC CTG AAC AC - #C ATC AAG GAG GACAAG 1151 Leu Lys Asp Phe Glu Glu Gln Phe Leu Asn Th - #r Ile Lys Glu Asp Lys 820 - # 825 - # 830 - - GGC TAC ATG AGC ACC AGC CTG AGC AGC GAG CG - #C CTG GCC GCC TTC GGC 1199 Gly Tyr Met Ser Thr Ser Leu Ser Ser Glu Ar - #g Leu Ala Ala Phe Gly 835 - # 840 - # 845 - - AGC CGC AAG ATC ATC CTG CGC CTG CAG GTG CC - #C AAG GGC AGC ACT GGT 1247 Ser Arg Lys Ile Ile Leu Arg Leu Gln Val Pr - #o Lys Gly Ser Thr Gly 850 - # 855 - # 860 - - GCC TAC CTG AGC GCC ATC GGC GGC TTC GCC AG - #C GAG AAG GAG ATC CTG 1295 Ala Tyr Leu Ser Ala Ile Gly Gly Phe Ala Se - #r Glu Lys Glu Ile Leu 865 - # 870 - # 875 - - CTG GAT AAG GAC AGC AAG TAC CAC ATC GAC AA - #G GTG ACC GAG GTG ATC 1343 Leu Asp Lys Asp Ser Lys Tyr His Ile Asp Ly - #s Val Thr Glu Val Ile 880 8 - #85 8 - #90 8 -#95 - - ATC AAG GGC GTG AAG CGC TAC GTG GTG GAC GC - #C ACC CTG CTG ACCAAC 1391 Ile Lys Gly Val Lys Arg Tyr Val Val Asp Al - #a Thr Leu Leu Thr Asn 900 - # 905 - # 910 - - TCC CGG GGG CCT TCT ACT CCC CCA ACT CCC TC - #T CCT AGC ACG CCT CCG 1439 Ser Arg Gly Pro Ser Thr Pro Pro Thr Pro Se - #r Pro Ser Thr Pro Pro 915 - # 920 - # 925 - - ACA CCT AGC GAT ATC GGA TCC ACC ATG AAG AC - #C AAC CAG ATC AGC ACC 1487 Thr Pro Ser Asp Ile Gly Ser Thr Met Lys Th - #r Asn Gln Ile Ser Thr 930 - # 935 - # 940 - - ACC CAG AAG AAC CAG CAG AAG GAG ATG GAC CG - #C AAG GGC CTG CTG GGC 1535 Thr Gln Lys Asn Gln Gln Lys Glu Met Asp Ar - #g Lys Gly Leu Leu Gly 945 - # 950 - # 955 - - TAC TAC TTC AAG GGC AAG GAC TTC AGC AAC CT - #G ACC ATG TTC GCC CCC 1583 Tyr Tyr Phe Lys Gly Lys Asp Phe Ser Asn Le - #u Thr Met Phe Ala Pro 960 9 - #65 9 - #70 9 -#75 - - ACG CGT GAC AGC ACC CTG ATC TAC GAC CAG CA - #G ACC GCC AAC AAGCTG 1631 Thr Arg Asp Ser Thr Leu Ile Tyr Asp Gln Gl - #n Thr Ala Asn Lys Leu 980 - # 985 - # 990 - - CTG GAC AAG AAG CAG CAG GAG TAC CAG AGC AT - #C CGC TGG ATC GGC CTG 1679 Leu Asp Lys Lys Gln Gln Glu Tyr Gln Ser Il - #e Arg Trp Ile Gly Leu 995 - # 1000 - # 1005 - - ATC CAG AGC AAG GAG ACC GGC GAC TTC ACC TT - #C AAC CTG AGC GAG GAC 1727 Ile Gln Ser Lys Glu Thr Gly Asp Phe Thr Ph - #e Asn Leu Ser Glu Asp 1010 - # 1015 - # 1020 - - GAG CAG GCC ATC ATC GAG ATC AAC GGC AAG AT - #C ATC AGC AAC AAG GGC 1775 Glu Gln Ala Ile Ile Glu Ile Asn Gly Lys Il - #e Ile Ser Asn Lys Gly 1025 - # 1030 - # 1035 - - AAG GAG AAG CAG GTG GTG CAC CTG GAG AAG GG - #C AAG CTG GTG CCC ATC 1823 Lys Glu Lys Gln Val