CA1341472C - Process for altering the host range of bacillus thuringiensis toxins, and novel toxins produced thereby - Google Patents

Abstract

The invention concerns an in vitro process for altering the insect host range (spectrum) of pesticidal toxins. The process comprises recombining in vitro the variable region(s) (non-homologous) of two or more genes encoding a pesticidal toxin. Specifically exemplified is the recombining of the variable regions of two genes obtained from well-known strains of Bacillus thuringiensis var. kurstaki. The resulting products are chimeric toxins which are shown to have an expanded and/or amplified insect host range as compared to the parent toxins.

Description

DESCRIPTION ~ ~ 4 1 4 7 2 This application is a division of Application No. 520,375 filed on October 14, 1986.
PROCESS FOR ALTERING THE HOST RANGE OF
BACILLUS THURINGIENSIS TOXINS, AND NOVEL
TOXINS PRODUCED THEREBY
Background of the Invention 15 The most widely used microbial pesticides are de-rived from the bacterium Bacillus thuringiensis. This bacterial agent is used to control a wide range of leaf-eating caterpillars, Japanese beetles and mos-quitos. Bacillus thuringiensis produces a proteina-20 ceous paraspore or crystal which is toxic upon ingestion by a susceptible insect host. For example, B. thur-ingiensis var. kurstaki HD-1 produces a crystal called a delta toxin which is toxic to the larvae of a number of lepido~teran insects. The cloning and expression 25 of this B.t. crystal protein gene in Escherichia coli has been described in the published literature (Schnepf, H.E. and Whiteley, H.R. [1981] Proc. Natl.
Acad. Sci. USA 78:2893-2897). U.S. Patent 4,448,885 and U.S. Patent 4,467,036 both disclose the expression 30 of B.t. crystal protein in E. coli. In U.S. 4,467,036 B. thuringiensis var. kurstaki HD-1 is disclosed as being available from the well-known NRRL culture reposi-tory at Peoria, Illinois. Its accession number there is NRRL B-3792. B, thuringiensis var. kurstaki HD-73 35 is also available from NRRL. Its accession number is NRRL B-4488.

1~4~47~
Brief Summarv of the Invention The subject invention concerns a novel process for altering the insect host range of Bacillus thurinQiensis toxins, and novel toxins produced as exemplification of this useful process. This altera-tion can result in expansion of the insect host range of the toxin, and/or, amplification of host toxicity. The process comprises recombining in vitro the variable regions) of two or more d-endotoxin genes.
Specifically exemplified is the recombining of portions of two Bacillus _thuringiensis var. kurstaki DNA
sequences, i.e., referred to herein as k-1 and k-73, to produce chimeric B.t. toxins with expanded host ranges as compared to the toxins produced by the parent DNA's.
"Variable regions," as used herein, refers to the non-homologous regions of two or more DNA
sequences. As shown by the examples presented herein, the recombining of such variable regions from two different _B._t. DNA sequences yields, unexpectedly, a DNA sequence encoding a 8-endotoxin with an expanded insect host range. In a related example, the recom-bining of two variable regions of two different B.t. toxin genes results in the creation of a chimeric toxin molecule with increased toxicity toward the target insect. The utility of this discovery by the inventorsis clearly broader than the examples disclosed herein. From this discovery, it can be expected that a large number of new and useful toxins will be produced. Thus, though the subject process is exemplified by construction of chimeric toxin-producing DNA sequences from two well-known B.t. kurstaki DNA
sequences, it should be understood that the process -3- 13~ 1~~~
is not limited to these starting DNA sequences. The invention process also can be used to construct chimeric toxins from any B. th uringiensis toxin-producing DNA sequence.
Description of the Drawines FIGURE 1: A schematic diagram plasmid pEWl which of contains the DNA seque nce encoding Bacillus thuringiensi s toxin 1.
k-FIGURE 2: A schematic diagram plasmid pEW2 which of contains the DNA seque nce encoding Bacillus thuringiensis 73.
toxin k-FIGURE 3: A schematic diagram plasmid pEW3 which of contains the DNA seque nce encoding Bacillus thuringiensi s chimerictoxin k-73/k -1 (pllY).

FIGURE 4: A schematic diagram plasmid pEW4 which of contains the DNA seque nce encoding Bacillus thuringiensi s chimerictoxin k-1/k- 73 (pYH).

Detailed Disclosure of the Invention Upon recombining in vitro the variable regions) of two or more d-endotoxin genes, there is obtained a genes) encoding a chimeric toxins) which has an expanded and/or amplified host toxicity as compared to the toxin produced by the starting genes. This recombination is done using standard well-known genetic engineering techniques.
The restriction enzymes disclosed herein can be purchased from Bethesda Research Laboratories, Gai-thersburg, MD, or New England Biolabs, Beverly, MA.
The enzymes are used accordi.np to the instructions provided by the supplier.

-4- ~3~~412 The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. These procedures are all described in Maniatis, ~I., Fritsch, E.F., and Sambrook, J.(1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Thus, it is within the skill of those in the genetic engineering art to extract DNA from microbial cells, perform restriction enzyme digestions, electrophorese DPdA
fragments, tail and anneal plasmid and insert DNA, ligate DNA, transform cells, prepare plasmid DNA, electrophorese proteins, and sequence DNA.
Plasmids pEWl , pEW2 , pEW3 , and pEW4 , constructed as described infra, have been deposited in E. coli hosts in the permanent collection (to be maintained for at least 30 years) of the Northern Regional Research Laboratory (NRRL), U.S. Department of Agriculture, Peoria, Illinois, USA. Their accession numbers and dates of deposit are as follows:
pEWl--NRRL B-18032; deposited on Nov. 29, 1985 pEW2--NRRL B-18033; deposited on Nov. 29, 1985 pEW3--NRRL B-18034; deposited on Nov. 29, 1985 pEW4--NRRL B-18035; deposited on Nov. 29, 1985 B. thuringiensis strain MTX-36, NRRL B-18101 was deposited on August 25, 1986.
Plasmid pBR322 is a well-known and available plasmid. It is maintained in the E. coli host ATCC
37017. Purified pBR322 DNA can be obtained as described in Bolivar, F., P,odriguez, R.L., Greene, P.J., Betlach, M.C., Heynecker, H.1., Boyer, H.W., Crosa, J.H. and Falkow, S. (1977) Gene 2:95-113; and Sutcliffe, J.G. (1978)Nucleic Acids Res. 5:2721-2728.
NRRL B-18032, NRRL B-18033, NRRI, B-18034 , NRRL B-180.35, and NRRL B-18101 are available to the public upon the grant of a patent which discloses these accession numbers in conjunction with the invention described herein. It should be understood that the availability of these deposits dies not constitute a license to practice the subject invention in derogation of patent rights granted for the subject invention by govern-mental action.
As disclosed above, a~ B. thuringiensis toxin-producing DNA sequence can be used as starting material for the subject invention. Examples of B. thuringiensis organisms, other than those previously given, are as follows:
Bacillus thuringiensis var. israelensis--ATCC 35646 Bacillus thuringiensis M-7--NRRT B-15939 Bacillus thuringiensis var. tenebrionis--DSM 2803 The following B. thuringiensis cultures are avail-able from the United States Department of Agriculture (USDA) at Brownsville, Texas. Requests should be made to Joe Garcia, USDA, ARS, Cotton Insects Research Unit, P.O. Box 1033, Brownsville, Texas 78520 USA.
B. thuringiensis HD2 B. thuringiensis var. finitimus HD3 B. thurin~iensis var. alesti HD4 B. thuringiensis var. kurstaki HD73 B. thuringiensis var. sotto HD770 B. thuringiensis var. dendrolimus HD7 B. thuringiensis var. kenyae HD5 B. thurinpiensis var. galleriae HD29 B. thuringiensis var. canadensis HD224 B. thuringiensis var. entomocidus HD9 B~ thurin~iensis var. subtoxicus HD109 B. thuringiensis var. aizawai HD11 B. thuringiensis var. morrisoni HD12 B. thuringiensis var. ostriniae HD501 B. thuringiensis var. tolworthi HD537 B. thuringiensis var. darmstadiensis HD146 B. thuringiensis var. toumanoffi HD201 B. thuringiensis var. kyushuensis HD541 B. thuringiensis var. thomosoni HD542 B. thurinniensis var. Pakistani HD395 B. thuringiensis var. israelensis HD567 B. thuringiensis var. Indiana HD521 B. thuringiensis var. dakota B. thuringiensis var. tohokuensis HD866 B. thuringiensis var. kumanotoensis HD867 B. thuringiensis var. tochigiensis HD868 B. thuringiensis var. colmeri HD847 B. thuringiensis var. wuhanensis HD525 Though the main thrust of the subject invention is direc=ed toward a process for altering the host range of B. thuringiensis toxins, the process is also applicable in the same sense to other Bacillus toxin-producing microbes. Examples of such Bacillus organisms which can be used as starting material are as follows:
Bacillus cereus--,'~'~'CC 21281 Bacillus moritai--ATCC 21282 Bacillus popilliae--ATCC 14706 Bacillus lentimorbus--ATCC 14707 Bacillus sphaericus--ATCC 33203 Bacillus thuringiensis M-7, exemplified herein, is a Bacillus thuringiensis isolate which, surprisingly, has activity against beetles of the order Coleoptera but not against Trichoplusia ni, Spodoptera exipua or Aedes aegypti. Included in the Coleoptera are various Diabrotica species (family Chrysomelidae) that are responsible for large agricultural losses, for example, D. undecimpunctata (western spotted cucumber beetle), D. longicornis (northern corn_ - rootworm), D.
virgitera (western corn rootworm), and D. undecimpunctata howardi (southern corn rootworm).
B. thurinaiensis M-7 is unusual in having a unique parasporal body (crystal) which under phase contrast microscopy is dark in appearance with a flat, square configuration.
The pesticide encoded by the DNA sequence used as starting material for the invention process can be any toxin produced by a microbe. For example, it can be a polypeptide which has toxic activity toward a eukaryotic multicellular pest, such as insects, e.g., coleoptera, lepidoptera, diptera, hemiptera, dermaptera, and orthoptera; or arachnids; gastropods; or worms, such as nematodes and platyhelminths. Various susceptible insects include beetles, moths, flies, grasshoppers, lice, and earwigs.
Further, it can be a polypeptide produced in active form or a precursor or proform requiring further processing for toxin activity, e.g., the novel crystal toxin of B. thuringiensis var. kurstaki, which requires processing by the pest.
The constructs produced by the process of the invention, containing chimeric toxin-producing DNA
sequences, can be transformed into suitable hosts by using standard procedures. Illustrative host cells may include either prokaryotes or eukaryotes, JJ:in 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 lower eukaryotes, such as fungi.
Illustrative pro;~caryotes, both Gram-negative and -positive, include Entereobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus;
Baceillaceae; Rhizobiaceae, 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 as Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula Aureaobasidium, ~orobolomyces, and the like.
Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the chimeric toxin-producing gene into the host, availability of expression systems, efficiency of expression, stability of the pesticide in the host, and the presence of auxiliary genetic capabilities.
Characteristics of interest for use as a pesticide micro-capsule include protective qualities for the pesticide, JJ:in i - 1~414~2 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., Saccharomvces sp., and ~orobolomyces sp.; phylloplane organisms such Pseudomonas sp., Erwinia sp. and Flavobacterium sp.; or such other organisms as Escherichia, Lactobacillus sp., Bacillus sp., and the like. Specific organisms include Pseudomonas aerucrinosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillus thurinc~iensis, Escherichia coli, Bacillus subtilis, and the like.
The chimeric toxin-producing genes) can be introduced into the host in any convenient manner, either providing for extrachromosomal maintenance or integration into the host genome.
Various constructs may be used, which include replication systems from plasmids, viruses, or centromeres in combination with an autonomous replicating segment (ars) for stable maintenance. Where only integration is desired, constructs can be used which may provide for replication, and are either transposons or have transposon-like insertion activity or provide for homology with the genome of the host. DNA sequences can be employed having the chimeric toxin-producing gene between sequences which are homologous with sequences in the genome of the host, either chromosomal or plasmid.
Desirably, the chimeric toxin-producing genes) will be present in multiple copies. See for example, U.S. Patent JJ:in -lo- ~~4~4~'~
No. 4,399,216. Thus, conjugation, transduction, transfection and transformation may be employed for introduction of the gene.
A large number of vectors are presently available which depend upon eukaryotic and prokaryotic replication systems, such as ColEl, P-1 incompatibility plasmids, e.g., pRK290, yeast 2m ~ plasmid, lambda, and the like.
Where an extrachromosomal element is employed, the DNA construct will desirably include a marker which allows for a selection of those host cells containing the construct. The marker is commonly one which provides for biocide resistance, e.g., antibiotic resistance or heavy metal resistance, complementation providing prototrophy to an auxotrophic host, or the like. The replication systems can provide special properties, such as runaway replication, can involve cos cells, or other special feature.
Where the chimeric toxin-producing genes) has transcriptional and translational initiation and termination regulatory signals recognized by the host cell, it will frequently be satisfactory to employ those regulatory features in conjunction with the gene.
However, in those situations where the chimeric toxin-producing gene is modified, as for example, removing a leader sequence or providing a sequence which codes for the mature form of the pesticide, where the entire gene encodes for a precursor, it will frequently be necessary to manipulate the DNA sequence, so that a transcriptional initiation regulatory sequence may be provided which is different from the natural one.
A wide variety of transcriptional initiation sequences exist for a wide variety of hosts. The sequence can provide for constitutive expression of the pesticide or regulated expression, where the regulation JJ:in _11- 7 2 1 ~ ~ ~ ~

