CA2103248C - Novel bacillus thuringiensis isolates active against hymenopteran pests and genes encoding hymenopteran-active toxins - Google Patents

Abstract

Novel Bacillus thuringiensis isolates with hymenopteran activity are described. Also described are toxins having the advantageous hymenopteran activity. This invention further concerns genes or gene fragments which have been cloned from the novel Bacillus thuringiensis isolates which have formicidal activity. These genes or gene fragments can be used to transform suitable hosts for controlling ants.

Description

DESCRIPTION
NOVEL, BACILLUS TRZWNGMM ISOLATES ACr1VE AGAINST
HYMENOPTERANPF= AND GENES ENCODING
HYMENOPTERAN-ACTIVE TONES
Background of the Invention The development of biological control agents as alternatives to chemical insecticides for the control of important pest species is a subject of increasing interest.
Concerns for the environment and exposure of man to harmful substances in air, food and water have stimulated legislation and restrictions regarding the use of chemical pesticides, particularly for pests found in the urban environment. Control of insect pests in urban areas is highly desirable but exposure to chemical pesticides in the household and from lawns and gardens is of great concern to the public. If given a choice, most people would use a non-toxic biological control rather than a toxic chemical to control insects in the urban environment. The problem is that very few biological alternatives to chemical insecticides are available for purchase and use by the public.
Bacillus thuringiensic (B.t) produces an insect toxin designated as b-endotoxln. It is synthesized by the B.t sporulating cell. The toxin, upon being ingested in its crystalline form by susceptible insects, is transformed into biologically active moieties by the insect gut juice proteases. Tice primary target is insect cells of the gut epithelium, which are rapidly destroyed.
The reported activity spectrum ofB.t, covers insect species within the order Lepidoptera, many of which are major pests in agriculture and forestry. The activity spectrum also includes the insect order Diptera, which includes mosquitos and black flies. See Couch, T.L
(1980) "Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis,"
Developments in Ltdustria!
Microbiology 22:61-76; Beegle, C.C., (1978) "Use of Entomogenous Bacteria in Agroecosystems,"
Developments in Industrial Microbiology 20:97-104. Krieg, et al (1983) Z ang.
Ent 96:500-508, describe a at isolate named Bacillus thwingiensis var. tenebrionis, which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle, Leptinotarsa decemlineara, and ABelasrica ab-i. In European Patent Application No. 0 202 739 there is disclosed a novel B.t isolate active against Coleoptera. It is known as A
thwirtgiensis var. san diego (BJ sd). U.S. Patent No. 4,966,765 discloses the eoleoptesan-active Bacillus t usingiensis isolate At PS86BL
Ants comprise a large group of insects (family Formic lddae) from the taxonomic order, Hymenoptera. They are among the most common house pests. In many situations, ants are a

2 PCF/US92/04316 nuisance pest. Foraging ants create problems with hygiene in hospitals and the food industry.
Ants also create problems in agriculture. Damage can be caused by direct feeding on plants.
Harvester and fire ants are commonly associated with this type of damage (Holldobler, B., E.O.
Wilson [1990] The Ants, Belkap Press, Cambridge, Mass. 732 pp.) Some ants cause indirect damage by nurturing and protecting sap feeding insects such as mealybugs and aphids. Ants, particularly in the genus Solenopsis are capable of producing extremely painful stings to humans.
It has been estimated that approximately 10,000 stings occur each year (Habermehl, G.G. [1981]
Venomous Animals and Their Taxies, Springer-Verlag, NY, 195 pp.). The pharaoh ant (Monomoriusm pharaonis) is primarily an urban pest. However, this species can also be an agricultural pest and damage to corn has been noted (Ebeling, W. [1978] Urban Entomology, UC
Press, Berkeley, Calif, 695 pp.).
Carpenter ants, Camponotus spp., are distributed throughout North America.
Some of the more common and/or studied species include C modoc in the Pacific northwest, C clarithorax in southern California, and the black, red, and Florida carpenter ants, C.
pennsylvanicus, C.
noveboracensis and C abdominalis, respectively, in the east (Ebeling, W.
[1978] Urban Entomology, Univ. Calif: Berkeley p. 209-213). Public concern over carpenter ants has been increasing due to the greater probability of structural infestations as suburban developments extend into the forest habitats of the ants.
Pestiferous species of carpenter ants may be considered nuisance pests because of their foraging activity inside homes More significant damage occurs when carpenter ants extend their nests into sound wood. Nesting sites may be located in live and dead trees, sometimes resulting in damage to shade trees. Nests may also be established in walls and support beams of structures, or in voids within doors, walls, and furniture. Preference for moist or decaying wood has been reported, but nesting sites are not restricted to such areas. Carpenter ant populations develop relatively slowly with colonies of 300-2,000 workers being produced over a 2 -year or longer period for various species. Thepresence of reproductives follows this slow development since their production has been reported only from well established colonies (Hansen, LD., R.D. Akre [1985]
Biology of carpenter ants in Washington state (Hymenoptera: Formicidae:
Camponotus).
Melanderia 43. 62 p.; Pricer, J.I.. [19081 Biol. Bull. 14:177-218). Despite the slow colony growth, large colonies with satellite colonies have been found. Worker movement occurs between the main colony and the satellites, which serve as areas for further brood development and colony expansion (Hansen and Akre [1985], supra).
Current methods for controlling structural infestations of carpenter ants include sanitation of potential and current nest sites, minimizing access to structures (eg.
preventing the contact of tree branches with a structure), and the application of insecticides to repel (perimeter spray barriers) and/or eliminate carpenter ants. The use of boric acid dust in dry, wall voids is reported to be effective for up to 20 years (Hansen and Akre, supra).
Recommendations for the chemical control of established structural infestations in the home are often accompanied with warnings of possible hazards to the applicator as well as .~'Y!:l l./:e:, ..; f.l...rr.... ..d'. -. ..`S~'J k.T.' f f .. .. .. .. .. ~
., .:~t. .= ..i. .5. t _...... .._. -. ~... .. - ..

3 children and pets. Alternative control methods such as effective biological control agents have not been found (Akre, R.D., L.D. Hansen, A.L. Antonelli [1989] Fat. Bull.
Washington State Univ. Coop. Fat. Serv.1989 rev no. EB 0818, 6 pp.).
A need clearly exists for a safe, effective biological control agent for carpenter ants-Pharaoh ants, Monomorium pharaonic, have been described as "... the most persistent and difficult of all our house-infesting ants to control or eradicate" (Smith, M.R. [1965] USDA-ARS Tech. Bull. No. 1326, 105 pp.). It is a tropical species which has extended its range to more temperate regions by establishing colonies in heated buildings. Pharaoh ants frequently infests buildings where food is prepared, and have been found to carry pathogenic organisms (Beatson, SH. [1972] Lancet 1425-427).
attributed to their inaccessible nesting The difficulty in controlling pharaoh ants may be and sites, rapid population growth, and dispersion of colonies. Their small size allows establishment of colonies in any suitable location, including unusual places such as between books and in stored clothing. With multiple queen colonies, and the warm (30 C), humid (63-80%
RH) conditions that favor pharaoh ants, large colonies can develop rapidly. Portions of these large colonies may disperse to form new colonies at any time, probably in response to overcrowding and unfavorable mic roenvironmental conditions. Unlike other ant species, pharaoh ants do not exhibit intercolony aggression. This permits the adoption of ants from other colonies and may further enhance the establishment of new colonies and reinfestations. Pharaoh ants also forage for food more than 35 m from the nest without distinct trail following, and thus make nests difficult to find and eradicate.
Control methods for pharaoh ants emphasize the use of insect growth regulators (IOR) or toxicants incorporated into baits. Properly implemented bait programs are effective, however it may take over a month to achieve control. Insecticide applications, while fast acting, usually do not eliminate colonies, and may be unacceptable in certain areas where toxic residues are a concern. In addition, insecticide applications are generally not compatible with bait programs.
A need exists for safe and effective biological control agents for pharaoh ants.

Brief Summary of the Im-ention The subject invention concerns novel Bacillus thunngiensis (Ba.) isolates and genes therefrom which encode novel hymenopteran-active proteins. The novel Rt.
isolates, known herein as Bacillus t/uaingiensis PS14OE2 (Bt PS14OE2), Bacillus duaingiensis PS86Q3 (Bt.
PS86Q3) and Bacillus duautgiens&c PS211B2 (Rt. PS211B2) have been shown to be active against, for example, the pharaoh ant (Monomorium pharaonic). Toxins of the subject invention control, for example, fire ants, carpenter ants, argentine ants, and pharaoh ants.
The subject invention also includes mutants of the above isolates which have substantially the same pesticidal properties as the parent isolate. Procedures for making mutants are well known in the microbiological art. Ultraviolet light and nitrosoguanidine are used extensively toward this end.

i1/L6IF.frr5.r.f rh!!,.~.t.....j. 9?r .,.ifs It ;.i~'=!",~. 1'. _. 1. 1.. ._ ~A=! ...L ..... ?~. E?f:rr. -......... .... ,,_,. },.... .. r'. u. ,.. .y-.,.
.., ... .

The subject invention also concerns novel toxins active against ants. A
further aspect of the invention concerns genes coding for these formicidal toxins. The subject invention provides the person skilled in this art with a vast array of formicidal toxins, methods for using these toxins, and genes that code for the toxins. The genes or gene fragments of the invention encode Bacillus thuringiensis d-endotoxins which have formicidal activity. The genes or gene fragments can be transferred to suitable hosts via a recombinant DNA vector.
One aspect of the invention is the discovery of a generalized chemical formula common to a wide range of formicidal toxins. This formula can be used by those skilled in this art to obtain and identify a wide variety of toxins having the desired formicidal activity. The subject invention concerns other teachings which enable the skilled practitioner to identify and isolate ant-active toxins and the genes which code therefor. For example, characteristic features of ant-active toxin crystals are disclosed herein. Furthermore, characteristic levels of ammo acid homology can be used to characterize the toxins of the subject invention. Yet another characterizing feature pertains to immunoreactivity with certain antibodies.
Also, nucleotide probes specific for genes encoding toxins with formicidal activity are described. Thus, the identification of toxins of the subject invention can be accomplished by sequence-specific motifs, overall sequence similarity, immunoreactivity, and ability to hybridize with specific probes.
In addition to the teachings of the subject invention which broadly define At.
toxins with advantageous formicidal activity, a further aspect of the subject invention is the provision of specific formicidal toxins and the nucleotide sequences which code for these toxins. One such toxin- is the gene expression product of isolate PS86Q3.

Brief Description of the Drawings Figure 1 is a photograph of a standard SDS polyacrylamide gel of At PS140E2, and8 t.
PS86Q3.
Figure 2 is a photograph of a standard SDS polyacrylamide gel showing alkali-soluble proteins of Bt PS211132 compared to a protein standard.
Figures 3-5 are transmission electron micrographs of ultrathin sections of the ant-active At. strains (Figure 3 is At. PS14E2, Figure 4 is At. PS86Q3; and Figure 5 is At. PS211B2). Cells were embedded in an epoxy resin and stained with uranyl acetate and lead citrate.

Brief Description of the Sequences SEQ ID NO.1 is the nucleotide sequence of gene 17a.
SEQ ID NO.2 is the amino acid sequence of protein 17a.
SEQ ED NO.3 is the nucleotide sequence of gene 17b.
SEQ ID NO.4 is the amino acid sequence of protein 17b.
SEQ ID NO.5 is the nucleotide sequence of gene 33F2.
SEQ ID NO.6 is the amino acid sequence of protein 33F2.
SEQ ID NO.7 is the nucleotide sequence of gene 8603(a).

SEQ ID NO.8 is the amino add sequence of protein 86Q3(a).
SEQ ID NO.9 is the nucleotide sequence of gene 63B.
SEQ ID NO. 10 is the amino acid sequence of protein 63B.
SEQ ID NO.11 is the amino acid sequence of a probe which can be used according to 5 the subject invention.
SEQ ID NO. 12 is DNA coding for the amino acid sequence of SEQ ID NO. 11.
SEQ ID NO. 13 is DNA coding for the amino add sequence of SEQ ID NO. 1L
SEQ ID NO.14 is the amino add sequence of a probe which can be used according to the subject invention.
SEQ ID NO. 15 is DNA coding for the amino add sequence of SEQ ID NO. 14.
SEQ ID NO. 16 is DNA coding for the amino add sequence of SEQ ID NO. 14.
SEQ ID NO. 17 is the N-terminal amino add sequence of 17a.
SEQ ID NO. 18 is the N-terminal amino add sequence of 17b.
SEQ ID NO. 19 is the N-terminal amino add sequence of 86Q3(a).
SEQ ID NO.20 is the N-terminal amino acid sequence of 63B.
SEQ ID NO.21 is the N-terminal amino acid sequence of 33F2.
SEQ ID NO.22 is an internal amino acid sequence for 63B.
SEQ ID NO.23 is a synthetic oligonudcotide derived from 17.
SEQ ID NO.24 is the forward oligonucleotide primer from 63B.
SEQ ID NO.25 is the reverse oligonucleotide primer from 63B.
SEQ ID NO.26 is oligonucleotide probe 33F2A.
SEQ ID NO.27 is oligonudeotide probe 33F2B.
SEQ ID NO. 29 is a reverse primer used according to the subject invention.
SEQ ID NO.29 is an oligonudeotide derived from the N-terminal amino acid sequence of 86Q3(a) (SEQ ID NO. 19).
SEQ ID NO. 30 is the amino acid sequence coded for by an oligonucleotide used according to the subject invention (SEQ ID NO. 31).
SEQ ID NO.31 is an oligonucleotidc which codes for the amino acid sequence of SEQ
ID NO. 30.
SEQ ID NO.32 is the amino acid sequence coded for by the oligonucleotide of SEQ ID
NO. 33.
SEQ ID NO.33 is a DNA sequence coding for the peptide of SEQ ID NO. 32.
SEQ ID NO.34 is the reverse complement primer to SEQ ID NO. 38, used according to the subject invention.
SEQ ID NO.35 is a forward primer according to the subject invention.
SEQ ID NO.36 is an amino acid sequence according to the subject invention.
SEQ ID No. 37 is a reverse primer according to the subject invention.
SEQ ID NO. 38 is the nematode (NEMI) variant of'region 5 of HSfte and Whiteley, Microbiological Reviews, (1989) Vol. 53, p. 242-255.

WO 92/20802 PCr/US92/04316 42 10312.18 6 Detailed Disclosure of the Invention One aspect of the subject invention is the discovery of Bacillus thuringiensis isolates having activity against ants. The novel Bacillus thuringiensis isolates of the subject invention have the following characteristics in their biologically pure form:
Characteristics of At PS14OE2 Colony morphology-large colony, dull surface, typical At.
Vegetative cell morphology typical At.
Culture methods-typical for At.
Inclusions-an elliptical coated inclusion outside the exosporium, and a long inclusion inside the exosporium Approximate molecular weight of alkaliVSDS-soluble polypeptides (kDa)-78, 70, Serotype-6, entomocidus.
15 Characteristics of At. PS86Q3 Colony morphology-large colony, dull surface, typical At.
Vegetative cell morphology-typical AL
Culture methods-typical for B.L
Inclusions-long amorphic inclusion and a small inclusion, both of which remain with the 20 spore after lysis Approximate molecular weight of alkali SDS-soluble polypeptides (kDa)-155, 135, 98, 62,58 Serotype-new serotype (not H-1 through H-27).
25 Characteristics of At. PS211132 Colony morphology: large colony, dull surface, typical R L
Vegetative cell morphology-typical BL
Culture methods-typical for At Inclusions-large round amorphic inclusion with coat, and elliptical inclusion 30 Approximate molecular weight of alkali/SDS-soluble polypeptides (kDa)--175,130, 100, 83, 69, 43, 40, 36, 35, 34 and 27 Serotype-6, entomocidus.

A comparison of the characteristics of B thuringiensis PS140E2 (BL PS140E2), B.
35 thuringiensis PS86Q3 (At. PS86Q3), B thuringiensis PS211B2 (Bt. PS211I32), B. thuringiensis var.
san diego (BL.sd), and B. thuringiensis var. Iwrstaki (HD-1) is shown in Table 1.

Table 1. Comparison of B.t PS14OE2, B.t. PS86Q3, Rt PS211B2, Bt.sd, and B.L HD-B.t. PS14OE2 Bt. PS86Q3 llt. PS211B2 B.L HD-1 B.ts d Inclusions: Ellipse and 2 1 long and 1 Large Bipyramid Flat square small or 2 small amorphic inclusions inclusions Approximate 78,000 155,000 175,000 130,000 72,000 molecular wt. of 70,000 135,000 130,000 68,000 64,000 proteins by 35,000 98,000 100,000 SDS-PAGE 62,000 83,000 58,000 69,000 43,000 40,000 36,000 35,000 34,000 27,000 Host range Hymenoptera Hymenoptera J Hymenoptera Lepidopteran Coleoptera (Colorado and Coleopteran Potato Beetle In addition to the ant-active B.t isolates described herein, the subject invention concerns a vast array of B.t b-endotoxins having formicidal activity. In addition to having formicidal activity, the toxins of the subject invention will have one or more of the following characteristics:
1. An amino acid sequence according to the generic formula disclosed herein.
2. A high degree of amino acid homology with specific toxins disclosed herein.
3. A DNA sequence encoding the toxin wherein said sequence hybridizes with probes or genes disclosed herein.

4. A nucleotide sequence which can be amplified using primers disclosed herein.

5. A crystal toxin presentation as described herein.

6. Immunoreactivity to an antibody raised to a toxin disclosed herein.
One aspect of the subject invention concerns the discovery of a generic chemical formula (hereinafter referred to as the Generic Formula) which can be used to identify toxins having activity against ants. This formula describes toxin proteins having molecular weights in excess of 130,000kDa. The Generic Formula below covers those amino acids in the N-terminal region extending two amino acids past the invariant proline residue encountered at amino acid number 695 in the sequence of 86Q3(a). The organization of the toxins within this class is delineated by the following generic sequence motif that is the ultimate determinant of structure and function.
1 MOXLUEBYPx BXYUBLXxxx xxxXXXXXXX XXXXXBXXxX EXXXKXXXKX
XxxxxxXJXX XXBXXXXXXX XXI-XXXXXXX XXLZBLZBxB PXXXXXXXXX
101 XXBBXXBXXX XXXXXXXXKX xxLBXXBXXXBXXBBXXXBX XXXXXXXUXX
BXZLUXXXXX XXXOBXXXX* XX*xxxxxxx 201 xxxxxXXUZX XOXXLXXBxx xxxxxxxXXE XXXXXxxxXL PXYOXBOXXH
LBLXJXXLxx xxxxxXKXXB XXJXXxBXXXK XXLXXXLXXX XLOBXXXBXX
301 XLXXXxXXXJ xXZXXXXXXY BJXBOXX*LEBXXXXPOBEX XXYXXxxxxx XLXXOKXLXZ XxxxxxXXXX BXXXXXZXXXX ZXXXXXXxXX XXXBXXXXXX

WO 92/2080a 3 ry PCT/US92/04316 401 XXXXBxxxxx xxxxXXXXXX LXXXXXXXXX XXX*xxXXXX Xxxxxxxxxx XXXXXXXXXX XXXXX*XXXX XXPLXXX*XJ XxXXXXXXXX XXXXXBXXXX
501 XXZXXXXXXX xx*x*XXXXX XXXXXXXxxx XXXXXXXLXX LYXXXXXXXJ
XXXUXBXBXB ZXXXXXEXXX XXBXZXXXXX XXBXXXXBXx xxXXLtxxxxx 601 XxxxxxxxxE XLUZXUXBXL XXXUXBXBXB XXXXXXXYXL K*KUPZXXXX
XXXBXBEXXX xUXBXXXXXX XZXXXXXXZx XXXXXXYXBX ZXOxxxxxxX
701 xXLXxxxxxx xxxXUXXXXB BLEKLEBBPX X
Numbering is for convenience and approximate location only.
Symbols used:
A = ala G = gly M = met S = ser C=cys H=his N=asn T=thr D=asp I=ile P=pro V=val E=glu K=lys Q=gin W=trp F = phe L = leu R = arg Y =tyr K=KorR
E=EorD
L=Lori B=M,L,1,V,orF
J=K,R,E,orD
O=AorT
U=Nor Q
Z=GorS
X any naturally occurring amino acid, except C.
= any naturally occurring amino acid.
x = any naturally occurring amino acid, except C (or complete omission of any amino acids).