Val His Leu Glu Lys Gl - #y Lys Leu Val Pro Ile 1040 1045 - # 1050 - # 1055 - - AAG ATC GAG TAC CAG AGC GAC ACC AAG TTC AA - #C ATC GAC AGC AAG ACC 1871 Lys Ile Glu Tyr Gln Ser Asp Thr Lys Phe As - #n Ile Asp Ser Lys Thr 1060 - # 1065 - # 1070 - - TTC AAG GAG CTG AAG CTT TTC AAG ATC GAC AG - #C CAG AAC CAG CCC CAG 1919 Phe Lys Glu Leu Lys Leu Phe Lys Ile Asp Se - #r Gln Asn Gln Pro Gln 1075 - # 1080 - # 1085 - - CAG GTG CAG CAG GAC GAG CTG CGC AAC CCC GA - #G TTC AAC AAG AAG GAG 1967 Gln Val Gln Gln Asp Glu Leu Arg Asn Pro Gl - #u Phe Asn Lys Lys Glu 1090 - # 1095 - # 1100 - - AGC CAG GAG TTC CTG GCC AAG CCC AGC AAG AT - #C AAC CTG TTC ACC CAG 2015 Ser Gln Glu Phe Leu Ala Lys Pro Ser Lys Il - #e Asn Leu Phe Thr Gln 1105 - # 1110 - # 1115 - - CAG ATG AAG CGC GAG ATC GAC GAG GAC ACC GA - #C ACC GAC GGC GAC AGC 2063 Gln Met Lys Arg Glu Ile Asp Glu Asp Thr As - #p Thr Asp Gly Asp Ser 1120 1125 - # 1130 - # 1135 - - ATC CCC GAC CTG TGG GAG GAG AAC GGC TAC AC - #C ATC CAG AAC CGC ATC 2111 Ile Pro Asp Leu Trp Glu Glu Asn Gly Tyr Th - #r Ile Gln Asn Arg Ile 1140 - # 1145 - # 1150 - - GCC GTG AAG TGG GAC GAC AGC CTG GCT AGC AA - #G GGC TAC ACC AAG TTC 2159 Ala Val Lys Trp Asp Asp Ser Leu Ala Ser Ly - #s Gly Tyr Thr Lys Phe 1155 - # 1160 - # 1165 - - GTG AGC AAC CCC CTG GAG AGC CAC ACC GTG GG - #C GAC CCC TAC ACC GAC 2207 Val Ser Asn Pro Leu Glu Ser His Thr Val Gl - #y Asp Pro Tyr Thr Asp 1170 - # 1175 - # 1180 - - TAC GAG AAG GCC GCC CGC GAC CTG GAC CTG AG - #C AAC GCC AAG GAG ACC 2255 Tyr Glu Lys Ala Ala Arg Asp Leu Asp Leu Se - #r Asn Ala Lys Glu Thr 1185 - # 1190 - # 1195 - - TTC AAC CCC CTG GTG GCC GCC TTC CCC AGC GT - #G AAC GTG AGC ATG GAG 2303 Phe Asn Pro Leu Val Ala Ala Phe Pro Ser Va - #l Asn Val Ser Met Glu 1200 1205 - # 1210 - # 1215 - - AAG GTG ATC CTG AGC CCC AAC GAG AAC CTG AG - #C AAC AGC GTG GAG AGC 2351 Lys Val Ile Leu Ser Pro Asn Glu Asn Leu Se - #r Asn Ser Val Glu Ser 1220 - # 1225 - # 1230 - - CAC TCG AGC ACC AAC TGG AGC TAC ACC AAC AC - #C GAG GGC GCC AGC GTG 2399 His Ser Ser Thr Asn Trp Ser Tyr Thr Asn Th - #r Glu Gly Ala Ser Val 1235 - # 1240 - # 1245 - - GAG GCC GGC ATC GGT CCC AAG GGC ATC AGC TT - #C GGC GTG AGC GTG AAC 2447 Glu Ala Gly Ile Gly Pro Lys Gly Ile Ser Ph - #e Gly Val Ser Val Asn 1250 - # 1255 - # 1260 - - TAC CAG CAC AGC GAG ACC GTG GCC CAG GAG TG - #G GGC ACC AGC ACC GGC 2495 Tyr Gln His Ser Glu Thr Val Ala Gln Glu Tr - #p Gly Thr Ser Thr Gly 1265 - # 1270 - # 1275 - - AAC ACC AGC CAG TTC AAC ACC GCC AGC GCC GG - #C TAC CTG AAC GCC AAC 2543 Asn Thr Ser Gln Phe Asn Thr Ala Ser Ala Gl - #y Tyr Leu Asn Ala Asn 1280 1285 - # 1290 - # 1295 - - GTG CGC TAC AAC AAC GTG GGC ACC GGC GCC AT - #C TAC GAC GTG AAG CCC 2591 Val Arg Tyr Asn Asn Val Gly Thr Gly Ala Il - #e Tyr Asp Val Lys Pro 1300 - # 1305 - # 1310 - - ACC ACC AGC TTC GTG CTG AAC AAC GAC ACC AT - #C GCC ACC ATC ACC GCC 2639 Thr Thr Ser Phe Val Leu Asn Asn Asp Thr Il - #e Ala Thr Ile Thr Ala 1315 - # 1320 - # 1325 - - AAG TCG AAT TCC ACC GCC CTG AAC ATC AGC CC - #C GGC GAG AGC TAC CCC 2687 Lys Ser Asn Ser Thr Ala Leu Asn Ile Ser Pr - #o Gly Glu Ser Tyr Pro 1330 - # 1335 - # 1340 - - AAG AAG GGC CAG AAC GGC ATC GCC ATC ACC AG - #C ATG GAC GAC TTC AAC 2735 Lys Lys Gly Gln Asn Gly Ile Ala Ile Thr Se - #r Met Asp Asp Phe Asn 1345 - # 1350 - # 1355 - - AGC CAC CCC ATC ACC CTG AAC AAG AAG CAG GT - #G GAC AAC CTG CTG AAC 2783 Ser His Pro Ile Thr Leu Asn Lys Lys Gln Va - #l Asp Asn Leu Leu Asn 1360 1365 - # 1370 - # 1375 - - AAC AAG CCC ATG ATG CTG GAG ACC AAC CAG AC - #C GAC GGC GTC TAC AAG 2831 Asn Lys Pro Met Met Leu Glu Thr Asn Gln Th - #r Asp Gly Val Tyr Lys 1380 - # 1385 - # 1390 - - ATC AAG GAC ACC CAC GGC AAC ATC GTG ACG GG - #C GGC GAG TGG AAC GGC 2879 Ile Lys Asp Thr His Gly Asn Ile Val Thr Gl - #y Gly Glu Trp Asn Gly 1395 - # 1400 - # 1405 - - GTG ATC CAG CAG ATC AAG GCC AAG ACC GCC AG - #C ATC ATC GTC GAC GAC 2927 Val Ile Gln Gln Ile Lys Ala Lys Thr Ala Se - #r Ile Ile Val Asp Asp 1410 - # 1415 - # 1420 - - GGC GAG CGC GTG GCC GAG AAG CGC GTG GCC GC - #C AAG GAC TAC GAG AAC 2975 Gly Glu Arg Val Ala Glu Lys Arg Val Ala Al - #a Lys Asp Tyr Glu Asn 1425 - # 1430 - # 1435 - - CCC GAG GAC AAG ACC CCC AGC CTG ACC CTG AA - #G GAC GCC CTG AAG CTG 3023 Pro Glu Asp Lys Thr Pro Ser Leu Thr Leu Ly - #s Asp Ala Leu Lys Leu 1440 1445 - # 1450 - # 1455 - - AGC TAC CCC GAC GAG ATC AAG GAG ATC GAG GG - #C TTG CTG TAC TAC AAG 3071 Ser Tyr Pro Asp Glu Ile Lys Glu Ile Glu Gl - #y Leu Leu Tyr Tyr Lys 1460 - # 1465 - # 1470 - - AAC AAG CCC ATC TAC GAG AGC AGC GTG ATG AC - #C TAT CTA GAC GAG AAC 3119 Asn Lys Pro Ile Tyr Glu Ser Ser Val Met Th - #r Tyr Leu Asp Glu Asn 1475 - # 1480 - # 1485 - - ACC GCC AAG GAG GTG ACC AAG CAG CTG AAC GA - #C ACC ACC GGC AAG TTC 3167 Thr Ala Lys Glu Val Thr Lys Gln Leu Asn As - #p Thr Thr Gly Lys Phe 