Bacillus thuringiensis toxins active against corn rootworm larvae

Disclosed and claimed are toxins and genes from Bacillus thuringiensis strains designated PS80JJ1, PS158D5, PS167P, PS169E, PS177F1, PS177G, PS204G4, PS204G6 which can be used to control corn rootworm. Mutants which retain the activity of the parent strain can be used to control the pest. Further, isolated spores or purified toxins from these isolates can be used to control corn rootworm. Genes encoding .delta.-endotoxins can be removed from these strains using standard well-known techniques, and transferred to other hosts. Expression of the .delta.-endotoxin in such hosts results in control of corn rootworm larvae.

BACKGROUND OF THE INVENTION 
The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, 
spore-forming bacterium characterized by parasporal crystalline protein 
inclusions. These inclusions often appear microscopically as distinctively 
shaped crystals. The proteins can be highly toxic to pests and specific in 
their toxic activity. Certain B.t. toxin genes have been isolated and 
sequenced, and recombinant DNA-based B.t. products have been produced and 
approved for use. In addition, with the use of genetic engineering 
techniques, new approaches for delivering these B.t. endotoxins to 
agricultural environments are under development, including the use of 
plants genetically engineered with endotoxin genes for insect resistance 
and the use of stabilized intact microbial cells as B.t. endotoxin 
delivery vehicles (Gaertner, F. H., L. Kim [1988] TIBTECH 6:S4-S7). Thus, 
isolated B.t. endotoxin genes are becoming commercially valuable. 
Until the last ten years, commercial use of B.t. pesticides has been 
largely restricted to a narrow range of lepidopteran (caterpillar) pests. 
Preparations of the spores and crystals of B. thuringiensis subsp. 
kurstaki have been used for many years as commercial insecticides for 
lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1 
produces a crystalline .delta.-endotoxin which is toxic to the larvae of a 
number of lepidopteran insects. 
In recent years, however, investigators have discovered B.t. pesticides 
with specificities for a much broader range of pests. For example, other 
species of B.t., namely israelensis and tenebrionis (a.k.a.B.t. M-7, 
a.k.a.B.t. san diego), have been used commercially to control insects of 
the orders Diptera and Coleoptera, respectively (Gaertner, F. H. [1989] 
"Cellular Delivery Systems for Insecticidal Proteins: Living and 
Non-Living Microorganisms," in Controlled Delivery of Crop Protection 
Agents, R. M. Wilkins, ed., Taylor and Francis, New York and London, 1990, 
pp. 245-255). See also Couch, T. L. (1980) "Mosquito Pathogenicity of 
Bacillus thuringiensis var. israelensis," Developments in Industrial 
Microbiology 22:61-76; Beegle, C. C., (1978) "Use of Entomogenous Bacteria 
in Agroecosystems," Developments in Industrial Microbiology 20:97-104. 
Krieg, A., A. M. Huger, G. A. Langenbruch, W. Schnetter (1983) Z. ang. 
Ent. 96:500-508, describe Bacillus thuringiensis var. tenebrionis, which 
is reportedly active against two beetles in the order Coleoptera. These 
are the Colorado potato beetle, Leptinotarsa decernlineata, and Agetastica 
alni. 
Recently, new subspecies of B. t. have been identified, and genes 
responsible for active .delta.-endotoxin proteins have been isolated 
(Hofte, H., H.R. Whiteley [1989] Microbiological Reviews 52(2):242-255). 
Hofte and Whiteley classified B.t. crystal protein genes into 4 major 
classes. The classes were CryI (Lepidoptera-specific), CrylI (Lepidoptera- 
and Diptera-specific), CryIII (Coleoptera-specific), and CryIV 
(Diptera-specific). The discovery of strains specifically toxic to other 
pests has been reported. (Feitelson, J. S., J. Payne, L. Kim [1992] 
Bio/Technology 10:271-275). 
The cloning and expression of a B.t. crystal protein gene in Escherichia 
coli has been described in the published literature (Schnepf, H. E., H. R. 
Whiteley [1981] Proc. Natl. Acad. Sci. USA 78:2893-2897). U.S. Pat. Nos. 
4,448,885 and 4,467,036 both disclose the expression of B.t. crystal 
protein in E. coli. U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose B. 
thuringiensis strain tenebrionis (a.k.a. M-7, a.k.a.B.t. san diego) which 
can be used to control coleopteran pests in various environments. U.S. 
Pat. No. 4,918,006 discloses B.t. toxins having activity against 
Dipterans. U.S. Pat. No. 4,849,217 discloses B.t. isolates which have 
activity against the alfalfa weevil. U.S. Pat. No. 5,208,077 discloses 
coleopteran-active Bacillus thudngiensis isolates. U.S. Pat. No. 5,151,363 
and U.S. Pat. No. 4,948,734 disclose certain isolates of B.t. which have 
activity against nematodes. As a result of extensive research and 
investment of resources, other patents have issued for new B.t. isolates 
and new uses of B.t. isolates. However, the discovery of new B.t. isolates 
and new uses of known B.t. isolates remains an empirical, unpredictable 
art. 
Approximately 9.3 million acres of U.S. corn are infested with corn 
rootworm species complex each year. The corn rootworm species complex 
includes the northern corn rootworm, Diabrotica barberi, the southern corn 
rootworm, D. undecimpunctata howardi, and the western corn rootworm, D. 
virgifera virgifera. The soil-dwelling larvae of these Diabrotica species 
feed on the root of the corn plant, causing lodging. Lodging eventually 
reduces corn yield and often results in death of the plant. By feeding on 
cornsilks, the adult beetles reduce pollination and, therefore, 
detrimentally effect the yield of corn per plant. In addition, adults and 
larvae of the genus Diabrotica attack cucurbit crops (cucumbers, melons, 
squash, etc.) and many vegetable and field crops in commercial production 
as well as those being grown in home gardens. 
Control of corn rootworm has been partially addressed by cultivation 
methods, such as crop rotation and the application of high nitrogen levels 
to stimulate the growth of an adventitious root system. However, chemical 
insecticides are relied upon most heavily to guarantee the desired level 
of control. Insecticides are either banded onto or incorporated into the 
soil. The major problem associated with the use of chemical insecticides 
is the development of resistance among the treated insect populations. 
BRIEF SUMMARY OF THE INVENTION 
The subject invention concerns novel materials and methods for controlling 
corn rootworm. The materials and methods of the subject invention result 
from the unexpected discovery that certain B.t. isolates, as well as 
toxins from these isolates, have activity against this pest. 
More specifically, the methods of the subject invention use B.t. microbes, 
or variants thereof, and/or their toxins, to control corn rootworms. 
Specific B.t. microbes useful according to the invention are B.t. PS80JJ1, 
B.t. PS158D5, B.t. PS167P, B.t. PS169E, B.t. PS177F1, B.t. PS177G, B.t. 
PS204G4, and B.t. PS204G6. Further, the subject invention also includes 
the use of variants of the exemplified B.t. isolates which have 
substantially the same corn rootworm-active properties as the specifically 
exemplified B.t. isolates. Such variants would include, for example, 
mutants. Procedures for making mutants are well known in the 
microbiological art. Ultraviolet light and nitrosoguanidine are used 
extensively toward this end. 
The subject invention also includes the use of genes from the B.t. isolates 
of the invention which genes encode the corn rootworm-active toxins. 