Where a stretch of wild-card amino acids are encountered (X(n) or x(n) where n>2), repetition of a given amino acid should be avoided. Similarly, P, C, E, D, K, or R utilization should be minimized.
Formicidal toxins according to the - Generic Formula of the subject invention are specifically exemplified herein by the toxin encoded by the gene designated 86Q3(a). Since this toxin is merely exemplary of the toxins represented by the Generic Formula presented herein, it should be readily apparent that the subject invention further comprises equivalent toxins (and nucleotide sequences coding for equivalent toxins) having the same or similar biological activity of 86Q3(a). These equivalent toxins will have amino acid homology with 86Q3(a). This amino acid homology will typically be greater than 50%, preferably be greater than 75%, and most preferably be greater than 90%. The amino acid homology will be highest in certain 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 2 provides a listing of examples of amino acids belonging to each class.

Table 2 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val, Leu, De, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, On 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 information presented in the generic formulae of the subject invention provides clear guidance to the person skilled in this art in malting various amino acid substitutions.
Further guidance for characterizing the formicidal toxins of the subject invention is provided in Tables 4 and 5, which demonstrate the relatedness among toxins within the formicidal toxins. Time tables show a numeric score for the best matching alignment between two proteins that reflects: (1) positive scores for exact matches, (2) positive or negative scores reflecting the likelihood (or not) of one amino acid substituting for another in a related protein, and (3) negative scores for the introduction of gaps. A protein sequence aligned to itself will have the highest possible score-i.e., all exact matches and no gaps. However, an unrelated protein or a randomly generated sequence will typically have a low positive score. Related sequences have scores between the random background score and the perfect match score.
The sequence comparisons were made using the local homology algorithm of Smith and Waterman ([1981] Advances in Applied Mathematics 2:482-489), implemented as the program "Bestfit" in the GCG Sequence Analysis Software Package Version ? April 1991.
The sequences were compared with default parameter values (comparison table: Swgappep.Cmp, Gap weight:3.0, Length weight-0.1) except that gap limits of 250 residues were applied to each sequence compared.
The program output value compared is referred to as the Quality score.

Tables 4 and 5 show the pairwise alignments between the indicated amino acids of the ant-active proteins and representatives of dipteran (CryIV; ISRH3 of Sen, K.
et aL [1988] Agric.
BioL Chem. 52:873-878), lepidopteran and dipteran (CryIIA; CryBi of Widner and Whiteley [1989]
J. Bacteriol 171:965-974), and lepidopteran (CryIA(c); Adang et aL [1981] Gene 36:289-300) 5 proteins.

Table 3 shows which amino acids were compared from the proteins of interest.
Table 3 10 Protein Amino acids compared 86Q3(a) 1-697 17a 1-677 17b 1-678 CryIV 1-633 CryIIA 1-633 CryIA(c) 1-609 Table 4 shows the scores prior to adjustment for random sequence scores.
Table 4 ` , _ 86Q3(a) 63B 33F2 17b 17a jCryIVA IQYIJA CryIA(c) 86Q3(a) 1046 389 310 342 340 236 237 238 17b 1017 1007 238 240 236 17a 1016 240 240 237 CiyIVA 950 245 325 CryII.A 950 244 CryIA(c) 914 Note that ant-active protein 86Q3(a) is more closely related to 63B, 17a, 17b, and 33F2 than it is to the CryIVA, CryILA, and CrylA(c) toxins.

5 Table 5 shows the same analysis after subtraction of the average score of 50 alignments of random shuffles of the column sequences with the row sequences.

Table 5 86Q3(a) 63B 1331z2 17b 17a CryNA CryIIA CryIA(c) 86Q3(a) 841 184 118 136 135 41 40 50 10 63B 831 81 133 130 40 33 4v 17b 811 798 42 44 47 17a 808 43 44 44 CryIVA 761 54 141 CryIIA 755 55 ( ryIA(c) 729 Note that in Table 5 the same relationships hold as in Table 4, i.e., 86Q3(a)'s highest score, aside from itself, is with 63B.
This degree of relatedness provides the basis for using common or similar sequence 5 elements from the previously-described known genes to obtain related, but non-identical genes from an ant-active isolate.
Thus, certain toxins according to the subject invention can be defined as those which have formicidal activity and have an alignment value (according to the procedures of Table 5) greater than 100 with 86Q3(a). As used herein, the term "alignment value" refers to the scores obtained using the methods described above which were used to create the scores reported in Table 5.
The toxins of the subject invention can also be characterized in terms of the shape and location of toxin inclusions.
Inclusion We PS86Q3-Long amorphic inclusion and a small inclusion, both of which remain with the spore after lysis. See Figure 3.
PS14OE2-An elliptical coated inclusion situated outside the exosporium, and a long inclusion inside the exosporium. See Figure 4.
PS211B2--Large round amorphic inclusion with coat, and an elliptical inclusion.
See Figure 5.

The genes and toxins according to the subject invention include not only the full length sequences disclosed herein but also fragments of these sequences, or fusion proteins, which retain the characteristic formicidal activity of the sequences specifically exemplified herein.
It should be apparent to a person skilled in this art that genes coding for ant-active toxins can be identified and obtained through several means. The specific genes may be obtained from a culture depository as described below. These genes, or portions thereof, may be constructed synthetically, for example, by use of a gene machine. Variations of these genes may be readily constructed using standard techniques for making point mutations. Also, fragments of these genes cann 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 code for active fragments may be obtained using a variety of other restriction enzymes.
Proteases may be..thtd to directly obtain active fragments of these toxins.
Equivalent toxins and/or genes encoding these equivalent toxins can also be located from B a isolates and/or DNA libraries using the teachings provided herein. There are a number of methods for obtaining the ant-active toxins of the instant invention which occur in nature. For example, antibodies to the ant-active 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 ant-active toxins which are most constant and most distinct from other At. toxins.
These antibodies can then be used to specifically identify equivalent toxins with the characteristic formicidal activity by immunoprecipitation, enzyme linked immunoassay (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 coding for these toxins can then be obtained from the microorganism.
A further method for identifying the toxins and genes of the subject invention is through the use of oligonucleotide probes. These probes are nucleotide sequences having a detectable label. As is well known in the art, if the probe molecule and nucleic acid sample hybridize by WO 92/20802 2.0324 PCT/US92/04316 forming a strong bond between the two molecules, it can be reasonably assumed that the probe and sample are essentially identical. The probe's detectable label provides a means for determining in a known manner whether hybridization has occurred. Such a probe analysis provides a rapid method for identifying formicidal endotoxin genes of the subject invention.
The nucleotide segments which are used as probes according to the invention can be synthesized by use of DNA synthesizers using standard procedures. In the use of the nucleotide segments as probes, the particular probe is labeled with any suitable label known to those skilled in the art, including radioactive and non-radioactive labels. Typical radioactive labels include 32P, 125j 3S, or the like. A probe labeled with a radioactive isotope can be constructed from a nucleotide sequence complementary to the DNA sample by a conventional nick translation reaction, using a DNase and DNA polymerase. The probe and sample can then be combined in a hybridization buffer solution and held at an appropriate temperature until annealing occurs.
Thereafter, the membrane is washed free of extraneous materials, leaving the sample and bound probe molecules typically detected and quantified by autoradiography and/or liquid scintillation counting.
Non-radioactive labels include, for example, ligands such as biotin or thyroxine, as well as enzymes such as hydrolases or perixodases, or the various chemiluminescers such as luciferin, or fluorescent compounds like fluorescein and its derivatives. The probe may also be labeled at both ends with different types of labels for ease of separation, as, for example, by using an isotopic label at the end mentioned above and a biotin label at the other end.
Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid, and, as noted above, a certain degree of mismatch can be tolerated.
Therefore, the probes of the subject invention include mutations (both single and multiple), deletions, insertions of the described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given polynucleotide sequence in many ways, and these methods are known to an ordinarily skilled artisan.
Other methods may become known in the future.
The known methods include, but are not limited to:
(1) synthesizing chemically or otherwise an artificial sequence which is a mutation, insertion or deletion of the known sequence;
(2) using a probe of the present invention to obtain via hybridization a new sequence or a mutation, insertion or deletion of the probe sequence, and (3) mutating, inserting or deleting a test sequence in vitro or in vivo.
It is important to note that the mutational, insertional, and deletional variants generated from a given probe may be more or less efficient than the original probe.
Notwithstanding such differences in efficiency, these variants are within the scope of the present invention.
Thus, mutational, insertional, and deletional variants of the disclosed test sequences can be readily prepared by methods which are well known to those skilled in the are. These variants 2103245'. :. ;.:

can be used in the same manner as the instant probes so long as the variants have substantial sequence homology with the probes. As used herein, substantial sequence homology refers to homology which is sufficient to enable the variant to function in the same capacity as the original probe. Preferably, this homology is greater than 50%; more preferably, this homology is greater than 75%; and most preferably, this homology is greater than 90%. The degree of homology needed for the variant to function in its intended capacity will depend upon the intended use of the sequence. It is well within the skill of a person trained in this art to make mutational, insertional, and deletional mutations which are designed to improve the function of the sequence or otherwise provide a methodological advantage.
Specific nucleotide probes useful, according to the subject invention, in the rapid identification of ant-active genes are (i) DNA coding for a peptide sequence whose single letter amino acid designation is "REWINGAN" (SEQ II) NO. 11) or variations thereof which embody point mutations according to the following: position 1, R or K, position 3, W or Y, position 4, I or L; position 7, A or N; position 8, N or Q; a specific example of such a probe is "AGA(A or G)T(G or A)(G or T)(A or T)T(A or T)AATGG(A
or T)GC(G or T)(A or C)A" (SEQ ID NO. 12); another example of such a probe is "GA(A or G)TGG(A or T)TAAATGGT(A or G)(A or C)(G or C)AA-" (SEQ 1D NO. 13);
(ii) DNA coding for a peptide sequence whose single letter amino acid designation is "PTFDPDLY" (SEQ ID NO. 14) or variations thereof which embody point mutations according to the following: position 3, f or L, position 4, I2 or Y;
position 5. P or T, position 6, D or It position 7, L or H or D or N; a specific example of such a probe is "CC(A or T)AC(C or T)TTT(T Tor G)ATCCAGAT(C or G)(T or A)(T or C)TAT" (SEQ ID NO. 15); another example of such a probe is "CC(T or A)AC(T or A)TT(T or C)GAT(C or A)CA(G or C)AT(C or A)(T or A)TTAT" (SEQ ID NO. 16);
(iii) additional useful probes for detecting ant-active At. genes include "GCAATTI'I'AA ATGAATTATA TCC" (SEQ ID NO. 23), "CAAYTACAAG
CWCAACC" (SEQ ID NO. 24), "AATGAAGTWT ATCCWGTWAA T"
(SEQ ID NO. 27), "GCAAGCGGCC GCTTATGGAA TAAATTCAAT
TYKRTCWA" (SEQ ID NO. 28), "AGACFGGATC CATGGCWACW
ATWAATGAAT TATAYCC" (SEQ ID NO. 29), "TAACGTGTAT
WCGST1TTAA TITWGAYTC' (SEQ ID NO. 31), "TGGAATAAAT
TCAATTYKRT CWA" (SEQ IDNO.33), "AGGAACAAAY TCAAKWCGRT
CTA" (SEQ ID NO. 34), and "TCTCCATCFT CTGARGWAAT" (SEQ ID
NO. 37).
The potential variations in the probes listed is due, in part, to the redundancy of the genetic code. Because of the redundancy of the genetic code, ie., more than one coding nucleotide triplet (codon) can be used for most of the amino adds used to make proteins.
Therefore different nucleotide sequences can code for a particular amino acid.
Thus, the amino acid sequences of the at toxins and peptides can be prepared by equivalent nucleotide sequences encoding the same amino acid sequence of the protein or peptide. Accordingly, the subject 5 invention includes such equivalent nucleotide sequences. Also, inverse or complement sequences are an aspect of the subject invention and can be readily used by a person skilled in this art. In addition it has been shown that proteins of identified structure and function may be constructed by changing the amino acid sequence if such changes do not alter the protein secondary structure (Kaiser, E.T., Kezdy, F.J. [1984] Science 223:249-255). Thus, the subject invention includes 10 mutants of the amino acid sequence depicted herein which do not alter the protein secondary structure, or if the structure is altered, the biological activity is substantially retained. Further, the invention also includes mutants of organisms hosting all or part of a toxin encoding a gene of the invention. Such microbial mutants can be made by techniques well known to persons skilled in the art. For example, UV irradiation can be used to prepare mutants of host organisms.
15 Likewise, such mutants may include asporogenous host cells which also can be prepared by procedures well known in the art.
The toxin genes or gene fragments exemplified according to the subject invention can be obtained from R thuringienth (Re) isolates designated PS17, PS33F2, PS63B, and PS86Q3.
Subcultures of the E colt host harboring the toxin genes of the invention were deposited in the permanent collection of the Northern Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois, USA. The accession numbers are as follows:
Culture Repository No. posit Date At PS140E2 NRRL B-18812 April 23, 1991 Rt. PS96Q3 NRRL B-18765 February 6, 1991 At. PS211B2 NRRL B-18921 November 15, 1991 Bt PS17 NRRL B-18243 July 28, 1987 At. PS33F2 NRRL B-18244 July 28, 1987 At PS63B NRRL B-18246 July 28, 1987 E cols NM522(pMYC2316)(33F2) NRRL B-18785 March 15, 1991 E coil NM522(pMYC2321) NRRL B-18770 February 14, 1991 B. tali NM522(pMYC2317) NRRL B-18816 April 24, 1991 B. coil NM522(pMYCI627)(17a) NRRL B-18651 May 11, 1990 E coil NM522(pMYC1628)(17b) NRRL B-18652 May 11, 1990 E coil NM522(pMYC1642)(63B) NRRL 13-18961 April 10, IM
E cols MR618(pMYC1647)(86Q3) NRRL B-18970 April 29, 1992 The subject 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.

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.
S Further, the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, ic., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures. The depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposit(s). All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.
1S The At isolates 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 theB.t spores and crystals from the fermentation broth by means well known in the att. The recovered Bar. 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 formulation and application procedures are all well known in the art Formulated products can be sprayed or applied as baits to control hymenopteran pests.
When applied with a bait, theB.x itself maybe used, or another suitable host, as described herein, may be transformed with a At. gene and used to express toxins. A vegetable off or other liquid substance can be added to a bait to make it more attractive to the pests.
Various attractants, including pheromone compounds, are well known to those skilled in the art and can be used as a component of the bait. The bait and toxin or toxin-producing microbe can be used as part of a trap.
The At cells of the invention can be treated prior to formulation to prolong the pesticidal activity when the cells are applied to the environment of a target pest. Such treatment 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 pesticide, nor diminish the cellular capability in protecting the pesticide. 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 formaldehyde and glutaraldehyde;
anti-infectives, such as tephiran chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Bouln's f xative and Helly's fixative (See: Humason, Gretchen. L,Animal T
Fssue Techniques, W.H.