1490 - # 1495 - # 1500 - - AAG GAC GTG AGC CAC CTG TAC GAC GTG AAG CT - #G ACC CCC AAG ATG AAC 3215 Lys Asp Val Ser His Leu Tyr Asp Val Lys Le - #u Thr Pro Lys Met Asn 1505 - # 1510 - # 1515 - - GTG ACC ATC AAG CTG AGC ATC CTG TAC GAC AA - #C GCC GAG AGC AAC GAC 3263 Val Thr Ile Lys Leu Ser Ile Leu Tyr Asp As - #n Ala Glu Ser Asn Asp 1520 1525 - # 1530 - # 1535 - - AAC AGC ATC GGC AAG TGG ACC AAC ACC AAC AT - #C GTG AGC GGC GGC AAC 3311 Asn Ser Ile Gly Lys Trp Thr Asn Thr Asn Il - #e Val Ser Gly Gly Asn 1540 - # 1545 - # 1550 - - AAC GGC AAG AAG CAG TAC AGC AGC AAC AAC CC - #C GAC GCC AAC CTG ACC 3359 Asn Gly Lys Lys Gln Tyr Ser Ser Asn Asn Pr - #o Asp Ala Asn Leu Thr 1555 - # 1560 - # 1565 - - CTG AAC ACC GAC GCC CAG GAG AAG CTG AAC AA - #G AAC CGC GAC TAC TAC 3407 Leu Asn Thr Asp Ala Gln Glu Lys Leu Asn Ly - #s Asn Arg Asp Tyr Tyr 1570 - # 1575 - # 1580 - - ATC AGC CTG TAC ATG AAG AGC GAG AAG AAC AC - #C CAG TGC GAG ATC ACC 3455 Ile Ser Leu Tyr Met Lys Ser Glu Lys Asn Th - #r Gln Cys Glu Ile Thr 1585 - # 1590 - # 1595 - - ATC GAC GGC GAG ATA TAC CCC ATC ACC ACC AA - #G ACC GTG AAC GTG AAC 3503 Ile Asp Gly Glu Ile Tyr Pro Ile Thr Thr Ly - #s Thr Val Asn Val Asn 1600 1605 - # 1610 - # 1615 - - AAG GAC AAC TAC AAG CGC CTG GAC ATC ATC GC - #C CAC AAC ATC AAG AGC 3551 Lys Asp Asn Tyr Lys Arg Leu Asp Ile Ile Al - #a His Asn Ile Lys Ser 1620 - # 1625 - # 1630 - - AAC CCC ATC AGC AGC CTG CAC ATC AAG ACC AA - #C GAC GAG ATC ACC CTG 3599 Asn Pro Ile Ser Ser Leu His Ile Lys Thr As - #n Asp Glu Ile Thr Leu 1635 - # 1640 - # 1645 - - TTC TGG GAC GAC ATA TCG ATT ACC GAC GTC GC - #C AGC ATC AAG CCC GAG 3647 Phe Trp Asp Asp Ile Ser Ile Thr Asp Val Al - #a Ser Ile Lys Pro Glu 1650 - # 1655 - # 1660 - - AAC CTG ACC GAC AGC GAG ATC AAG CAG ATA TA - #C AGT CGC TAC GGC ATC 3695 Asn Leu Thr Asp Ser Glu Ile Lys Gln Ile Ty - #r Ser Arg Tyr Gly Ile 1665 - # 1670 - # 1675 - - AAG CTG GAG GAC GGC ATC CTG ATC GAC AAG AA - #A GGC GGC ATC CAC TAC 3743 Lys Leu Glu Asp Gly Ile Leu Ile Asp Lys Ly - #s Gly Gly Ile His Tyr 1680 1685 - # 1690 - # 1695 - - GGC GAG TTC ATC AAC GAG GCC AGC TTC AAC AT - #C GAG CCC CTG CAG AAC 3791 Gly Glu Phe Ile Asn Glu Ala Ser Phe Asn Il - #e Glu Pro Leu Gln Asn 1700 - # 1705 - # 1710 - - TAC GTG ACC AAG TAC GAG GTG ACC TAC AGC AG - #C GAG CTG GGC CCC AAC 3839 Tyr Val Thr Lys Tyr Glu Val Thr Tyr Ser Se - #r Glu Leu Gly Pro Asn 1715 - # 1720 - # 1725 - - GTG AGC GAC ACC CTG GAG AGC GAC AAG ATT TA - #C AAG GAC GGC ACC ATC 3887 Val Ser Asp Thr Leu Glu Ser Asp Lys Ile Ty - #r Lys Asp Gly Thr Ile 1730 - # 1735 - # 1740 - - AAG TTC GAC TTC ACC AAG TAC AGC AAG AAC GA - #G CAG GGC CTG TTC TAC 3935 Lys Phe Asp Phe Thr Lys Tyr Ser Lys Asn Gl - #u Gln Gly Leu Phe Tyr 1745 - # 1750 - # 1755 - - GAC AGC GGC CTG AAC TGG GAC TTC AAG ATC AA - #C GCC ATC ACC TAC GAC 3983 Asp Ser Gly Leu Asn Trp Asp Phe Lys Ile As - #n Ala Ile Thr Tyr Asp 1760 1765 - # 1770 - # 1775 - - GGC AAG GAG ATG AAC GTG TTC CAC CGC TAC AA - #C AAG TAGATCTGAG 4029 Gly Lys Glu Met Asn Val Phe His Arg Tyr As - #n Lys 1780 - # 1785 - - CT - # - # - # 4031 - - - - (2) INFORMATION FOR SEQ ID NO:50: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1338 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: - - Met Lys Arg Met Glu Gly Lys Leu Phe Met Va - #l Ser Lys Lys Leu Gln 1 5 - # 10 - # 15 - - Val Val Thr Lys Thr Val Leu Leu Ser Thr Va - #l Phe Ser Ile Ser Leu 20 - # 25 - # 30 - - Leu Asn Asn Glu Val Ile Lys Ala Glu Gln Le - #u Asn Ile Asn Ser Gln 35 - # 40 - # 45 - - Ser Lys Tyr Thr Asn Leu Gln Asn Leu Lys Il - #e Thr Asp Lys Val Glu 50 - # 55 - # 60 - - Asp Phe Lys Glu Asp Lys Glu Lys Ala Lys Gl - #u Trp Gly Lys Glu Lys 65 - # 70 - # 75 - # 80 - - Glu Lys Glu Trp Lys Leu Thr Ala Thr Glu Ly - #s Gly Lys Met Asn Asn 85 - # 90 - # 95 - - Phe Leu Asp Asn Lys Asn Asp Ile Lys Thr As - #n Tyr Lys Glu Ile Thr 100 - # 105 - # 110 - - Phe Ser Ile Ala Gly Ser Phe Glu Asp Glu Il - #e Lys Asp Leu Lys Glu 115 - # 120 - # 125 - - Ile Asp Lys Met Phe Asp Lys Thr Asn Leu Se - #r Asn Ser Ile Ile Thr 130 - # 135 - # 140 - - Tyr Lys Asn Val Glu Pro Thr Thr Ile Gly Ph - #e Asn Lys Ser Leu Thr 145 1 - #50 1 - #55 1 -#60 - - Glu Gly Asn Thr Ile Asn Ser Asp Ala Met Al - #a Gln Phe Lys GluGln 165 - # 170 - # 175 - - Phe Leu Asp Arg Asp Ile Lys Phe Asp Ser Ty - #r Leu Asp Thr His Leu 180 - # 185 - # 190 - - Thr Ala Gln Gln Val Ser Ser Lys Glu Arg Va - #l Ile Leu Lys Val Thr 195 - # 200 - # 205 - - Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Th - #r Lys Ala Gly Val Ile 210 - # 215 - # 220 - - Leu Asn Asn Ser Glu Tyr Lys Met Leu Ile As - #p Asn Gly Tyr Met Val 225 2 - #30 2 - #35 2 -#40 - - His Val Asp Lys Val Ser Lys Val Val Lys Ly - #s Gly Val Glu CysLeu 245 - # 250 - # 255 - - Gln Ile Glu Gly Thr Leu Lys Lys Ser Leu As - #p Phe Lys Asn Asp Ile 260 - # 