AU668687B2 - Novel (bacillus thuringiensis) isolates active against hymenopteran pests and genes encoding hymenopteran-active toxins - Google Patents

Description

i=i" IIIPJ~~.IFT~-~ ;Rl~l~ OPI, DATE 30/12/92 APPLN. ID 21847/92 llllll lll AOJ1P DATE 11/02/93 PCT NUMBER PCT/US92/04316 1111111111111 AU9221847 (51) International Patent Classification 5 C12N 15/32, A01N 63/02 C12P 21/02, C12N 1/21 (C12P 1/04, C12R 1:07) (C12N 1/21, C12R 1:19) Y (PCT) (I11) International Publication Number: WO 92/20802 A2 (43) International Publication Date: 26 November 1992 (26.11.92) (21) International Application Number: (22) International Filing Date: PCT/US92/04316 22 May 1992 (22.05.92) Priority data: 703,977 22 May 1991 (22.05.91) US 797,645 25 November 1991 (25.11.91) US 92304228.7 12 May 1992 (12.05.92) EP 3 4 Countries for which the regional or international application wasfiled: GB et al.

(71) Applicant: MYCOGEN CORPORATION [US/US]; 5451 Oberlin Drive, San Diego, CA 92121 (US).

(72) Inventors: PAYNE, Jewel, M. 7984 Hemphill Drive, San Diego, CA 92126 KENNEDY, Keith 2 Landsdale Lane, Racine, WI 53402 RANDALL, John, B. 1729 Centenial Lane, Racine, W1 53406 MEI- ER, Henry 2827 Santa Fe Trail, Racine, WI 53404 UICK, Heidi, J. 139 Greshell Lane, Racine, WI 53406 (US).

(74) Agents: SALIWANCHIK, David, R. et al.; Saliwanchik Saliwanchik, 2421 N.W. 41st Street, Suite A-I, Gainesville, FL 32606 (US).

(81) Designated States: AT (European patent), AU, BE (European patent), BR, CA, CH (European patent), DE (European patent), DK (European patent), ES (European patent), FR (European patent), GB (European patent), GR (European patent), HU, IT (European patent), JP, KR, LU (European patent), MC (European patent), NL (European patent), RU, SE (European patent).

Published Without international search report and to be republished upon receipt of that report.

All" (54) Title: NOVEL BACILLUS THURINGIENSIS ISOLATES ACTIVE AGAINST HYMENOPTERAN PESTS AND GEN- ES ENCODING HYMENOPTERAN-ACTIVE TOXINS (57) 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.

WO 92/20802 PCT/US92/04316 1

DESCRIPTION

NOVEL BACILLUS THURINGIENSIS ISOLATES ACTIVE AGAINST HYMENOPTERAN PESTS AND GENES ENCODING HYMENOPTERAN-ACIVE TOXINS Cross-Reference to a Related Application This is a continuation-in-part of co-pending application Serial No. 07/703,977, filed on May 22, 1991. This is also a continuation-in-part of application Serial No. 07/797,645, filed on November 25, 1991.

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 thuringiensis produces an insect toxin designated as 5-endotoxin. 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. The primary target is insect cells of the gut epithelium, which are rapidly destroyed.

The reported activity spectrum of B.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 Industrial Microbiology 22:61-76; Beegle, (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 B.t. isolate named Bacillus thuringiensis var. tenebrionis, which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle, Leptinotarsa decemlineata, and Agelastica alni. In European Patent Application No. 0 202 739 there is disclosed a novel B.t isolate active against Coleoptera. It is known as B. thuringiensis var. san diego U.S. Patent No. 4,966,765 discloses the coleopteran-active Bacillus thuringiensis isolate B.t. PS86B1.

Ants comprise a large group of insects (family Formicidae) from the taxonomic order, Hymenoptera. They are among the most common house pests. In many situations, ants are a 1 ii WO 92/20802 PCT/US92/04316 2 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, 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 Solcnopsis are capable of producing extremely painful stings to humans.

j It has been estimated that approximately 10,000 stings occur each year (Habermehl, G.G. [1981] Venomous Animals and Their Toxins, Springer-Verlag, NY, 195 The pharaoh ant (Monomorium 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. The presence 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 Pricer, J.L [1908] 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 I ~I WO 92/20802 PCT/US92/04316 3 children and pets. Alternative control methods such as effective biological control agents have not been found (Akre, LD. Hansen, A.L Antonelli [1989] Ext. Bull. Washington State Univ. Coop. Ext. 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 pharaonis, 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 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, S.H. [1972] Lancet 1:425-427).