WO 92/20802 Q PCr/US92/04316 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). Examples of physical means are short wavelength radiation such as gamma-radiation and X-radiation, freezing, UV irradiation, lyophilization, and the like.
Genes encoding toxins having activity against the target susceptible pests can be isolated from the At. isolate of the invention by use of well known procedures.
The toxin genes of the subject invention can be introduced into a wide variety of microbial hosts. Expression of the toxin gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. With suitable hosts, e.g., Pseudomonas, the microbes can be applied to the situs of hymenopteran insects where they will proliferate and be ingested by the insects. The result is a control of the unwanted insects.
Alternatively, the microbe hosting the toxin gene can be treated under conditions that prolong the activity of the toxin produced in the cell. The treated cell then can be applied to the environment of target pest(s).
The resulting product retains the toxicity of the At toxin.
Where the At. 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 occ'ipy 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 varidy of important crops. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e g., genera Pseudomonas, Erwinia, Serratia, Kiebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophzhus, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azetobacter, d euconostoc, and Alcabgenes; fungi, particularly yeast, e.g., genera Saccharomyces, Coptococcus, Kfuyveromyces, Sporobolomyces, P.haodotorula, and Aureobas dawn Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serrana marcescens, Acetobacter xylinum, Agrobacteririm tumefaciens, Rhodopseudomonas spherofdes, Xanthomonas campestris, Rhizobium melon, Alcafigenes entrophus, and Acetobacter vinlandu, and phytosphere yeast species such as Ri xdotorula rubra, R gluwus, R marina, R auranhaca, Cryptococcus albidus, C.
difuens, C.
laumuu, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Khqveromyces veronae, and Aureobasidium poflulans. Of particular interest are the pigmented microorganisms.
A wide variety of ways are available for introducing the At gene expressing the toxin into the microorganism host under conditions which allow for stable maintenance and expression of .1..S1:e.....Ltx.:J'e!a..r..~. ". rf,1:Yd~=eu.:r~ie~W:.... ?!t i !
'fii...n..... r..r r.lr. .I~I .uSõ l:J{:.1 {fi.M , A`..,, . .., jo ~.."~.~....
.. ...... ...

the gene. One can provide for DNA constructs which include the transcriptional and translational regulatory signals for expression of the toxin gene, the toxin gene under their regulatory control and a DNA sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system which is functional in the host, whereby integration or stable maintenance will occur.
The transcriptional initiation signals will include a promoter and a transcriptional initiation start site. In some instances, it may be desirable to provide for regulative expression of the toxin, where expression of the toxin will only occur after release into the environment. This can be achieved with operators or a region binding to an activator or enhancers, which are capable of induction upon a change in the physical or chemical environment of the microorganisms. For example, a temperature sensitive regulatory region may be employed, where the organisms may be grown up in the laboratory without expression of a toxin, but upon release into the environment, expression would begin. Other techniques may employ a specific nutrient medium in the laboratory, which inhibits the expression of the toxin, where the nutrient medium in the environment would allow for expression of the toxin. For translational initiation, a ribosomal binding site and an initiation codon will be present.
Various manipulations may be employed for enhancing the expression of the messenger, particularly by using an active promoter, as well as by employing sequences, which enhance the stability of, the messenger RNA. The initiation and translational termination region will involve stop codon(s), a terminator region, and optionally, a polyadenylation signal.
In the direction of transcription, namely in the 5' to 31 direction of the coding or sense sequence, the construct will involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either 5' or 3' of the promoter, the ribosomal binding site, the initiation codon, the structural gene having an open reading frame in phase with the initiation codon, the stop codon(s),'the polyadenylation signal sequence, if any, and the terminator region.
This sequence as a double strand may be used by itself for transformation of a microorganism host, but will usually be included with a DNA sequence involving a marker, where the second DNA sequence may be joined to the toxin expression construct during introduction of the DNA
into the host.
By a marker is intended a structural gene which provides for selection of those hosts which have been modified or transformed. The marker will normally provide for selective advantage, for example, providing for biocide resistance, e.g., resistance to antibiotics or heavy metals; complementation, so as to provide prototropy to an auxotrophic host, or the like.
Preferably, complementation is employed, so that the modified host may not only be selected, but may also be competitive in the field. One or more markers may be employed in the development of the constructs, as well as for modifying the host. The organisms may be further modified by providing for a competitive advantage against other wild-type microorganisms in the field. For example, genes expressing metal chelating agents, e.g., siderophores, may be introduced into the host along with the structural gene expressing the toxin. In this manner, the enhanced expression r ~.;. ~~ . 11 ..,l Sr .. .. .. ,..,., ./. t.rr {., ,. . f1,.A y7~ ,. r.. . ., ' ~y .. ~. . . ~~. may. ..+. x. r , ~

of a siderophore may provide for a competitive advantage for the toxin-producing host, so that it may effectively compete with the wild-type microorganisms and stably occupy a niche in the environment.
Where no functional replication system is present, the construct will also include a 5 sequence of at least 50 basepairs (bp), preferably at least about 100 bp, and usually not more than about 1000 bp of a sequence homologous with a sequence in the host. In this way, the probability of legitimate recombination is enhanced, so that the gene will be integrated into the host and stably maintained by the host. Desirably, the toxin gene will be in close proximity to the gene providing for complementation as well as the gene providing for the competitive advantage.
10 Therefore, in the event that a toxin gene is lost, the resulting organism will be likely to also lose the complementing gene and/or the gene providing for the competitive advantage, so that it will be unable to compete in the environment with the gene retaining the intact construct.
A large number of transcriptional regulatory regions are available from a wide variety of microorganism hosts, such as bacteria, bacteriophage, cyanobacteria, algae, fungi, and the like.
Various transcriptional regulatory regions include the regions associated with the trp gene, lac gene, gal gene, the lambda left and right promoters, the tac promoter, the naturally-occurring promoters associated with the toxin gene, where functional in the host. See for example, U.S.
Patent Nos. 4,332,898, 4,342,832 and 4,356,270. The termination region may be the termination region normally associated with the transcriptional initiation region or a different transcriptional initiation region, so long as the two regions are compatible and functional in the host.
Where stable episomal maintenance or integration is desired, a plasmid will be employed which has a replication system which is functional in the host. The replication system may be derived from the chromosome, an episomal element normally present in the host or a different host, or, a replication system from a virus which is stable in the host. A
large number of plasthills are available, such as pBR322, pACYC184, RSF1010, pRO1614, and the lace. See for example, Olson et aL (1982) J BacterioL 150:6069; Bagdasarian et aL (1981) Gene 16:237, and US. Patent Nos. 4,356,270, 4,362,817, and 4,371,625.
The At. gene can be introduced between the transcriptional and translational initiation region and the transcriptional and translational termination region, so as to be under the regulatory control of the initiation region. This construct will be included in a plasmid, which will include at least one replication system, but may include more than one, where one replication system is employed for cloning during the development of the plasmid and the second replication system is necessary for functioning in the ultimate host. In addition, one or more markers may be present, which have been described previously. Where integration is desired, the plasmid will desirably include a sequence homologous with the host genome.
The transformants can be isolated in accordance with conventional ways, usually employing a selection technique, which allows for selection of the desired organism as against unmodified organisms or transferring organisms, when present. The transformants then can be tested for pesticidal activity.

Suitable host cells, where the pesticide-containing cells will be treated to prolong the activity of the toxin in the cell when the then treated cell is applied to the environment of target pest(s), may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms 5 which produce substances toxic to higher organisms could be used, where the toxin is unstable or the level of application sufficiently low as to avoid any possibility of toxicity to a mammalian host.
As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi.
Illustrative prokaryotes, both Gram-negative and -positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; BadIlaceae;
Rhizobiceae, such as 10 Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibno, 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, Aureobasidium, 15 Sporobolomyces, and the like.
Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the Bt. gene into the host, availability of expression systems, efficie icy 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 20 qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity, lack of mammalian toxicity;
attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.
Host organisms of particular interest include yeast, such as Rhodotorulkl'sp., Aureobasid um sp., Saccharomyces sp., and Sporobolomyces sp.; phylloplane organisms such as Pseudomonas sp., Erwura sp. and Flavobacterium sp.; or such other organisms 'as Escherichia, Lactobacillus sp., Bacillus sp., Streptomyces sp., and the like. Specific organisms include Pseudomonas aeruginosa, Pseudomonas fkeorescens, Saccharomyces cerevisiae, Bacilkts thunngtensis, Escherichia cob, Bacillus subtilis, Streptomyces lividans, and the like.
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 recombinant microbial cell can be done as disclosed infra.
The treated cells generally will have enhanced structural stability which will enhance resistance to environmental conditions. 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 a polypeptide pesticide. The method of inactivation or killing retains at least a substantial portion of the bio-availability or bioactivity of the toxin.

The cellular host containing the B.L 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 Rt.
gene. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.
The Rt. cells may be formulated in a variety of ways. They may be employed as wettable powders, baits, 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 pesticidal additives, 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 rheological agents, surfactants, emulsifiers, dispersants, or polymers.
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 102 to about 104 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 hymenopteran pest(s), e.g., plants, soil or water, by spraying, dusting, sprinkling, baits or the like.

Following are examples which illustrate procedures, including the best mode, or 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 B.t Isolates of the Invention A subculture of a Bt. isolate can be used to inoculate the following medium, a peptone, glucose, salts medium.
Bacto Peptone 7.5 g/l Glucose 1.0 gIl KH2PO4 3.4 g/l K2HPO4 435 g/l Salts Solution 5.0 ml/1 Ca%72 Solution 5.0 ml/1 Salts Solution (100 ml) MgSO4.7H20 2.46 g MnSO4.1-120 0.04 g ZnSO4.7H20 0.28 g FeSO4=7H20 0.40 g CaC12 Solution (100 ml) CaC12.2H20 3.66 g pH 7.2 The salts solution and CaC12 solution are filter-sterilized and added to the autoclaved and cooked broth at the time of inoculation. Flasks are incubated at 30 C on a rotary shaker at 200 rpm for 64 hr.
E nple 2 - Purification of Protein and Amino Acid Sequencing The At. isolates PS86Q3, PS17, PS63B, and PS33F2 were cultured as described in Example 1. The parasporal inclusion bodies were partially purified by sodium bromide (28-38%) isopycnic gradient centrifugation (Pfannenstiel, M.A, E.J. Ross, V.C. Kramer, K.W. Nickerson [1984] FEMS MicrobioL Lett. 21:39). The proteins were bound to PVDF membranes (Millipore, Bedford, MA) by western blotting techniques (Towbin, H., T. Staehlelin, K.
Gordon [1979] Prot.
NatL Acad. Sci. USA 76:4350) and the N-terminal amino acid sequences were determined by the standard Edman reaction with an automated gas-phase sequenator (Hunkapiller, LW., R.M.
Hewick, W.L. Dreyer, and L.E. Hood [1983] Meth. Enrymol 91:399). The sequences obtained were:
17a: AILNELYPSVPYNV(SEQIDNO.17) 17b. AILNELYPSVPYN V (SEQIDNO.18) 86Q3(a): MATINELYPNVPYNVL(SEQIDNO.19) 63B: QLQAQPLIPYNVLA(SEQIDNO.20) 33F2: A TL N E V Y P V N (SEQ ID NO. 21) In addition, internal amino acid sequence data were derived for 63B. The toxin protein was partially digested with Staphylococcus aureus V8 protease (Sigma Chem.
Co., St. Louis, MO) essentially as described (Cleveland, D.W., S.G. Fischer, M.W. Kirschner, U.K.
Laemmli [1977] J
BioL Chem. 252:1102). The digested material was blotted onto PVDF membrane and a ca. 28 kDa limit peptide was selected for N-terminal sequencing as described above. The sequence obtained was:
63B(2) VQRILDEKLSFQLIK(SEQIDNO.22) From these sequence data oligonucleotide probes were designed by utilizing a codon frequency table assembled from available sequence data of other Bt. toxin genes. The probes were synthesized on an Applied Biosystems, Inc. DNA synthesis machine.
Protein purification and subsequent amino acid analysis of the N-terminal peptides listed above has led to the deduction of several oligonucleotide probes for the isolation of toxin genes from formicidal B.L isolates. RFLP analysis of restricted total cellular DNA
using radiolabeled oligonucleotide probes has elucidated different genes or gene fragments.

Example 3 - Cloning of Novel Toxin Genes and Transformation into Escherichia coli Total cellular DNA was prepared by growing the cells At. PS17 to a low optical density (ODD = 1.0) and recovering the cells by centrifugation. The cells were protoplasted in TES
buffer (30 mM Tris-Cl, 10 mM EDTA, 50 mM NaCl, pH = &0) containing 20 %
sucrose and 50 mg/ml lysozyme. The protoplasts were lysed by addition of SDS to a final concentration of 4%.
The cellular material was precipitated overnight at 4 C in 100 mM (final concentration) neutral potassium chloride. The supernate was extracted twice with phenol/chloroform (1:1). The DNA
was precipitated with ethanol and purified by isopycnic banding on a cesium chloride-ethidium bromide gradient.
Total cellular DNA from PS17 was digested with EcoRI and separated by electrophoresis on a 0.8% (w/v) Agarose-TAE (50 mM Tris-HCI, 20 mM NaOAc22.5 mM EDTA, pH=8.0) buffered gel. A Southern blot of the gel was hybridized with a [32P]-radiolabeled oligonucleotide probe derived from the N-terminal amino acid sequence of purified 130 kDa protein from PS17.
The sequence of the oligonucleotide synthesized is (GCAATTTTAAATGAATTATATCC) (SEQ
ID NO. 23). Results showed that the hybridizing EcoRl fragments of PS17 are 5.0 kb, 4.5 kb, 2.7 kb and 1.8 kb in size, presumptively identifying at least four new ant-active toxin genes, 17d, 17b, 17a and 17e, respectively.
A library was constructed from PS17 total cellular DNA partially digested with Sau3A and size fractionated by electrophoresis. The 9 to 23 kb region of the gel was excised and the DNA
was electroeluted and then concentrated using an Elutip7m ion exchange column (Schleicher and Schuel, Keene NH). The isolated Sau3A fragments were ligated into LambdaGEM-11Tt''I
(PROMEGA). The packaged phage were plated on KW251 E. cold cells (PROMEGA) at a high titer and screened using the above radiolabeled synthetic oligonucleotide as a nucleic acid hybridization probe. Hybridizing plaques were purified and rescreened at a lower plaque denary.
" Single isolated purified plaques that hybridized with the probe were used to infect KW251 E coli cells in liquid culture for preparation of phage for DNA isolation. DNA was isolated by standard procedures.
Recovered recombinant phage DNA was digested with EcoRI and separated by electrophoresis on a 0.8% agarose-TAE gel. The gel was Southern blotted and hybridized with the oligonucleotide probe to characterize the toxin genes isolated from the lambda library. Two patterns were present, clones containing the 4.5 kb (17b) or the 2.7 kb (17a) EcoRI fragments.
Preparative amounts of phage DNA were digested with Sall (to release the inserted DNA from lambda arms) and separated by electrophoresis on a 0.6% agarose-TAE gel. The large fragments, electroeluted and concentrated as described above, were ligated to SaII-digested and dephosphorylated pBClac, an coli/Rz shuttle vector comprised of replication origins from pBC16 and pUC19. The ligation mix was introduced by transformation into NM522 competent E. coli cells and plated on LB agar containing ampicillin, isopropyl-(Beta)-D-thiogalactoside (IPTG) and 5-Bromo-4-Chloro-3-indolyl-(Beta)-D-galactoside (XGAL).. White colonies, with putative insertions in the (Beta)-galactosidase gene of pBClac, were subjected to standard rapid plasmid purification procedures to isolate the desired plasmids. The selected plasmid containing the 2.7 kb EcoRI fragment was named pMYC1627 and the plasmid containing the 4.5 kb EcoRI
fragment was called pMYCI628.
The toxin genes were sequenced by the standard Sanger dideoxy chain termination method using the synthetic oligonucleotide probe, disclosed above, and by "walking"
with primers made to the sequence of the new toxin genes.
The PS17 toxin genes were subcloned into the shuttle vector pHT3101 (Lereclus, D. et al [1989] FMS Microbiol Lett 60:211-218) using standard methods for expression in B.t. Briefly, Salt fragments containing the 17a and 17b toxin genes were isolated from pMYC1629 and pMYC1627, respectively, by preparative agarose gel electrophoresis, electroelution, and concentrated, as described above. These concentrated fragments were ligated into Sail-cleaved and dephosphorylated pI TT3101. The ligation mixtures were used separately to transform frozen, competent E cob NM522. Plasmids from each respective recombinant E cob strain were prepared by alkaline lysis and analyzed by agarose gel electrophoresis. The resulting subclones, pMYC2311 and pMYC23O9, harbored the 17a and 17b toxin genes, respectively.
These plasmids were transformed into the acrystalliferous B.t strain, HD-1 cryB (Aronson, A., Purdue University, West Lafayette, IN), by standard electroporation techniques (Instruction Manual, Biorad, Richmond, CA).
Recombinant At strains HD-1 cryB [pMYC2311] and [pMYC2309] were grown to sporulation and the proteins purified by NaBr gradient centrifugation as described above for the wild-type Bt. proteins.

Example 4 -- Molecular Cloning of a Gene Encoding a Novel Toxin from Bacillus thunngiensis Strain PS63B
Example 2 shows the aminotermiml and internal polypeptide sequences of the 63B
toxin protein as determined by standard Edman protein sequencing. From these sequences, two oligonucleotide primers were designed using a colon frequency table assembled from B.tw genes encoding 6-endotoxins. The sequence of the forward primer (63B-A) was complementary to the predicted DNA sequence at the 5' end of the gene:
63B-A - 5' CAA T/CTA CAA GCA/T CAA CC 3' -(SEQ ID NO. 24) The sequence of the reverse primer (63B-IN) was complementary to the inverse of the internal predicted DNA sequence:
63B-INT - 5' TTC ATC TAA AAT TCT TTG ATFAC 3' (SEQ ID NO. 25) These primers were used in standard polymerase chain reactions (Cetus Corporation) to amplify an approximately 460 bp fragment of the 63B toxin gene for use as a DNA
cloning probe.
Standard Southern blots of total cellular DNA from 63B were hybridized with the radiolabeled PCR probe. Hybridizing bands included an approximately 4.4 kbp XbaI fragment, an approximately 2.0 kbp HindiII fragment, and an approximately 6.4 kbp Spel fragment.

Total cellular DNA was prepared from Bau7bsi lava ginsir (8t) calls grown to an optical density of L0 at 600 nm. The cells were recovered by centrifugation and protoplasts were prepared in lysis mix (300 mM sucrose, 25 mM Tris-HCI, 25 mM EDTA, pH = &0) and lysozyme at a concentration of 20 mg/mL The protoplasts were ruptured by addition of ten volumes of 0.1 5 M NaCl, 0.1 M Tris-HCl pH 8.0, and 0.1% SDS. The cellular material was quickly frozen at 70 C and thawed to 37 C twice. The supernatant was extracted twice with phenol/chloroform (1:1). The nucleic acids were precipitated with ethanol. To remove as much RNA
as possible from the DNA preparation, RNase at final concentration of 200 ug/ml was added.
After incubation at 37 C for 1 hour, the solution was extracted once with pbenollchloroform and 10 precipitated with ethanol A gene library was constructed from 63D total cellular DNA partially digested with NdeII
and size fractioned by gel electrophoresis. The 9-23 kb region of the gel was excised and the DNA
was electroeluted and then concentrated using an Elutip-d ion exchange column (Schleicher and Schuel, Keene, NH). The isolated NdeII fragments were ligated into BamHl-digested 15 LambdaGEM-11 (PROMEGA). The packaged phage were plated on 8 coli KW251 cells (PROMEGA) at a high titer and screened using the radiolabeled approximately 430 bp fragment probe amplified with the 63B-A and 63B internal primers (SEQ ID NOS. 27 and 28, respectively) by polymerase chain reaction. Hybridizing plaques were purified and rescreened at a lower plaque density . Single isolated, purified plaques that hybridized with the probe were used to infect 20 KW251 cells in liquid culture for preparation of phage for DNA isolation.
DNA was isolated by standard procedures (Maniatis, T., E.F. Fritsch, J. Sambrook [1982] Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, New York). Preparative amounts of DNA
were digested with Sall (to release the inserted DNA from lambda sequences) and separated by electrophoresis on a 0.6% agarose-TAE gel. The large fragments were purified by Ion exchange *
25 chromatography as above and ligated to Sall-digested, dephosphorylated pHTBlueII (anE. coWB.t *
shuttle vector comprised of pBlueScript S/K [Stratagene, San Diego, CA] and the replication origin from a resident AL plasmid [Lereclus, D. et aL (1989) FEMS MicrobioL
Lett. 60:211-218]).
The ligation mix was introduced by transformation into competent F. coil NM522 cells (ATCC
47000) and plated on LB agar containing ampiclllin (100 rughnl), IPTG (2%), and XGAL (2%).
White colonies, with putative restriction fragment insertions in the (Beta)-galactosidase gene of pHlBludIU, were subjected to standard rapid plasmid purification procedures (Maniatis er aL, supra). Plasmids ere analyzed by Sall digestion and agarose gel electrophoresis. The desired plasmid construct, pMYC1641, contains an approximately 14 kb Sail insert.
For subcioning, preparative amounts of DNA were digested with Xbal and electrophoresed on an agarose gel. The approximately 4.4 kbp band containing the toxin gene was e :ised from the gel, electroeluted from the gel slice, and purified by ion exchange chromatography as above. This fragment was ligated into Xbal cut pHTBlueII and the resultant plasmid was designated pMYC1642.