265 - # 270 - - Asn Ala Glu Ala His Ser Trp Gly Met Lys As - #n Tyr Glu Glu Trp Ala 275 - # 280 - # 285 - - Lys Asp Leu Thr Asp Ser Gln Arg Glu Ala Le - #u Asp Gly Tyr Ala Arg 290 - # 295 - # 300 - - Gln Asp Tyr Lys Glu Ile Asn Asn Tyr Leu Ar - #g Asn Gln Gly Gly Ser 305 3 - #10 3 - #15 3 -#20 - - Gly Asn Glu Lys Leu Asp Ala Gln Ile Lys As - #n Ile Ser Asp AlaLeu 325 - # 330 - # 335 - - Gly Lys Lys Pro Ile Pro Glu Asn Ile Thr Va - #l Tyr Arg Trp Cys Gly 340 - # 345 - # 350 - - Met Pro Glu Phe Gly Tyr Gln Ile Ser Asp Pr - #o Leu Pro Ser Leu Lys 355 - # 360 - # 365 - - Asp Phe Glu Glu Gln Phe Leu Asn Thr Ile Ly - #s Glu Asp Lys Gly Tyr 370 - # 375 - # 380 - - Met Ser Thr Ser Leu Ser Ser Glu Arg Leu Al - #a Ala Phe Gly Ser Arg 385 3 - #90 3 - #95 4 -#00 - - Lys Ile Ile Leu Arg Leu Gln Val Pro Lys Gl - #y Ser Thr Gly AlaTyr 405 - # 410 - # 415 - - Leu Ser Ala Ile Gly Gly Phe Ala Ser Glu Ly - #s Glu Ile Leu Leu Asp 420 - # 425 - # 430 - - Lys Asp Ser Lys Tyr His Ile Asp Lys Val Th - #r Glu Val Ile Ile Lys 435 - # 440 - # 445 - - Gly Val Lys Arg Tyr Val Val Asp Ala Thr Le - #u Leu Thr Asn Ser Arg 450 - # 455 - # 460 - - Gly Pro Ser Thr Pro Pro Thr Pro Ser Pro Se - #r Thr Pro Pro Thr Pro 465 4 - #70 4 - #75 4 -#80 - - Ser Asp Ile Gly Ser Thr Met Lys Thr Asn Gl - #n Ile Ser Thr ThrGln 485 - # 490 - # 495 - - Lys Asn Gln Gln Lys Glu Met Asp Arg Lys Gl - #y Leu Leu Gly Tyr Tyr 500 - # 505 - # 510 - - Phe Lys Gly Lys Asp Phe Ser Asn Leu Thr Me - #t Phe Ala Pro Thr Arg 515 - # 520 - # 525 - - Asp Ser Thr Leu Ile Tyr Asp Gln Gln Thr Al - #a Asn Lys Leu Leu Asp 530 - # 535 - # 540 - - Lys Lys Gln Gln Glu Tyr Gln Ser Ile Arg Tr - #p Ile Gly Leu Ile Gln 545 5 - #50 5 - #55 5 -#60 - - Ser Lys Glu Thr Gly Asp Phe Thr Phe Asn Le - #u Ser Glu Asp GluGln 565 - # 570 - # 575 - - Ala Ile Ile Glu Ile Asn Gly Lys Ile Ile Se - #r Asn Lys Gly Lys Glu 580 - # 585 - # 590 - - Lys Gln Val Val His Leu Glu Lys Gly Lys Le - #u Val Pro Ile Lys Ile 595 - # 600 - # 605 - - Glu Tyr Gln Ser Asp Thr Lys Phe Asn Ile As - #p Ser Lys Thr Phe Lys 610 - # 615 - # 620 - - Glu Leu Lys Leu Phe Lys Ile Asp Ser Gln As - #n Gln Pro Gln Gln Val 625 6 - #30 6 - #35 6 -#40 - - Gln Gln Asp Glu Leu Arg Asn Pro Glu Phe As - #n Lys Lys Glu SerGln 645 - # 650 - # 655 - - Glu Phe Leu Ala Lys Pro Ser Lys Ile Asn Le - #u Phe Thr Gln Gln Met 660 - # 665 - # 670 - - Lys Arg Glu Ile Asp Glu Asp Thr Asp Thr As - #p