The difficulty in controlling pharaoh ants may be attributed to their inaccessible nesting 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 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 microenvironmental 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 (IGR) 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 Invention The subject invention concerns novel Bacillus thuringiensis isolates and genes therefrom which encode novel hymenopteran-active proteins. The novel B.t isolates, known herein as Bacillus thuringiensis PS140E2 (B.t PS140E2), Bacillus thuringiensis PS86Q3 (B.t.

PS86Q3) and Bacillus thuringiensis PS211B2 (B.t PS211B2) have been shown to be active against, for example, the pharaoh ant (Monomorium pharaonis). 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.

In rrr 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 6-endotoxins which have formicidal activity. The genes or -ene 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 o1 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 amino 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, immunreactivity, and ability to hybridize with specific probes.

In addition to the teachings of the subject invention which broadly define B.t. 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.

According to the first embodiment of this invention, there is provided a substantially pure toxin protein which is toxic to ants and which has at least one characteristic selected from: the amino acid sequence of said toxin conforms to the following Generic Formula: MOXLUEBYPx 1 XxxxxxXJXX 101 XXBBXXBXXX

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xxXXKxxxxx K*KUPZXXxx ZXOxxxxxxX IGAWPUSER\LIBRR]003 i7:TCWtAR 1 4A wher-ein A is ala, G is gly, M is Met, S is ser, C is cys, H is his, N is asn, T thr, D is asp, I is ile, P is pro, V is val, E is glu, K is lys, Q is gin, W is trp, F is phe, L is leu, R is argY is tyr, K is K or R, E is E or D, L is L or I, B is M, L, I, V, or F, J is K, R, E, or D, O is A or T, U is N or Q, Z is G or S, X is any naturally occurring amino acid, except C, is any naturally occurring amino acid, x is any naturally occurring amino acid, except C, ur complete omission of any amino acids.

the amino acid sequence of said toxin is at least 50% homologous with toxin 86Q3(a) as herein defined; the amino acid sequence of said toxin has an alignment value of at least 100 with toxin 86Q3(a); the DNA which codes for said toxin hybridizes with DNA which codes for all or part of toxin 86Q3(a); the DNA which codes for said toxin hybridizes with a probe selected from the group consisting of SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 34, SEQ ID NO. 33, SEQ ID NO. 31, SEQ ID NO.

27, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 29, and SEQ ID NO. 37; a portion of the nucleotide sequence coding for said toxin can be amplified from total cellular DNA from a Bacillus thuringiensis strain using polymerase chain reaction with a reverse primer selected from SEQ ID NO. 34, SEQ ID NO. 33, SEQ ID NO. 31, SEQ ID NO. 37, or the complements of SEQ ID NO. 12 or SEQ ID NO. 13; and a forward primer selected from SEQ ID NO.

12, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 27, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 335, or SEQ ID NO. 29; or said toxin is immunoreactive with an antibody which immunoreacts with a toxin selected from toxins expressed by PS86Q3, toxins expressed by PS140E2, or toxins expressed by PS211B2, as herein defined.

According to the second embodiment of this invention, there is provided a nucleotide sequence encoding an ant toxin as defined in the first embodiment.

According to the third embodiment of this invention, there is provided a host 30 comprising a nucleotide sequence which codes for an ant toxin as defined in the first embodiment.

According to the fourth embodiment of this invention, there is provided a process for controlling ants, wherein said process comprises contacting said ants with an antcontrolling effective amount of a toxin as defined in the first embodiment.

According to the fifth embodiment of this invention, there is provided a biologically pure culture of Bacillus thuringiensis PS140E2, having the identifying characteristic of activity against hymenopteran pests of NRRL B-18812, or mutants thereof.

According to the sixth embodiment of this invention, there is provided a biologically pure culture of Bacillus thuringiensis PS211B2, having the identifying characteristic of 40 activity against hymenopteran pests of NRRL B-18921, or mutants thereof.

[G:\WPUSER\LIBRR]00317:TCW:IAR 4B According to the seventh embodiment of this invention, there is provided a biologically pure culture of Bacillus thuringiensis PS86Q3, having the identifying characteristic of activity against hymenopteran pests of NRRL B-18765, or mutants thereof.