*Trade-mark 2103248 :: .

Example 5 - Cloning of a Novel Toxin Gene From At PS33F2 and Transformation into Escherichia coli Total cellular DNA was prepared from B.t PS33F2 cells grown to an optical density, at 600 nm, of 1Ø Cells were pelleted by centrifugation and resuspended in protoplast buffer (20 mg/ml lysozyme in 0.3 M sucrose, 25 mM Tris-Cl [pH 8.0], 25 mM EDTA). After incubation at 37 C for 1 hour, protoplasts were lysed by the addition of nine volumes of a solution of 0.1 M
NaCI, 0.1% SDS, 0.1 M Tris-Cl followed by two cycles of freezing and thawing.
The cleared lysate 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 10 mM Tris-Cl, 1 mM EDTA ('TE) and RNase was added to a final concentration of 50 ug/mL After incubation at 37 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 3 M NaOAc and two volumes of ethanol DNA was pelleted by centrifugation, washed with 70% ethanol, dried, and resuspended in TE.
Plasmid DNA was extracted from protoplasts prepared as described above.
Protoplasts were lysed by the addition of nine volumes of a solution of 10 mM Tris-Cl, 1 mM EDTA, 0.085 N NaOH, 01% SDS, PH=&O. SDS was added to 1% final concentration to complete lysis. One-half volume of 3 M KOAc was then added and the cellular material was precipitated overnight at 4 C. After centrifugation, the DNA was precipitated with ethanol and plasmids were purified by isopycnic centrifugation on cesium chloride-ethidium bromide gradients.
Restriction Fragment Length Polymorphism (RFLP) analyses were performed by standard hybridization of Southern blots of PS33F2 plasmid and total cellular DNA with 32P-labelled oligonucleotideprobes designed to the N-terminal amino acid sequence disclosed in Example 2.
Probe 33F2A: 5' GCA/T ACA/T TTA AAT GAA GTA/T TAT 3' (SEQ ID NO 261 Probe 33F2B 5' AAT GAA GTA/T TAT CCAIT GTA/T AAT 3' (SEQ ID NO 27) Hybridizing bands included an approximately 5.85 kbp EcoRI fragment. Probe 33F2A and a reverse PCR primer were used to amplify a DNA fragment of approximately 1.8 kbp for use as a hybridization probe for cloning the 33F2 toxin gene. The sequence of the reverse primer was:
5' GCAAGCGGCCGCTI'ATGGAATAAATTCAATT C/T T/G A/G TC T/A A 3' (SEQ ID
NO. 28).
A gene library was constructed from 33F2 plasmid DNA digested with EcoRL
Restriction digests were fractionated by agarose gel electrophoresis. DNA fragments 4.3-6.6 kbp were excised from the gel, electroeluted from the gel slice, and recovered by ethanol precipitation after purification on an Elutip-D ion exchange column (Schleicher and Schuel, Keene NH). The EcoRI
inserts were ligated into EcoRl-digested pHTBluell (an E. cob/E thuringiensis shuttle vector comprised of pBluescript S/K [Stratagene] and the replication origin from a resident Rt plasmid (Lereclus, A et aL [1989] FEMS Microbial Lett 60:211-218]). The ligation mixture was transformed into frozen, competent NM522 cells (ATCC 47000). Transformants were plated on LB agar containing ampicillin, isopropyl-(Beta)-D-thiogalactoside (IPTG), and 5-bromo-4-chloro-d 3-indolyl-(Beta)-D-galactoside (XGAL). Colonies were screened by hybridization with the radiolabeled PCR amplified probe described above. Plasmids were purified from putative toxin gene clones by alkaline lysis and analyzed by agarose gel electrophoresis of restriction digests. The desired plasmid construct, pMYC2316, contains an approximately 5.85 kbp Eco4RI
insert; the toxin gene residing on this DNA fragment (33F2a) is novel compared to the DNA
sequences of other toxin genes encoding formicidal proteins.
Plasmid pMYC2316 was introduced into the acrystalliferous (Cry-) Bt host, HD-1 CryB
(A. Aronson, Purdue University, West Lafayette, IN) by electroporation.
Expression of an approximately 120-140 kDa crystal protein was verified by SDS-PAGE analysis.
Crystals were purified on NaBr gradients N.A. Pfannenstiel et aL [1984] FEMS MicrobioL Lett 21:39) for determination of toxicity of the cloned gene product to Pratylenchus spp.

Example 6 - Cloning of a Novel Toxin Gene from at Isolate PS86Q3 Total cellular DNA was prepared from Bacillus th gienszs (B t.) cells grown to an optical density of 1.0 at 600 nm. The cells were recovered by centrifugation and protoplasts were prepared in lysis mix (300 mM sucrose, 25 mM Tris-HCI, 25 mM EDTA, pH = &0) containing lysozyme at a concentration of 20 mglml. The protoplasts were ruptured by addition of ten volumes of 0.1 M NaCl, 0.1% SDS, 0.1 M Tris-CI, pH = 8Ø The cleared lysate was quickly frozen at --70 C and thawed to 37 C twice The supernate was extracted twice with phenoichloroform (1:1). The pellet was resuspended in 10 mM Tris-CI, 1 mM
EDTA,pH = 8.0 (TB), and RNase was added to a final concentration of 50 4uglml. After incubation at 37 C for one hour, the solution was extracted once with phenol:chloroform (1:1) and then with 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, was'h$d with 70% ethanol, dried, and resuspended in TB.
Total cellular DNA from isolate PS86Q3 was used as template for polymerase chain reaction (PCR) analysis. according to protocols furnished by Perkin Elmer Cetus. An oligonucleotide derived from the N-terminal amino acid sequence of the toxin protein was used as a 5' primer. The sequence of this oligonucleotide is:
5'- AGACTGGATCCATGGC(A or T)AC(A or T)AT(A or T)AATGAATTATA (T or C)CC-3' (SEQ ID NO. 29).
An oligonucleotide coding for the amino acid sequence "ESKLKPNTRY" (SEQ ID NO.
30) can be used as the reverse 3' primer. The sequence of this oligonucleotide can be: "5'-TAACGTGTAT(A or T)CG(C or G)TITTAATIT(T or A)GA(C or T)TC-3"(SEQ ID NO.
31).
The reverse "YIDKIEFIP" (SEQ ID NO. 32) oligonucleotide was also used as a reverse 3' primer in conjunction with the above mentioned 5' primer. The sequence of the reverse primer can be: "5'-TGGAATAAATTCAATT(C or T)(T or G)(A or G)TC(T or A)A-3"' (SEQ
ID NO. 33).

w_.,." '-..-.. ._,_,.,... '__ -.. .. --ramõ ..... . ,...-_.. :`r. .. ..,. .:.
r:,..... :-._.. -. .. ..r..,....,... ... ..., r;,.... _ ...

21x3248 Amplification with the 5' primer and SEQ ID NO. 31 generates an approximately 2.3 kbp DNA fragment and an approximately 4.3 kbp DNA fragment. Amplification with the 5' primer and SEQ ID NO. 33 generates an approximate 1.8 kbp DNA fragment and an approximately 3.7 kbp DNA fragment. The approximately 2.3 kbp fragment was radiolabeled with 32P and used as a hybridization probe to generate restriction fragment polymorphism (RFLP) patterns and to screen recombinant phage libraries.
A Southern blot of total cellular DNA digested with EcoRV was probed with the radiolabeled 2.3 kbp probe described above. The resultant RFLP includes 9.5 kbp, 6.4 kbp, and 4.5 kbp hybridizing fragments.
A gene library was constructed from PS86Q3 total cellular DNA partially digested with Ndell and size fractioned by gel electrophoresis. The 9-23 kb region of the gel was excised and the DNA was electroeluted and then concentrated using an Elutip-d ion exchange column (Schleicher and Schuel, Keene, NH). The isolated Ndel fragments were ligated into BamHI-digested LambdaGEM-11 (PROMEGA). The packaged phage were plated on R coil KW251 cells (PROMEGA) at a high titer and screened using the radiolabeled probe described above.
Hybridizing plaques were purified and rescreened at a lower plaque density Single isolated, purified plaques that hybridized with the probe were used to infect KW251 cells in liquid culture for preparation of phage for DNA isolation. DNA was isolated by standard procedures (Maniatis et aL, supra). Preparative amounts of DNA were digested with Sall (to release the inserted DNA
from lambda sequences) and separated by electrophoresis on a 0.6% agarose-TAE
gel. The large fragments were purified by ion exchange chromatography as above and ligated to Sall-digested, dephosphorylated pHTBlueU (an E. cok/8.L shuttle vector comprised of pBluescript S/K
[Stratagene, San Diego, CA]) and the replication origin from a resident At.
plasmid (Lereclus et aL [19891, supra). The ligation mix was introduced by transformation into competent E-`coli NM522 cells (ATCC 47000) and plated on LB agar containing ampicillin, IPM, and KGAL-White colonies, with putative restriction fragment insertions in the (Beta)-galactosidase gene of pHTBluell, were subjected to standard rapid plasmid purification procedures (Maniatis et aL, supra). Plasnud DNA was analyzed by Sall digestion and agarose gel electrophoresis. The desired plasmid construct, pMYC1647, contains an approximately 12 kb Sall insert.
Plasmid pMYC1647 was introduced by electroporation into an acrystalliferous (Cry) At., HD-1 CryB (AL Aronson, Purdue University) host to yield MR515, a recombinant At. clone of 86Q3(a). Expression of an approximately 155 kDa protein was verified by SDS-PAGE. Spores and crystals were removed from broth cultures and were used for determination of toxicity to pharaoh ants.
Example 7 - Activity of the Rt. Toxin Protein and Gene Product Aeainst Ants Broths were tested for the presence of fi-exotoxin by a larval house fly 'bioassay (Campbell, D.P., Dieball, D.E., Bracket, J.M. = [1987] "Rapid HPLC assay for the fi-exotoxin of Bacillus thuringiensi c, ' J. Agric. Food Chem. 35:156-158). Only isolates which tested free of 9-exotoxin were used in the assays against ants.
A bait was made consisting of 10% Bacillus thuringiensis isolates of the invention and Crosse and Blackwell mint apple jelly. Approximately 100 ants were placed in each plastic test chamber replicate with the baits. Control experiments were performed with untreated mint apple jelly. Each test was replicated a minimum of 10 times. Mortality was assessed at 7, 14 and 21 days after introduction of the bait to the ants. Results are shown below:

Table 6. Toxicity of B. thunngiensis Isolates to the Pharaoh Ant (Monomoruum Pharaonic) At. Isolate Percent Mortality Control 11 PS211B2 90.0 Control 3.8 Exjrle 8 Activity Against Pharaoh Ants Mint apple jelly containing 10% AL (100,000 ppm) was fed to 5 replicates of approximately 100 worker ants for 21 days. Total mortality (in %) over the test period is compared to control.

Table 7. Three week mortality on pharaoh ant workers.

Sample Rate ppm Percent Mortality MR315 100000 40.1 86Q3 100000 29.2 211B2 100000; 58.5 MAJ Blank 25.0 Control Blank 14.4 MR515 a recombinant At. clone of 86Q3(a) gene, 10% in MAT (Example 6) 86Q3 = spray dried powder of At. PS86Q3, 10% in MAJ
211B2 = spray dried power of At. PS211B2,10% in MAT
MAJ = Mint apple jelly, Crosse & Blackwell Control = rearing diet of water, frozen flies, mealworms/honey agar Table & Three week mortality (%) on pharaoh ant workers.

Sample Rate ppm Percent Mortality 50000 100.0 86Q3 50000 99.6.
211B2 50000 100.0 MAJ Blank 753 Control Blank 39.0 140E2 = 5% 140E2 purified protein in MAJ
86Q3 = 5% 86Q3 purified protein in MAJ
211B2 5% 211B2 purified protein in MAJ
MAJ = Mint apple jelly, Crosse & Blackwell Control = rearing diet of water, frozen flies, mealworms/honey agar Example 9 - Cloning of Novel Ant-Active Genes Using Generic Oligonucleotide Primers The formicidal gene of a new formicidal At. can be obtained from DNA of the strain by performing the standard polymerase chain reaction procedure as in Example 6 using the ohgonucleotides of SEQ ID NO. 33 or AGGAACAAAYTCAAKWCGRTCTA (SEQ ID NO. 34) as reverse primers and SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO.
16, SEQ
ID NO. 23, SEQ ID NO. 27, SEQ ID NO. 29, or SEQ ID NO. 24 as forward primers.
The expected PCR fragments would be approximately 330 to 600 bp with either reverse primer and SEQ ID NO. 12 or SEQ ID NO.13,1000 to 1400 bp with either reverse primer and SEQ ID NO.
15, or SEQ ID NO. 16, and 1800 to 2100 bp with either reverse primer and any of the thr N
terminal primers, SEQ ID NO. 27, SEQ ID NO. 23, SEQ ID NO. 29, and-SEQ ID NO.
24.
Alternatively, a complement from the primer family described by SEQ ID NO. 12 and SEQ ID
NO. 13 can be used as reverse primer with SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID
NO. 23, SEQ ID NO. 27, SEQ ID NO. 29, or SEQ ID NO. 24 as forward primers. The expected PCR
fragments would be approximately 650 to 1000 bp with SEQ ID NO. 15 or SEQ ID
NO. 16, and 1400 to 1800 bp for the four N-terminal primers (SEQ ID NO. 27, SEQ ID NO. 23, SEQ ID NO.
29, and SEQ ID NO. 24).
As another alternative, the reverse primer SEQ ID NO. 31 can be used with any of the four N-terminal forward primers to yield fragments of approximately 2550-3100 bp; 1750-2150 bp with the forward primers SEQ ID NOS. 15 or 16; 850-1400 bp with SEQ ID NOS. 12 or 13; and 550-1050 bp with the forward primer TITAGATCGT(A or C)TTGA(G or A)TTT(A or G)T(A
or T)CC (SEQ ID NO. 35).
As yet another alternative, the ITSED (SEQ ID NO 37) reverse primer (TCfCCATCTTCTGA(G or A)G(T or A)AAT) (SEQ ID NO. 37) can be used with the N-terminal forward primers (SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 27, and SEQ
ID NO.
SUBSTITUTE SHEET

ID NOS. 15 or 16;1800-2400 bp with forward primers SEQ ID NOS. 12 or 13; and 1500-2050 bp with forward primer SEQ ID NO. 35.
Amplified DNA fragments of the indicated sizes can be radiolabeled and used as probes to clone the entire gene as in Example 6.
Example 10 - Insertion of Toxin Gene Into Plants One aspect of the subject invention is the transformation of plants with genes coding for a formicidal toxin. The transformed plants are resistant to attack by ants.
Genes coding for formicidal 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 cob 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, pUC series, M13mp series, pACYC184, etc.
Accordingly, the sequence coding for the At. toxin can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E
cold. 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 right 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. -c4 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. T., Alblasserdam, Chapter 5; Fraley et aL, Cut.
Rev. Plant Sci. 4:1-46;
and An et aL (1985) EMBO 14:277-287.
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 confers on the transformed plant cells resistance to a biocide or an antibiotic, such as kanamycin, G 418, bieomycin, hygromycin, or chloramphemcol, inter aba. 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 tumefaciew or Agrobacterium rhrzogenes 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. cold 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 [19781 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 'I -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 Agrobacterdum tumefaciens or Agrobacterium rhdzogmes 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 plasnuds, 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.
P. xample 11- Cloning of Novel B. thunnsiensis Genes Into Insect Viruses , d 4 A number of viruses are known to infect insects. These viruses include, for example, baculoviruses and entomopoxviruses. In one embodiment of the subject invention, ant-active genes, as described herein, can be placed with the genome of the insect virus, thus enhancing the pathogenicity of the virus. Methods for constructing insect viruses which comprise at. toxin genes are well known and readily practiced by those skilled in the art. These procedures are described, for c zample, in Meriyweather et aL (Merryweather, A.T., U. Weyer, M.F.G.
Harris, M. Hirst, T.
Booth, R.D. Possee (1990) J. Gen. YtroL 71:1535-1544) and Martens et aL
(Martens, J.W.M., G.
Honee, D Zuidema, JWM. van Lent, B. Visser, J.M. Vlak (1990) AppL
Environmental MurobwL
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.

WO 92/20802 Pcr/US92/04316 BUDAPEST VdZATZ ON TYPE INTRPHATIONAL
RECOGIlTION OF TUC DWOSI? OF 1NICROORCAUISMS
FOR THE PURPOSES OIF PATENT PROCEDURZ

INTZRN TIONAL 1'OPl1 rT0 RECEIPT IN THE CASE OP AN ORIGINAL DEPOSIT
Dr. Jewel Payne issued pursuant to Rule 7.1 by the Mycogen Corporation INTERNATIONAL DEPOSITARY AUTHORITY
5451 Cberlin Drive Identified at the bottom of this page San Diego, CA 92121 NAME AND ADDRESS
OP DEPOSITOR

I I. IDENTIFICATION Or THR MXCROORCANISN

Identification reference given by the Accession number given by the DEPOSITORS IRtZKNATIONAL DEPOSITARY AUTHORITY1 Bacillus thuri><S iensis PS86Q3 NRRL B-19765 Xi. SCXX=XPIC OESCI%XX TION AND/OR PROPOSED TAXONOMIC DZSZCASATION
The main. uorgnni>vae Identified under a above vas aeco.pe*da4 bys a scientific description a proposed taxonomic designation (Mark with a er'oen whore applicable) Ill. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the faicroorganiss identified under I above, which was received by it on Feb.6,1991' (date of the original deposit)), IV. RECEIPT Or R }UZST FOR CONVERSION

The microorganism identified under I above was received by this International Depositary Authority on (date of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (ants of receipt of request for conversion) V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: AOricu3.tural Research Culture Signature(s) of person(s) having the power Collection (NRRL) to represent the International Depositary International Depositary Authorit Authors of uthoriaed official(s):
LAddress: 1615 N. University Street Date, Peoria, Illinois 61604 U.S.A. 1J / y Where Rule 6.4(d) applies, such date Is the date on which the status of international 1eponitary authority was acquired.
SUBSTITUTE SHED'' 2suwu'isS'l 'A'f+a..l va ana lava+a....ea i. ..
RECOGNITION OF THE DEPOSIT OF MICR.OOi3CAMISi4S
FOR Tat PURPOSES or PATEN? PROCCOURE

INNTERHATIOHAL FORM

FT-' RECEIPT IN THE CASE Or AN ORIGINAL DEPOSIT
Dr. Jecl Payne issued pursuant to Rule 7.1 by the Mycogen Corporation INTERNNATIONA DEPOSITARY AUTHORITY
5451 Oberlin Drive identified at the bottom of this page San Diego, CA 92121 NAME AND ADDRESS
OF DEPOSITOR

1. ID0fZFICATION Of THE MICROORGANISM

Identification teterence given by the Accession number given by the DEPOSITORa INTSMATIONNAL DEPOSITARY AUTHORITY!
Bacillus illus thuurin i~ s PS140E2 NRRL 8-18812 II. SCIZTYIPIC D&SCRIPTION AND/OR PROPOSED TAXOSOHIC DESIGNATYON
The microorganism identified under I above was accompanied bys EJ a scientific description t.. .~ 4 proposed tauonoeic designation (Mark with a cross where applicable) Ill. Ractipt. ATE ACCEPTANCB

This International Depositary Authority accepts the Microorganism identified under I above, which was received by it onflpr.23,199(date of the original deposit) 2V. RECEI or REQUEST FOR CONVERSION

The microorganism identified under I above vas received by this International Depositary Authority on (date of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion) V. IirrERNATIOHAL DEPOSITARY 'AUTHORITY

Names Agricultural Research Culture Signature(s) of person(s) having the power Collection (NRRL) to represent the International Depositary International Depositary Authorit Authority Or Of author204 official(s):
Addreass 1815 N. University Street Datei Peoria, Illinois 61604 U.S.A.