Gly Asp Ser Ile Pro 675 - # 680 - # 685 - - Asp Leu Trp Glu Glu Asn Gly Tyr Thr Ile Gl - #n Asn Arg Ile Ala Val 690 - # 695 - # 700 - - Lys Trp Asp Asp Ser Leu Ala Ser Lys Gly Ty - #r Thr Lys Phe Val Ser 705 7 - #10 7 - #15 7 -#20 - - Asn Pro Leu Glu Ser His Thr Val Gly Asp Pr - #o Tyr Thr Asp TyrGlu 725 - # 730 - # 735 - - Lys Ala Ala Arg Asp Leu Asp Leu Ser Asn Al - #a Lys Glu Thr Phe Asn 740 - # 745 - # 750 - - Pro Leu Val Ala Ala Phe Pro Ser Val Asn Va - #l Ser Met Glu Lys Val 755 - # 760 - # 765 - - Ile Leu Ser Pro Asn Glu Asn Leu Ser Asn Se - #r Val Glu Ser His Ser 770 - # 775 - # 780 - - Ser Thr Asn Trp Ser Tyr Thr Asn Thr Glu Gl - #y Ala Ser Val Glu Ala 785 7 - #90 7 - #95 8 -#00 - - Gly Ile Gly Pro Lys Gly Ile Ser Phe Gly Va - #l Ser Val Asn TyrGln 805 - # 810 - # 815 - - His Ser Glu Thr Val Ala Gln Glu Trp Gly Th - #r Ser Thr Gly Asn Thr 820 - # 825 - # 830 - - Ser Gln Phe Asn Thr Ala Ser Ala Gly Tyr Le - #u Asn Ala Asn Val Arg 835 - # 840 - # 845 - - Tyr Asn Asn Val Gly Thr Gly Ala Ile Tyr As - #p Val Lys Pro Thr Thr 850 - # 855 - # 860 - - Ser Phe Val Leu Asn Asn Asp Thr Ile Ala Th - #r Ile Thr Ala Lys Ser 865 8 - #70 8 - #75 8 -#80 - - Asn Ser Thr Ala Leu Asn Ile Ser Pro Gly Gl - #u Ser Tyr Pro LysLys 885 - # 890 - # 895 - - Gly Gln Asn Gly Ile Ala Ile Thr Ser Met As - #p Asp Phe Asn Ser His 900 - # 905 - # 910 - - Pro Ile Thr Leu Asn Lys Lys Gln Val Asp As - #n Leu Leu Asn Asn Lys 915 - # 920 - # 925 - - Pro Met Met Leu Glu Thr Asn Gln Thr Asp Gl - #y Val Tyr Lys Ile Lys 930 - # 935 - # 940 - - Asp Thr His Gly Asn Ile Val Thr Gly Gly Gl - #u Trp Asn Gly Val Ile 945 9 - #50 9 - #55 9 -#60 - - Gln Gln Ile Lys Ala Lys Thr Ala Ser Ile Il - #e Val Asp Asp GlyGlu 965 - # 970 - # 975 - - Arg Val Ala Glu Lys Arg Val Ala Ala Lys As - #p Tyr Glu Asn Pro Glu 980 - # 985 - # 990 - - Asp Lys Thr Pro Ser Leu Thr Leu Lys Asp Al - #a Leu Lys Leu Ser Tyr 995 - # 1000 - # 1005 - - Pro Asp Glu Ile Lys Glu Ile Glu Gly Leu Le - #u Tyr Tyr Lys Asn Lys 1010 - # 1015 - # 1020 - - Pro Ile Tyr Glu Ser Ser Val Met Thr Tyr Le - #u Asp Glu Asn Thr Ala 1025 1030 - # 1035 - # 1040 - - Lys Glu Val Thr Lys Gln Leu Asn Asp Thr Th - #r Gly Lys Phe Lys Asp 1045 - # 1050 - # 1055 - - Val Ser His Leu Tyr Asp Val Lys Leu Thr Pr - #o Lys Met Asn Val Thr 1060 - # 1065 - # 1070 - - Ile Lys Leu Ser Ile Leu Tyr Asp Asn Ala Gl - #u Ser Asn Asp Asn Ser 1075 - # 1080 - # 1085 - - Ile Gly Lys Trp Thr Asn Thr Asn Ile Val Se - #r Gly Gly Asn Asn Gly 1090 - # 1095 - # 1100 - - Lys Lys Gln Tyr Ser Ser Asn Asn Pro Asp Al - #a Asn Leu Thr Leu Asn 1105 1110 - # 1115 - # 1120 - - Thr Asp Ala Gln Glu Lys Leu Asn Lys Asn Ar - #g Asp Tyr Tyr Ile Ser 1125 - # 1130 - # 1135 - - Leu Tyr Met Lys Ser Glu Lys Asn Thr Gln Cy - #s Glu Ile Thr Ile Asp 1140 - # 1145 - # 1150 - - Gly Glu Ile Tyr Pro Ile Thr Thr Lys Thr Va - #l Asn Val Asn Lys Asp 1155 - # 1160 - # 1165 - - Asn Tyr Lys Arg Leu Asp Ile Ile Ala His As - #n Ile Lys Ser Asn Pro 1170 - # 1175 - # 1180 - - Ile Ser Ser Leu His Ile Lys Thr Asn Asp Gl - #u Ile Thr Leu Phe Trp 1185 1190 - # 1195 - # 1200 - - Asp Asp Ile Ser Ile Thr Asp Val Ala Ser Il - #e Lys Pro Glu Asn Leu 1205 - # 1210 - # 1215 - - Thr Asp Ser Glu Ile Lys Gln Ile Tyr Ser Ar - #g Tyr Gly Ile Lys Leu 1220 - # 1225 - # 1230 - - Glu Asp Gly Ile Leu Ile Asp Lys Lys Gly Gl - #y Ile His Tyr Gly Glu 1235 - # 1240 - # 1245 - - Phe Ile Asn Glu Ala Ser Phe Asn Ile Glu Pr - #o Leu Gln Asn Tyr Val 1250 - # 1255 - # 1260 - - Thr Lys Tyr Glu Val Thr Tyr Ser Ser Glu Le - #u Gly Pro Asn Val Ser 1265 1270 - # 1275 - # 1280 - - Asp Thr Leu Glu Ser Asp Lys Ile Tyr Lys As - #p Gly Thr Ile Lys Phe 1285 - # 1290 - # 1295 - - Asp Phe Thr Lys Tyr Ser Lys Asn Glu Gln Gl - #y Leu Phe Tyr Asp Ser 1300 - # 1305 - # 1310 - - Gly Leu Asn Trp Asp Phe Lys Ile Asn Ala Il - #e Thr Tyr Asp Gly Lys 1315 - # 1320 - # 1325 - - Glu Met Asn Val Phe His Arg Tyr Asn Lys 1330 - # 1335__________________________________________________________________________

Number	Name	Date
3632747	Satohiro et al.	Jan 1972
3651215	Satohiro et al.	Mar 1972
4996155	Sick et al.	Feb 1991
5262323	Baird et al.	Nov 1993

Number	Date	Country
0498537A2	Jan 1992	EPX
0501650A2	Feb 1992	EPX
WO 8808880	Nov 1988	WOX
WO 9013651	Nov 1990	WOX
WO 9116434	Oct 1991	WOX
WO 9116432	Oct 1991	WOX
US9403131	Jul 1994	WOX
WO 9421795	Sep 1994	WOX

	Number	Date
Parent	314594	Sep 1994
Parent	218018	Mar 1994
Parent	037057	Mar 1993

Genes encoding insecticidal proteins

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Priority Claims (1)

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US Referenced Citations (4)

Foreign Referenced Citations (8)

Non-Patent Literature Citations (33)

Divisions (1)

Continuations (1)

Continuation in Parts (3)

Entry
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