Still further, the invention includes the treatment of substantially intact 
B.t. cells, and recombinant cells containing the genes of the invention, 
treated to prolong the corn rootworm activity when the substantially 
intact cells are applied to the environment of a target pest. Such 
treatment can be by chemical or physical means, or a combination of 
chemical and physical means, so long as the chosen means do not 
deleteriously affect the properties of the pesticide, nor diminish the 
cell's capability of protecting the pesticide. The treated cell acts as a 
protective coating for the pesticidal toxin. The toxin becomes active upon 
ingestion by a target insect. 
Finally, the subject invention concerns plants cells transformed with genes 
of the subject invention which encode corn rootworm-active toxins.

DETAILED DISCLOSURE OF THE INVENTION 
Certain Bacillus thuringiensis stains useful according to the subject 
invention are disclosed in U.S. Pat. No. 5,151,363. The disclosure of the 
cultures and their taxonomic characteristics are incorporated herein by 
reference to said patent. 
The B.t. isolates of the subject invention have been deposited in the 
permanent collection of the Agricultural Research Service Patent Culture 
Collection (NRRL), Northern Regional Research Center, 1815 North 
University Street, Peoria, Ill. 61604, USA. The culture repository numbers 
of the B.t. strains are as follows: 
______________________________________ 
Culture Repository No. 
Deposit Date 
______________________________________ 
B.t. strain PS80JJ1 
NRRL B-18679 
July 17, 1990 
B.t. strain PS158D5 
NRRL B-18680 
July 17, 1990 
B.t. strain PS167P 
NRRL B-18681 
July 17, 1990 
B.t. strain PS169E 
NRRL B-18682 
July 17, 1990 
B.t. strain PS177F1 
NRRL B-18683 
July 17, 1990 
B.t. strain PS177G 
NRRL B-18684 
July 17, 1990 
B.t. strain PS204G4 
NRRL B-18685 
July 17, 1990 
B.t. strain PS204G6 
NRRL B-18686 
July 17, 1990 
E. coli NM522 (pMYC2365) 
NRRL- 
E. coli NM522 (pMYC2379) 
NRRL B-21155 
Nov. 3, 1993 
______________________________________ 
Certain of these culture deposits are now available to the public by virtue 
of the issuance of U.S. Pat. No. 5,151,363. 
Other cultures have been deposited under conditions that assure that access 
to the cultures will be available during the pendency of this patent 
application to one determined by the Commissioner of Patents and 
Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. The 
deposits are available as required by foreign patent laws in countries 
wherein counterparts of the subject application, or its progeny, are 
filed. However, it should be understood that the availability of a deposit 
does not constitute a license to practice the subject invention in 
derogation of patent rights granted by governmental action. 
Genes and toxins. The genes and toxins useful according to the subject 
invention include not only the full length sequences disclosed but also 
fragments of these sequences, variants, mutants, and fusion proteins which 
retain the characteristic pesticidal activity of the toxins specifically 
exemplified herein. As used herein, the terms "variants" or "variations" 
of genes refer to nucleotide sequences which encode the same toxins or 
which encode equivalent toxins having pesticidal activity. As used herein, 
the term "equivalent toxins" refers to toxins having the same or 
essentially the same biological activity against the target pests as the 
claimed toxins. 
It should be apparent to a person skilled in this art that genes encoding 
active toxins can be identified and obtained through several means. The 
specific genes exemplified herein may be obtained from the isolates 
deposited at a culture depository as described above. These genes, or 
portions or variants thereof, may also be constructed synthetically, for 
example, by use of a gene synthesizer. Variations of genes may be readily 
constructed using standard techniques for making point mutations. Also, 
fragments of these genes can be made using commercially available 
exonucleases or endonucleases according to standard procedures. For 
example, enzymes such as Ba131 or site-directed mutagenesis can be used to 
systematically cut off nucleotides from the ends of these genes. Also, 
genes which encode active fragments may be obtained using a variety of 
restriction enzymes. Proteases may be used to directly obtain active 
fragments of these toxins. 
Equivalent toxins and/or genes encoding these equivalent toxins can be 
derived from B.t. isolates and/or DNA libraries using the teachings 
provided herein. There are a number of methods for obtaining the 
pesticidal toxins of the instant invention. For example, antibodies to the 
pesticidal toxins disclosed and claimed herein can be used to identify and 
isolate other toxins from a mixture of proteins. Specifically, antibodies 
may be raised to the portions of the toxins which are most constant and 
most distinct from other B.t. toxins. These antibodies can then be used to 
specifically identify equivalent toxins with the characteristic activity 
by immunoprecipitation, enzyme linked immunosorbent assay (ELISA), or 
western blotting. Antibodies to the toxins disclosed herein, or to 
equivalent toxins, or fragments of these toxins, can readily be prepared 
using standard procedures in this art. The genes which encode these toxins 
can then be obtained from the microorganism. 
Fragments and equivalents which retain the pesticidal activity of the 
exemplified toxins would be within the scope of the subject invention. 
Also, because of the redundancy of the genetic code, a variety of 
different DNA sequences can encode the amino acid sequences disclosed 
herein. It is well within the skill of a person trained in the art to 
create these alternative DNA sequences encoding the same, or essentially 
the same, toxins. These variant DNA sequences are within the scope of the 
subject invention. As used herein, reference to "essentially the same" 
sequence refers to sequences which have amino acid substitutions, 
deletions, additions, or insertions which do not materially affect 
pesticidal activity. Fragments retaining pesticidal activity are also 
included in this definition. 
A further method for identifying the toxins and genes of the subject 
invention is through the use of oligonucleotide probes. These probes are 
detectable nucleotide sequences. These sequences may be detectable by 
virtue of an appropriate label or may be made inherently fluorescent as 
described in International Application No. WO93/16094. As is well known in 
the art, if the probe molecule and nucleic acid sample hybridize by 
forming a strong bond between the two molecules, it can be reasonably 
assumed that the probe and sample have substantial homology. Preferably, 
hybridization is conducted under stringent conditions by techniques 
well-known known in the art, as described, for example, in Keller, G. H., 
M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pp. 
169-170. Detection of the probe provides a means for determining in a 
known manner whether hybridization has occurred. Such a probe analysis 
provides a rapid method for identifying toxin-encoding genes of the 
subject invention. The nucleotide segments which are used as probes 
according to the invention can be synthesized using DNA synthesizer and 
standard procedures. These nucleotide sequences can also be used as PCR 
primers to amplify genes of the subject invention. 
Certain toxins of the subject invention have been specifically exemplified 
herein. Since these toxins are merely exemplary of the toxins of the 
subject invention, it should be readily apparent that the subject 
invention comprises variant or equivalent toxins (and nucleotide sequences 
coding for equivalent toxins) having the same or similar pesticidal 
activity of the exemplified toxin. Equivalent toxins will have amino acid 
homology with an exemplified toxin. This amino acid homology will 
typically be greater than 75%, preferably be greater than 90%, and most 
preferably be greater than 95%. The amino acid homology will be highest in 
critical regions of the toxin which account for biological activity or are 
involved in the determination of three-dimensional configuration which 
ultimately is responsible for the biological activity. In this regard, 
certain amino acid substitutions are acceptable and can be expected if 
these substitutions are in regions which are not critical to activity or 
are conservative amino acid substitutions which do not affect the 
three-dimensional configuration of the molecule. For example, amino acids 
may be placed in the following classes: non-polar, uncharged polar, basic, 
and acidic. Conservative substitutions whereby an amino acid of one class 
is replaced with another amino acid of the same type fall within the scope 
of the subject invention so long as the substitution does not materially 
alter the biological activity of the compound. Table 1 provides a listing 
of examples of amino acids belonging to each class. 