Brief Description of the Drawings Figure 1 is a photograph of a standard SDS polyacrylamide gel of B.t. PS140E2, and B.t. PS86Q3.

Figure 2 is a photograph of a standard SDS polyacrylamide gel showing alkalisoluble proteins of B.t. PS211B2 compared to a protein standard.

Figures 3-5 are transmission electron micrographs of ultrathin sections of the antactive B.t. strains (Figure 3 is B.t. PS14E2; Figure 4 is B.t. PS86Q3; and Figure 5 is B.t.

PS211B2). Cells were embedded in an epoxy resin and stained with uranyl acetate and lead citrate.

SEQ ID NO. 1 is SEQ ID NO. 2 is SEQ ID NO. 3 is SEQ ID NO. 4 is SEQ ID NO. 5 is SEQ ID NO. 6 is SEQ ID NO. 7 is Brief Description of the Sequences the nucleotide sequence of gene 17a.

the amino acid sequence of protein 17a, the nucleotide sequence of gene 17b.

the amino acid sequence of protein 17b.

the nucleotide sequence of gene 33F2.

the amino acid sequence of protein 33F2.

the nucleotide sequence of gene 86Q3(a).

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i i t e" [o:\wPUSER\LI3RR]003 17.TCNWAR ,WO 92/20802 PCT/US92/04316 SEQ ID NO. 8 is the amino acid 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 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 acid sequence of SEQ ID NO. 11.

SEQ ID NO. 14 is the amino acid sequence of a probe which can be used according to the subject invention.

SEQ ID NO. 15 is DNA coding for the amino acid sequence of SEQ ID NO. 14.

SEQ ID NO. 16 is DNA coding for the amino acid sequence of SEQ ID NO. 14.

SEQ ID NO. 17 is the N-terminal amino acid sequence of 17a.

SEQ ID NO. 18 is the N-terminal amino acid sequence of 17b.

SEQ ID NO. 19 is the N-terminal amino acid 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 oligonucleotide 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 oligonucleotide probe 33F2B.

SEQ ID NO. 28 is a reverse primer used according to the subject invention.

SEQ ID NO. 29 is an oligonucleotide 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 oligonucleolide which codes for the amino acid sequence of SEQ ID NO. 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 HOfte and Whiteley., WO 92/20802 PC/US92/04316 6 Detailed Disclosure of the IDvention One aspect of the subject invention is the discovery of Bacillus thuringiensis isolates having activity against ants. The novel Bacillus thuringionsis isolates of the subject invention have the following characteristics in their biologically pure form: Characteristics of B.t. PS140E2 Colony morphology-large colony, dull surface, typical B.t.

Vegetative cell morphology--typical B.t Culture methods-typical for B.t.

Inclusions--an elliptical coated inclusion outside the exosporium, and a long inclusion inside the exosporium Approximate molecular weight of alkali/SDS-soluble polypeptides (kDa)--78, Serotype--6, entomocidus.

Characteristics of B.t. PS86Q3 Colony morphology--large colony, dull surface, typical B.t.

Vegetative cell morphology--typical B.t.

Culture methods--typical for B.t.

Inclusions--long amorphic inclusion and a small inclusion, both of which remain with the spore after lysis Approximate molecular weight of akall/SDS-soluble polypeptides (kDa)--155, 135, 98, 62, 58 Serotype--new serotype (not H-1 through H-27) Characteristics of B.t. PS211B2 Colony morphology--large colony, dull surface, typical B.t.

Vegetative cell morphology--typical B.t.

Culture methods--typical for B.t.

Inclusions--large round amorphic inclusion with coat, and elliptical inclusion Approximate molecular weight of alkalilSDS-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 231402), B.

thuringiensis PS86Q3 (B.t PS86Q3), B. thuringlensis PS211iB2 PS211B2), B. thuringiensis var.

san diego and B. thuringiensis var. kurstaki (HD-1) is shown in Table 1, WO 92/20802 PCr/US92/04316 7 Table 1. Comparison of At. PS140E32, B.t. PS86Q3, B.t. PS211B2, Rts.d, and B.t. HD-1 PS14OE2 Atj. PS86Q3 BRt PS211B2 B.t. ND-i Bats.d Inclusions: Ellipse and 2 1 long and 1 Large Bipyramid Flat square small or 2 small amorphic inclusions inclusions Approxcimate 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 69,000J 43,000 40,000 36,000 35,000 34,000 Host range Hymenoptera 1Hymenoptera Hymenoptera Lepidopteran Coleoptera (Colorado -and Coleopteran [Potato Beetle In addition to the ant-active BR. isolates described herein, the subject invention concerns a vast array of .BLt 6-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 130,000 kDa. 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 sequenc motif that is the ultimate determinant of structure and function.