may be inducible by a chemical, e.g., a metabolite, by temperature, or by a regulatable repressor. See for example, U.S. Patent No. 4,374,927. The particular choice of the promoter will depend on a number of factors, the strength of the promoter, the interference of the promoter with the viability of the cells, the effect o.f regulatory mechanisms endogenous to the cell on the promoter, and the like. A large number of promoters are available from a variety of sources, including commercial sources.

The cellular host containing the chimeric toxin-producing pesticidal gene may be grown in any convenient nutrient medium, where the DNA construct provides a selective advantage, providing for a selective medium so that substantially all or all of the cells retain the chimeric toxin-producing gene. These cells may then be harvested in accordance with conventional ways and modified in the various manners described above.

Alternatively, the cells can be fixed prior to harvesting.

Host cells transformed to contain chimeric toxin-producing DNA sequences can be treated to prolong pesticidal activity when the cells are applied to the environment of a target pest. This treatment can involve the killing of the host cells under protease deactivating or cell wall strengthening conditions, while retaining pesticidal activity.

The cells may be inhibited from proliferation in a variety of ways, so long as the technique does not deleteriously affect the properties of the pesticide, nor diminish the cellular capability in protecting the pesticide. The techniques ;nay involve physical treatment, chemical treatment, changing the physical JJ:in character of the cell or leaving the physical character of the cell substantially intact, or the like.
Various techniques for inactivating the host cells include heat, usually 50°C to 70°C; freezing; W
irradiation; lyophilization; toxins, e.g., antibiotics;
phenols; anilides, e.g., carbanilide and salicylanilide;
hydroxyurea; quaternaries; alcohols; antibacterial dyes;
EDTA and amidines; non-specific organic and inorganic chemicals, such as halogenating agents, e.g., chlorinating, brominating or iodinating agents;
aldehydes, e.g., glutaraldehyde or formaldehyde; toxic gases, such as ozone and ethylene oxide; peroxide;
psoralens; desiccating agents; or the like, which may be used individually or in combination. The choice of agent will depend upon the particular pesticide, the nature of the host cell, the nature of the modification of the cellular structure, such as fixing and preserving the cell wall with cross-linking agents, or the like.
The cells generally will have enhanced structural stability which will enhance resistance to environmental degradation in the field. Where the pesticide is in a proform the method of inactivation should be selected so as not to inhibit processing of the proform to the mature form of the pesticide by the target pest pathogen. For example, formaldehyde will crosslink proteins and could inhibit processing of the proform of ~~ polypeptide pesticide. The method of inactivation or killing retains at least a substantial portion of the bioavailability or bioacLivity of the toxin.
JJ:in -13- '~34~472 The method of treating the organism can fulfill a number of functions. First, it may enhance structural integrity. Second, it may provide for enhanced proteo-lytic stability of the toxin, by modifying the toxin so as to reduce its susceptibility to proteolytic degrada-tion and/or by reducing the proteolytic activity of proteases naturally present in the cell. The cells are preferably modified at an intact stage and when there has been a substantial build-up of the toxin protein. These modifications can be achieved in a variety of ways, such as by using chemical reagents having a broad spectrum of chemical reactivity. The intact cells can be combined with a liquid reagent medium containing the chemical reagents, with or without agitation at temperatures in the range of about -10 to 60°C. The reaction time may be determined empirically and 4ii11 vary widely with the reagents and reaction conditions. Cell concentrations will vary from about 10E2 to 10E10 per ml.
Of particular interest as chemical reagents are halogenating agents, particularly halogens of atomic no. 17-80. More particularly, iodine can be used under mild conditions and for sufficient time to achieve the desired results. Other suitable techniques include treatment with aldehydes, such as formaldehyde and glutaraldehyde; anti-infectives, such as zephiran chloride and cetylpyridinium chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Bouin's fixative and Helly's fixative (See:
Humason, Gretchen L., Animal Tissue Techniques, W.H.
Freeman and Company, 1967); or a combination of physical (heat) and chemical agents that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of the target pest(s).
For halogenation with iodine, temperatures will generally range from about 0 to 50°C, but the reaction can be conveniently carried out at room temperature.

-~4- 141472 Conveniently, the iodination may be performed using triiodide or iodine at 0.5 to 5% in an acidic aqueous medium, particularly an aqueous carboxylic acid solution that may vary from about 0.5-5M. Conveniently, acetic acid may be used, although other carboxylic acids, generally of from about 1 to 4 carbon atoms, may also be employed. The time for the reaction will generally range from less than a minute to about 24 hrs, usually from about 1 to 6 hrs. Any residual iodine may be removed by reaction with a reducing agent, such as dithionite, sodium thiosulfate, or other reducing agent compatible with ultimate usage in the field.
In addition, the modified cells may be subjected to further treatment, such as washing to remove all of the reaction medium, isolation in dry form, and formulation with typical stickers, spreaders, and adjuvants generally utilized in agricultural applications, as is well known to those skilled in the art.
Of particular interest are reagents capable of crosslinking the cell wall. A number of reagents are known in the art for this purpose. The treatment should result in enhanced stability of the pesticide.
That is, there should be enhanced persistence or residual activity of the pesticide under field conditions.
Thus, under conditions where the pesticidal activity of untreated cells diminishes, the activity of treated cells remains for periods of from 1 to 3 times longer.
The cells can be formulated for use in the environ-ment in a variety of ways. They can be employed as wettable powders, granules, or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, or phosphates) or botanical materials (powdered corncobs, rice hulls, or walnut shells). The formulations can include spreader/
sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations can be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, and the like. The ingredients can include rheological agents, surfactants, emulsifiers, dispersants, polymers, and the like.
The pesticidal concentration will vary depending upon the nature of the particular formulation, e.g., whether it is a concentrate or to be used undiluted.
The pesticide will generally be present at a concentra-tion of at least about 1% by weight, but can be up to 100% by weight. The dry formulations will have from about 1 to 95% by weight of the pesticide, while the liquid formulations will generally be from about 1 to 607 by weight of the solids in the liquid phase. The formulations will generally have from about lE2 to lE8 cells /mg.
The formulations can be applied to the environment of the pest(s), e.g., plants, soil or water, by spray-ing, dusting, sprinkling, or the like. These formula-tions can be administered at about 2 oz (liquid or dry) to 2 or more pounds per hectare, as required.
Following are examples which illustrate oroce-dures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
Example 1--Construction of plasmid pEWl The k-1 gene is the hd-1 gene described by Schnepf et al. (J. Biol. Chem. 260:6264-6272 1985).