TABLE 1 
______________________________________ 
Class of Amino Acid 
Examples of Amino Acids 
______________________________________ 
Nonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp 
Uncharged Polar 
Gly, Ser, Thr, Cys, Tyr, Asn, Gln 
Acidic Asp, Glu 
Basic Lys, Arg, His 
______________________________________ 
In some instances, non-conservative substitutions can also be made. The 
critical factor is that these substitutions must not significantly detract 
from the biological activity of the toxin. 
The toxins of the subject invention can also be characterized in terms of 
the shape and location of toxin inclusions, which are described above. 
Recombinant Hosts. The toxin-encoding genes harbored by the isolates of the 
subject invention can be introduced into a wide variety of microbial or 
plant hosts. Expression of the toxin gene results, directly or indirectly, 
in the intracellular production and maintenance of the pesticide. With 
suitable microbial hosts, e.g., Pseudomonas, the microbes can be applied 
to the situs of the pest, where they will proliferate and be ingested. The 
result is a control of the pest. Alternatively, the microbe hosting the 
toxin gene can be treated under conditions that prolong the activity of 
the toxin and stabilize the cell. The treated cell, which retains the 
toxic activity, then can be applied to the environment of the target pest. 
Where the B.t. toxin gene is introduced via a suitable vector into a 
microbial host, and said host is applied to the environment in a living 
state, it is essential that certain host microbes be used. Microorganism 
hosts are selected which are known to occupy the "phytosphere" 
(phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one or more 
crops of interest. These microorganisms are selected so as to be capable 
of successfully competing in the particular environment (crop and other 
insect habitats) 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. 
A large number of microorganisms are known to inhabit the phylloplane (the 
surface of the plant leaves) and/or the rhizosphere (the soil surrounding 
plant roots) of a wide variety of important crops. These microorganisms 
include bacteria, algae, and fungi. Of particular interest are 
microorganisms, such as bacteria, e.g., genera Pseudomonas, Erwinia, 
Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, 
Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, 
Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; 
fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus, 
Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of 
particular interest are such phytosphere bacterial species as Pseudomonas 
syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter 
xylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroides, 
Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, and 
Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula 
rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. 
diffiuens, C. laurenti, Saccharomyces rosei, S. pretoriensis, S. 
cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and 
Aureobasidium pollulans. Of particular interest are the pigmented 
microorganisms. 
A wide variety of ways are available for introducing a B.t. gene encoding a 
toxin into a microorganism host under conditions which allow for stable 
maintenance and expression of the gene. These methods are well known to 
those skilled in the art and are described, for example, in U.S. Pat. No. 
5,135,867, which is incorporated herein by reference. 
Treatment of cells. As mentioned above, B.t. or recombinant cells 
expressing a B.t. toxin can be treated to prolong the toxin activity and 
stabilize the cell. The pesticide microcapsule that is formed comprises 
the B.t. toxin within a cellular structure that has been stabilized and 
will protect the toxin when the microcapsule is applied to the environment 
of the target pest. Suitable host cells 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 toxic substances are 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. 
The cell will usually be intact and be substantially in the proliferative 
form when treated, rather than in a spore form, although in some instances 
spores may be employed. 
Treatment of the microbial cell, e.g., a microbe containing the B.t. toxin 
gene, can be by chemical or physical means, or by a combination of 
chemical and/or physical means, so long as the technique does not 
deleteriously affect the properties of the toxin, nor diminish the 
cellular capability of protecting the toxin. Examples of 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 glutaraldehyde; anti-infectives, such as 
zephiran chloride and cetylpyridinium chloride; alcohols, such as 
isopropyl and ethanol; various histologic fixatives, such as Lugol iodine, 
Bouin's fixative, various acids and Hetty'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 preserve and 
prolong the activity of the toxin produced in the cell when the cell is 
administered to the host environment. Examples of physical means are short 
wavelength radiation such as gamma-radiation and X-radiation, freezing, UV 
irradiation, lyophilization, and the like. Methods for treatment of 
microbial cells are disclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462, 
which are incorporated herein by reference. 
The cells generally will have enhanced structural stability which will 
enhance resistance to environmental conditions. Where the pesticide is in 
a proform, the method of cell treatment 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 a polypeptide 
pesticide. The method of treatment should retain at least a substantial 
portion of the bio-availability or bioactivity of the toxin. 
Characteristics of particular interest in selecting a host cell for 
purposes of production include ease of introducing the B.t. 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 
microcapsule include protective qualities for the pesticide, such as thick 
cell walls, pigmentation, and intracellular packaging or formation of 
inclusion bodies; survival in aqueous environments; 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. 
Growth of cells. The cellular host containing the B.t. insecticidal 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 B.t. gene. These cells 
may then be harvested in accordance with conventional ways. Alternatively, 
the cells can be treated prior to harvesting. 
The B.t. cells of the invention can be cultured using standard art media 
and fermentation techniques. Upon completion of the fermentation cycle the 
bacteria can be harvested by first separating the B.t. spores and crystals 
from the fermentation broth by means well known in the art. The recovered 
B.t. spores and crystals can be formulated into a wettable powder, liquid 
concentrate, granules or other formulations by the addition of 
surfactants, dispersants, inert carriers, and other components to 
facilitate handling and application for particular target pests. These 
formulations and application procedures are all well known in the art. 
Formulations. Formulated bait granules containing an attractant and spores 
and crystals of the B.t. isolates, or recombinant microbes comprising the 
genes obtainable from the B.t. isolates disclosed herein, can be applied 
to the soil. Formulated product can also be applied as a seed-coating or 
root treatment or total plant treatment at later stages of the crop cycle. 
Plant and soil treatments of B.t. cells may be employed as wettable 
powders, granules or dusts, by mixing with various inert materials, such 
as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, 
and the like) or botanical materials (powdered corncobs, rice hulls, 
walnut shells, and the like). The formulations may include 
spreader-sticker adjuvants, stabilizing agents, other pesticidaladditives, 
or surfactants. Liquid formulations may be aqueous-based or non-aqueous 
and employed as foams, gels, suspensions, emulsifiable concentrates, or 
the like. The ingredients may include theological agents, surfactants, 
emulsifiers, dispersants, or polymers. 
As would be appreciated by a person skilled in the art, the pesticidal 
concentration will vary widely depending upon the nature of the particular 
formulation, particularly whether it is a concentrate or to be used 
directly. The pesticide will be present in at least 1% by weight and may 
be 100% by weight. The dry formulations will have from about 1-95% by 
weight of the pesticide while the liquid formulations will generally be 
from about 1-60% by weight of the solids in the liquid phase. The 
formulations will generally have from about 10.sup.2 to about 10.sup.4 
cells/mg. These formulations will be administered at about 50 mg (liquid 
or dry) to 1 kg or more per hectare. 
The formulations can be applied to the environment of the pest, e.g., soil 
and foliage, by spraying, dusting, sprinkling, or the like. 
Mutants. Mutants of the isolates of the invention can be made by procedures 
well known in the art. For example, an asporogenous mutant can be obtained 
through ethylmethane sulfonate (EMS) mutagenesis of an isolate. The 
mutants can be made using ultraviolet light and nitrosoguanidine by 
procedures well known in the art. 