1 MOXLUEBYPx BXYUBLXxxx xxxxXXXXXX XXXXXBXXxX EXXXKXXXKX Xx;xxXJXX XXBX)XXXXX XXLXXXXXXX XXLZBLZBxB VXXXXXXXXX 101 XXBBXXBXXX XXXXXXXXKX xxLBXXBXXX B:XXBBXXXBX XXXXXXXUXX BX-ZIUXXXXX XXXOBXXXX* XxXXXXXXXX xxxxxxxxxX XX*XXXXXXX 201 xxxxxXXUZX XOXXLXXBxx xxxxxxxXXE XXXXXxxxXL PXYOXBOXXH LBLXJXXLxx xxxxxXKXXB XXJXxBXXXK XXLXXXLXXX XLOBXXXBXX 301 XLXXXxXXXJ xXZXXXXXXY BJXBOXX*JE BXXXXPOBEX XXYXXxxxxx XYL-XXOKXLXZ XxxxxxXXXX BXXXXXZXX ZXXXXXXxXX XXXBXXXXXX WO 92/20802 WO 9220802PCU/US92/0431 6 401 XXXXBXXXXX xxxkXXXXXX LXXXXXXXXX XXX*xXXXXX XXXXXXXXXX XXXUX*XXXX XXPLXXX*XJ XxXXXXXXXXX 501 XXZXXxxxxx xx*X*XXXXX XXXXXXXxx XXXXXXXLXX XXXxXBXxBB ZXXXXXEXXX XXBXZXXXXX XXBXXXXBXx 601 XxxxxxxxxE XLUZXUXBXL XXXUXBXBXB XXXXXXXYXL XXXBXBEXXX AUXBXXXXXX XZXXXXXXZX XXXXXXYXBX 701 xXLXxxxxxx xxxX.UXXXXB BLEKLEBBPX X Numbering is for convenienco and approximate location only.

Symbols used: A =ala G =gly M =met S =ser C =cys H =his asn T =thr D =asp I He P =pro V =val E gu K lys Q =gln W =trp F =phe L=Ieu R =arg Y =tyr

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K =K or R E E or D L L or I B L, I, V, or F J K, R, E, or D 0 A or T U N or Q Z G or S X =any naturally occurring amino acid, except any naturally occurring amino acid.

x any naturally occurring amino acid, except acids).

C (or complete omission of any amino Where a stretch of wild-card amino acids are encountered 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 sh-ailar biological activity of 86Q3(a). These equivalent toxins yill have amino acid homology with 86Q3(a). This amino acid homology wifll 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

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WO 92/20802 WO 9220802PCT/US92/043J 6 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] Agic.

IBioL Chem. 52:873-878), lepidopteran and dipteran (CryIIA, CryBI of Widner and Whiteley [1989] I. BacterioL 171:965-974), and lepidopteran (CryIA(c); Adang et aL [1981] Gene 36:289-300) proteins.

Table 3 shows which amino acids were compared from the proteins of interest.

Table 3 Protein Amino acids compared 86Q3(a) 1-697 63B 1-692 33F'2 1-618 17a 1-677 17b 1-678 CrylV 1-633 CryllA 1-633 CryIA(c) 1-609 M_ WO 92/20802 PCTI/US92/04316 11 Table 4 shows the scores prior to adjustment for random sequence scores.

Table 4 Tal 117b 117 I cr Icyi~ )I ::SSI:i f 86Q3(a) 63B 133F2 17b 17a CryIVA CryIIA CryIA(c) 86Q3(a) 1046 389 310 342 340 236 237 238 63B 1038 274 339 338 235 228 232 33F2 927 323 322 '251 232 251 17b 1017 1007 238 240z 236 17a 1016 240 240 237 CryIVA 245 325 0 CryILkA 950 244 914 Note that ant-active protein 86Q3(a) is more closely related to 63B, 17a, 17b, and 33F2 than it is to the CryIVA, CrylIA, and CryIA(c) toxins.