-16- 1 3 4 1 4 ~ 2 The k-1 gene was resected from the 5' end with Ba131 up to position 504. To this position was added a Sall linker (5'GTCGACC3'). The 3' end of the gene was cleaved at position 4211 with the enzyme Ndel and blunt ended with the Klenow fragment of DNA polymerase.
The cloning vector pUC8 (Messing, J. and Vieira, J. [1982] Gene 19:269-276) which can be purchased from Pharmacia, Piscataway, NJ, was cleaved with Sall and EcoRI and cloned into plasmid pBR322 which had been cut with the same enzymes. The trp promoter (Genblock, available from Pharmacia) was blunt ended at the S' end with Klenow and inserted into this hybrid vector by blunt end ligation of the 5' end to the Smal site of the vector, and by insertion of the 3' end at the Sall site of the vector. The k-1 gene was then inserted using the Sall site at the 5' end and by blunt end ligation of the 3' end to the PvuII site of the vector.
A schematic drawing of this construct, called pEWl, is shown in Fig. 1 of the drawings.
Plasmid pEWl contains the DNA sequence encoding Bacillus thurin~iensis toxin k-1.
Example 2--Construction of plasmid pEW2 The k-73 gene is the HD-73 gene described by Adang et al. (Gene 36:289-300 1985). The k-73 gene was cleaved at position 176 with Nsil. The sequence was then cleaved at position 3212 with HindIII and the 3036 base fragment consisting of residues 176-3212 was isolated by agarose gel electrophoresis.
Plasmid pEWl, prepared as described in Example 1, was also cleaved with HindIII (position 3345 in Table 1) and partially digested with NsiI (position 556 in Table 1). The 3036 base fragment from k-73, disclosed above, was inserted into the Nsil to HindIII
region of pEWl replacing the comparable fragment of the k-1 gene, and creating plasmid pEW2. A schematic diagram of pEW2 is shown in Fig. 2 of the drawings.
Plasmid pEW2 contains the DNA sequence encoding Bacillus thurinQiensis toxin k-73.
Example 3--Construction of plasmid pEW3 The k-1 gene was cut with Sacl at position 1873.
The gene was then submitted to partial digestion with HindIII and the 1427 base fragment consisting of residues 1873 to 3345 was isolated by agarose gel electrophoresis. Plasmid pEW2 was cut with SacI
and HindIII and the large fragment representing the entire plasmid minus the SacI to HindIII fragment of the k-2 gene was isolated by agarose gel electrophore-sis. The 1427 base fragment from the k-1 gene was then ligated into the Sacl to HindIII region of pEW2, creat-ing plasmid pEW3. A schematic diagram of pEW3 is shown in Fig. 3 of the drawings.
Plasmid pEW3 contains the DNA sequence encoding Bacillus thuringiensis chimeric toxin k-73/k-1 (pHy), The nucleotide sequence encoding the chimeric toxin is shown in Table 1. The deduced amino acid sequence is shown in Table lA.
Example 4--Construction of Dlasmid pEW4 The k-1 gene was cut at position 556 with NsiI.
The gene was then cut with Sacl at position 1873 and the 1317 base fragment from Nsil to Sacl was isolated by agarose gel electrophoresis. Plasmid pEW2 was cut with Sacl and then submitted to partial digestion with Nsil. The large fragment representing the entire 1~~+1472 plasmid, minus the Nsil to Sacl region of the k-73 gene, was isolated by agarose gel electrophoresis.
The 1317 base NsiI to Sacl fragment of gene k-1 was then ligated into Nsil to SacI region of pEW2 to S create plasmid pEW4. A schematic diagram of pEW4 is shown in Fig. 4 of the drawings.
The nucleotide sequence encoding the chimeric toxin is shown in Table 2. The deduced amino acid sequence is shown in Table 2A.
Plasmid pEW4 contains the DNA sequence encoding Bacillus thuringiensis chimeric toxin k-1/k-73 (PYH).
Example 5--Insertion of Chimeric Toxin Genes Into Plants Genes coding for chimeric insecticidal toxins, as disclosed herein, can be inserted into plant cells using the Ti plasmid from Agrobacter tumefaciens.
Plant cells can then be caused to regenerate into plants (Zambryski, P., Joos, H., Gentello, C., Leemans, J., Van Montague, M. and Schell, J. [1983] EMBO J.

2:2143-2150; Bartok, K., Binns, A., Matzke, A. and Chilton, M-D. [1983] Cell 32:1033-1043). A particu-larly useful vector in this regard is pEND4K (Klee, H.J., Yanofsky, M.F. and Nester, E.W. [1985] Bio/
Technology 3:637-642). This plasmid can replicate both in plant cells and in bacteria and has multiple cloning sites for passenger genes. Toxin genes, for example, can be inserted into the BamHI site of pEND4K, propagated in E, coli, and transformed into appropriate plant cells.
Example 6--Cloning of B. thuringiensis genes into baculoviruses Genes coding for Bacillus thuringiensis chimeric toxins, as disclosed herein, can be cloned a into baculoviruses such as Autographa californica nuclear polyhedrosis virus (AcNPV). Plasmids can be constructed that contain the AcNPV genome cloned into a commercial cloning vector such as pUC8. The AcNPV genome is modified so that the coding region of the polyhedrin gene is removed and a unique cloning site for a passenger gene is placed directly behind the polyhedrin promoter. Examples of such vectors are nGP-B6874,described by Pennock et al. (Pennock, G.D., Shoemaker, C. and Miller, L.K. [1984] Mol. Cell. Biol.
4:399-406), and pAC380,described by Smith et al. (Smith, G.E., Summers, M.D. and Fraser, M.J. [1983] Mol. Cell.
Biol. 3:2156-2165). The genes coding for k-1, k-73, k-73/k-1, k-1/k-73, or other B.t. genes can be modified with BamHI linkers at appropriate regions both up-stream and downstream from the coding regions and inserted into the passenger site of one of the AcNPV
vectors.
Example 7--Chimeric Toxin Denoted ACB-1 Enhanced toxicity against all three insects tested was shown by a toxin denoted ACB-1. The toxin ACB-1 (Table 3A) is encoded by plasmid pACB-1 (Table 3).
The insecticidal activity encoded by pACB-1, in com-parison with pEW3 (Example 3), is as follows:
LC50 (O.D.575/ml) Clone T. ni H. zea S. exigua pEW3 4.3 23.0 12.3 pACB-1 1.2 3.9 1.2 -20- ~~4'~4~~
The above test was conducted using the conditions described previously.
The above results show that the ACB-1 toxin has the best composite activity as compared to the other S toxins tested herein against all three insects.
Plasmid pACB-1 was constructed between the variable region of MTX-36, a wild B. thuringiensis strain, having the deposit accession number NRRL B-18101, and the variable region of HD-73 as follows: MTX-36;
N-terminal to Sacl site. HD-73; Sacl site to C-terminal.
Total plasmid DNA was prepared from strain MTX-36 by standard procedures. The DNA was submitted to complete digestion by restriction enzymes SpeI and Dral.
The digest was separated according to size by agarose gel electrophoresis and a 1962 by fragment was purified by electroelution using standard procedures.
Plasmid pEW2 was purified and digested completely with Spel and then submitted to partial digestion with Dral. The digest was submitted to agarose gel electro-phoresis and a 4,138 by fragment was purified by electroelution as above.
The two fragments (1962 by from MTX-36 and 4138 by from pEW2 were ligated together to form construct pACB.
Plasmid DNA was prepared from pACB, digested completely with Sacl and Ndel and a 3760 by fragment was isolated by electroelution following agarose gel electrophoresis.
Plasmid oEWl was digested completely with Sacl and NdeI and a 2340 b~ fragment was isolated by electroelution following agarose gel electrophoresis.
The two fragments (3760 bn from pACB and 2340 from pEWl) were ligated together to form construct pACB-1.

The complete nucleotide sequence of the ACB-1 gene was determined and the deduced amino acid sequence of the toxin was compared with that determined for the toxin encoded by pEW3 (EW3). The result was that the deduced amino acid sequence of the ACB-1 toxin was identical to that of EW3 with two exceptions: (1) Aspartic acid residue 411 in EW3 was changed to asparagine in ACB-1 and (2) glycine residue 425 in EW3 was changed to glutamic acid in ACB-1. These two amino acid changes account for all of the changes in insect toxicity between these strains. The amino acid sequence of the EW3 toxin is as reported in Table 1. A schematic representation of these two toxins is as follows:

411 - Asp X11 - Asn 425 - Gly 425 - Glu COON COOH

-22- ~ ~ 4 1 4 7 2 The above disclosure is further exemplification of the subject invention process for altering the host range of Bacillus toxins which comprises recombining in vitro the variable region of two or more toxin genes.
Once a chimeric toxin is produced, the gene encoding the same can be sequenced by standard procedures, as disclosed above. The sequencing data can be used to alter other DNA by known molecular biology procedures to obtain the desired novel toxin. For example, the above-noted changes in the ACB-1 gene from HD-73, makes it possible to construct the ACB-1 gene as follows:
Plasmid pEW3, NRRL B-18034, was modified by altering the coding sequence for the toxin. The 151 by DNA fragment bounded by the Accl restriction site at nucleotide residue 1199 in the coding sequence, and the Sacl restriction site at residue 1350 were removed by digestion with the indicated restriction endonu-cleases using standard procedures. The removed 151 by DNA fragment was replaced with the following synthetic DNA oligomer by standard procedures:
A TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG AAT GAA
ATA CCG CCA CAG AAT AAC AAC GTG CCC CCG AGG CAA
GAA TTT AGT CAT CGA TTA AGC CAT GTT TCA ATG TTT
AGA TCT GGC TTT AGT AAT AGT AGT GTA AGT ATA ATA
AGA GCT
The net result of this change is that the aspartic residue at position 411 in the toxin encoded by pEW3 (Table lA) is converted to asparagine, and the glycine residue at position 425 is converted to a glutamic residue. All other amino acids encoded by these genes are identical.

The changes made at positions 411 and 425, dis-cussed above, clearly illustrate the sensitivity of these two positions in toxin EW3. Accordingly, the scope of the invention is not limited to the particular amino acids depicted as participating in the changes.
The scope of the invention includes substitution of all 19 other amino acids at these positions. This can be shown by the following schematic:

411 - Asp ~ 411 - X
425 - Gly ~ 425 - Y
COON COOH

wherein X is one of the 20 common amino acids except Asp when the amino acid at position 425 is Gly; Y is one of the 20 common amino acids except Gly when the amino acid at position 411 is Asp. The 20 common amino acids are as follows: alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phertylalanine, proline, serine, threonine, tryotophan, tyrosine, and valine.