A smaller percentage of the asporogenous mutants will remain intact and not 
lyse for extended fermentation periods; these strains are designated lysis 
minus (-). Lysis minus strains can be identified by screening asporogenous 
routants in shake flask media and selecting those mutants that are still 
intact and contain toxin crystals at the end of the fermentation. Lysis 
minus strains are suitable for a cell treatment process that will yield a 
protected, encapsulated toxin protein. 
To prepare a phage resistant variant of said asporogenous mutant, an 
aliquot of the phage lysate is spread onto nutrient agar and allowed to 
dry. An aliquot of the phage sensitive bacterial strain is then plated 
directly over the dried lysate and allowed to dry. The plates are 
incubated at 30.degree. C. The plates are incubated for 2 days and, at 
that time, numerous colonies could be seen growing on the agar. Some of 
these colonies are picked and subcultured onto nutrient agar plates. These 
apparent resistant cultures are tested for resistance by cross streaking 
with the phage lysate. A line of the phage lysate is streaked on the plate 
and allowed to dry. The presumptive resistant cultures are then streaked 
across the phage line. Resistant bacterial cultures show no lysis anywhere 
in the streak across the phage line after overnight incubation at 
30.degree. C. The resistance to phage is then reconfirmed by plating a 
lawn of the resistant culture onto a nutrient agar plate. The sensitive 
strain is also plated in the same manner to serve as the positive control. 
After drying, a drop of the phage lysate is placed in the center of the 
plate and allowed to dry. Resistant cultures showed no lysis in the area 
where the phage lysate has been placed after incubation at 30.degree. C. 
for 24 hours. 
Following are examples which illustrate procedures, 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--Culturing of B.t. Isolates of the Invention 
A subculture of the B.t. isolates, or mutants thereof, can be used to 
inoculate the following medium, a peptone, glucose, salts medium. 
______________________________________ 
Bacto Peptone 7.5 g/l 
Glucose 1.0 g/l 
KH.sub.2 PO.sub.4 3.4 g/l 
K.sub.2 HPO.sub.4 4.35 g/l 
Salt Solution 5.0 ml/l 
CaCl.sub.2 Solution 5.0 ml/l 
pH 7.2 
Salts Solution (100 ml) 
MgSO.sub.4.7H.sub.2 O 2.46 g 
MnSO.sub.4.H.sub.2 O 0.04 g 
ZnSO.sub.4.7H.sub.2 O 0.28 g 
FeSO.sub.4.7H.sub.2 O 0.40 g 
CaCl.sub.2 Solution (100 ml) 
CaCl.sub.2.2H.sub.2 O 3.66 g 
______________________________________ 
The salts solution and CaCl.sub.2 solution are filter-sterilized and added 
to the autoclaved and cooked broth at the time of inoculation. Flasks are 
incubated at 30.degree. C. on a rotary shaker at 200 rpm for 64 hr. 
The above procedure can be readily scaled up to large fermentors by 
procedures well known in the art. 
The B.t. spores and/or crystals, obtained in the above fermentation, can be 
isolated by procedures well known in the art. A frequently-used procedure 
is to subject the harvested fermentation broth to separation techniques, 
e.g., centrifugation. 
Example 2--Purification of Protein and Amino Acid Sequencing 
The Bacillus thuringiensis (B.t.) isolates were cultured as described in 
Example 1 or can be cultured using other standard media and fermentation 
techniques well-known in the art. Delta-endotoxins were isolated and 
purified by harvesting toxin protein inclusions by standard sedimentation 
centrifugation. Recovered parasporal inclusion bodies of some of the 
isolates were partially purified by sodium bromide (26-40%) isopycnic 
gradient centrifugation (Pfannenstiel, M. A., E. J. Ross, V. C. Kramer, K. 
W. Nickerson [1984] FEMS Microbiol. Lett. 21:39). Thereafter the 
individual toxin proteins were resolved by solubilizing the crystalline 
protein complex in alkali buffer and fractionating the individual proteins 
by DEAE-sepharose CL-6B (Sigma Chem. Co., St. Louis, Mo.) chromatography 
by step-wise increments of increasing concentrations of an NaCl-containing 
buffer (Reichenberg, D., in Ion Exchangers in Organic and Biochemistry [C. 
Calmon and T. R. E. Kressman, eds.], Interscience, New York, 1957). 
Fractions containing a protein toxic to corn rootworm were bound to PVDF 
membrane (Millipore, Bedford, Mass,) by western blotting techniques 
(Towbin, H., T. Staehelin, K. Gordon [1979] Proc. Natl. Acad. Sci. USA 
76:4350) and the N-terminal amino acids determined by the standard Edman 
reaction with an automated gasphase sequenator (Hunkapiller, M. W., R. M. 
Hewick, W. L. Dreyer, L. E. Hood [1983]Meth. Enzyrnol. 91:399). 
The sequence obtained from the PS204G6 20-25 kDa polypeptide was: 
##STR1## 
where X represents an amino acid residue with an undetermined identity. 
From this sequence data oligonucleotide probes were designed by utilizing 
a codon frequency table assembled from available sequence data of other 
B.t. toxin genes. The probes can be synthesized on an Applied Biosystems, 
Inc. DNA synthesis machine. 
Example 3--Molecular Cloning and Expression of Gene Encoding a Toxin from 
Bacillus thudngiensis Strain PS204G6 
Total cellular DNA was prepared from Bacillus thuringiensis (B.t.) cells 
grown to an optical density, at 600 nm, of 1.0. Cells were pelleted by 
centrifugation and resuspended in protoplast buffer (20 mg/ml lysozyme in 
0.3M sucrose, 25 mM Tris-Cl (pH 8.0), 25 mM EDTA). After incubation at 
37.degree. C. for 1 hour, protoplasts were lysed by two cycles of freezing 
and thawing. Nine volumes of a solution of 0.1M NaCl, 0.1% SDS, 0.1M 
Tris-Cl were added to complete lysis. The cleared lystate was extracted 
twice with phenol:chloroform (1:1). Nucleic acids were precipitated with 
two volumes of ethanol and pelleted by centrifugation. The pellet was 
resuspended in TE buffer and RNase was added to a final concentration of 
50/.mu.g/ml. After incubation at 37.degree. C. for 1 hour, the solution 
was extracted once each with phenol:chloroform (1:1) and TE-saturated 
chloroform. DNA was precipitated from the aqueous phase by the addition of 
one-tenth volume of 3M NaOAc and two volumes of ethanol. DNA was pelleted 
by centrifugation, washed with 70% ethanol, dried, and resuspended in TE 
buffer. 
An oligonucleotide probe with the following sequence was synthesized based 
on the PS204G6 20-25 kDa toxin peptide sequence: 
##STR2## 
This oligonucleotide contains a 5' BamHI cloning site and is mixed at 
three positions as shown. This probe was radiolabeled with .sup.32 P and 
used in standard hybridizations of Southern blots of PS204G6 total 
cellular DNA. Hybridizing bands included an approximately 2.4 kbp HindIII 
fragment. This DNA fragment contains all or a fragment of this PS204G6 
toxin gene. 