Where Rule 6.4(d) applies, such data is the data on which the statue ofinternationaL depositary authority was acquired.
SUBSTITUTE SHE

.- .... :-.,, :, ,-..-:. ,.:-:: -.. _., ..,.-.rs: -, r, zr.. .::r .m....
........,. ., F.....:,.,, i:. z, .r .'..'r. ..., o...ii , 2(03248 BUDAPEST TBATY ON THE INTERNATIONAL
RECOCNITIOH Of Tans DEPOSIT or NICROOR ZSN.S
FOR THE pwpoSz$ or PATS" PROCIMU

INTLPR3rtATIOWAL FORM

' ~ r~ Jewel Payne RECEIPT IN THE CASE OF AN ORXGINAL DEPOSIT
Corporation issued pur'uant to Rule 7.1 by the Mycogeri po SPPP ATIONJd7. DEPOSITARY AUTHORITY
5451 Oberlin Drive identified at the bottom of this page San Diego, CA 92121 NME AND ADDRESS
OF DEPOSITOR

1. IDOMITICATION OF THE NICROORGA)USH

saentification reference given by the Accession number given by the DaDFOSI?ORz IN7@ -TIGt1AL DQOSITARY AUTHORITY:
Bacillus thuringiensis PS211B2 NRRL B-16921 II. SCIENTIPIC DESCRIPTION AND/OR PROPOSED TA39o1fof{IC DESIe iATION
The microorganism identified under I above was accompanied bys a scientific description m a proposed taxonomic designation (Mark with a cross where applicable) -III. RECEIPT AND ACCtpTANCZ

This International Depositary Authority accepts the microorganism identified under I above, which was received by it onNOV. 15,1991 (date of the original deposit)l 2'V. RECEIPT OF REQUEST ?OR CONY SXOH

The microorganism Identified under I above was received by this International Depositary Authority on (date of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion) V. INTERNATIONAL DEPOSITARY AUTHORITY

Names Agricultural Research Culture Signature(s) of parson(s) having the power Collection (NRRL) to represent the International Depositary International Depositary Authority Authority or Of authorizg4 otiieial(s)e Addsesas 1815 N. University Street Data ~ `-Peoria, Illinois 61604 U.S.A. e~y Where Rule 6.4(d) applies, such date is the data on which the status of international depositary authority was acquired.
SUBSTITUTE SHE

WO 92/20802 PC r/US92/04316 page 14 BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORH
TO
Dr. Jewel Payne RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
Entomology issued pursuant to Rule 7.1 by the Pal Corporation INTERNATIONAL DEPOSITARY AUTHORITY
identified at the bottom of this page 5457 Oberlin Dr.
San Diego, CA 92121 HAMS AND ADDRESS
L = OF DEPOSITOR

1. IDENTIFICATION OF THE MICWORG)WISM

Identification reference given by the Accession number given by the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:
Bacillus thuriiensis PS17 NRRL B-18243 3g. s IC oN AND/OR F 'i71I DMIC DESIGNATION
The microorganism identified under I above was accaosvpanied by:

a scientific description .~4 a proposed taxonomic designation -.~(l4ar& with a cross where applicable) III. RECEIPT AM ACCEPT NCE

This International Depositary Authority accepts the microorganism identified under I above, which was received by it on July 28 ,1987(date of the original deposit)1 IV. IONAL DEPOSITARY AUTHORITY

tee: Agricultural Research Culture Signature(s) of person(s) having the power Collection (Mn) to represent the T!!15021 ational Depositary International Depositary Authority Authority o~roff 4iorize d official (s) :
Address. 1815 N. University Street 1 ~1+4, _ _ a--Peoria, Illinois 61604 U.S.A. Date: ~( f iU ds7 Where Rule 6.4(d) applies, such date is the date on which the status of international depositary authority was acquired; where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by the international depositary authority.

Form BP/4 (sole page) SUBSTITUTE SHE

page 14 BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM
FTO
Dr. Jewel Payne RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
tolocgy issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY
Mycogen Corporation identified at the bottom of this page 5457 Oberlin Dr.
.can Diego, CA 92121 NAME AND ADDRESS
t OF DEPOSITOR

1. IDENTIFICATION OF THE MXCRCORGANISM

Identification reference given by the Accession number given by the DEPOSMTOR: INTERNATIONAL DEPOSIU AUTHORITY:
Bacillus thuringiensis ps33F2 NRRL B-18244 II. s C DESCRIPTION AND/OR PROPOSED TAXON02ZC DESIMFATION
The microorganism identified under I above was accompanied by:
~- a scientific description -.a a proposed taxonomic designation (21axk with a cross where applicable) IYI.' '1 PT ANA ACC'EPTANcz This International Depositary authority accepts the microorganism identified under I above, 1.
which was received by it on July 28,1987(date of the original deposit) TV. ' ZNTEFWATIOUAL DEPOSITARY AUTHORITY

e: Agricultural Research Culture Signature(s) of person(s) having the power Collection (NRRL) to represent the International Depositary International Depositary Authority Authority or of uthorized official(s):
Address: 1815 N. University Street Peoria, Illinois 61604 U.S.A. Date:
r~) l Where Rule 6.4(d) applies, such data is the date an which the status of international depositary authority was acquired; where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by the international depositary authority.

`Fora BP/4 (sole page) SUBSTITUTE SHMET

page 14 BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM
TO
'Dr. Jewel Payne RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
Entomology issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY
Mycogen Corporation identified at the bottom of this page 5457 Oberlin Dr.
San Diego, CA 92121 j NAME AND ADDRESS
OF DEPOSITOR

1. IDENTIFICATION OF TEE MICROORGANISM

Identification reference given by the Accession number given by the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:
Bacillus thurinSiensis PS63B NRRL B-18246 11. SCI IFIC DESCRIPTION AM/oR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under I above was accompanied by:

a scientific description a proposed taxonomic designation f1ara with a cross where applicable) xxl. RECt.IIPT AND ACCEPTANCE

This International Depositary authority accepts the microorganism identified under I above, which was received by it on July 28 ,1987(date of the original deposit)1 IV. INTERNATIONAL DEPOSITARY AUTHORITY

..ame; Agricultural Research Culture Signature(s) of person (a) having the power Collection (NRRL) to represent the ternational Depositary International Depositary Authority Authority or of au riled official (s) radr..a: 1815 N. University Street Peoria, Illinois 61604 U.S.A. Date:

1 Where Rule 6.4 (d) applies, such date is the date on which the status of international depositary authority was acquired; where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by the international depositary authority.

q Form BP/4 (sole page) SUBSTITUTE SHEET

r r = T r f 5 ryt vl w r ~ i ! i f i'' y <4 WO 92/20802 2:103248 PCT/US92/04316 BUDAPEST TREATY ON THE INTERNATIONAL
ECOGNITION OF THE DEPOSIT OF MICAC ;ANISMS
FOR THE PURPOSES OF PATENT PRO(. IRE

INTERNATIONAL FORM

` s. L?nore Linda R. N d RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
issued pursuant to Rule 7.1 by the Mycogen Corporation INTERNATIONAL DEPOSITARY AUTHORITY
5451 Oberlin Dr. identified at the bottom of this page San Diego, CA 92121 NAME AND ADDRESS
L OF DEPOSITOR

I. IDENTIFICATION OF THE MICROORGANISM

Identification reference given by the Accession number given by the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:
Escherichia cols NM522/pMYC2316 MR608 NRRL B-18785 It. SCIENTIFIC DESCRIPTION AaO/Oft PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under I above was accompanied byt a scientific description a proposed taxonomic designation (Mark with a cross where applicable) III RECEIPT AM ACCEPTANCE

This international Depositary_ Auth r4laccepts the microorganism identiified under I above, which was received by it an i~`ilar.ll~Ss (date of the original deposit) IV. RECEIPT OF REOUZST FOR CONVERSION

The microorganism identified under I above was received by this International Depositary Authority on (date of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion) V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: Agricultural Research Culture Signature(s) of rson(s) having the power Collection (NRRL) to represent the ternational Depositary International Depositary Authorit Authors to au ized attic al(s):
Address: 1815 N. University Street Date:
Peoria, Illinois 61604 frJU.S.A.

Where Rule 6.4(d) applies, such date is the date on which the status of international depositar authority was acquired. SUBSTITUTE SUBSTITUTE SHEET

,BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICR-"M JISXS
FOR THE PURPOSES OF PATENT PRE JURE

INTERNATIONAL FORM

Linda R. Nyga d RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
i' I~ issued pursuant to Rule 7.1 by the My en enOr Corporation R. INTERNATIONAL DEPOSITARY AUTHORITY
5451 Oberlin Dr. identified at the bottom of this page San Diego, CA 92121 NAME AND ADDRESS
OF DEPOSITOR

1. IDENTIFICATION OF THE MICROORGANISM

Identification reference given by the Accession number given by the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:
Escherichia coil NM522/PM"1C 2321 MR607 NRRL B-18770 II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAEONONIC DESIGNATION
The nieroorganisn identified under I above, was accompanied byt a scientific description a proposed taxonomic designation (Mark with a cross where applicable) III. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I above, which was received by it on Feb.14,1991 (date of the original deposit)1 IV. RECEIPT OF REOUEST FOR CONVERSION

The microorganism identified under I above was received by this International -Depositary Authority on (date of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion) V. INTERNATIONAL DEPOSITARY AUTHORITY

Names Agricultural Research Culture Signature(s) of person(s) having the power Collection (NRRL) to represent the International Depositary International Depositary Authority Authori o of a horized official(s):
Address: 1815 N. University Street Date:
Peoria, Illinois 61604 U.S.A. ~ycfr Wbere Rule 6.4(d) applies. such date is the data on which the status of international depositary authority was acquired.
Form ~/4 (sole page) SUBSTITUTE SHEET

N-Y
err:~Ar eta rh.~ rI fr ~<<i ~~ A`rt. ~f ` . , ., BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORa"ISMS
POR 72EC PURPOSES OF PAT3WT PROCEDdm..

INTERNATIONAL FORM

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
issued pursuant to Rule 7.1 by the i4s Lenore Linda R. Nygaard INTERtiATIONAL DEPOSITARY AUTHORITZ
Mycogen Corporation identified at the bottom of this page 5451 Oberlin Dr.
San Diego, CA 92121 NAHE AND ADDRESS
OF DEPOSITOR

1. IDE33TZrICATION Or T3lE MICROORGANISM

Identification reference given by the Accession number given by the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:
Eseherichia coli 19-1522/pMYC2317 MR609 NRRL B-18816 II. SCIglITZFZC DESCR ION AN/OR PROPOSED TAXONOMIC DESIC ATION
The microorganism identified under I above was accompanied bys a scientific description fl : 1, a proposed taxonomic designation (Mark with a cress where applicable) 111. RECZXPT AND AOCETAaaCL

This International Depositary Authority accepts the microorganism identified under I above, which was received by it on Apr.24,1991 (date of the original depokit)1 W. RECEIPT or REQUEST FOR CONVSSIOM

The microorganism identified under I above was received by this International I Depositary Authority on (date of.the original deposit) and a requestt to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion) V. INTERUATIONAL DEPOSITARY AUTHORITY

Names Agricultural Research Culture Signature(s) of person(s) having the power Collection (NRRL) to represent the International Depositary International Depositary Authorit Authority or of authrariz d officiatts):
( Address: 1815 N. University Street Dac9: Peoria, Illinois G-1604 U.S.A.

Where Rule 6.4(d) applies. such dat',is the date on which the status of international. depositar=
authority was acquired. SUBSTITUTE SHE

page 14 BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

14s[ enure Linda R. Nygaard 1 RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
Mycogen corporation issued pursuant to Rule 7.1 by the 5451 Oberlin Dr. INTERNATIONAL DEPOSITARY AUTHORITY
San Diego, CA 92121 identified at the bottom of this page.
L NAME AND ADDRESS
OF DEPOSITOR

a. IDENTIFICATION OF THE MICROORGANISM

identification reference given by the Accession number given by the DPOSZ=Rt INTERNATIONAL DEPOSITARY AUTHORITY:
Escherichia coli NM522/pMYC1627 MR398 NRRL B-18651 I2. S C DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under I above was accompanied by.
ED a scientific description a proposed . taxonomic designation (Mark with a cross where applicable) Z=- R CEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I above, which was received by it on May 11,1990 (date of the original deposit) Iv. X TZRNATZONAL DEPOSITARY AUTHORITY

mez Agricultural Research Culture Signature(s) of person(s) having the power Collect on (NRRL) to represent the I ornational Depositary International Depositary Authority Authority or f au riaed official(s):
Address:.1815 N. University, Street.'' Peoria, Illinois 61604 U.S.A. Date:

1 Where Rule 6.4(d) applies, such date is the date on which the status of international depositary authority was acquired; where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by the international depositary-authority.

Form BP/4 (sole page) SUBSTITUTE SH

=..., rr~' ,,,,,. .., fxr+ ~;~:.,. ..,<., ..=,..,:~a=.:~+r:.;.a=~ ........, ,.........., ............::.Y*"r.r=,.;,--t+f rr;. ..,...... ~rs,S+..;2,, F......... .........F....... .3r... ... . .. ..., page 14 BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Ms1 -Lenore Linda R. Nygaard RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
Mycogen Corporation issued pursuant to Rule 7.1 by the 5451 Oberlin Dr. INTERNATIONAL DEPOSITARY AUTHORITY
San Diego, CA 92121 identified at the bottom of this page.
NAME AND ADDRESS
L OF DEPOSITOR

1. IDENTIFICATION OF THE MICROORGANISM

Identification reference given by the Accession number given by the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:
Eseberichia coli NM522/pMYC1628 MR399 NRRL B-18652 XI. S C DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under I above was aeeowpanied by:

a scientific description X(~ a proposed taxonomic designation (Mark with a cross where applicable) III. RECEIPT ANDACCEpTANCE

This International Depositary Authority accepts the microorganism identified under I above, which was received by it ca May 11,1990 (date of the original deposit)1 IV. INTERNATIONAL DE'POSITVM AUTHORITY

boa; Agricultural Research Culture Signature(s) of person(s) having the power Collection (NRR,L) to represent the ternational Depositary International Depositary Authority Authority or f au prized offi (s) :
Address: 1815 N. University Street ~/~'' Peoria, Illinois 61604 U.S.A. Date:
t. Ill'/yv l Where Rule 6.4(d) aspplles, such date is the date on which the status of international depositary authority was acquired; where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by the International depositary =authority.

Form BP/4 (sole page) SUBSTITUTE SHEET

21O 2~0 32-12 BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF NICR006IGA (zsMa FOR THE PURPOSES OF PATEN? FROC=URE

INTERNATIONAL FORM

1=" RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
Ms. Lenore Linda R. Nygaard Issued pursuant to Rule 7.1 by the Mycogen Corporation INTERNATIONAL DEPOSITARY AUTHORITY
5451 Oberlin Drive Identified at the bottom of this page San Diego, CA 92121 HAMS AND ADDRESS
OF DEPOSITOR

I. IDENTI?ICATION OF THE MICROORGANISM

Identification reference given by the Accession number given by the DEPOSITORa INTERNATIONAL DEPOSITARY AUTHORITY:
Escherichia coli NM 522/pMYC 1642 MR626 NRRL B-18961 EI. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism Identified under I above was accompanied byt a, scientific description a. a proposed taxonomic designation dfark with a cross where applicable) 1 X I . RECEIPT AND ACCEPTANCE

This International Depositary authority accepts the microorganism identified under I above, which was received by it on 4-10-92 (date of the original deposit)l IV. RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under I above was received by this International Depositary Authority on (date of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by It on (date of receipt of request for conversion) V. INTERNATIONAL DEPOSITARY AUTHORITY

names Agricultural Research Culture Signature(s) of person(s) having the power Collection (NRRL) to represent the International Depositary International Depositary Authorit Authority or of A thosizoo official(s)a Addressi 1815 N. University Street Date: 4 -%
Peoria, Illinois 61604 U.S.A. ((,-y')-Where Rule 6.4(d) applies, such date is the date on which the status of International depositary authority was acquired. -~^ SHEET
Forte BP/4 (sole page) SUBSTI i UTE

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Payne, Jewel M.
Kennedy, M. Keith Randall, John Brooks Meier, Henry Uick, Heidi Jane Foncerrada, Luis Schnepf, Harry E.
Schwab, George E.
(ii) TITLE OF INVENTION: Novel Bacillus thuringiensis isolates Active Against Hymenopteran Pests and Genes Encoding Hymenopteran Active Toxins (iii) NUMBER OF SEQUENCES: 38 (iv) CORRESPONDENCE ADDRESS:
A) ADDRESSEE: David R. Saliwanchik B) STREET: 2421 N.W. 41st Street, Suite A-1 C CITY: Gainesville D STATE: FL
E COUNTRY: USA
F ZIP: 32606 (v) COMPUTER ) MDIUMATYYPPE Floppy disk B) COMPUTER: IBM PC compatible C OPERATING SYSTEM: PC-DOS/MS-DOS
D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
JA APPLICATION NUMBER: US
B FILING DATE:
C CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
JA NAME: Saliwanchik, David R.
B REGISTRATION NUMBER: 31,794 C REFERENCE/DOCKET NUMBER: M/SCJ 104 (ix) TELECOMMUNICATION INFORMATION:
M TELEPHONE: 904-375-8100 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS
JA LENGTH: 4155 base pairs B TYPE: nucleic acid C STRANDEDNESS: double D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
JA~ ORGANISM: Bacillus thuringiensis BSTRAIN: PS17 C) INDIVIDUAL ISOLATE: PS17a (vii) IMMEDIATE SOURCE:
(B) CLONE: E. soli NM522(pMYC1627) NRRL B-18651 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

GGTTTAAGTT TAATTACATT AGCTGTACCG GAAATTGGTA TTTTTACACC TTTCATCGGT .300 SUBSTITUTE SHEET

TATCAAACTT CTGATAACTA TTCTGGTCAC,GTTGGTGCAT TGGTAGGTGT GAGTACGCCT 1560 GTAGATAATA ATACGGGAGT ACAAGGAGCA AATGGTGTCT ATGTAGTCAA ATCTATTGCT .1920 ATATATCATG GAAGTTATAA TACTTCATCA GGTGCAGATG ATGTTTTATG GTCTTCTTCA 210D.--t TCTATGCATT TTGTTAATGA AGGAAAAGTG ATAAAAACAA TTGATATTCC AGGGCATTCG .2220 TC'1"ZTAAGCG GATCTGATCA TACGACTATT TATCATGGAA AACTTGAAAC TGGGATTCAT 2460 (2) INFORMATION FOR SEQ ID AO:2 (i) S CE CHARACTERISTICS:
JjUZ
LENGTH: 1385 amino acids 8 TYPE: amino acid C STRANDEDNESS single D TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
IAI ORGANISM: BACILLUS THURINGIENSIS .-rt BSTRAIN: PS17 C INDIVIDUAL' ISOLATE: PS17a (vii) IMMEDIATE SOURCE:
(8)CLONE: E. cols NM522(pMYC1627) NRRL B-18651 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Not Ala Ile Lou Asn Glu Leu Tyr Pro Ser Val Pro Tyr Asn Val Leu Ala Tyr Thr Pro Pro Ser Phe Leu Pro Asp Ala Gly Thr Gin Ala Thr 25 Pro Ala Asp Leu Thr Ala Tyr Glu Gln Leu Leu Lys Asn Leu Glu Lys Gly Ille Asn Ala Gly Thr, Tyr Ser Lys Ala Ile Ala Asp Val Leu Lys s 55 Gly Ile Phe Ile Asp Asp Thr Ile Asn Tyr Gln Thr Tyr Val Asn Ile Gly Len Ser Leu 8ie Thr Leu Ala Val PO Glu Ile Gly Ile Phe Thr Pro Phe Ile Gl09 Leu Phe Phe Ala Ala Leu Asn Lys His Asp Ala Pro 1 Pro Pro Pro Asn Ala Lys Asp 1iee Phe Glu Ala Met LYS Pro Ala Ile Gin Glu Net Ile Asp Arg Thr Leu Thr Ala Asp Glu Gin Thr Phe Leu ~... '',, .....A _7,f ~1r........ ...;!-a.L. .. ..., 4'T
:r . C._.i, ..,`~ t,._ . ..