-24- 1 3 4 1 ~+ 7 2 Example 8--Chimeric Toxin Denoted SYW1 Enhanced toxicity against tested insects was shown by a toxin denoted SYW1. The toxin SYW1 (Table 4A) is encoded by plasmid pSYWl (Table 4). The insecticidal activity encoded by pSYWl, in comparison with pEWl (Example 1) and pEW2 (Example 2), is as follows:
LC50 (O.D.575~m1) Clone T. ni H. zea S, exigua pEWl 3.5 12.3 18.8 pEW2 1.4 52.3 5.9 pSYWl 0.7 1.9 12.0 The above test was conducted using the conditions described previously.
Plasmid pSYW1 was constructed as follows:
Plasmid DNA from pEW2 was prepared by standard procedures and submitted to complete digestion with restriction enzyme AsuII followed by partial digestion with EcoRI. A 5878 by fragment was purified by electroelution following agarose gel electrophoresis of the digest by standard procedures.
Plasmid DNA from strain HD-1 was prepared and submitted to complete digestion with restriction enzymes AsuII and EcoRI. A 222 by fragment was purified by electroelution following agarose gel electrophoresis of the digest.
The two fragments (5878 by from pEW2 and 222 by from HD-1) were ligated together, by standard proce-dures, to form construct pSYWI.
The amino acid changes (3) in toxin SYWl from EW3 are as follows: (1) Arginine residue 289 in EW3 was _25_ ~ ~ 4 ~ ~t changed to glycine in SYWl, (2) arginine residue 311 in EW3 was changed to lysine in SYW1, and (3) the tyrosine residue 313 was changed to glycine in SYW1.
A schematic representation of these two toxins is as follows:

289 - Arg 289 - Gly 311 - Arg 311 - Lys 313 - Tyr 313 - G1u COON COON

The changes made at positions 289, 311, and 313, discussed above, clearly illustrate the sensitivity of these three positions in toxin EW3. Accordingly, the scope of the invention is not limited to the parti-cular amino acids depicted as participating in the changes. The scope of the invention includes substitution of all the common amino acids at these positions. This can be shown by the following schematic:

2 89 - Arg '~ 289 -X

311 - Arg ~ 11 -Y

313 - Tyr '~ 313 - Z

COOH COON

wherein X is one of the 20 common amino acids except Arg when the amino acid at position 311 is Arg and the amino acid at position 313 is Tyr; Y is one of the 20 common amino acids except Arg when the amino acid at position 289 is Arg and the amino acid at position 313 is Tyr; and Z is one of the 20 common amino acids except Tyr when the amino acid at position 289 is Arg and the amino acid at position 311 is Arg.
Construction of the SYW1 gene can be carried out by procedures disclosed above for the construction of the ACB-1 gene from plasmid pEW3 with appropriate changes in the synthetic DNA oligomer.

~~41472 As is well known in the art, the amino acid sequence of a protein is determined by the nucleotide sequence of the D;IA. Because of the redundancy of the genetic code, i.e., more than one coding nucleotide triplet (codon) can be used for most of the amino acids used to make proteins, different nucleotide sequences can code for a particular amino acid. Thus, the genetic code can be depicted as follows:
Phenylalanine (Phe) TTK Histidine (His) CAK
Leucine (Leu) XTY Glutamine (Gln) CAJ
Isoleucine (Ile) ATM Asparagine (Asn) AAK
Methionine (I~fet ) ATG Lys ine (Lys ) AAJ
Valine (Val) GTL Aspartic acid (Asp) GAK
Serine (Ser) QRS Glutamic acid (Glu) GAJ
Proline (Pro) CCL Cysteine (Cys) TGK
Threonine (Thr) ACL Tryptophan (Trp) TGG
Alanine (Ala) GCL Arginine (Arg) WGZ
Tyrosine (Tyr) TAB Glycine (Gly) GGL
Termination signal TAJ
Key: Each 3-letter deoxynucleotide trivlet corresponds to a trinucleotide of mRNA, having a 5'-end on the left and a 3'-end on the right. All DNA sequences given herein are those of the strand whose sequence corresponds to the mRNA sequence, with thymine substi-tuted for uracil. The letters stand for the purine or.
pyrimidine bases forming the deoxynucleotide sequence.
A = adenine G - guanine C = cytosine T = thymine X = T or C if Y is A or G
X = C if Y is C or T
Y = A, G, C or T if X is C
Y = A or G if X is T

W = C or A if Z is A or G
W = C if Z is C or T
Z = A, G, C or T if W is C
Z - A or G if W is A
QR = TC if S is A, G, C or T; alternatively QR =
AG if S is T or C
J = A or G
K = T or C
L = A, T, C ar G
M = A, C or T
The above shows that the novel amino acid sequence of the chimeric toxins, and other useful proteins, can be prepared by equivalent nucleotide sequences encoding the same amino acid sequence of the proteins.
Accordingly, the subject invention includes such equivalent nucleotide sequences. In addition it has been shown that proteins of identified structure and function may be constructed by changing the amino acid sequence if such changes do not alter the protein secondary structure (Kaiser, E.T. and Kezdy, F.J.
11984] Science 223:249-255). Thus, the subject inven-tion includes muteins of the amino acid sequences depicted herein which do not alter the protein secondary structure.
The one-letter symbol for the amino acids used in Tables lA and 2A is well known in the art. For convenience, the relationship of the three-letter abbreviation and the one-letter symbol for amino acids is as follows:
Ala A
Arg R
Asn N
Asp D

Cys C

Gln Q

Glu E

Gly G

His H

Ile I

Leu L

Lys K

Met M

Phe F

Pro P

Ser S

Thr T

Trp W

Tyr Y

Val V

The work described herein was all done in conformity with physical and biological containment requirements specified in the NIH Guidelines.

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~34fi~~2 Table 1 Nucleotide Sequence of Plasmid pEW3 Encoding Chimeric Toxin Numbering of the nucleotide bases is the same as Schnepf et al. (J. Biol. Chem. 260:6264-6272 [1985]) for HD-1 and Adang et al. (Gene 36:289-300 [1985]) for HD-73. Only protein coding sequences are shown.
( st art HO-73 ) ATG GATAACAATC 4CiC~
CGAACATCAA TGAATGCATT CCTTATAATT GTTTAAGTAA CCCTGAAGTA
GAAGTATTAG GTGGAGAAAG AATAGAAACT GGTTACACCC CAATCGATAT 50C~
TTCCTTGTCG CTAACGCAAT TTCTTTTGAG TGAATTTGTT CCCGGTGCTG
GATTTGTGTT AGGACTAGTT GATATAATAT GGGGAATTTT TGGTCCCTCT 6C~C~
CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA ACCAAAGAAT
AGAAGAATTC GCTAGGAACC AAGCCATTTC TAGATTAGAA GGACTAAGCA 7C~C~
ATCTTTATCA AATTTACGCA GAATCTTTTA GAGAGTGGGA AGCAGATCCT

CAGTGCCCTT ACAACCGCTA TTCCTCTTTT TGCAGTTCAA AATTATCAAG
TTCCTCTTTT ATCAGTATAT GTTCAAGCTG CAAATTTACA TTTATCAGTT 9C~C~
TTGAGAGATG TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC
TATCAATAGT CGTTATAATG ATTTAACTAG GCTTATTGGC AACTATACAG iCrUCr ATTATGCTGT ACGCTGGTAC AATACGGGAT TAGAACGTGT ATGGGGACCG
GATTCTAGAG ATTGGGTAAG GTATAATCAA TTTAGAAGAG AATTAACACT IloC~
AACTGTATTA GATATCGTTG CTCTGTTCCC GAATTATGAT AGTAGAAGAT

GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC AGGGCATAGA
AAGAAGTATT AGGAGTCCAC ATTTGATGGA TATACTTAAC AGTATAACCA l3CrCr TCTATACGGA TGCTCATAGG GGTTATTATT ATTGGTCAGG GCATCAAATA
ATGGCTTCTC CTGTAGGGTT TTCGGGGCCA GAATTCACTT TTCCGCTATA 14C~~
TGGAACTATG GGAAATGCAG CTCCACAACA ACGTATTGTT GGTGAACTAG

AATATAGGGA TAAATAATCA ACAACTATCT GTTCTTGACG GGACAGAATT
TGCTTATGGA ACCTCCTCAA ATTTGCCATC CGCTGTATAC AGAAAAAGCG l6Crp GARCGGTAGA TTCGCTGGAT GAAATACCGC CACAGAATAA CAACGTGCCA

AGGCTTTAGT AATAGTAGTG TAAGTATAAT AAGAGCT (end hd-73) (start HD-1) CCAACGT TTTCTTGGCA GCATCGCAGT l9Un GCTGAATTTA ATAATATAAT TCCTTCATCA CAAATTACAC AAATACCTTT
AACAAAATCT ACTAATCTTG GCTCTGGAAC TTCTGTCGTT AAAGGACCAG 2UUCr GATTTACAGG AGGAGATATT CTTCGAAGAA CTTCACCTGG CCAGRTTTCA
ACCTTAAGAG TAAATATTAC TGCACCATTA TCACAAAGAT ATCGGGTAAG 2lCrCr AATTCGCTAC GCTTCTACTA CAAATTTACA ATTCCATACA TCAATTGACG
GAAGACCTAT TAATCAGGGT AATTTTTCAG CAACTATGAG TAGTGGGAGT 220Cr AATTTACAGT CCGGAAGCTT TAGGACTGTA GGTTTTACTA CTCCGTTTAA
CTTTTCAAAT GGATCAAGTG TATTTACGTT AAGTGCTCAT GTCTTCAATT 23«C~
CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC GGCAGAAGTA

~CTGTTTACT TCTTCCAATC AAATC~GGTT AAAAACAGAT GTGACGGATT

Table 1 (cont.) ATCATATTGA TCAAGTATCC AATTTAGTTG AGTGTTTATC AGATGAATTT 25C>U
TGTCTGGATG AAAAACAAGA ATTGTCCGAG AAAGTCAAAC ATGCGAAGCG
ACTTAGTGAT GAGCGGAATT TACTTCAAGA TCCAAACTTC AGAGGGATCA ~b~rir ATAGACAACT AGACCGTGGC TGGAGAGGAA GTACGGATAT TACCATCCAA
GGAGGCGATG ACGTATTCAA AGAGAATTAC GTTACGCTAT TGGGTACCTT ~7C>0 TGATGAGTGC TATCCAACGT ATTTATATCA AAAAATAGAT GAGTCGAAAT
TAAAAGCCTA TACCCGTTAT CAATTAAGAG GGTATATCGA AGATAGTCAA 28r=r«
GACTTAGAAA TCTATTTAAT TCGCTACAAT GCAAAACATG AAACAGTAAA

GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA ATGGAATCCT
GACTTAGATT GTTCGTGTAG GGATGGAGAA AAGTGTGCCC ATCATTCGCA 3Cr00 TCATTTCTCC TTAGACATTG ATGTAGGATG TACAGACTTA AATGAGGACC