A gene library was constructed from PS204G6 DNA partially digested with 
Sau3A. Partial restriction digests were fractionated by agarose gel 
electrophoresis. DNA fragments 9.3 to 23 kbp in size were excised from the 
gel, electroeluted from the gel slice, purified on an Elutip-D ion 
exchange column (Schleicher and Schuell, Keene, N.H.), and recovered by 
ethanol precipitation. The Sau3A inserts were ligated into BamHI-digested 
LambdaGem-11 (Promega, Madison, Wis.). Recombinant phage were packaged and 
plated on E. coli KW251 cells. Plaques were screened by hybridization with 
the radiolabeled probe described above. Hybridizing phage were 
plaque-purified and used to infect liquid cultures of E. coli KW251 cells 
for isolation of DNA by standard procedures (Maniatis et al, supra. ). 
For subcloning the gene encoding the PS204G6 toxin, preparative amounts of 
phage DNA were digested with EcoRI+Sal1 and electrophoresed on an agrose 
gel. The approximately 5.5 kbp band containing the toxin gene was excised 
from the gel, electroeluted from the gel slice, and purified by ion 
exchange chromatography as described above. The purified DNA insert was 
ligated into EcoRI+Sal1-digested pHTBlueII (an E. coli/B. thuringiensis 
shuttle vector comprised of pBluescript S/K (Stratagene, La Jolla, Calif.) 
and the replication origin from a resident B.t. plasmid [D. Lereclus et 
al. (1989) FEMS Microbiology Letters 60:211-218]). The ligation mix was 
used to transform frozen, competent E. coli NM522 cells (ATCC 47000). 
.beta.-galactosidase.sup.- transformants were screened by restriction 
digestion of alkaline lysate plasmid minipreps. The desired plasmid 
construct, pMYC2365, contains a toxin gene that is novel compared to other 
.delta.-endotoxin genes. 
pMYC2365 was introduced into the acrystalliferous (Cry.sup.-) B.t. host, 
CryB (A, Aronson, Purdue University, West Lafayette, Ind.), by 
electroporation. Expression of an approximately 75-85 kDa toxin was 
demonstrated by SDS-PAGE analysis. The polypeptide profile of the cloned 
toxin was similar to that of purified native PS204G6 crystals. In addition 
to the 75-85 kDa polypeptide, both native and cloned toxins exhibited the 
approximately 20-25 kDA polypeptide. 
Example 4--Cloning and Expression of a Novel Toxin Gene from Bacillus 
thudngiensis strain PS80JJ1 
Total cellular DNA was prepared from B.t. cells as described in Example 3. 
An approximately 700-800 bp DNA fragment from a novel PS80JJ1 130 kDa 
toxin gene was obtained by polymerase chain reaction (PCR) amplification 
using PS80JJ1 cellular DNA and the following primers: 
##STR3## 
The DNA fragment was cloned into pBluescript S/K (Stratagene, LaJolla, 
Calif.) and partially sequenced by dideoxynucleotide DNA sequencing 
methodology (Sanger et al. [1977] Proc. Natl. Acad. Sci. USA 74:5463-5467) 
using Sequenase (US Biochemicals, Cleveland, Ohio). DNA sequences unique 
to at least one PS80JJ1 toxin gene were identified by computer comparison 
with other known .delta.-endotoxin genes. 
The 700-800 bp DNA fragment was radiolabelled with .sup.32 P and used in 
standard hybridizations of Southern blots of PS80JJ1 total cellular DNA. 
Hybridizing bands included an approximately 1.8 kbp EcoRI fragment and an 
approximately 9.5 kbp HindIII fragment. These hybridizing DNA bands 
contain toxin genes or restriction fragments of toxin genes from PS80JJ1. 
A gene library was constructed from PS80JJ1 DNA partially digested with 
NdeII. Partial restriction digests were fractionated by agarose gel 
electrophoresis. DNA fragments 93 to 23 kbp in size were excised from the 
gel, electroeluted from the gel slice, purified on an Elutip-D ion 
exchange column (Schleicher and Schuell, Keene, N.H.), and recovered by 
ethanol precipitation. The NdeII inserts were ligated into BamHI-digested 
LambdaGem-11 (Promega, Madison, Wis.). Recombinant phage were packaged and 
plated on E. coil KW251 cells. Plaques were screened by hybridization with 
the probe described above. Hybridizing phage were plaque-purified and used 
to infect liquid cultures of E. coli KW251 cells for isolation of DNA by 
standard procedures (Maniatis et al., supra). 
For subcloning the gene encoding the PS80JJ1 130 kDa toxin, preparative 
amounts of phage DNA were digested with XhoI and electrophoresed on an 
agarose gel. The approximately 12 kbp band containing the toxin gene was 
excised from the gel, electroeluted from the gel slice, and purified by 
ion exchange chromatography as described above. The purified DNA insert 
was ligated into XhoI-digested pHTBluelI (an E. coli/B. thuringiensis 
shuttle vector comprised of pBluescript S/K [Stratagene, La Jolla, Calif.] 
and the replication origin from a resident B.t. plasmid [D. Lereclus et 
al. [1989] FEMS Microbiology Letters 60:211-218]). The ligation mix was 
used to transform frozen, competent E. coli NM522 cells (ATCC 47000). 
.beta.-galactosidase-transformants were screened by restriction digestion 
of alkaline lysate plasmid minipreps as above. The desired plasmid 
construct, pMYC2379, contains a toxin gene that is novel compared to other 
toxin genes containing insecticidal proteins. 
Sequence analysis of the toxin gene revealed that it encodes a protein of 
approximately 130,000 daltons, deduced from the DNA sequence. The 
nucleotide and deduced amino acid sequences are shown in SEQ ID NOS. 5 and 
6, respectively. 
pMYC2379 was introduced into the acrystalliferous (Cry.sup.-) B.t. host, 
CryB (A. Aronson, Purdue University, West Lafayette, Ind.), by 
electroporation. Expression of the 130kDa toxin was demonstrated by 
SDS-PAGE analysis. 
The PS80JJ1 toxin gene encoded by pMYC2379 was sequenced using the ABI373 
automated sequencing system and associated software. 
Example 5--Restriction Fragment Length Polymorphism Analysis of 
.delta.-endotoxin Genes From Bacillus thuringiensis strain PS167P 
Total cellular DNA was prepared from Bacillus thuringiensis (B.t.) cells as 
described in Example 3. 
An approximately 700-800 bp DNA fragment from novel PS167P 130 kDa toxin 
genes was obtained by polymerase chain reaction (PCR) amplification using 
PS167P cellular DNA and the primers shown in SEQ ID NO. 3 and SEQ ID NO. 
4. This DNA fragment was cloned into pBluescript S/K (Stratagene, LaJolla, 
Calif.) and partially sequenced by dideoxynucleotide DNA sequencing 
methodology (Sanger et al, supra) using Sequenase (US Biochemicals, 
Cleveland, Ohio). DNA sequences unique to at least two PS167P toxin genes 
were identified by computer comparison with other known .delta.-endotoxin 
genes. 
The 700-800 bp DNA fragment was radiolabeled with .sup.32 P and used in 
standard hybridizations of Southern blots of PS167P total cellular DNA. 
Hybridizing bands included approximately 1.8 kbp and 2.3 kbp EcoRI 
fragments and approximately 5.5 kbp and 8.0 kbp HindIII fragments. These 
DNA fragments contain toxin genes or restriction fragments of toxin genes 
unique to PS167P. 
Example 6--Insertion of Toxin Genes Into Plants 
One aspect of the subject invention is the transformation of plants with 
genes encoding the insecticidal toxin. The transformed plants are 
resistant to attack by the target pest. 
Genes encoding pesticidal toxins, as disclosed herein, can be inserted into 
plant cells using a variety of techniques which are well known in the art. 