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 sequeaces.

I jQ3a Table 5 la l 86Q3(a) 63B 33F2 17b 17a CryIVA Cry flA [CryIA(c) 86Q3(a) 841 184 118 136 135 41 40 .0 63B 831 81 133 130 40 33 43 33F2 740 130 128 65 50 71 17b 811 798 42 44 47 17a 808 43 44 44 CryIVA 761 54 141 CryIIA CryIA(c) ILL_ 729 Note that in Table 5 the same relationships hold as in Table 4, 86Q3(a)'s highest score, aside from itself, is with 63B.

This degree of relatedness provides thc basis for using common or similar sequence 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 fonrmicidal activity and have an alignment value (according to the procedures of Table 5) greater WO 92/20802 PCT/US92/04316 12 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 The toxins of the subject invention can also be characterized in terms of the shape and location of toxin inclusions.

Inclusion type PS86Q3--Long amorphic inclusion and a small inclusion, both of which remain with the spore after lysis. See Figure 3.

PS140E2--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 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 can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Also, genes which code for active fragments may be obtained using a variety of other restriction enzymes. Proteases may be used to directly obtain active fragments of these toxins.

Equivalent toxins and/or genes encoding these equivalent toxins can also be located from B.t. 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 B.t. toxins.

These antibodies can then be used to specifically identify equivalent toxins with the characteristic formicidal activity by immunoprecipitation, enzyme linked im:niunoassay (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 knovn in the art, if the probe molecule and nucleic acid sample hybridize by

I

WO 92/20802 PCT/US92/04316 13 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 32 p, 125, 35S, or the like. A probe labeled with a radioactive isotope can be constructed from a nucleotide sequence complementary to the DNA sample by a conventional nick translation reaction, using a DNase and DNA polymerase. The probe and sample can then be combined in a hybridization buffer solution and held at an appropriate temperature until annealing occurs.

Thereafter, the membrane is washed free of extraneous materials, leaving the sample and bound probe molecules typically detected and quantified by autoradiography and/or liquid scintillation counting.

Non-radioactive labels include, for example, ligands such as biotin or thyroxine, as well as enzymes such as hydrolases or 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: synthesizing chemically or otherwise an artificial sequence which is a mutation, insertion or deletion of the known sequence; 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 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 art. These variants

I

aiego (Bl.ts.a). U.S. Patent No. 4,966,765 discloses the coleopteran-active Bacillus thuringiensis isolate B.t PS86B1.

Ants comprise a large group of insects (family Formicidae) from the taxonomic order, Hymenoptera. They are among the most common house pests. In many situations, ants are a WO 92/20802 PC/US92/04316 14 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 DNA coding for a peptide sequence whose single letter amino acid designation is "REWINGAN" (SEQ ID 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)GIC(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 ID 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, D or Y; position 5, P or T; position 6, D or H; 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)TIT(T or 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)TTI'(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 B.t genes include "GCAATTITAA 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), "AGACTGGATC CATGGCWACW ATWAATGAAT TATAYCC" (SEQ ID NO. 29), "TAACGTGTAT WCGSTITrAA TITWGAYTC" (SEQ ID NO. 31), "TGGAATAAAT TCAATTYKRT CWA" (SEQ ID NO. 33), "AGGAACAAAY TCAAKWCGRT CTA" (SEQ ID NO. 34), and "TCTCCATCTT 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, more than one coding L I WO 92/20802 PCT/US92/04316 nucleotide triplet (codon) can be used for most of the amino acids used to make proteins.

Therefore different nucleotide sequences can code for a particular amino acid. Thus, the amino acid sequences of the B.L toxins and peptides can be prepared by equivalent nucleotide sequences encoding the same amino acid sequence of the protein or peptide. Accordingly, the subject invention includes such equivalent nucleotide sequences. Also, inverse or complement sequences are an aspect of the subject invention and can be readily used by a person skilled in this art. In addition it has been shown that proteins of identified structure and function may be constructed by changing the amino acid sequence if such changes do not alter the protein secondary structure (Kaiser, Kezdy, F.J. [1984] Science 223:249-255). Thus, the subject invention includes mutants of the amino acid sequence depicted herein which do not alter the protein secondary structure, or if the structure is altered, the biological activity is substantially retained. Further, the invention also includes mutants of organisms hosting all or part of a 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.