CTAGGGAATC TAGAGTTTCT CGAAGAGAAA CCATTAGTAG GAGRAGCGCT

TGGAATGGGA AACAAATATC GTTTATAAAG AGGCAAAAGA ATCTGTAGAT

TGCCATGATT CATGCGGCAG ATAAACGTGT TCATAGCATT CGAGAAGCTT
ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG TCAATGCGGC TATTTTTGAA 34C>Cr GAATTAGAAG GGCGTATTTT CACTGCATTC TCCCTATATG ATGCGAGAAA
TGTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTGC TGGAACGTGA 350?
AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGTTC GGTCCTTGTT

TCGTGGCTAT ATCCTTCGTG TCACAGCGTA CAAGGAGGGA TATGGAGAAG

AGCAACTGCG TAGAAGAGGA AATCTATCCA AATRACACGG TAACGTGTAA

ATCGAGGATA TAACGAAGCT CCTTCCGTAC CAGCTGATTA TGCGTCAGTC

TAACAGAGGG TATAGGGATT ACACGCCACT ACCAGTTGGT TATGTGACAA
ARGAATTAGA ATACTTCCCA GAAACCGATA AGGTATGGAT TGAGATTGGA 4~~C~0 GAAACGGAAG GAACATTTAT CGTGGACAGC GTGGAATTAC TCCTTATGGA
GGAA (end HD-1>

~34~47~
Table lA
Deduced Amino Acid Sequence of Chimeric Toxin Produced by Plasmid pEW3 M D t~! N F N I N E C I F Y N C L S N F E V E V L G r, E R I E
T r, Y T P I L~ I S L S L T C, F L L S E F V F G A G F V L G L
V Li I I W r, I F G F' S ~! W Li A F L V i i I E 6t L I N n F I E E
F A R ~J t:! A I S R L E G L S t~J L 'r G! I 'f A E S F F: E 4J E A L~
F' T t~l F' A L Fs E E M R I G! F tJ D M N S A L T T A I F' L F A V
i! N Y n' V F' L L S V Y V i=! A A N L H L. S V L F D V S V F G i!
F; bJ r, F Lt A A T I N S F~ Y N L~ L T R L I r, tJ Y T L~ Y A V R l~J
Y N T G L E F; V W G F Ci S R L~ W V R Y N ~:! F F F E L T L T V
L D I V A L F F' N Y C~ S R R Y F' I R T' V S G: L T R E I Y T N
F' V L E N F D G S F R G S A ~! G I E R S I R S F' H L M L~ I L
N S I T I Y T Li A H F; G Y Y 'i bJ S G H G! T M A S F V G F S G
F'EFTt= F'LYGTM13NAAFilaR I VAG!LGn!GVYF
T L S S T L Y R R F F N I G I N N i' ~t L S V L Li G T E F A Y
G T S S N L F S A V Y F. K S r, T V L~ S L D E i F F n! N N N V
F F F f! r, F S H R L S ti V 5 M F fi S G F S N 5 S V S I I R A
F' T F S lJ l' H R S A E F N N I I F' S S G! I T C! I F L T K S T
N L r, S r, T S V V K G F G F T r, G D I L F R T S F G r! I S T
L R V N I T A F L S r. F Y F V R I F Y A S T T N L n! F H T S
I L~ r, fi F' I N ~; G N F S A T M S S G S N L ~' S G S F R T V r, F T T F' F N F S N G S S V F T L S A H V F PJ S G N E V Y I D
R I E F V F' A E V T F E A E Y Lt L E R A i! K A V N E L F T S
S N n:; I G L K T Li V T G Y H I to n' V S N L V E C L S Li E F C
L L~ E K n~ E L 5 E K V K H A K F L S C~ E R N L L G! Li F' N F R
G I ~J R ~? L D R G W R G S T D I T I n! r, r, L~ L~ V F K E N Y V
T L L G T F D E C Y F' T Y L Y G' K I Lt E S K L K A Y T F; Y i-!
L R G '~( I E D S [! L~ L E I Y L I F; Y N A K H E T V N V F' G T
S L W F' L S A i'! S F I G K C G E F ~d R C A F H L E 4J N F I;i L D C S ~ F; I) r, E K C A H H 5 H H F S L C~ I L~ V ~ C T L~ L N
E Li L G ~' W V I F K I K T G! Li G H A R I_ 6 N L E F L E E K F' L V r E A L A R V K fi A E K K W R D K R E K L E W E T N I V
Y K E A K E S V Li A L F V N S t! Y D l! L n! A D T ~d i A M I H
A A D K R V H S I R E A Y L F' E L S V I F' G V N A A I F E E
L E ~ R I F T A F S L Y D A Ft ~J V I K N r, D F N N r, L S C W
N V K G H V L~ V E E n! ~d N i! F, S V L V L F' E W E A E V S u! E
V R V C F' r, F G Y I L R V T A Y K E G Y G E G C V T I H E I
E N N T Lt E L K F S N C V E E E I Y F' N N T V T C N Li Y T V
N a E E Y r, r, A Y T S F N R G Y N E A P 5 V F' A P Y A S V Y
E E K S Y T L~ G R R E N F C E F N R G Y R L~ Y T F L F' V ~ Y
V T K E L E Y F F E T D K V W I E I G E T E ~ T F I V Lt S V
E L L L M E E

~~4~~72 Table 2 Nucleotide Sequence of Plasmid pEW4 Encoding Chimeric Toxin Numbering of nucleotide bases is the same as Schnepf et al. (J. Biol. Chem. 260:6264-6272 [1985)) for HD-1 and Adang et al. (Gene 36:289-300 [1985]) for HD-73. Only protein coding sequences are shown.
tstart HD-1) ATGG ATAACAATCC GAACATCAAT
GAATGCATTC CTTATAATTG TTTAAGTAAC CCTGAAGTAG AAGTATTAGG 6C~0 TGGAGAAAGA ATAGAAACTG GTTACACCCC AATCGATATT TCCTTGTCGC

GGACTAGTTG ATATAATATG GGGAATTTTT GGTCCCTCTC AATGGGACGC

CTAGGAACCA AGCGATTTCT AGATTAGAAG GACTAAGCAA TCTTTATCAA

ATTAAGAGAA GAGATGCGTA TTCAATTCAA TGACATGAAC AGTGCCCTTA
CAACCGCTAT TCCTCTTTTG GCAGTTCAAA ATTATCAAGT TCCTCTTTTA 100Cr TCAGTATATG TTCAAGCTGC AAATTTACAT TTATCAGTTT TGAGAGATGT

GTTATAATGA TTTAACTAGG CTTATTGGCA ACTATACAGA TTATGCTGTG

TTGGGTAAGG TATAATCAAT TTAGAAGAGA GCTAACACTT ACTGTATTAG

ACAGTTTCCC AATTAACAAG AGAAATTTAT ACGAACCCAG TATTAGAAAA
TTTTGAT~GT AGTTTTCGTG GRATGGCTCA GAGAATAGAA CA~AATATTA 1400 GGCAACCACA TCTTATGGAT ATCCTTAATA GTATAACCAT TTATACTGAT
GTGCATAGAG GCTTTAATTA TTGGTCAGGG CATCAAATAA CAGCTTCTCC l5ui~
TGTAGGGTTT TCAGGACCAG AATTCGCATT CCCTTTATTT GGGAATGCGG
GGAATGCArC TCCACCCGTA CTTGTCTCAT TAACTGGTTT GGGGATTTTT ibC>C~
AGAACATTAT CTTCACCTTT ATATAGAAGA ATTATACTTG GTTCAGGCCC
AAATAATCAG GAACTGTTTG TCCTTGATGG AACGGAGTTT TCTTTTGCCT 17C»~
CCCTAACGAC CAACTTGCCT TCCACTATAT ATAGACAAA~ GGGTACAGTC
GATTCACTA6 ATGTAATACC GCCACAGGAT AATAGTGTAC CAGCTCGTGC 180Cr GGGATTTAGC CATCGATTGA GTCATGTTAC AATGCTGAGC CAAGCAGCTG
GAGCAGTTTA CACCTTGAGA GCTCAACGT (stop HD-11 (start HLv-73) CCT ATGTTCTCTT

ACTCAAATCC CTGCAGTGAA GGGAAACTTT CTTTTTAATG GTTCTGTAAT
TTCAGGACCA GGATTTACTG GTGGGGACTT AGTTAGATTA AATAGTAGTG l9UCa GAAATAACAT TCAGAATAGA GGGTATATTG AAGTTCCAAT TCACTTCCCA
TCGACATCTA CCAGATATCG AGTTCGTGTA CGGTATGCTT. CTGTAACCCC 2000 GATTCACCTC AACGTTAATT GGGGTAATTC ATCCATTTTT TCCAATACAG
TACCAGCTAC AGCTACGTCA TTAGATAATC TACAATCAAG TGATTTTGGT ~~10~?
TATTTTGAAA GTGCCAATGC TTTTACATCT TCATTAGGTA ATATAGTAGG

TTATTCCAGT TACTGCAACA CTCGAGGCTG AATATAATCT GGRAAGAGCG

d Table 2 (cont.) CAGAAGGCGG TGAATGCGCT GTTTACGTCT ACAAACCAAC TAGGGCTAAA 23U«
AACAAATGTA ACGGATTATC ATATTGATCA AGTGTCCAAT TTAGTTACGT
ATTTATCGGA TGAATTTTGT CTGr,ATGAAA AGCGAGAATT GTCCGAGAAA 'r4C>0 GTCAAACATG CGAAGCGACT CAGTGATGAA CGCAATTTAC TCCAAGATTC

CAGGGATTAC CATCCAAGGA GGGGATGACG TATTTAAAGA AAATTACGTC
ACACTATCAG GTACCTTTGA TGAGTGCTAT CCAACATATT TGTATCAAAA ~6C~C>
AATCGATGAA TCAAAATTAA AAGCCTTTAC CCGTTATCAA TTAAGAGGGT

AAACATGAAA CAGTAAATGT GCCAGGTACG GGTTCCTTAT GGCCGCTTTC

ACCTTGAATG GAATCCTGAC TTAGATTGTT CGTGTAGGGA TGGAGAAAAG

AGACTTAAAT GAGGACCTAG GTGTATGGGT GATCTTTAAG ATTAAGACGC

TTAGTAGGAG AAGCGCTAGC TCGTGTGAAA AGAGCGGAGA AAAAATGGAG

CAAAAGAATC TGTAGATGCT TTATTTGTAA ACTCTCAATA TGATCAATTA
CAAGCGGATA CGAATATTGC CATGATTCAT GCGGCAGATA AACGTGTTCA 3200 _ TAGCATTCGA GAAGCTTATC TGCCTGAGCT GTCTGTGATT CCGGGTGTCA