For example, a large number of cloning vectors comprising a replication 
system in E. coli and a marker that permits selection of the transformed 
cells are available for preparation for the insertion of foreign genes 
into higher plants. The vectors comprise, for example, pBR322, pUG series, 
M13mp series, pACYC184, etc. Accordingly, the sequence encoding the B.t. 
toxin can be inserted into the vector at a suitable restriction site. The 
resulting plasmid is used for transformation into E. coli. The E. coli 
cells are cultivated in a suitable nutrient medium, then harvested and 
lysed. The plasmid is recovered. Sequence analysis, restriction analysis, 
electrophoresis, and other biochemical-molecular biological methods are 
generally carried out as methods of analysis. After each manipulation, the 
DNA sequence used can be cleaved and joined to the next DNA sequence. Each 
plasmid sequence can be cloned in the same or other plasmids. Depending on 
the method of inserting desired genes into the plant, other DNA sequences 
may be necessary. If, for example, the Ti or Ri plasmid is used for the 
transformation of the plant cell, then at least the fight border, but 
often the right and the left border of the Ti or Ri plasmid T-DNA, has to 
be joined as the flanking region of the genes to be inserted. 
The use of T-DNA for the transformation of plant cells has been intensively 
researched and sufficiently described in EP 120 516; Hoekema (1985) In: 
The Binary Plant Vector System, Offset-durkkerij Kanters B. V., 
Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci. 4:1-46; and 
An et al. (1985) EMBO J. 4:277-287. 
Once the inserted DNA has been integrated in the genome, it is relatively 
stable there and, as a rule, does not come out again. It normally contains 
a selection marker that coffers on the transformed plant cells resistance 
to a biocide or an antibiotic, such as kanamycin, G 418, bleomyein, 
hygromycin, or chloramphenicol, inter alia. The individually employed 
marker should accordingly permit the selection of transformed cells rather 
than cells that do not contain the inserted DNA. 
A large number of techniques are available for inserting DNA into a plant 
host cell. Those techniques include transformation with T-DNA using 
Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation 
agent, fusion, injection, or electroporation as well as other possible 
methods. If agrobacteria are used for the transformation, the DNA to be 
inserted has to be cloned into special plasmids, namely either into an 
intermediate vector or into a binary vector. The intermediate vectors can 
be integrated into the Ti or Ri plasmid by homologous recombination owing 
to sequences that are homologous to sequences in the T-DNA. The Ti or Ri 
plasmid also comprises the vir region necessary for the transfer of the 
T-DNA. Intermediate vectors cannot replicate themselves in agrobacteria. 
The intermediate vector can be transferred into Agrobacterium tumefaciens 
by means of a helper plasmid (conjugation). Binary vectors can replicate 
themselves both in E. coli and in agrobacteria. They comprise a selection 
marker gene and a linker or polylinker which are framed by the right and 
left T-DNA border regions. They can be transformed directly into 
agrobacteria (Holsters et al. [1978] Mol. Gen. Genet. 163:181-187). The 
agrobacterium used as host cell is to comprise a plasmid carrying a vir 
region. The vir region is necessary for the transfer of the T-DNA into the 
plant cell. Additional T-DNA may be contained. The bacterium so 
transformed is used for the transformation of plant cells. Plant explants 
can advantageously be cultivated with Agrobacterium tumefaciens or 
Agrobacterium rhizogenes for the transfer of the DNA into the plant cell. 
Whole plants can then be regenerated from the infected plant material (for 
example, pieces of leaf, segments of stalk, roots, but also protoplasts or 
suspension-cultivated cells) in a suitable medium, which may contain 
antibiotics or biocides for selection. The plants so obtained can then be 
tested for the presence of the inserted DNA. No special demands are made 
of the plasmids in the case of injection and electroporation. It is 
possible to use ordinary plasmids, such as, for example, pUC derivatives. 
The transformed cells grow inside the plants in the usual manner. They can 
form germ cells and transmit the transformed trait(s) to progeny plants. 
Such plants can be grown in the normal manner and crossed with plants that 
have the same transformed hereditary factors or other hereditary factors. 
The resulting hybrid individuals have the corresponding phenotypic 
properties. 
In a preferred embodiment of the subject invention, plants will be 
transformed with genes wherein the codon usage has been optimized for 
plants. Also, advantageously, plants encoding a truncated toxin will be 
used. The truncated toxin typically will encode about 55% to about 80% of 
the full length toxin. Methods for creating synthetic B.t. genes for use 
in plants are known in the art. 
Example 7--Cloning of B.t. Genes Into Insect Viruses 
A number of viruses are known to infect insects. These viruses include, for 
example, baculoviruses and entomopoxviruses. In one embodiment of the 
subject invention, genes encoding the insecticidal toxins, as described 
herein, can be placed within the genome of the insect virus, thus 
enhancing the pathogenicity of the virus. Methods for constructing insect 
viruses which comprise B.t. toxin genes are well known and readily 
practiced by those skilled in the art. These procedures are described, for 
example, in Merryweather et al. (Merryweather, A. T., U. Weyer, M. P. G. 
Harris, M. Hirst, T. Booth, R. D. Possee (1990) J. Gen. Virol. 
7/:1535-1544) and Martens et al. (Martens, J. W. M., G. Honee, D. Zuidema, 
J. W. M. van Lent, B. Visser, J. M. Vlak (1990) Appl. Environmental 
Microbid. 56(9):2764-2770). 