Asn Gly Glu Ile Ser Gly Lau Gin Asn Leu Ala Ala Arg Tyr Gln Ser Thr Met Asp Asp lie Gln Ser His Gly lly Phe Asn Lys Val Asp Ser Gly Leu Ile Lys Lye Pile Thr Asp Glu Valll Leu Ser Lou Ann Ser Pile Tyr Thr Asp Arg Leu Pro Val Phe Ile Thr Asp Asn Thr Ala Asp Arg Thr Lau Leu Gly Leu Pro Tyr Tyr Ala Ile Lau Ala Ser Met His Lau Met Lau Lau Arg Asp Ile Ile Thr Lys Gly Pro Thr Trp Asp Ser Lys Ile Asn Phe Thr Pro Asp Ala Ile Asp Ser Phe Lys Thr Asp Ile Lys Asn Asn Ile Lys Lou s Tyr Ser Lys 265 Thr Ile Tyr Asp Val 27e Gin Lys Gly Lou Ala Ser Tyr Gly Thr PPrro Ser Asp Leu Glu SSerr Phe Ala Lys Lys Gln Lye Tyr Ile Gin Ile Met Thr Thr His s Lau Asp Phe Ala Arg Lau The Pro Thr The Asp Pro Asp Leu Tyr Pro Thr Gly Ser Giy Asp Ile Ser Leu G32n Lys Thr Arg Arg lie e Lau Ser Pro Phe 335 Ile Ile Arg Thr 3laa Asp Gly Leu Thr Len Ann Asn Thr'Ser lie e Asp Thr 345 Ser Asn Tr Pro Ann Tyr Gin A36n Giy Asn Gly Ala 36e Pro Asn Pro Lys Glu Arg Ile Lau Lys G35 in The Lys Leu Tyr Prroo Ser Trp Arg Ala G1 Gin Tyr Gly Gly Lau Lou Gin Pro Tyr Lau Trp Ala-Ile Gin Val Gin Asp Ser Val GG01u5 Thr Arg Lou Tyr G1 Gin Lau Pro Ala VVal Asp 45 Pro Gin Ala Glyy Pro Ann Tyr Val Ser Ileee Asp Ser Ser Ann Pro Ile Ile Gin lie Asn Met Asp Thr Trp Lys Thr Pro Pro 44n Gly Ala-Ser 43; 4ib Gly Trp Asn Thr Asn Lau 4ett Arg Giy Ser Val Seer Gly Lou Ser Pile Lau Gin Arg Asp Gly Thr Arg Lau Ser Ala Glq Met Gly Gly Gly Phe Ala Asp Thr Ile Tyrr Ser Lou Pro Ala 4Thr His Tyr Lau Ser Tyr Lou Tyr Gly Thr PPro ro Tyr Gin Thr SerA0p Ann Tyr Ser G1y His Val Gly Ala Lau Val 1l Gly Val Ser Thr Proo Gin Glu Ala Thr 52eu Pro Ann Ile Ile Glyy Gin Pro Asp Glu Gin Gly Asn Val Ser Thr Met Gly Phe Pro Phe Glu Lys Ala Ser Tyr Gly Gly Thr Val Val Lys Glu Trp Leu Asn Gly Ala Asn Ala Met Lys Lau Ser Pro 5G ly Gin Ser Ile G1y lie e Pro Ile Thr Asn Val Thr Ser Gly Glu Tyr Gin Ile Arg Cys Sg Tyr Ala Ser Asn 59p Asn Thr Asn Val Pile Phe Asn Val Asp Thr Gly Gly Ala WO 92/20802 210324 8 PCi'/US92/04316 Asn PProo Ile Phe Gln Gin 6le Asn Phe Ala Ser Thr Val Asp Asn Asn Thr Gly Val Gln Gly Ala Asn Gly Val Tyr Val Val Lys Ser Ile Ala Thr Thr Asp Asn Ser Phe Thr Glu Ile Pro Ala Lys Thr Ile Asn Val His Lau Thr AAenn Gin Gly Ser Ser Asp Val Phe Leu Asp 6ra Ile Glu 660 6 Phe Ile Pro Phe Ser Leu Pro Lau Ile Tyr His Gly Ser Tyrr Asn Thr Ser Ser Gly Ala Asp Asp Val Leu Trp Ser Ser Ser Asn Met Asn Tyr Tyr Asp Ile Ile Val Asn Gly Gin Ala Asn Ser Ser Ser Ile Ala Ser Ser Met His Lau Len Asn Lys Gly Lys Val Ile Lys Thr Ile Asp Ile Pro Gly His Ser Glu Thr Phe Phe Ala Thr Phe Pro Val Pro Glu Giy The Asn Glu Val Arg Ile Leu Ala Giy Lau Pro Glu Val Ser Gly Asn Ile Thr Val Gin Ser Asn Asn Pro Pro Gin Pro Ser Asn Asn Gly Gly G1y Asp Gly Gly Gly Asn Gly Gly Gly Asp Gly Gly Gin Tyr Asn Phe Ser Lau Ser Gly Ser Asp His Thr Thr lie Tyr His Gly Lys L81u Gin Thr Gly Ile HHis Val GinGly AsnTyr Thr Tyr Thr Gly Thr Pro Val Lau Ile Leu Asn Ala Tyr Arg Asn Asn Thr Val Val Ser Ser Ile Pro Val Tyr Ser Pro Phe Asp I815e Thr Ile Gln Thr Gin Ala Asp Ser Lau Gin Lau Glu Lau Gin Pro Arg TyrGly Phe Ala Thr Val Asn Gly Thr Ala Thr Val Lys Ser Pro Asn Val Asn Tyr Asp Arg Ser Phe Lps Lau 885 890 895 ,e=~
Pro Ile Asp Leu Gin Aan Ile Thr Thr Gln Val Asn Ala Lau Phe-Ala Ser Gly TThr5 Gln Asn Met Lau 92aa His Asn Val Ser Asp His Asp Ile Glu GG310 Val Val Leu Lys Val Asp Ala Lau Ser Asp Glu Val Phe.Gly 935 0 Asp Glu Lys Lys Ala Leu Arg Lys Lau Val Asn Gln Ala Lys Arg Lau Ser Arg Ala Arg A65 Lau Lau Ile Gly 9G ly Ser Phe Glu Asn Trp Asp Ala Trp Tyr Lyas Gly Avg Asn Val Val Thr Val Ser Asp Hiss Gin Lau 95 Phe Lys Ser Asp His Val Leu Leu Pro Pro Pro Giy Lau Ser Pro Ser Tyr Ile Phe Gln Lys Val Glu Glu Ser Lys Lau Lyys Pro Asn Thr Arg Tyr Ile Val Ser Gly Phe Ile Ala His Gly Lys Asp Lau Gin Ile Val Val Ser Arg Tyr G1y Gin Glu Val Gin Lys Val Val Gin Val Pro Tyr Gly Glu Ala Phe Pro Lau Thr Ser Asn Gly Pro Val Cys Cvs Pro Pro Arg Ser Thr Ser Asn Gly Thr Lou Gly Asp Pro His Phe Phe Ser Tyr Ser Ile Asp Val Gly Ala Lou Asp Lou Gln Ala Asn Pro Gly Ile Glu Phe Gly Lou Arg Ile Val Asn Pro Thr Gly Met Ala Arg Val Ser Asn Lou Glu Ile Arg Glu Asp Arg Pro Lou Ala Ala Asn Glu Ile Ar Gln Val Gln Arg V140 Ala Arg Asn Trp AArrg5Thr Glu Tyr Glu L150G1u Arg Ala Glu Val Thr Ser Lou Ile Gin Pro Val Ile Asn Ara Ile Asn Gly Lou TTyyr Glu Asn Gly Asn Trp Asa Gly Ser Ile Arg Ser Asp Ile Ser Tyr Gln Asn Ile Asp Ala Ile Val Lou Pro Thr Lou Pro Lys Lou Arg His Trp Phe Met Ser Asp Arg Phe Ser Glu Gln Gly Asp Ile Met Ala Lys Phe Gln Gly Ala Lou Asn Arg Ala Tyr Ala Gin, Leu Glu Gin Ser Thr Lou Lou His Asn Gly His Phe Thr Lys Asp Ala Ala Asn Trp Thr Ile Glu Gly Asp Ala His Gin Ile Thr Lou Glu Asp Gly Arg Arg Val Lou Arg Lou Pro Asp Trrpp Ser Ser Ser Val Ser Gin Met lie Gin Ile Glu Asn Phe Asn Pro Asp Lys Glu Tyr Asn Lou Val Phe His G1y Gin Gly Glu Gly Thr Val Thr Lou Glu His Gly Glu Glu Thr Lys Tyr Ile Glu Thr His Thr His His Phe Ala Asn Phe Thr Thr Sex Gln Arg Gln Gly Leu Thr Phe Glu Ser Asn Lys Val Thr Val Thr Ile Ser Ser Glu Asp Gly Glu Phe Lou Val Asp,Asn Ile Ala Lou Val Glu Ala Pro Lou Pro Thr Asp Asp G1n-Asn Ser Glu Gly Asn Thr Ala Ser Ser Thr Asn Ser Asp Thr S380 Met Asn Asn Asn Gins (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 3867 base pairs B TYPE: nucleic acid C STRANDEDNESS: double D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
M A ORGANISM: Bacillus thuringiensis B STRAIN: PS17 C INDIVIDUAL ISOLATE: PS17b (vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC1628) NRRL B-18652 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:

GCGAAGCGCT TAAGCAAGGC GCGTAATCTC CTGGTAGGAG GCAATTTTGA TAACCATGAT. 2640 (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
JA LENGTH: 1289 amino acids B TYPE: amino acid C STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE-JAI ORGANISM: BACILLUS THURINGIENSIS
B STRAIN: PS17 CINDIVIDUAL ISOLATE: PSI7b (Vii) IMMEDIATE SOURCE:
(B)-CLONE: E. coli NM522(pMYC1628) NRRL B-18652 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Ala Ile Leu Aan Glu Leu Tyr Pro Ser Val Pro Tyr Asn Val Leu Ala Tyr Thr Prro Pro Ser Phe Leu 2ro Asp Ala Gly Thr Gan Ala Thr Pro Ala 3sp Leu Thr Ala Tyr l40 U GGin Leu Len Lys 45n Leu Gin Lys Gly Sae Aen Ala Gly Thr Tyr Ser Lys Ala Ile Ala Asp Val Len Lye Ely Ile Phe Ile ASP ASP Thr Ile Asn Tyr 75n Thr Tyr Val Asn Sae Gly Leu Ser Leu Ile Thr Leu Ala Val Pro Glu Ile Gly Ile Phe Thr Pro Phe Ile Gly Leu Phe Phe Ala Ala Len Asn Lys His sp Ala Pro 105 Pro Pro Pro Asn Ala Lys Asp 1iee Phe Glu Ala Met Lye Pro Ala Ile Gln Glu Met Ile Asp Arg Thr Leu Thr Ala Asp Giu Gln Thr Phe Leu Aen Gly Glu Ile Ser Gly Len Gin Asn Leu Ala Ala Arg Tyr Gin Ser Thr Met Asp Asp lie Gin Ser His Gly lly Phe Asn Lys Val AAsp Ser Gly Leu Ile lye Lys Phe Thr Asp G1 85 Val Leu Ser Len lean Ser Phe Tyr Thr 1sp Arg Leu Pro Val Phe Ile Thr Asp Asn 205Thr Ala Asp Arg Thr Lelu0 Leu Gly Leu Pro T
22yr Tyr Ala Ile Leu Alaa Ser Met His Leu Met Len Leu Arg Asp Ile 1111e Thr Lys Gly Pro Thr Trp Asp Ser Lys Ile Asn Phe Thr Pro Asp Ala Ile Asp Ser Phe Lys Thr Asp lie Lys Asn Asn Ile Lye Leu TyrSer Lys 26 Thr Ile Tyr Asp Val 2Phe 7e Gin Lys Gly Leu Ala Ser Tyr ply Thr Pro Ser Asp Leu Glu Ser Phe Ala Lys Lys Gin Lys Tyr Ile Giu lie e Met Thr Thr His 300 Leu Asp Phe Ala Arqq Len Phe Pro Thr Phe Asp Pro Asp Len Tyr Pro Thr Gly Ser Giy Asp Ile Ser Leu Gin Lye Thr Arg Arg lie Len Ser Pro Phe lie Pro 33 335 Ile Arg Thr Ala Asp Gly Len Thr LLeu5 Asn Asn Thr Ser lie Asp Thr 3 35 Ser ken 3rp Pro Asn Tyr Glu sn Gly Asn Gly Ala P65 Pro Asn Pro Lys Glu Arg Ile Leu Lys Gin Phe Lys Leu Tyr Prroo Ser Trp Arg Ala 370 5 Ala Gln Tyr Gly Gly Leu Leu Gin Pro Tyr Leu Trp Ala Ile Glu Val Gln Asp Ser Val 405 Thr Arg Len Tyr 4ly Gin Len Pro Ala 41V 5 Asp Pro Gin Ala 42y Pro'Asn Tyr Val Ser Ile Asp Ser Ser Asn Pro Ile 425 430 Ile Gln 4iee Asn Met Asp Thr 4Trp Lys Thr Pro Pro Gin G1y Ala Ser Gly Trp ken Thr Asn Leu M4 t Arg Gly Ser Val 460 Gly Leu Ser Phe 4~b 55 Leu Gln Arg Asp Gly Thr Arg Leu Ser Ala Giy Met Gly Gly Gly Phe Ala Asp Thr Ile Tyr Ser Leu Pro Ala Thr His Tyr Len Ser Tyr Len Tyr Gly Thr Pro Tyr Gin Thr Ser Asp Asn Tyr Ser Gly His Val Gly Ala Leu Val Gly Val Ser Thr 5Pro 20 Gln Glu Ala Thr L25 Pro Asn Ile Ile Gay Gin Pro Asp Glu Gin Gly Asn Val Ser Thr Met Gly Phe Pro 55 540 Phe Gin Lys Ala Ser Tyr Giy G1y Thr Val Val Lys Glu Trp Leu Asn Gly Ala Asn Ala Met Lys Len Ser Pro Giy Gin Ser Ile Gly lie Pro Ile Thr Asn Vaal Thr Ser Gly Glu Tyr Gin Ile Arg Cys Ar0 Tyr Ala Ser Asn Assp Asn Thr Asn Val Phe Phe Asn Val Asp T605 Gly Gly Ala 600 Asn Pro Ile Phe Gln Gln lie Asn Phe Ala Ser T6hrr Val Asp Asn Asn Thr Gly Val Gln Gly Ala Asn Gly Val Tyr Val Val Lys Ser Ile Ala Thr Thr Asp Asn Ser Phe Thr Val Lys Glee Pro Ala Lys Thr lie Asn 645 Val His Lau Thr Asn Gln Gly Ser Ser Asp Val Phe Lau AAsp Arg Ile Glu Phe Val Pro Ile Lau Gin Ser Asn Thr Val Thr Ile Phe Asn Asn Ser Tyr Thr Thr Gly Ser Ala Asn Leu Ile Pro Ala Ile Ala Pro Leu Trp Ser Thr Ser Ser 710 Lys Ala Lau Thr GGly Ser Met Ser Ile 720 705 Gly Arg Thr Thr P
Lys 725 73 ro Asn Ser Asp Asp AAla Lau Lau Arg Phe 7Phe 35 Thr Asn Tyr AAssp Thr Gin Thr Ile Pro Ile Pro Gly Ser 7Gly 50 Lys Asp Phe Thr Asn T Dhr Lau Glu Ile Gin Asp Ile Val Ser Ile Asp Ile Phe Val 77y Ser Gly Lau His yly Ser Asp Gly Ser 1810 Lys Lau Asp Phe Thr Asn Asn Asn Ser Gly Ser Gly Gly Ser Pro Lys Ser Phe Thr Gin Gin Asn Asp Lau G05 Asn Ile Thr Thr G10n Val Asn Ala Leu Phe Thr Ser Asn Thr Gin Asp Ala Lau Ala Thr Asp Val Ser Asp His Asp Ile Gin Gin VVaallVal Lau Lys Val Asp Ala Lau Ser Asp Gin Val Phe Gly Lys GG5u0 Lys Lys Thr Leu 8 g Lys Phe Val Asn G16nn Ala Lys Arg Leu Ser Lys Ala Arg Asn Lau Leu Val Gly Gly Asn Phe Asp Asn Leu Asp 865 870 875 880 ..- ' Ala Trp Tyr Arg G11 Arg Asn Val Val ken Val Ser Asn His G] Lau Sal 895 Lau Lye Ser 9 p His Val Lau Lau PPrro Pro Pro Gly Lau Ser Pro Ser Tyr Ile PPhe Gin Lys ValGlu Gin Ser Lys Lau Lys 9 g Asn Thr Arg 91 920 Tyr TT3 Val Ser Gly Phe IIle Ala His Ala Thr As Lau Glu Ile Val Val Ser ArgTyr Gly Gin Gin Ile Lys Lys Val VaD0l Gin Val Pro Tyr Gly Glu Ala Phe Pro Lau Thr Ser Ser G7lg Pro Val Cys Cys lie Pro 975 Asn Pro His Phe PPhhe Ser Tyr His Ser Thr SSerr Asn Gly Thr Leu Gly Ser Ile Asp Val Gly Ala Lau Asp Val Asp Thr Asn Pro Gly Ile Glu Phe Gly Lau Arg Ile Val Asn Pro Thr Gly Met Ala Arg Val Ser Asn Lau Glu Ile Arg Gin Asp Arg Pro Lau Ala Ala Asn Glu Ile Arg Gln Val Gin Arg Val Ala Arg Asn Trp Arg Thr Glu Tyr Glu Lys Gin Arg Ala Glu Val Thr Ser Leu Ile Gln Pro Val Ile Asn Arg Ile Asn Gly Leu Tyr Asp Asn Gly Asn Trp Asn Gly Ser Ile Arg Ser Asp Ile Ser Tyr Gln Asn Ile Asp Ala Ile Val Leu Pro Thr Leu Pro Lys Leu Arg His Trp Phe Met Ser Asp Arg Phe Ser Glu Gln Gly Asp Ile Met Ala Lys Phe Gln Gly Ala Leu Asn Arg Ala Tyr Ala Gln Leu Glu Gln Asn Thr Leu Leu His Asn Gly His Phe Thr Lys Asp Ala Ala Asn Trp Thr Val Glu Gly Asp Ala His Gln Val Val Leu Glu Asp Gly Lys Arg Val Leu Arg Leu Pro Asp Trp Ser Ser Ser Val Ser Gin Thr Ile Glu Ile Glu Asn Phe Asp Pro Asp Lys Glu Tyr Gin Leu Val Phe His Gly Gin Gly Glu Gly Thr Val Thr Leu Glu His G1 Glu Glu Thr Lys Tyr Ile Glu Thr His Thr His His Phe Ala Asn Phe Thr Thr Ser Gln Arg Gin Gly Lou Thr Phe Glu Ser Asn Lys Val Thr Val Thr Ile Ser Ser Giu Asp Glyy Glu Phe Leu Val Asp Asn Ile Ala Leu Val Glu Ala Pro Leu Pro Thr Asp Asp Gln Asn Ser Glu Gly Asn Thr Ala Ser Ser Thr Asn Ser Asp Thr Ser Met Asn Asn Asn Gln (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS
LENGTH: 37?1 base pairs B TYPE: nucleic acid C STRANDEDNESS: double D TOPOLOGY: linear . f~
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
SA ORGANISM: Bacillus thuringiensis !C) INDIVIDUAL ISOLATE: 33F2 (vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC2316) B-18785 (ix) FEATURE:
JA) NAME/KEY: misc feature Bj)}) LOCATION: 4..24 D OTHER INFORMATION: /function= "oligonucleotide hybridization probe-/,Product= "GCA/T ACA/T TTA AAT GAA GTA/T TAT"
/standard name= "probe an /note= "P?obe A"
(ix) FEATURE :
A NAME/KEY: misc feature B LOCATION: 13..33 D OTHER INFORMATION: /function= "oligonucleotide hybridization probe"
/product= "AAT GAA GTA/T TAT CCA/T GTA/T AAT"
/standard name= "Probe B"
/label= probe-b /note= "Probe b"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:

CAGCAAACAT TAATATTCGA ATTTCATGCT TCAAAAACAG CTCAATATAC CATTCTAAAA 1620;4 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
I LENGTH: 1257 amino acids B TYPE: amino acid C STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
V
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thurin iensis (C)INDIVIDUALISOLATE: PS33F2 (vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC2316) B-18785 (ix) FEATURE:
M NAME/KEY: Protein LOCATION: 1..1257 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Ala Thr Leu Asn Glu Val Tyr Pro Val Asn Tyr Asn Val Leu Ser Ser Asp Ala Phhe Gin Gin Leu Asp Thr Thr Gly Phe Lys Ser Lys Tyr Asp Glu Net Ile Lys Ala Phe Glu Lys Lys Trp Lys Lys Gly Ala Lys Gly Lps Asp Leu Leu Asp Val Ala Trp Thr Tyr sole Thr Thr Gly Glu lie Asp Pro Leu Asn 7aal Ile Lys Gly Val 7eeu Ser Val Leu Thr Leu 65 Ile Pro Glu Val Gly Thr Val Ala Ser Ala Ala Ser Thr Ile Val Ser Phe Ile Trp Pro Lys Ile Phe Gly Asp Lys Pro Asn Ala Lye Asn Ile Phe Glu Glu Leu Lys Pro Gln Ile Glu Ala Leu Ile Gln Gln Asp Ile Thr Asn Tyr Gin Asp Ala Ile Asn Gin Lys Lys Phe Asp Ser Leu Gln LLye Thr Ile Asn Len Tyrr Thr Val Ala Ile Asp Asn Asn Asp Tyr Val Thr Ala Lys Thr Gin Leu Glu Asn Leu Asn Ser Ile Leu Thr Ser Asp Ile Ser Ile Phe Ile Pro Glu Gly Tyr Glu Thr Gly Gly Leu Pro Tyr Tyr Ala Met Val Ala Asn Ala Hiss Ile Leu Leu Leu 205 Asp Ala Ile Val Asn Ala Glu Lys Len G1y Phe Ser Asp Lys Gin Val Asp Thr His Lys Lys Tyr Ile Lys Met Thrr Ile His Asn His Thr Glu Ala Val Ile Lys Ala Phe Leu Aen Gly Leu Asp Lys Phe Lys Ser Leu Asp Val Asn Ser Tyr Asn Lye Lys Ala Asn Tyr lie e Lys Gly Met Thr GG in Met Val Leu Asp Leu Val Ala Leu TrpPro Thr Phe Asp Pro Asp His Tyr Gin Lys Gin Val Glu Ile Glu PPhhe Thr Arg Thr Ile Seer Ser Pro Ile Tyr Gln Pro Val Pro Lys Asn Met Gin Asn Thr Ser Ser Ser Ile Val Pro '305 310 315 320 Ser Asp Leu Phe His Tyr Gin Gly Asp Leeuu Val Lys Leu Glu Phe Ser 3 335 Thr Arg Thr 3sp Asn Asp GlyLeu 3A4aa Lys Ile Phe Thr 359 Ile Arg Asn Thr 35e Tyr Lys Ser Pro Aen Thr His Glu Thr Tyr His Val Asp 360 365 Phe 3err Tyr Asn Thr Gin Ser Ser Gly Asn Ile Seer Arg Gly Ser Ser Asn Pro Ile Pro Ile Asp Lou Asn Asn Pro Ile Ile"Ser Thr Cys Ile Arg Asn Ser Phe Tyr Lye Ala Ile AlaG41y Ser Ser Val Leu 4aall Asn 405, Phe Lys Asp 4ly Thr Gin Gly Tyr Ala Phe Ala Gin Ala PPrroo Thr Gly Gly Ala 4Trp Asp His Ser Phelie Glu Ser Asp Gly Alaa Pro Glu Gly His Lyee Len Asn Tyr Ile Tyr Thr Ser Pro Gly AAssp Thr Leu Arg Asp Phe Ile Asn; Val Tyr; Thr Len Ile Ser Thr Pro Thar Ile Asn Glu Leu Ser Thr Glu Lys I18e e Lys Gly Phe Pro Ala Glu Lys Gly Tyr lie Lys Asn Gln Gly I0e e Met Lys Tyr Tyr 5G ly Lys Pro Glu Tyr lie e Asn Gly Ala Gin Pro Val Asn Leu Glu Asn Gin Gin Thr Leu Ile Phe Gin Phe His Ala Ser Lys Thr Ala Gln Tyr Thr Ile Arg Ile Arg Tyr Ala Ser Thr Gln Gly Thr Lys Gl Tyr Phe Arg Leu Asp Asn Gin Gin Len Gin Thr Leu, Asn Ile Pro Thr Ser His Asn sly Tyr Val Thr Gly A5e5 Ile Gly Glu Asn Tyr Asp Lau Tyr Thr Ile Gly Ser Tyr Thr Ile Thr Gin Gly Asn His Thr Lau Gln Ile Gln His Asn Asp Lys Asn Gly Met Val Lau AAsp Arg Ile Glu Phe Val Pro Lys Asp Ser LLauu Gln Asp Ser Pro Gln Asp Ser Pro Pro Glu Val His Glu Ser Thr Ile Ile Phe Asp Lys Ser Ser Pro Thr IIllee Trp Ser Ser Asn L6ye His Ser Tyr Ser His s Ile His Lou Glu GG16y Ser Tyr Thr Ser Gin Gly Ser Tyr Pro His Asn Leu Lau Ile Asn Leu Phe His Pro Thr Asp Pro Asn Arg Asn His Thr Ile His Vaal Asn Asn Gly Asp MMeett Asn Val Asp Tyr Goy Lys Asp Ser Val Ala Asp Gly Lau Asn Phe Asn Lys Ile Thr Ala Thrrr Ile Pro Ser Asp Ala Trp Tyr Ser GGly Thr Ile Thr Ser Met His Lou Phe Asn Asp Asn 72 7 Asn Phe Lys Thr Ile Thr Pro Lys Phe Glu Leu Ser Asn Gin Lau Glu Asn Ile Thr Thr Gin Val Asn.Allaa Leu Phe Ala Ser S65 Ala Gln Asp Thr Lou Ala Ser Asn Val Ser Asp Tyr Trp Ile Glu Gln Val Val Met L s Val Asp Ala Lau Ser Asp Glu Val Phe Gly Lys Glu Lys Lys Ala Lou Arg Lys Lau Val Asn Gin Ala Lys $1g Lau Ser Lys Ile A Asn 819 Lau Lou Ile Gly Gly Asn Phe Asp 825 Lau Val Ala Trp Tyr Met Gly Lys Asp VVall Val Lys Giu Ser Asp His Glu Lou Phe Lye Ser Asp His 83 Val Lou oLen Pro Pro Pro ass Thr Phe His Pro Ser Tyr Ile Phe Gin Lys 86 Val Gin Glu Ser Lys Leu Lys Pro Asn Thr Arg Tyr Thr Ile Ser Giy Phe Ile Ala His GGlly Glu Asp Val Glu Lau Val Val Ser Arg Tvr Gly Gin Glu Ile GGinn Lys Val Met Gin Val Pro Tyr Gin Gin 91aa Lau Pro 9 Lau Thr Ser Glu Ser Asn Ser SSerr Cys Cys Val Pro As 925n Lau Asn Ile Asn Glu ThrLau Ala Asp Pro His Phe Phe Ser Tyr Ser Ile Asp Val Glp Ser Leu Gin Met Glu Ala Asn Pro Gly Ile Glu Phe Giy Leu Arg Ile Val Lys Pro T965 Gly Met Ala Arg 9al Ser Asn Lau Glu lie Arg Glu Asp Arg Prro Leu Thr Ala Lys 9Glu 85 Ile Arg Gin Val Ginn Arg Ala Ala Arg Asp Trp Lys Gin Asn Ty00Glu Gin Gin Arg l r Glu Ile Thr Ala Ile Ile Gin Pro Val Lau Assn Gin Ile Asn Ala Lau Tyr Giu Asn Gin Asp Trp Asn Gly Ser Ile Arg Ser Asn Val Ser Tyr His Asp Lau WO 92/20802 PCl'/US92/04316 Glu Gln Ile Met Leu Pro Thr Len Leu Lys Thr Glu Gin Ile Asn Cys Asn Tyr Asp His Pro Ala Phe Leu Leu Lys Val Tyr His Trp Phe Net Thr Asp 1075I1e Gly Glu His GGly0Thr Ile Leu Ala OgSPhe Gin Glu 10 Ala Leu Asp Arg Ala Tyr Thr Ginn Leu Glu Ser Arg Asn Leu Len His Asn Gly His Phe Thr Thr Asp Thr Ala Asn Trp Thr Ile Glu Gly Asp Ala His His Thr Ile Leu Glu Asp Gly Arg Arg Val Leu Arg Leu Pro Asp Trp Ser Ser Asn Ala Thr Gin Thr Ile Gin Ile Glu Asp Phe Asp Leu Asp Gin Gin Tyr Gin Leu Leu Ile His Ala Lys G1y Lys Gly Ser Ile Thr Leu Gln His Gly Glu Glu Asn Gin Tyr Val Glu Thr His Thr His His Thr Asn Asp Phe Ile Thr Ser Gln Asn Ile Pro Phe Thr Phe Lys Gly Asn Gin Ile Gin Val His Ile Thr Ser Glu Asp Gly Glu Phe Leu Ile Asp His Ile Thr Val Ile Gin Val Ser Lys Thr Asp Thr Asn Thr Asn Ile Ile Glu Asn Ser Pro lie Asn Thr Ser Met Asn Ser Asn Val rgoVal Asp Ile Pro AArrg5Ser Leu (2) INFORMATION FOR SEQ ID NO:7:
(1) SEQUENCE CHARACTERISTICS:
A) LENGTH: 3738 base pairs B TYPE: nucleic acid C STRANDEDNESS: double D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL : NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:
1 A ORGANISM: Bacillus thuringiensis C) INDIVIDUAL ISOLATE: PS86Q3 (Vii) IMMEDIATE SOURCE:
(A) LIBRRARY: Lambdagem (TM) - 11 LIBRARY
(E: 86Q3a (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

SUBSTITUTE SHEET

TTAAAGGGCT`TTCCATTTGA AAAATATGGT TCTGAGTATA ATAATCGGGG TATCTCTCTP 1740 ATACAAATTA CGAATCAAAC CAAACAAAAA TATGAAA".CAC GTTGCCGTTA TGCGAGTAAA 1860 TGTACGCTAT GTTGTCAGGT AGAAAATCAG'CTACCTTCTT TTGTGACACT TACAGATTTA 22W' GGCCGAAATG.TAGTAAACGT ATCTGATCAT GAACTATTTA AGAGTGATCATGTATTATTG 2580 213248 so (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 1245 amino acids B TYPE: amino acid C STRANDEDNESS: single D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(Vi) ORIGINAL SOURCE:
(A) ORGANISM: BACILLUS THURINGIENSIS
(C) INDIVIDUAL ISOLATE: PS86Q3 (vii) IMMEDIATE SOURCE:
((A LIBRARY: LAMBDAGEM (tm) - 11 library B~ CLONE: 86Q3A
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Ala Thr Ile Assn Glu Lou Tyr Pro Val Pro Tyr Asn Val Lou Ala His Pro Ile LLys Gin Val Asp Asp Pro Tyr Ser Trp Ser Assn Leu Lou Lys Gly 15e Gln Gin Gly Trp G40 lu Glu Trp Gly Lys 4Thr 5 Gly Gin Lys Lys Lou Phe Glu Asp His Len Thr Ile Ala Trp Asn Lou Tyr Lys Thr Gly Lys Lou Asp Tyr Phe Ala Lou Thr Lys Ala Ser Ile Ser Lou Ile =~'~
65 .70 75 80 Gly Phe Ile Pro Gly Ala Gin Ala Ala Val Pro Phe Ile Asn Met Phe Val Asp Phe ~aall Trp Pro Lys Lou Pile Gly Ala Asn Thr Glluu Gly Lys 10 Asp Gln Gln Lou Phe Asn Ala Ile Met Asp Ala Val Asn Lys Met Val Asp Asn Lys Phe Lou Ser Tyr Asn Lou Ser Thr Len Asn Lys Thr Ile 130 135 140 ' Gin Gly Lou Gln Gly'Asn Len Gly Lou Phe Gln Asn Ala Ile Gln Val Ala Ile Cys Gln Zly Ser Thr Pro Glu A1rrg Val Asn Phe Asp Ginn Asn 175 Cys Thr Pro Cys Asn Pro Asn Gin Pro Cysss Lys Asp Asp Lou Asp Arg Val Ala Ser Arg Phe Asp Thr Ala Asn Ser Gln Phe Thr Gln His Lou Pro Gin Phe Lys Asn Pro Trp Ser Asp Glu Asn Ser Thr Gin Glu Pile 210 Lys Arg Thr Ser Val Glu Lou Thr Lou Pro Met Tyr Thr Thr Val Ala Thr Lou His Lou Lou Lou Tyr Glu Gly Tyr Ile Gin Phe Met Thr Lys SUBSTITUTE SHEET