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 B. thuringiensis isolates designated PS17, PS33P2, PS63B, and PS86Q3.

Subcultures of the E. coli host harboring the toxin genes of the invention were deposited in the permanent collection of the Northern Research Laboratory, Peoria, Illinois, USA. The accession numbers are as follows: U.S. Department of Agriculture, Culture PS140E2 B.t. PS86Q3 B.t. PS211B2 B.t PS17 B. PS33F2 B.t PS63B E. coi NM522(pMYC2316)(33F2) E. coli NM522(pMYC2321) E. coli NM522(pMYC2317) E. coli NM522(pMYC1627)(17a) E. coli NM522(pMYC1628)(17b) E. coil NM522(pMYC1642)(63B) E. coli MR618(pMYC1647)(86Q3) Repository No.

NRRL B-18812 NRRL B-18765 NRRL B-18921 NRRL B-18243 NRRL B-18244 NRRL B-18246 NRRL B-18785 NRRL B-18770 NRRL B-18816 NRRL B-18651 NRRL B-18652 NRRL B-18961 NRRL B-18970 Deposit Date April 23, 1991 February 6, 1991 November 15, 1991 July 28, 1987 July 28, 1987 July 28, 1987 March 15, 1991 February 14, 1991 April 24, 1991 May 11, 1990 May 11, 1990 April 10, 1992 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 under 37 CFR 1.14 and 35 USC 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.

"(I

WO 92/20802 PCT/US92/04316 16 122. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action, 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, 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.

The B.t 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 the B.t. spores and crystals from the fermentation broth by means well known in the art. The recovered B.t. spores and crystals can be fonrulated 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, the B.t. itself may be used, or another suitable host, as described herein, may be transformed with a B.t. gene and used to express toxins. A vegetable oil 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 B.t. 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 chbmical 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 zephiran chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Bouin's fixative and Helly's fixative (See: Humason, Gretchen. L,Animal Tissue Techniques, W.H.

L

601 XxxxxxxxxE XLUZXUXBXL XXXUXBXBXB XXXXXXXYXL K*EUPZXXXX XXXBXBEXXX xUXBXXXXXX XZXXXXXXZx XXXXXXYXBX ZXOxxxxxxX 701 xXLXxxxxxx xxxXUXXXXB BLEKLEBBPX X,

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P4 [G:\WPUSER\LIBRR]00317:TCW:IAR r WO 92/20802 PCT/US92/04316 17 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 gammaradiation 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 B.t 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, 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 B.t. toxin.

Where the B.t toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, it is essential that certain host microbes be used. Microorganism hosts are selected which are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops of interest.

These microorganisms are selected so as to be capable of successfully competing in the particular environment (crop and other insect habitats) with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane (the surface of the plant leaves) and/or the rhizosphere (the soil surrounding plant roots) of a wide variety of important crops. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, genera Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C.

laurentii, Saccharonyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms.

A wide variety of ways are available for introducing the B.t. gene expressing the toxin into the microorganism host under conditions which allow for stable maintenance and expression of L_ According to the sixth embodiment of this invention, there is provided a biologically pure culture of Bacillus thuringiensis PS211B2, having the identifying characteristic of 40 activity against hymenopteran pests of NRRL B-18921, or mutants thereof.

[G:\WPUSER\LIBRR]00317:TCW:IAR WO 92/20802 PCT/US92/04316 18 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 i 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 i be grown up in the laboratory without expression of a toxin, but upon release into the S|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 3' 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 th& 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, 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, siderophores, may be introduced into the host along with the structural gene expressing the toxin. In this manner, the enhanced expression i I I WO 92/20802 PCT/US92/04316 19 of a siderophore may provide for a competitive advantaga 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 sequence of at least 50 basepairs 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.

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 plasmids are available, such as pBR322, pACYC184, RSF1010, pRO1614, and the like. See for example, Olson et at (1982) J. Bacteriol 150:6069; Bagdasarian et aL (1981) Gene 16:237; and U.S. Patent Nos. 4,356,270, 4,362,817, and 4,371,625.

The B.t 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 developmet 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.

1 U zv ur iuJ. a Ua aunul aiu sequence accoramg to te 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 Hofte and Whiteley.