It should be understood that the examples and embodiments described herein 
are for illustrative purposes only and that various modifications or 
changes in light thereof will be suggested to persons skilled in the art 
and are to be included within the spirit and purview of this application 
and the scope of the appended claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 6 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GlyAsnPheAsnXaaGluLysAspTyrAsp 
510 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (synthetic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
AGACGTGGATCCGGAAATTTTAATTTTGAAAARGAYTAYGA41 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (synthetic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GGACCAGGATTTACAGGWGGRRA23 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (synthetic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
TAACGTGTATWCGSTTTTAATTTWGAYTC29 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3561 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
ATGGATTGTAATTTACAATCACAACAAAATATTCCTTATAATGTATTAGCAATACCAGTA60 
TCTAATGTTAATGCGTTGGTTGATACAGCTGGAGATTTAAAAAAAGCATGGGAAGAATTT120 
CAAAAAACTGGTTCTTTTTCATTAACAGCTTTACAACAAGGATTTTCTGCCTCACAAGGA180 
GGAGCATTCAATTATTTAACATTATTACAATCAGGAATATCATTAGCTGGTTCTTTTGTC240 
CCTGGAGGTACTTTTGTAGCACCCATTGTTAATATGGTTATTGGTTGGTTATGGCCACAT300 
AAAAACAAGACAGCGGATACAGAAAATTTAATAAAATTAATTGATGAAGAAATTCAAAAA360 
CAATTAAACAAAGCCTTATTAGACCAAGATAGAAACAATTGGACCTCTTTTTTAGAAAGT420 
ATATTTGATACTTCAGCTACAGTAAGTAATGCAATTATAGATGCACAGTGGTCAGGTACT480 
GTAGATACTACAAATAGACAACAAAAAACTCCAACAACATCAGATTATCTAAATGTTGTT540 
GGAAAATTTGATTCAGCGGATTCTTCAATTATAACTAATGAAAATCAAATAATGAATGGC600 
AACTTTGACGTAGCTGCAGCACCCTATTTTGTTATAGGAGCAACATTACGTCTTTCATTA660 
TATCAATCTTATATTAAATTTTGTAATAGTTGGATTGATGCAGTTGGATTTAGTACAAAT720 
GATGCTAATACACAAAAAGCTAATTTAGCTCGTACGAAATTAACTATGCGTACTACAATT780 
AATGAATATACACAAAGAGTTATGAAAGTTTTTAAAGATTCCAAGAATATGCCTACAATA840 
GGTACTAATAAATTTAGTGTTGATGCTTATAATGTATATGTTAAAGGAATGACATTAAAT900 
GTTTTAGATATGGTAGCAATATGGTCTTCATTATATCCAAATGATTATACTTCACAAACA960 
GCCATAGAACAAACACGTGTCACTTTTTCAAATATGGTTGGACAAGAAGAAGGTACAGAT1020 
GGAACCCTAAAAATTTACAATACTTTTGATTCTCTTAGTTATCAACATAGCCTAATACCT1080 
AATAATAATGTTAATTTAATTTCTTATTATACTGATGAATTGCAAAATCTAGAATTAGCA1140 
GTATATACTCCTAAAGGTGGAAGTGGATACGCTTATCCTTATGGATTTATTTTAAATTAT1200 
GCAAACAGCAACTACAAATATGGTGATAATGATCCAACAGGCAAACCATTAAATAAACAA1260 
GATGGACCTATACAACAAATAAATGCAGCAACTCAAAACAGTAAATATCTAGATGGAGAA1320 
ACAATAAATGGAATAGGGGCATCCTTACCTGGTTATTGTACTACAGGATGTTCAGCAACA1380 
GAACAACCTTTTAGTTGTACTTCTACTGCTAATAGCTATAAAGCAAGCTGTAATCCTTCA1440 
GATACTAATCAAAAAATTAATGCTTTATATGCTTTTACACAAACTAATGTAAAGGGAAGC1500 
ACGGGGAAATTAGGAGTACTGGCAAGTCTTGTTCCATATGATTTAAATCCTAAAAATGTA1560 
TTTGGTGAATTAGATTCAGATACAAATAATGTTATCTTAAAAGGAATTCCTGCAGAAAAA1620 
GGGTATTTTCCTAATAATGCGCGACCTACTGTTGTAAAAGAATGGATTAATGGTGCAAGT1680 
GCTGTACCATTTTATTCAGGAAATACTTTATTTATGACGGCTACGAATTTAACAGCTACT1740 
CAATATAAAATTAGAATACGTTATGCAAATCCAAATTCAGATACTCAAATCGGTGTACTA1800 
ATTACGCAAAATGGTTCTCAAATTTCCAATAGTAATCTAACACTTTATAGTACTACTGAT1860 
TCAAGTATGAGTAGTAATTTACCACAAAATGTATATGTCACAGGGGAAAATGGAAATTAT1920 
ACACTTCTAGATTTATATAGTACTACTAATGTTTTATCAACAGGAGATATTACATTAAAA1980 
CTTACAGGAGGAAATCAAAAAATATTTATTGATCGAATAGAATTTATTCCTACTATGCCT2040 
GTACCTGCTCCTACTAATAACACTAATAACAATAACGGCGATAACGGCAATAACAATCCC2100 
CCACACCACGGTTGTGCAATAGCTGGTACACAACAACTTTGTTCTGGACCACCTAAGTTT2160 
GAACAAGTAAGTGATTTAGAAAAAATTACAACGCAAGTATATATGTTATTCAAATCTTCT2220 
TCGTATGAAGAATTAGCTCTAAAAGTTTCTAGCTATCAAATTAATCAAGTGGCATTGAAA2280 
GTTATGGCACTATCTGATGAAAAGTTTTGTGAAGAAAAAAGATTGTTACGAAAATTAGTC2340 
AATAAAGCAAACCAATTACTAGAAGCACGTAACTTACTAGTAGGTGGAAATTTTGAAACA2400 
ACTCAAAATTGGGTACTTGGAACAAATGCTTATATAAATTATGATTCGTTTTTATTTAAT2460 
GGAAATTATTTATCCTTACAACCAGCAAGTGGATTTTTCACATCTTATGCTTATCAAAAA2520 
ATAGATGAGTCAACATTAAAACCATATACACGATATAAAGTTTCTGGATTCATTGGGCAA2580 
AGTAATCAAGTAGAACTTATTATTTCTCGTTATGGAAAAGAAATTGATAAAATATTAAAT2640 
GTTCCATATGCAGGGCCTCTTCCTATTACTGCTGATGCATCGATAACTTGTTGTGCACCA2700 
GAAATAGACCAATGTGATGGGGGGCAATCTGATTCTCATTTCTTCAACTATAGCATCGAT2760 
GTAGGTGCACTTCACCCAGAATTAAACCCTGGCATTGAAATTGGTCTTAAAATTGTGCAA2820 
TCAAATGGTTATATAACAATTAGTAATCTAGAAATTATTGAAGAACGTCCACTTACAGAA2880 
ATGGAAATTCAAGCAGTCAATCGAAAAGATCACAAATGGAAAAGAGAAAAACTTCTAGAA2940 
TGTGCAAGTGTTAGTGAACTTTTACAACCAATCATTAATCAAATCGATTCATTGTTCAAA3000 
GATGCAAACTGGTATAATGATATTCTTCCTCATGTCACATATCAAACTCTAAAAAATATT3060 
ATAGTACCCGATTTACCAAAATTAAAACATTGGTTCATAGATCATCTCCCAGGTGAATAT3120 
CATGAAATTGAACAACAAATGAAAGAAGCTCTAAAACATGCATTTACACAATTAGACGAG3180 
AAAAATTTAATCCACAATGGTCACTTTGCAACTAACTTAATAGATTGGCAAGTAGAAGGT3240 
GATGCTCGAATGAAAGTATTAGAAAATAATGCTTTGGCATTACAACTTTCCAATTGGGAT3300 
TCTAGTGTTTCACAATCTATTGATATATTAGAATTTGATGAAGATAAAGCATATAAACTT3360 
CGCGTATATGCTCAAGGAAGCGGAACAATCCAATTTGGAAACTGTGAAGATGAAGCCATC3420 
CAATTTAATACAAACTCATTCGTATATAAAGAAAAAATAATCTATTTCGATACCCCATCA3480 