CTATATGATG CGAGAAATGT CATTAAAAAT GGTGATTTTA ATAATGGCTT

AACGTTCGGT CCTTGTTGTT CCGr,AATGrr, AAr,~AGAArT GTCACAAGAA

GGAGGGATAT GGAGAAGGTT GCGTAACCAT TCATGAGATC GAGAACAATA
CAGACGAACT GAAGTTTAGC AACTGCr,TAG AAGAGGAAAT CTATCCAAAT 360?
AACACGGTAA CGTGTAATGA TTATACTGTA AATCAAGAAG AATACGGAGG
TGCGTACACT TCTCGTAATC GAGGATATAA C~AAGCTCCT TCCGTACGAG 370C>
CTGATTATGC GTCAGTCTAT GAAGAAAAAT CGTATACAGA TGGACGAAGA
GAGAATCCTT GTGAATTTAA CAGAGGGTAT AGGGATTACA CGCCACTACC 38i~i~
AGTTGGTTAT GTGACAAAAG AATTAGAATA CTTCCCAGAA ACCGATAAGG

GAAT'('ACTCC TTATGGAGGA A (end HD-73) i -3s- 1 3 4 1 4 7 2 Table 2A
Deduced Amino Acid Sequence of Chimeric Toxin Produced by Plasmid pEW4 M G tJ N F N T N E C i F Y N C L S N F E V E V L r, r, E R I E
TT3YT~F I G I SLSLTr_.r.FLLSEFVF'G~AGFVLGL
V D I I ld G I F G F S n W D A F F' V n I E G! L I N r! R I E E
F A R N r! A I S R L E ~ L 5 tJ L Y >>! I Y A E S F R E 4J E A D
F' T N F' A L R E E M R I r. F N D M N 5 A L T T A I F L L A V
r~ N Y G! V F' L L S V Y V G! A A N L H L S V L R G V S V F G G!
R W r, F G A A T I N S R Y N D L T R L. I G N Y T Lt Y A V R W
Y N T G L E R V W r, F' G S R D W V iY N L; F R R E L T L T V
L D I V A L F S N Y D S R R Y F I F T V S ('~ L T R E I Y T N
F V L E N F D G S F F i3 M A i! R I E ~~! N I R ~? F' H L M D I L
N S I T I Y T D V H F ~3 F N Y W S G H r. I T A S F V G F S G
F E F A F F' L F G N A r; to A A F' F' v L V S L T G L r, I F F T
L S S F L Y F: F; I I L G S G F' N N n! E L F V L D r, T E F S F
A S L T T PJ L F' S T I Y F; i>! F; G T V D S L D V I ~ F' G! D N S
V F F R A G F S H R L '= H 'J T P1 l_ S n! A A G A V Y T L R A n!
RF'~iFSW I HRSAEF NfJ I I ASDS I Ty! I F'AVK G
l'J F L F N ~ S V I S G P ~ F T G G D L. V R L N S S G N PJ I n!
N R r, Y I E V F' I H F P S T S T F; Y f; V R V R Y A S V T F' I
H L tJ V N W G PJ S S I F S N T V F' A T A T S L Lt PJ L G! S S D
F G Y F E S A N A F T S S L G N I V r-, V F; N F S G T A G V I
I G R F E F I F' V T A T L E A E Y N L E R A l.! K A V N A L F
T S T N ~7 L G L K T N V T G Y H I D n V S N L v T Y L S D E
F C L D E K R E L S E K V K H A K R L S D E F N L L G D S N
F K G I N R G! F E R G W r, G S T G I T I ~: G G G G V F K E N
Y V T L S G T F Lt E C Y F' T Y L Y i=! K I D E S K L K A F T F~
Y a L R G Y I E D S G G L E I Y L i R Y N A K H E T V N V F
G T G S L W F L S A G S F' I G K C G E F N R C A F H L E W,N
P L~ L D C S C R D r, E K C A H H S H H F S L D I D V G C T G
L N E D L G V W V I F K I K T G! G r, H A R L G N L E F L E E
K F L V r, E A L A R V K R A E K K W R G K R E K L E W E T N
I V Y K E A K E S V D A L F V N S i! Y G ~:? L l~ A D T N I A M
I H A A D K R V H S I R E A Y L F' E L 5 V I F' G V N A A I F
E E L E G R I F T A F S L Y D A R N V I K N G G F N N G L S
C ld N V K r, H V G V E E G! N N G! R S V L V V F E W E A E V S
t:! E V R V C F ~ R G Y I L R ',i T A Y K E 6 Y G E ~ C V T I H
E I E N N T G E L K F S N C V E E E I Y F' N N T V T C N D Y
T V N n! E E Y r, r, A Y T S R t'J R ~ Y N E A F' S V F A G Y A S
V Y E E K S Y T D G R F.' E N F C E F N R G Y R G Y T F L F V
G Y V T K E L E Y F F F_ T L~ K V W I E I G E T E G T F I V G
S V E L L_ L M E E

Table 3 Nucleotide Sequence of Plasmid pACB-1 Encoding Chimeric Toxin ACB-1 The nucleotide differences as compared to the sequence shown in Table 1 are underlined at Dositions 1618 and 1661 and code for amino acid changes at positions 411 and 425 as shown in Table 3A.
' (start HD-73) ATG GATAACAATC ~i00 CGAACATCAA TGAATGCATT CCTTATAATT GTTTAAGTAA CCCTGAAGTA
GAAGTATTAG GTGGAGAAAG AATAGAAACT GGTTACACCC CAATCGATAT SC~C~
TTCCTTGTCG CTAACGCAAT TTCTTTTGAG TGAATTTGTT CCCGGTGCTG
GATTTGTGTT AGGACTAGTT GRTATAATAT GGGGARTTTT TGGTCCCTCT 6««
CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA ACCAAAGART
AGAAGAATTC GCTAGGAACC AAGCCATTTC TAGATTRGAA GGACTAAGCA 7C~C~
ATCTTTATCA AATTTRCGCA GRATCTTTTA GRGAGTGGGA RGCAGRTCCT

CAGTGCCCTT ACAACCGCTA TTCCTCTTTT TGCAGTTCAA AATTATCAAG

TTGAGAGRTG TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC

ATTATGCTGT ACGCTGGTAC AATACGGGAT TAGAACGTGT ATGGGGACCG

AACTGTATTA GATATCGTTG CTCTGTTCCC GRATTATGAT AGTAGAAGAT
ATCCAATTCG~AACAGTTTCC CAATTAACAA GAGAAATTTA TACAAACCCA 1200 GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC AGGGCATAGA
AAGAAGTATT AGGAGTCCAC ATTTGATGGA TATACTTARC AGTATAACCA 130«
TCTATACGGA TGCTCATAGG GGTTATTATT:ATTGGTCAGG GCATCAAATA
ATGGCTTCTC.CTGTAGGGTT TTCGrrr,CCA GAATTCACTT TTCCGCTATA 1400 TCiGAACTATG GGAAATGCAG CTCCACAACA ACGTATTGTT GCTCAACTAG , AATATAGGGA TAAATRATCA ACAACTATCT GTTCTTGACG GGACAGAATT

GAACGGTAGA TTCGCTG_AAT GAAATACCGC CACAGAATAA CAACGTGCCA
CCTAGGCAAG _AATTTAGTCA TCGATTARGC CATGTTTCAA TGTTTCGTTC 1700 AGGCTTTAGT AATAGTAGTG TAAGTATAAT AAGAGCT (end hd-73) (start Hti-1) CCARCGT TTTCTTGGCA GCATCGCRGT 1900 GCTGAATTTA ATAATATAAT TCCTTCATCA CAAATTACAC AAATACCTTT
AACRAAATCT ACTAATCTTG GCTCTGGAAC TTCTGTCGTT AAAGGACCAG ~~OC>0 GATTTACAGG AGGAGATATT CTTCGAAGAA CTTCACCTGG CCAGATTTCA
ACCTTAAGAG TAAATATTAC TGCACCATTA TCACAAAGAT ATCGGGTAAG ~1c'>~~
AATTCGCTAC GCTTCTACTA CAAATTTACA ATTCCATACA TCAATTGACG
GAAGACCTAT TAATCAGGGT AATTTTTCAG CAACTATGAG TAGTGGGAGT ~~00 AATTTACAGT~CCGGAAGCTT TAGGACTGTA GGTTTTACTA CTCCGTTTAA

CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC GGCAGAAGTA

GCTGTTTACT TCTTCCAATC AAATCGGGTT AAAAACAGAT GTGACGGATT
ATCATATTGA TCRAGTATCC AATTTAGTTG AGTGTTTATC AGATGAATTT L5V'J
TGTCTGGaTG AAAAACAAGA ATTGTCCGAG AAAGTCRAAC ATGCGAAGCG

134~~~~
Table 3 (cont.) ACTTAGTGAT GAGCGGAATT TACTTCAAGA TCCAAACTTC AGAGGGATCA ~bGC~
ATAGACAACT AGACCGTGGC TGGAGAGGAA GTACGGATAT TACCATCCAA
GGAGGCGATG ACGTATTCAA AGAGAATTAC GTTACGCTAT TGGGTACCTT i7U~
TGATGAGTGC TATCCAACGT ATTTATATCA AAAAATAGAT GAGTCGAAAT
TAAAAGCCTA TACCCGTTAT CAATTAAGAG GGTATATCGA AGATAGTCAA ~8C~o GACTTAGAAA TCTATTTAAT TCGCTACAAT GCAAAACATG AAACAGTAAA
TGTr,CCAr,~;T ACGGGTTCCT TATGGCCGCT TTCAGCCCfIA AGTCCAATCG ~9CW
GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA ATGGAATCCT
~3r=CTTAGATT GTTCGTGTAG GGATGGAGAA AAGTGTGCCC ATCATTCGCA 3C~C~i>
~TCAT~fTCTCC TTAGACATTG ATGTAGGATG TACAGACTTA AATGAGGACC
TAGGTGTATG GGTGATCTTT AAGATTAAGA CGCAAr"~Trr GCACGCAAGA 3100 CTAGGr~AATC TAGAGTTTCT CGAAGAGAAA CCAT1A~TIaG GHGAAGCGCT
AGCTC~TGT~ AAAAGAGCGG AGAAAAAATG GAGAGHCAAA CG-fGAAAAAT 3~oio TGGAATGGGA AACAAATATC GTTTATAAAG AGGCAA~aAr,A ATCTGTAGAT
GCTTTATTTG TAAACTCTCA A-fATGATCAA TTACAAr;CGr, ATACGAATAT 33~:>i>
IGCCATGATT CATGCGrCAG ATAAACGTGT TCATAGCA1-T CGAGAAGCTT
ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG TCAATGCGGC TATTTTTGAA 34C»~
GAATTAGAAG GGCGTATTTT CACTGCATTC TCCCTATATG ATGCGAGAAA
1'GTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTGC TG~;Ar=aCGTGA 35C~«
AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGT'I'C GGTCCTTGTT
CTTCCGGAAT GGGAAGCAGA AGTGTCACAA GAAGTTCGTG TCTGTCCGGG 36C~C~
TCGTGGCTAT ATCCTTCGTG TCACAGCGTA CAAGGAGGGA TATGGAGAAG
~TTGCGTAAC CATTCATGAG ATCGAGAACA ATACAGACGA ACTGAAGTTT 37«c>
~1~~CAACT~Cr, TAGAAGAGGA AATCTATCCA AATAACACGG TAACGTGTAA
TGATTATACT GTAAATCAAG AAGAATACGG AGGTGC,3TAC ACTTCTCGTA 38oC~
ATCGAG;aT;'~ TAACGAAGCT CCTTCCGTAC CAGCTGATTA TGCGTCAGTC
TATGAAGAAA AATCGTATAC F'GATGGACGA AGAGAGAATC CTTGTGAATT 39C~C>
TAACAGAGGG TATAGGGATT ACACGCCACT ACCAGTTGGT TATGTGACAA
l-;AGAATTAGA ATACTTCCCA GAAACCGATA AGGTATGGAT TGAGATTGGA 4C~CeC~
~AAACGr,AAG GAACATTTAT CGTGGACAGC GTGGAATTAC TCCTTATGGA
GGAA tend HG-1>