Trp Asn Phe His Asn Gin Gin Tyr Leu ken Asn Leu Lys Val Glu Leu Gin Gln Leu Ile His Ser Tyr Ser Glu Thr Val Arg Thr Ser Phe Leu Gln The Leu Pro Thr Leu Asn Asn Arg Ser Lys Ser Ser Val Asn Ala Tyr Asn Arg Tyr Val Ar Asn Met Thr Val Asn Cys Leu Asp Ile Ala Ala Thr Trp Pro Thr Phe Asp Thr His 33nn Tyr His Gin Gly G35 Lys Leu Asp Leu Thr Arg Ile Ile Leu Ser Asp Thr Ala G1y Pro Ile Glu Glu Tyr Thr Thr Gly Asp Lys Thr Ser Gly Pro Glu His Ser Asn Ile Thr 3ro Asn Asn Ile Len AAsp Thr Pro Ser Pro Thr Tyr Gin His Ser 365 Val Ser Val Asp Seerr Ile Val Tyr Ser 39g Lys Glu Leu Gin Gin Leu Asp Ile Ala Thr Tyr Ser Thr Asn Asn Ser Asn Asn Cys His Pro Tyr Gly Leu 4 g LeuSer Tyr Thr AAsp Gly Ser Arg Tyr 4Asp Tyr Sly Asp ken 4 5 Pro Asp Phe Thr 440 Ser Asn Asn Asn Tyr Cys His Asn Ser Tyrr Thr Ala Pro Ile 4Thr Leu Val Asn Ala 46g His Len Tyr Asn 4555 Ala Lys Giy Ser Len Gin Assn Val Glu Ser Len Val l Val Ser Thr Val Asn Gly Gly Ser 48y Ser Cys Ile Cys 4sp Ala Trp Ile Asn TVr Len 49 Arg Pro Pro Gin Thr Ser Lys ken 5Glu 05 Ser Arg Pro Asp Gin Lys Ile Asn Val Len Tyr Pro,Ile Thr Gin Thr Val Asn Lys G2y Thr Gly Sly Asn LLeu0 Gly Val Ile Ser Ala Tyr Val Pro Met GG41uu Leu Vai Pro Glu Asn Val Ile Gly Asp Vai Asn Ala Asp Thr L YS Leu Pro Len Thr-Gin Leu Lys Gly The Pro Phe Glu Lys Tyr GG7y Ser Gin Tyr Asn 57n Arg 56 Gly Ile Ser L80 Val Arg Gin Trp lie Asn Gly Asn Asn laa Val Lys Leu Ser 5Asn 95 Ser Gln Ser Val G60y Ile Gin Ile Thr 60n Gin Thr Lys Gin LLyss Tyr Glu Ile Arg Cys Arg Tyr Ala Ser L a Gly Asp Asn Asn Val Tyr Phe ken Val Asp Leu Ser Glu Asn Pro Phe Arg Asn Ser Ile Ser Phe Gly Ser T64hr Glu Ser Ser Vai Val50 G1y Val Gin Sly Gin Asn Gly Lys Tyr lie Leu Lys Ser Ile Thr Thr Vai Glu Ile Pro o Ala Gly Ser Phe TVr Val His Ile Thr 6Asn 80 Gln Gly Ser Ser Asp Leu Phe Leu Asp Ar Ile Glu The Val Pro Lys Ile Gln Phe Gin Phe Cys Asp Asn Asn Asn Leu His Cys Asp Cys Asn Asn Pro Val Asp Thr Asp Cys Thr The Cys Cys Val Cvs Thr Ser Leu Thr 73p Cys Asp Cys Asn 73n Pro Arg Gly Leu Assp eye Thr Leu Cys Is Gln Val Glu Asn 75 nn Len Pro 74 Ser Phe Val Thr Leu Thr Asp Leu Gin Asn Ile Thr Thr Gin Val Asn Ala Leu Val Ala Ser Ser Glu His Asp Thr Leu Ala Thr Asp Val Ser Asp Tyr Glu Ile Glu Glu Val Val Leu Lys Val Asp Ala Leu Ser Gl 785 790 795 80~
Glu Val Phe Gly Lys Glu Lys Lys Ala Leu Arg Lys Leu Val Asn His Thr Lys Arg LLeen Ser Lys Ala Arg 825 Len Leu Ile Gly Gly Asn The Asp Asn L3e5 Asp Ala Trp Tyr 8Arrg Giy Arg Asn Val Vaal Asn Val Ser 845 Asp His Glu Len The Lys Ser Asp His Val Len Leon Pro Pro Pro Thr ass 8 Leu Tyr Ser Ser Tyr Met Phe Gin Lys Val Glu Glu Ser Lys Leu Lys Ala Aan Thr Arg Tyr Thr Val Ser Gly The Ile Ala His Ala Glu Asp Leu Glu Ile Val Val Ser Arg Tyr GGiy Gln Glu Val Lys Lyes Val Val Gln ValP1o Tyr Gly Glu Ala Pao Pro Len Thr Ser 9 g Gly Ala Ile 95 Cys C
ons Pro Pro Arg Ser Thr Ser Asn Gly Lys Pro Ala Asp Pro His The Phe Ser Tyr Ser Ile Asp Val Gly Thr Lei Asp Val Glu Ala Asn Pro Gly Ile Glu Leu Gly Leu Arg Ile Val Glu Arg Thr Gly Met Ala Arg Val Ser AA8sn Lou Gin Ile Arg Giu Asp Arg Pro Leu LLye Lys Asn Glu Leu Ara lien Val Gin Arg Ala Ala Arg Asn Trp Ara Thr Ala Tyr Asp Gin Gin Arg Ala Gin Val Thr Ala Leu Ile Gin Pro Val Leu Asn Gin Ile Asn Ala Leu Tyr Glu Asn Glu Asp Trp Asn Gly Ala Ile Ara Ser Gly Val Ser THis Asp Len Glu Ala Ile Val Len Pro Thr Len Pro Lys Leu Aen His Trp Phe Met Ser Asp Met Leu Gly Glu Gin Gly Ser Ile Leu Ala Gin Phe Gin Glu Ala Leu Asp Arg Ala Tyr Thr Gin Leu Glu Glu Ser Thr Ile Leu His Asn Gly His Phe Thr Thr Asp Ala Ala Asn Trp Thr Ile Glu Giy Asp Ala His His Ala Ile Leu Glu Asp Gly Arg Arg Val Leu Arg Leu Pro Asp Trp Ser Ser Ser Val Ser Gln Thr Ile Glu Ile Gin Asn Phe Asp Pro Asp Lys Glu Tyr Gin Leu Val The His Ala Gln Gly Glu Gly Thr Val Ser Leu Gln His Gly Glu Gin G1y Gin Tyr Val Giu Thr His Pro His Lys Ser Ala Asn The Thr Thr WO 92/20802 219 43 2 4 8 PCI'/US92/04316 Ser His Arg Gin Gly Val Thr Phe Glu Thr Asn Lys Val Thr Val Glu Ile Thr Ser Glu Asp Gly Glu Phe Leu Val Asp His Ile Ala Leu Val Glu Ala Pro Leu Pro Thr Asp Asp Gln Ser Ser Asp Gly Asn Thr Thr Ser Asn Thr Asn Ser Asn Thr Ser Met Asn Asn Asn Gin (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
JA LENGTH: 2412 base pairs B TYPE: nucleic acid C STRANDEDNESS: double D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
1A1 ORGANISM: Bacillus thuringiensis C; INDIVIDUAL ISOLATE: PS63B
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. soli NM522(pMYCI642) NRRL B-18961 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

CGTTTGGAAG AAGTAATAAT AGATGCAACT TTCGAGAATC ACAAGCCTGT ACTACAAGTA, 540 C:CAGATGACT ATTCGTCTCA GATAAAAATG GAGAAAACAC GCGTGATCTT TTCAGATATG 1020 AATAGTACTG GATATGGAGA AAGTTGCAAT CAATCACTTC CAGGTCAAAA AATACATGCA 1.500 (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 803 amino acids B TYPE: amino acid C STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
A ORGANISM: Bacillus thuringiensis (C) INDIVIDUAL ISOLATE: PS63B
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC1642) NRRL B-18961 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Net Thr Cys Gln Leu Gln Ala Gln Pro Leu Ile Pro Tyr Asn Val Leu AlaGly Val Pro ThrSer Asn Thr Gly Ser Pro Ile Gly 3A sn Ala Gly 25 Asn Gin Phe Asp Gin Phe Glu Gln Thr Val Lys Glu Leu Lys Glu Ala Trp Gin Ala Phe Gin Lye Asn Gly Ser Phe Ser Len Ala Ala Leu Glu LLys G1y Phe Asp Ala Ala Ile Gly Gly Gly 7S 5er Phe Asp Tyr Leu Gly 6 so Leu Val Gin Ala G85y Leu Gly Len Val Gly Thr Len Gly Ala Ala Ile 90 95 Pro Gly Val Ser Val Ala Val Pro Leu Ile Ser Met Leu Val Gly Val Phe Trp Pro Lys Gly Thr Asn Asn Gln Glu Asn Leu Ile Thr Val Ile Asp Lys0 Glu Val Gin Arg 13e Len Asp Glu Lys Len Ser Asp Gin Leu Ile Lys Lys Leu Asn Ala Asp Len Asn Ala Phe Thr Asp Leu Val Thr Arg Leu Glu Glu Vaal Ile Ile Asp Ala T hr Phe Glu Asn His LL ye Pro Val Leu Gin Val Ser Lys Ser Asn Tyr Met Lys Val Asp Ser Ala Tyr Phe Ser Thr Gly Gly Ile Leu Thr Leu Gly Met Ser AsOp Phe Leu Thr 200 2 Asp Thr Tyr Ser Lys Len ZT1r Phe Pro Leu Tyr Val Len Gly Ala Thr Met Lys Leu Ser Ala Iyr r His Ser Tyr Ile Gin Phe Gly Asn Thr T
225 233 235 2rp Leu Asn Lys Val Tyr Asp Leu Ser Ser Asp Glu Gly Lys Thr Met Seer Gin Ala Leu Ala Arg Ala Lys Gin His Met Arg Gin Asp Ile Ala Phe Tyr Thr Ser Gin Ala Leu Asn MMeett Phe Thr Gly Asn Leu Pro Ser Leu Ser Ser Asn Lys Tyr Ala Ile Asn Asp Tyr Asn Val Tyr Thr Arg Ala Met Val Leu Asn Gly Leu Asp Ile Val Ala Thr Trp Pro Thr Leu Tyr Pro Asp Asp Tyr Ser Ser Gin Ile Lys 3eu0 Glu Lys Thr Arg Val Ile Phe Ser Asp Met Val Gly Gin Ser Glu Ser Arg Asp Gly Ser Val Thr 3 345 Ile Lys Asn Ile Phe Asp Asn Thr Asp Ser His Gin His Gly Ser Ile Gly Leu0 Asn Ser Ile Ser Tyr Phe Pro Asp Glu Leu Gin Lys Ala Gin Leu e5 Arg Met Tyr Asp Tv0 Asn His Lys Pro Tyr Cys Thr Asp Cys Phe j9 400 Cys Trp Pro Tyr GGly Val Ile Leu Asn Tyr Asn Lye Asn Thr Phe Arg Tyr Gly Asp Asn Asp Pro Giy Leu Ser GlyAsp Val Gin Leu Pro Ala Pro Met Ser Val Val Asn Ala GGiin Thr Gin Thr Ala G44nn Tyr Thr Asp Gly Glu Asn Ile Trp Thr AAsp Thr Gly Arg Ser Trp Len Cys Thr Leu Ar Gly Tyr Cys Thr Thr As55n Cys Phe Pro.Gly,Arg Gly Cys Tyr Asn 465 470 475 480 = -r Asn Ser ThrGly Tyr Gly Gin Ser Cys sn Gin Ser Leu Pro G4I9K -Gin Lys Ile His Ala Leu Tyr Pro Phe T 505 hr Thr Asn Val 510 Giy Gin Ser Gly LyL e Leu Giy Leu Leu 52Ala 0 Ser His Ile Pro Tyr r Asp Leu Ser Pro,53snn ken Thr Ile Gly Asp Lys Asp Thr Asp Ser Thr Asn Ile Val 5 540 ;Ala Lys Gly Ile Pro ValGlu Lys Gly Tyr Ala Ser Ser Gly Gin Lys Val Gin Ile Ile AArra Glu Trp Ile Asn Gly Ala Asn Val Val G7n Leu 5 Ser Pro Gly Gin Ser Trp Gly Met Ass? Phe Thr Asn Ser SThr Gly Giy Gin Tyr 9Met 5 Val Arg Cys Arg Tyrr Ala Ser Thr Asn Asp Thr Pro Ile Phe Phe Asn Leu Val Tyr 6A 13 Gly Gly Ser Asn PP2o Ile Tyr Asn Gin Met Thr Phe Pro Ala Thr Lys Glu Thr Pro Ala His Asp Ser Val Aspp Asn Lys Ile Leu 6lly Ile Lys Gly Ile 55n Gly Asn Tyr Ser Leu Met 655 WO 92/20802 PCr/US92/04316 Asn Val Lys Asp Ser Val Glu Leu Pro Ser Gly Lys Phe 67ss Val Phe Phe Thr Asn Asn Gly Ser Ser AAl8aa Ile Tyr Leu Asp Arrg Leu Glu Phe 675 Val Pro Leu Asp Gin Pro Ala Ala Pro Thr Gin Ser Thr55 Gln Pro Ile Asn Tyr Pro Ile Thr Ser Arg Leu Pro-His Arg Ser Gly Glu Pro Pro Ala Ile Ile Trp Glu Lys Ser Gly Asn Val Arg Gly Asn Gln Leu Thr Ile Ser Ala G41nn Gly Val Pro Glu 745 Ser Gin Ile Tyr Leon Ser Val

7 Gly Gly As Arg Gln lie Leu Asp Arg Ser Asn Gly ?65 Lys Len Val 70 Asn Tvr Ser Pro Thr Tyr Ser Phe Thr Asn Ile G8 0n Ala Ser Ser Ser 775 Asn Leu Val Asp Ile Thr Ser Gly Thr Ile Thr Gly Gin Val Gin Val Ser Aen Leu (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 8 amino acids B TYPE: amino acid C STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
A1rg Glu Trp Ile. Asn Gly Ala Asn (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE-CHARACTERISTICS:
A LENGTH: 21 bases B TYPE: nucleic acid C STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12 (2)INFORMATION.FOR SEQ ID NO:13 (i.) SEQUENCE CHARACTERISTICS:
A LENGTH: 20 bases B TYPE: nucleic acid C STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi.) SEQUENCE DESCRIPTION: SEQ ID NO:13 (2) INFORMATION FOR SEQ ID NO:14 (i) SEQUENCE CHARACTERISTICS:
A LENGTH: 8 amino acids B TYPE: amino acid C STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

iO3N4V
Pro Thr Phe Asp Pro Asp Leu Tyr (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
IAI LENGTH: 24 bases B TYPE: nucleic acid C STRANDEDNESS: single D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
A) LENGTH: 24 bases B TYPE: nucleic acid C STRANDEDNESS: single D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION SEQ ID NO:16:

(2) INFORMATION FOR SEQ ID NO:17:
(1.) SEQUENCE CHARACTERISTICS:
A) LENGTH: 14 amino acids B) TYPE: amino acid C STRANDEDNESS single D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Ala Ile Leu Asn Glu Leu Tyr Pro Ser Val Pro Tyr Asn Val (2) INFORMATION FOR SEQ ID NO 18 :
(i) SEQUENCE CHARACTERISTICS:
Al LENGTH: 14 amino acids = -''~
B) TYPE: amino acid C j single D) TOPOLOGY linear (ii) MOLECULE TYPE: protein (xi.)" SEQUENCE DESCRIPTION SEQ ID NO:18:
Ala Ile Leu Asn Glu Leu. Tyr Pro Ser Val Pro Tyr Asn Val l 5 10 (2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
Al ENGTH: 16 amino acids B) TYPE: amino acid C j single ID ) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE.DESCRIPTION: SEQ ID NO:19:
Met Ala Thr Ile Asn Glu Leu Tyr Pro Asn Val Pro Tyr Asn Val Leu (2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:'14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single SUSSTiTUTE SHEET

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Gin Leu Gln Ala Gln Pro Leu Ile Pro Tyr Asn Val Leu Ala (2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 10 amino acids B TYPE: amino acid C STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Ala Thr Leu Asn Glu Val Tyr Pro Val Asn (2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
A) LENGTH: 15 amino acids B) TYPE: amino acid C STRANDEDNESS: single D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Val Gin Arg Ile Leu AspGlu Lys Leu Ser Phe Gln Leu Ile Lys (2) INFORMATION FOR SEQ ID NO:23:
(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:23:

(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
JA LENGTH: 17 bases B TYPE: nucleic acid C STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEAUELENGTH: R RISTICS:
s B TYPE: nucleic acid C STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

WO 92/20802 210324 " PCT/US92/04316 (2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 21 bases B TYPE: nucleic acid C STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 21 bases B TYPE: nucleic acid C STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 38 bases B TYPE: nucleic acid C STRANDEDNESS: single D TOPOLOGY: linear (Li) MOLECULE TYPE DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH 37 bases B TYPE: nucleic acid C STRANDEDNESS: single .1 , D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
Al ENGTH: 10 amino acids B) TYPE: amino acid C j single ID ) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Glu Ser Lys Leu Lys Pro Asn Thr Arg Tyr (,2) INFORMATION FOR SEQ, ID NO:31:
(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:31:

(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 9 amino acids B TYPE: amino acid C STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Tyr Ile Asp Lys lie Glu Phe Ile Pro (2) INFORMATION FOR SEQ ID NO:33:
(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:33:

(2) INFORMATION FOR SEQ ID NO:34:
(k) 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:34:

(2) INFORMATION FOR SEQ ID NO:35:

(xi) SEQUENCE DESCRIPTION : SEQ ID NO-.35: TTTAGATCGT MTTGARTTTR TWCC 24 (2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 5 amino acids B TYPE: amino acid C STRANDEDNESS single D TOPOLOGY: linear (ii} MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Ile Thr Ser Glu Asp.

(2) INFORMATION FOR SEQ ID NO:37:
(i) SE - CHARACTERISTICS:
LENGTH: 20 bases B TYPE: nucleic acid C STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 8 amino acids B TYPE: amino acid C STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
feu Asp Arg Ile Glu Phe Val Pro -.4

Claims (17)

CLAIMS:

1. A substantially pure toxin protein which is toxic to hymenopteran pests and which has at least one characteristic selected from the group consisting of:
(a) the amino acid sequence of said toxin has at least 90% identity with the 86Q3(a) protein of SEQ ID NO. 8; and (b) the DNA which codes for said toxin hybridizes with the complement of a polynucleotide that codes for an amino acid sequence which has at least 90% identity with the 86Q3(a) protein of SEQ ID NO. 8 wherein hybridization occurs in a prehybridization solution of 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.1% SDS, and 100 µg/ml denatured salmon sperm DNA, at 15 °C, and wherein hybridization is maintained with a wash of 1x SSC and 0.1% SDS at 68°C.

2. The hymenopteran toxin, according to claim 1, wherein the DNA coding for said toxin hybridizes with the complement of a polynucleotide that codes for all or a hymenopteran-toxic part of toxin 86Q3(a) wherein hybridization occurs in a prehybridization solution of 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.1%
SDS, and 100 µg/ml denatured salmon sperm DNA, at 15 °C, and wherein hybridization is maintained with a wash of 1x SSC and 0.1% SDS at 68°C.

3. The hymenopteran toxin according to claim 1 wherein the DNA coding for said toxin hybridizes with the complement of a polynucleotide that codes for all or a hymenopteran-toxic part of toxin 86Q3(a) wherein hybridization occurs in a prehybridization solution of 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.1%
SDS, and 100 µg/ml denatured salmon sperm DNA, at 15°C, and wherein hybridization is maintained with a wash of 0.2x SSC and 0.1% SDS at 68°C.

4. The hymenopteran toxin, according to claim 1, wherein the amino acid sequence of said toxin has at least 90% identity with the 86Q3(a) protein of SEQ ID
NO. 8.

5. The toxin according to claim 1, wherein said toxin is 86Q3(a).

6. A nucleotide sequence encoding the hymenopteran toxin as defined in claim 1.

7. The nucleotide sequence according to claim 6 which encodes 86Q3(a).

8. A unicellular host comprising a nucleotide sequence which codes for the hymenopteran toxin as defined in Claim 1.

9. The host according to claim 8, wherein said host expresses a toxin wherein the toxin is a hymenopteran-active toxin obtained from a Bacillus thuringiensis isolate which is PS86Q3.

10. The host according to claim 8, which is a Bacillus thuringiensis.

It. The host according to claim 8, wherein said nucleotide sequence is a heterologous sequence which has been transformed into said host.

12. The host according to claim 11, wherein said host is capable of inhabiting the phylloplane or rhizosphere of a plant or is capable of survival in a baited trap.

13. The host according to claim 11, which is transformed with a nucleotide sequence which codes for 86Q3(a).

14. A process for controlling ants, wherein said process comprises contacting said ants with an ant-controlling effective amount of the toxin as defined in claim 1.

15. A formicidal composition comprising a suitable carrier and substantially intact cells which express the toxin as defined in claim 1.

16. The formicidal composition according to claim 15, wherein said cells have been treated to prolong their formicidal activity.

17. A biologically pure culture of Bacillus thuringiensis PS86Q3 (NRRL B-18765).