ATTAACTTACACATACAATCAGAAGGTTCTGAATTCGTTGTAAGTAGTATCGACCTCGTT3540 
GAATTATCAGACGACGAATAA3561 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1186 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
MetAspCysAsnLeuGlnSerGlnGlnAsnIleProTyrAsnValLeu 
151015 
AlaIleProValSerAsnValAsnAlaLeuValAspThrAlaGlyAsp 
202530 
LeuLysLysAlaTrpGluGluPheGlnLysThrGlySerPheSerLeu 
354045 
ThrAlaLeuGlnGlnGlyPheSerAlaSerGlnGlyGlyAlaPheAsn 
505560 
TyrLeuThrLeuLeuGlnSerGlyIleSerLeuAlaGlySerPheVal 
65707580 
ProGlyGlyThrPheValAlaProIleValAsnMetValIleGlyTrp 
859095 
LeuTrpProHisLysAsnLysThrAlaAspThrGluAsnLeuIleLys 
100105110 
LeuIleAspGluGluIleGlnLysGlnLeuAsnLysAlaLeuLeuAsp 
115120125 
GlnAspArgAsnAsnTrpThrSerPheLeuGluSerIlePheAspThr 
130135140 
SerAlaThrValSerAsnAlaIleIleAspAlaGlnTrpSerGlyThr 
145150155160 
ValAspThrThrAsnArgGlnGlnLysThrProThrThrSerAspTyr 
165170175 
LeuAsnValValGlyLysPheAspSerAlaAspSerSerIleIleThr 
180185190 
AsnGluAsnGlnIleMetAsnGlyAsnPheAspValAlaAlaAlaPro 
195200205 
TyrPheValIleGlyAlaThrLeuArgLeuSerLeuTyrGlnSerTyr 
210215220 
IleLysPheCysAsnSerTrpIleAspAlaValGlyPheSerThrAsn 
225230235240 
AspAlaAsnThrGlnLysAlaAsnLeuAlaArgThrLysLeuThrMet 
245250255 
ArgThrThrIleAsnGluTyrThrGlnArgValMetLysValPheLys 
260265270 
AspSerLysAsnMetProThrIleGlyThrAsnLysPheSerValAsp 
275280285 
AlaTyrAsnValTyrValLysGlyMetThrLeuAsnValLeuAspMet 
290295300 
ValAlaIleTrpSerSerLeuTyrProAsnAspTyrThrSerGlnThr 
305310315320 
AlaIleGluGlnThrArgValThrPheSerAsnMetValGlyGlnGlu 
325330335 
GluGlyThrAspGlyThrLeuLysIleTyrAsnThrPheAspSerLeu 
340345350 
SerTyrGlnHisSerLeuIleProAsnAsnAsnValAsnLeuIleSer 
355360365 
TyrTyrThrAspGluLeuGlnAsnLeuGluLeuAlaValTyrThrPro 
370375380 
LysGlyGlySerGlyTyrAlaTyrProTyrGlyPheIleLeuAsnTyr 
385390395400 
AlaAsnSerAsnTyrLysTyrGlyAspAsnAspProThrGlyLysPro 
405410415 
LeuAsnLysGlnAspGlyProIleGlnGlnIleAsnAlaAlaThrGln 
420425430 
AsnSerLysTyrLeuAspGlyGluThrIleAsnGlyIleGlyAlaSer 
435440445 
LeuProGlyTyrCysThrThrGlyCysSerAlaThrGluGlnProPhe 
450455460 
SerCysThrSerThrAlaAsnSerTyrLysAlaSerCysAsnProSer 
465470475480 
AspThrAsnGlnLysIleAsnAlaLeuTyrAlaPheThrGlnThrAsn 
485490495 
ValLysGlySerThrGlyLysLeuGlyValLeuAlaSerLeuValPro 
500505510 
TyrAspLeuAsnProLysAsnValPheGlyGluLeuAspSerAspThr 
515520525 
AsnAsnValIleLeuLysGlyIleProAlaGluLysGlyTyrPhePro 
530535540 
AsnAsnAlaArgProThrValValLysGluTrpIleAsnGlyAlaSer 
545550555560 
AlaValProPheTyrSerGlyAsnThrLeuPheMetThrAlaThrAsn 
565570575 
LeuThrAlaThrGlnTyrLysIleArgIleArgTyrAlaAsnProAsn 
580585590 
SerAspThrGlnIleGlyValLeuIleThrGlnAsnGlySerGlnIle 
595600605 
SerAsnSerAsnLeuThrLeuTyrSerThrThrAspSerSerMetSer 
610615620 
SerAsnLeuProGlnAsnValTyrValThrGlyGluAsnGlyAsnTyr 
625630635640 
ThrLeuLeuAspLeuTyrSerThrThrAsnValLeuSerThrGlyAsp 
645650655 
IleThrLeuLysLeuThrGlyGlyAsnGlnLysIlePheIleAspArg 
660665670 
IleGluPheIleProThrMetProValProAlaProThrAsnAsnThr 
675680685 
AsnAsnAsnAsnGlyAspAsnGlyAsnAsnAsnProProHisHisGly 
690695700 
CysAlaIleAlaGlyThrGlnGlnLeuCysSerGlyProProLysPhe 
705710715720 
GluGlnValSerAspLeuGluLysIleThrThrGlnValTyrMetLeu 
725730735 
PheLysSerSerSerTyrGluGluLeuAlaLeuLysValSerSerTyr 
740745750 
GlnIleAsnGlnValAlaLeuLysValMetAlaLeuSerAspGluLys 
755760765 
PheCysGluGluLysArgLeuLeuArgLysLeuValAsnLysAlaAsn 
770775780 
GlnLeuLeuGluAlaArgAsnLeuLeuValGlyGlyAsnPheGluThr 
785790795800 
ThrGlnAsnTrpValLeuGlyThrAsnAlaTyrIleAsnTyrAspSer 
805810815 
PheLeuPheAsnGlyAsnTyrLeuSerLeuGlnProAlaSerGlyPhe 
820825830 
PheThrSerTyrAlaTyrGlnLysIleAspGluSerThrLeuLysPro 
835840845 
TyrThrArgTyrLysValSerGlyPheIleGlyGlnSerAsnGlnVal 
850855860 
GluLeuIleIleSerArgTyrGlyLysGluIleAspLysIleLeuAsn 
865870875880 
ValProTyrAlaGlyProLeuProIleThrAlaAspAlaSerIleThr 
885890895 
CysCysAlaProGluIleAspGlnCysAspGlyGlyGlnSerAspSer 
900905910 
HisPhePheAsnTyrSerIleAspValGlyAlaLeuHisProGluLeu 
915920925 
AsnProGlyIleGluIleGlyLeuLysIleValGlnSerAsnGlyTyr 
930935940 
IleThrIleSerAsnLeuGluIleIleGluGluArgProLeuThrGlu 
945950955960 
MetGluIleGlnAlaValAsnArgLysAspHisLysTrpLysArgGlu 
965970975 
LysLeuLeuGluCysAlaSerValSerGluLeuLeuGlnProIleIle 
980985990 
AsnGlnIleAspSerLeuPheLysAspAlaAsnTrpTyrAsnAspIle 
99510001005 
LeuProHisValThrTyrGlnThrLeuLysAsnIleIleValProAsp 
101010151020 
LeuProLysLeuLysHisTrpPheIleAspHisLeuProGlyGluTyr 
1025103010351040 
HisGluIleGluGlnGlnMetLysGluAlaLeuLysHisAlaPheThr 
104510501055 
GlnLeuAspGluLysAsnLeuIleHisAsnGlyHisPheAlaThrAsn 
106010651070 
LeuIleAspTrpGlnValGluGlyAspAlaArgMetLysValLeuGlu 
107510801085 
AsnAsnAlaLeuAlaLeuGlnLeuSerAsnTrpAspSerSerValSer 
109010951100 
GlnSerIleAspIleLeuGluPheAspGluAspLysAlaTyrLysLeu 
1105111011151120 
ArgValTyrAlaGlnGlySerGlyThrIleGlnPheGlyAsnCysGlu 
112511301135 
AspGluAlaIleGlnPheAsnThrAsnSerPheValTyrLysGluLys 
114011451150 
IleIleTyrPheAspThrProSerIleAsnLeuHisIleGlnSerGlu 
115511601165 
GlySerGluPheValValSerSerIleAspLeuValGluLeuSerAsp 
117011751180 
AspGlu 
1185 
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