Table 3A
Deduced Amino Acid Sequence of Chimeric Toxin M D I'J N P N I N E C I P Y N C L S N F' E V E V L G G E R I E
T G Y~~~T P t D I S L S L T CI F L L S E F V P G A G F V L G L
V Lv I I W G I F G P S C~ W D A F L V et I E Ct L I N CI R I E E
F .A R N ft A I S .fi L E G L S N L Y Gt I Y A E S F R E W E A D
P T N P A L R E E M R I G? F N D M N S A L T T A I P L.F A V
C: N Y G! V P L L S V Y V G A A 1J L H L S V L Ft D V S V F G C?
fi~4Jr,F C~AAT I NSF:YNDLTfiL I GNYTDYAVfiW
Y N,T G L E fi V 4J G P D S fi D W V fi Y N Gt F R R E L T L T V
L D I V A L F F' N Y D S fi fi Y F I R T V S G L T R E I Y T N
P V L E N F D G S F R G S A C: G I E fi S I fi S F H L M D I L
N 5 I T I Y T D A H R G Y Y Y W S r, H r,! I M A S P V G F S G
P E F~ T F F L Y: G .T M G N A A F 6t a R I V A ~~ L G Ct G V Y fi T L S S T L Y R F; F' F N I G I N N G Ct L S V L Lt G T E F A Y
G T S S N L F' S A V Y R K S G T V D S L N E I P P r~ PJ N N V
P P F: t? E F S H fi L S H V S M F R S G F s N S 5 V S I I fi A
F' T F S l~l f! H R S A E F N N I I F' S S r! I T Ct I F L T K S T
N L G S G T S V V K G P G F T r, r D I L R fi T S F G n; I S T
L R V N I T A F L S G! Fs Y F; V R I R Y A S T T N L i=! F H T S
I D G R P I N It G N F S A T M S S G S N L G! S G S F R T V G
F T T F' F N F S N G S S V F T L S A H 'J F N S G N E V Y I L~
R I E F V F' A E V T F E A E Y Li L E Ft A t; K A V N E L F T S
S N rt I G L K T D V T f.~ Y H I D ~; V S N L V E C L S D E F C
L D E K ~:~ E L S E K V K H A K R L S L~ E F~ N L L C? L~ F' N F R
G I N R ~! L L~ fi G 4J R G S T C~ I T I n! r, r, L~ L V F K E N Y V
T- L L r, T F D E C Y F' T Y L Y f! K I C~ E S K L K A Y T R Y
L fi G Y I E D S ft Li L E I Y L I R Y N A K H E T V N V F' G T
r, S L DJ F' L S A G! S F' I r, K C G E F ~J F: C A F H L E ~J N P D
L D C S C fi D G E K C A H H S H H F S L Ci I D V G C T L~ L N
E D L G V 4J V I F K I K T r. L~ r, H A R L G N L E F L E E K P
L V G E A L A R V K fi A E K K 4J F; D K R E K L E W E T N I V
Y K E A K E 5 V L~ A L F V N 5 L; Y L~ G! L _n.., A D T .i'J I A M I H
A A D K R V H S I R E A Y L F' E L 5 V I F G V N A A I F E E
'L.E G R I F T A.F S L Y D A R N V I K N G Li F N N.G L S C W
N V K r, .H V D V E E Ct N N it R S V L V L F E W E 'A E V S G! E
V fi.V C.P G R G Y I L R V T A Y K E G Y G E G C V T I H E I
E N N T L~ E L K F S N C V E E E I Y F N N T V T C N Ll Y T V
N C? E E Y G G A Y T S fi N fi G Y N E A P S V F' A D Y A S V Y
E E K S Y'vT D G R R E N P C E F N fi G Y R L~ Y T P L P ~V G Y
V T K E L E Y F P E T~D K V W i E I G E T E G T F I V Li S V
E L L L N E E

~i Table 4 Nucleotide Sequence of Plasmid pSYWI Encoding Chimeric Toxin SYW1 The nucleotide differences as compared to the sequence shown in Table 1 are underlined at positions 1252, 1319, 1320, 1323, 1324, and 1326; and code for amino acid changes at positions 289, 311, and 313, as shown in Table 4A.
( st art HLi-73 ATG GATAACAATC4<.~C~
>

CGAACATCAATGAATGCATTCCTTATAATTGTTTAAGTAACCCTGAAGTA

GAAGTATTAGGTGGAGAAAGAATAGAAACTGGTTACACCCCAATCGATAT5C~

TTCCTTGTCGCTAACGCAATTTCTTTTGAGTGAATTTGTTCCCGGTGCTG

GATTTGTGTTAGr,ACTA~TTGATATAATATGrr,GAATTTTTGGTCCCTCT6 CAATGGGACGCATTTCTTGTACAAATTGAACAGTTAATTAACCAAAGAAT

AGAAGAATTCGCTAGGAACCAAGCCATTTCTAGATTAGAAGGACTAAGCA7C~

ATCTTTATCA'AATTTACGCAGAATCTTTTAGAGAGTGGGAAGCAGATCCT

ACTAATCCAGCATTAAGAGAAGAGATGCGTATTCAATTCAATGACATGAABCKr CAGTGCCCTTACAACCGCTATTCCTCTTTTTGCAGTTCAAAATTATCAAG

TTCCTCTTTTATCAGTATATGTTCAAGCTGCAAATTTACATTTATCAGTT9C~C~

TTGAGAGATGTTTCAGTGTTTGGACAAAGGTGGGGATTTGATGCCGCGAC

TATCAATAGTCGTTATAATGATTTAACTAGGCTTATTGGCAACTATACAGlr?C

ATTATGCTGTACGCTGGTAGAATACGGGATTAGAACGTGTATGGGGACCG

GATTCTAGAGATTGGGTAAGGTATAATCAATTTAGAAGAGAATTAACACT11<r~r AACTGTATTAGATATCGTTGCTCTGTTCCCGAATTATGATAGTAGAAGAT

ATCCRATTCGAACAGTTTCCCAATTARCAAGAGAAATTTATACAAACCCA1'Cr GTATTAGAAAATTTTGATGGTAGTTTTCGAGGCTCGGCTCAGGGCATAGA

A

_ ~AA GGGGAATATTATTGGTCAGGGCATCAAATA
TCTATACGGAT~GCTCATA

ATGGCTTCTC_ TTCGGr,GCCAGAAT1'CACTTTTCCGCTATA1~Cr CTGTAGGGTT

TGGAACTATGGGAAATGCAGCTCCACAACAACGTATTGTTGCTCAACTAG

GTCAGGGCGTGTATAGAACATTATCGTCCACTTTATATAGAAGACCTTTT1~<?

AATATAGGGATAAATAATCAACAACTATCTGTTCTTGACGGGACAGAATT

TGCTTATGGAACCTCCTCAAATTTGCCATCCGCTrTATACAGAAAAAGCG16x, GAACGGTAGATTCGCTGGATGAAATACCGCCACAGAATAACAACGTGCCA

CCTAGGCAAGGATTTAGTCATCGAT'fAAGCCATGTTTCAATGTTTCGTTC170 AGGCTTTAGTAATAGTAGTGTAAGTATAATAAGAGCT
tend hd-73) (sta.rt .HLi-i) CCAACGT TTTCTTGGCAGCATCGCAGT19GC~

GCTGAATTTAATAATATAATTCCTTCATCACAAATTACACAAATACCTTT

AACAAAATCTACTAATCTTGGCTCTGGAACTTCTGTCGTTAAAGGACCAG~~?C~

GATTTACAGG~AGGAGATATTCTTCGAAGAACTTCACCTGGCCAGATTTCA

ACCTTAAGAGTAAATATTACTGCACCATTATCACAAAGATATCGGGTAAG2lU~r AATTCGCTACGCTTCTACTACAAATTTACAATTCCATACATCAATTGACG

GAAGACCTA'f-TAATCAGGGTAATTTTTCAGCAACTATGAGTAGTGGGAGT~~Cr AATTTACAGT~CCGGAAGCTTTAGGACTGTAGGTTTTACTACTCCGTTTAA

CTTTTCAAAT TATTTACGTTAAGTGCTCATGTCTTCAATT23~r0 GGATCAAGTG

CAGGCAATGA AATTTGTTCCGGCAGAAGTA
AGTTTATATA
GATCGAATTG

ACCTTTGAGG CGGTGAATGA

TTTA~AAAGA
r,CACAAAA6G

GCTGTTTACT AAAAACAGATGTGACGGATT
TCTTCCAATC
AAATCGGGTT

-43- 1 3 4 ~ 4 7 2 Table 4 (cont.) ATCATATTGATCAAGTATCCAATTTAGTTGAGTGTTTATCAGATGAATTT~5C7 TGTCTGGATGAAAAACAAGAATTGTCCGAGAAAGTCAAACATGCGAAGCG

ACTTAGTGATGAGCGGAATTTAGTTCAAGATCCAAACTTCAGAGGGATCA~bvn ATAGACAACTAGACCGTGGCTGGA~A~GAAGTACGGATATTACCATCCAA

r,GAGGCGATGACGTATTCAAAGAGAATTACGTTACGCTATTGGGTACCTT27C>

TGATGAGTGCTATCCAACGTATTTATATCAAAAAATAGATGAGTCGAAAT

TAAAAGCCTATACCCGTTATCAATTAAGAGGGTATATCGAAGATAGTCAA~~8C~

GACTTAGAAATCTATTTAATTCGCTACAATGCAAAACATGAAACAGTAAA

GAAAGTGTGGAGAGCCGAATCGATGCGCGCCACACCTTGAATGGAATCCT

TCATTTCTCCTTAGACATTGATGTAGGATGTACAGACTTAAATGAGGACC

TAGGTGTATGGGTGATCTTTAAGATTAAGACGCAAGATGGGCACGCAAGA31C>ci CTAGGGAATCTAGAGTTTCTCGAAGAGAAACCATTAGTAGGAGAAGCGCT

TGGAATGGGAAACAAATATCGTTTATAAAGAGGCAAAAGAATCTGTAGAT

TGCCATGATTCATGCGGCAGATAAACGTGTTCATAGCATTCGAGAAGCTT

ATCTGCCTGAGCTGTCTGT.GATTCCGGGTGTCAATGCGGCTATTTTTGAA3400 GAATTAGAAGGGCGTATTTTCACTGCATTCTCCCTATATGATGCGAGAAA

AAGGGCATGTAGATGTAGAAGAACAAAACAACCAACGTTCGGTCCTTGTT

TCGTGGCTATATCCTTCGTGTCACAGCGTACAAGGAGGGATATGGAGAAG

AGCAACTGCGTAGAAGAGGAAATCTATCCAAATAACACGGTAACGTGTAA

TGATTATACTGTAAATCAAGAAGAATACGGAGGTGCGTACACTTCTCGTA3BC>0 ATCGAGGATATAACGAAGCTCCTTCCGTACCAGCTGATTATGCGTCAGTC

TAACAGAGGGTATAGGGATTACACGCCACTACCAGTTGGTTATGTGACAA

AAGAATTAGAATACTTCCCAGAAACCGATAAGGTATGGATTGAGATTGGA4C~OC~

GAAACGGAAGGAACATTTATCGTGGACAGCGTGGAATTACTCCTTATGGA

GGAA (endHG-1) tified B. t. crystal protein 1 toxicity against at least Table 4A
ny of said crystal protein Amino Acid Sequence of Chimeric Toxin SYW1 hereby the recombinant protein toxin is identifiedN I N EC i F YN C LS N F EV E VL G G ER I E
~

I D I SL S L Tr!F LL S E FV F'r,A G F VL G L

in having an altered G I F GF S C7WD A FL V itIE r7LI N r,,~RI E E
host A I S RL E r,LS t~JLY CtI YA E SF R E ~JE A D

L R E EM R I G!F N LiM N S AL T TA I F'LF A V

F' L L SV Y V ~tA A NL H L SV L C;D V S.VF G i=!

A A T IN S R YN D LT R L IG CJYT D Y AV R l~J

E R V WG P D SR D WV R Y NC?F RR E L TL T V

'3. t. crystal proteinL F F NY D S RR Y FI R T VS C!LT R E iY T N
toxin F D G SF R G SA G GI E G SI R SP H L MD I L

t one target lepidopteranY T D AH K G EY Y WS G _ ~I M AS P V GF S G
H

F L Y GT _ G _A A-Fr?Q R IV A nL G C~GV Y R
rocess of: M N
the b p L Y R RP F N Ir,I NN C!C7LS V LD G T EF A Y
y 5t arent DNA se uence L F S AV Y R KS G TV D S LD E IP P G,NN N V
p q F S H RL S H VS M ~'R 5 G FS N 5S V S II R A

~

xin with at least a ~ H R 5A E F NN I IF S S ~!I T ~I P L TK 5 T
part of T S V VK G P GF T GG D I LR R TS F G GI S T

A sequence encoding T A F LS G7R YR V RI R Y AS T TN L CtFH T S
a I N C7GN F S AT M SS G S NL G;SG S F RT V G

~ to obtain a recombinantN F 5 NG S S VF T LS A H VF N SG N E VY I D

F A E VT F E AE Y LtL E R.A6!K AV N E LF T S

protein toxin which L K T DV T D YH I DG?V S NL V EC L S L~E F C
is E L 5 EK V K HA K RL S D ER N LL L!D F'N F R

~ded by said parent L D R GW R G S.T D IT I C,GG D DV F K EN Y V
DNA

F D E CY F T YL Y G!K I D ES K LK A Y TR Y~G!

E D S (?D L E .IY L~IR Y N AK H ET V N VF G T

from said recombinant L S A QS P I GK C GE F N R.CA F.H L E WN F D
F S D V G C TD L N

R D G EK C A HH S HH L I D

L A R VK R A EK ~KWR D K RE K LE W E TN I V

to verify whether saidE 5 V DA L F VN S C,Y D C;LG A DT N I AM I H
E

V H S IR E A YL P EL S V IF G VN A A IF E

host range or increasedF T A FS L Y DA R NV I K N.GD FN N G LS C W

V D V EE Q N Nn R SV L V LF E WE A E VS C~E

! host as compared G R G YI L R VT A YK E G YG E GC V T IH .EI
to any E L K'FS .NC VE E EI Y F NN T VT C N CiY T V

it DNA sequences; : G G A YT ~ R :R G YN E A FS V FA D Y AS V Y
S N

T D G RR E N FC E FN R G YR D YT F L F'V r,Y

lifted B. t. crystal ~E Y F PE T D KV W IE I G ET E GT F I VD S ' protein V

E E

i toxicity against at least ny of said crystal protein hereby the recombinant motein toxin is identified

Claims (4)

CLAIMS:

1. A process for identifying a recombinant DNA sequence encoding a modified B.t. crystal protein toxin which has an altered host range comprising the steps of:
(a) replacing at least a part of a variable region of a first parent DNA
sequence encoding a lepidopteran active B.t. crystal protein toxin with at least a part of a variable region of at least one other parent DNA sequence encoding a different lepidopteran active B.t. crystal protein toxin to obtain a recombinant DNA sequence encoding a modified B.t. crystal protein toxin which is different from any of said crystal protein toxins encoded by said parent DNA
sequences;
(b) producing said modified B.t. crystal protein toxin from said recombinant DNA sequence; and (c) assaying said modified B.t. crystal protein toxin to verify whether said modified B.t. crystal protein toxin has an altered host range or increased toxicity against at least one target lepidopteran insect host as compared to any of said crystal protein toxins encoded by said parent DNA sequences:
whereby, if verified, said recombinant DNA sequence is identified as one encoding a modified B.t. crystal protein toxin having an altered host range or increased toxicity.

2. A recombinant DNA sequence encoding a modified B.t. crystal protein toxin which has an altered host range wherein said recombinant DNA sequence is produced by the process of:
(a) replacing at least a part of a variable region of a first parent DNA
sequence encoding a lepidopteran active B.t. crystal protein toxin with at least a part of a variable region of at least one other parent DNA sequence encoding a different lepidopteran active B.t. crystal protein toxin to obtain a recombinant DNA sequence encoding a modified B.t. crystal protein toxin which is different from any of said crystal protein toxins encoded by said parent DNA

sequences;
(b) producing said modified B.t. crystal protein toxin from said recombinant DNA sequence;
(c) assaying said modified B.t. crystal protein toxin to verify whether said modified B.t. crystal protein toxin has an altered host range or increased toxicity against at least one target lepidopteran insect host as compared to any of said crystal protein toxins encoded by said parent DNA sequences;
whereby, if verified, said recombinant DNA sequence is identified as one encoding a modified B.t. crystal protein toxin having an altered host range or increased toxicity; and (d) obtaining additional copies of said recombinant DNA sequence.

3. A process for identifying a recombinant DNA sequence encoding a modified B.t. crystal protein toxin which has an altered host range comprising the steps of:
(a) replacing at least a part of a variable region of a first parent DNA
sequence encoding a lepidopteran active B.t. crystal protein toxin with at least a part of a variable region of at least one other parent DNA sequence encoding a different lepidopteran active B.t. crystal protein toxin to obtain a recombinant DNA sequence encoding a modified B.t. crystal protein toxin which is different from any of said crystal protein toxins encoded by said parent DNA
sequences;
(b) producing said modified B.t. crystal protein toxin from said recombinant DNA sequence;
(e) assaying said modified B.t. crystal protein toxin to verify whether said modified B.t. crystal protein toxin has an altered host range or increased toxicity against at least one target lepidopteran insect host as compared to any of said crystal protein toxins encoded by said parent DNA sequences; and (d) repeating the foregoing steps (a) - (c) until said modified B.t. crystal protein is verified to have an altered host range or increased toxicity against at least one target lepidopteran insect host as compared to any of said crystal protein toxins encoded by said parent DNA sequences. whereby the recombinant DNA sequence encoding said modified B.t. crystal protein toxin is identified as one encoding a modified B.t. crystal protein toxin having an altered host range or increased toxicity.

4. A recombinant DNA sequence encoding a modified B.t. crystal protein toxin which has an altered host range wherein said recombinant DNA sequence is produced by the process of:
(a) replacing at least a part of a variable region of a first parent DNA
sequence encoding a lepidopteran active B.t. crystal protein toxin with at least a part of a variable region of at least one other parent DNA sequence encoding a different lepidopteran active B.t. crystal protein toxin to obtain a recombinant DNA sequence encoding a modified B.t. crystal protein toxin which is different from any of said crystal protein toxins encoded by said parent DNA
sequences;
(b) producing said modified B.t. crystal protein toxin from said recombinant DNA sequence;
(c) assaying said modified B.t. crystal protein toxin to verify whether said modified B.t. crystal protein toxin has an altered host range or increased toxicity against at least one target lepidopteran insect host as compared to any of said crystal protein toxins encoded by said parent DNA sequences;
(d) repeating the foregoing steps (a) - (c) until said modified B.t. crystal protein is verified to have an altered host range or increased toxicity against at least one target lepidopteran insect host as compared to any of said crystal protein toxins encoded by said parent DNA sequences, whereby the recombinant DNA sequence encoding said modified B.t. crystal protein toxin is identified as one encoding a modified B.t. crystal protein toxin having an altered host ranger or increased toxicity; and (e) obtaining additional copies of said recombinant DNA sequence.