WO 92/20802 PCT/US92/04316 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 which produce substances toxic to higher organisms could be used, where the toxin is unstable or the level of applcation sufficiently low as to avoid any possibility of toxicity to a mammalian host.

As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi.

Illustrative prokaryotes, both Gram-negative and -positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter, Azotobacteraceae and Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the gene into the host, availability of expression systems, efficiency of expression, stability of the pesticide in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.

Host organisms of particular interest include yeast, such as Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.; phylloplane organisms such as Pseudomonas sp., Erwinia sp. and Flavobacterium sp.; or such other organisms as Escherichia, Lactobacillus sp., Bacillus sp., Streptomyces sp., and the like. Specific organisms include Pseudomonas aeruginosa, Pseudomonasfluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis, Escherichia coll, 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 preform, 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.

L

san diego and B. thuringiensis var. kurstala is suwu in L.

WO 92/20802 PCr/US92/04316 21 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 B.t gene. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.

The cells may be formulated in a variety of ways. They may be employed as wettable powder s, 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 wid'Ay 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 wil 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, sprinlding, baits or the like.

Following are examples which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example I Culturing Bt. Isolates of the Invention A subculture of a isolate can be used to inoculate the following medium, a peptone, glucose, salts medium.

Bacto Peptone 7.5 g/l Glucose 1.0 g/l

KH

2

PO

4 3.4 g/l K 2HPO 4 4.35 g/l Salts Solution 5.0 mI/ CaCI 2 Solution 5.0 mli Salts Solution (100 ml) MgSO 4 .7H20 2.46 g MnSO 4

,H

2 0 0.04 g 201 xxxxXXUZX XOXXLXXBxx xxxxxXJXE XXXXXxxxXL PXYOXBOXXH LBLXJXXLxx xxxXXXKXXB XXJXxBXXXK XXLXXXLXX-X XLOBXXXBXX 301 XLXXXxXXXJ xXZXXXXXXY BJXBOXX*LE BXXXXPOBEX XXYXXxxxxx IXXKXILXZ XxxxxxXXXX BXXXXXZXXX ZXXXXXXXXX XXXBXXXXXX WO 92/20802 PCT/US92/0431 6 22 ZnSO 4 '7H 2 0 0.28 g FeSO 4 '7H 2 0 0.40) g CaCI 2 Solution (100 ml) CaCl -2H0 3.66 g pH 7.2 The salts solution and CaCI 2 solution are filter-sterilized and added to the autoclaved and cooked broth at the time of inoculation. Flasks are incubated at 3(rC on a rotary shaker at 200 rpm for 64 hr.

Example 2 Purification of Protein and Amino Acid Sequencing The At. isolates PS8603, 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, 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, T. Staehlelin, K Gordon [19791 Proc.

Nat!. Acad. Sc!. USA 76:4350) and the N-terminal amino acid sequences were determined by the standard Edman reaction with an automated gas-phase sequenator (Hunkapiller, R-M.

Hewick, W.L. Dreyer, and L.E. Hood [1983] Meth. Enzyrnol 91:399), The sequences obtained were: 17a: AILNELYPSVPYNV(SEQIDNO.17) 17b: AILNELYPSVPYNV(SEQ ID NO. 18) 86Q3(a): MATINELYPNVPYNVL(SEQ ID NO. 19) 63B: 33F2:A TL NE V YP VN (SEQ IDNO. 21) In addition, internal amino acid sequence data were derived for 63B. The toxin protein was partially digested with Staphylococcus aurcus V8 protease (Sigma Chem. Co., St. Louis, MO) essentially as described (Cleveland, S.G. Fischer, M.W. Kirschner, U.K. Laernnii [1977] J.

BWo! C/zelL 252:1102). The digested material was blotted onto PVDF membrane and a ca. 28 kDa limit peptidc was selected for N-termninal sequencing as described above. The sequence obtained was: 63B(2) VQRILDE-KLSFQLIK(SEQIDNO.22) From these sequence data oligonucleotide probes were designed by utilizing a codon frequency table assemblcd from available sequence data of other B. toxin genes. The probes were synthesized on an Applied Blosystems, 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 isolates. R.FLP analysis of restricted total cellular DNA using radiolabeled ofigonucleotide probes has elucidated different genes or gene fragments.

nucleotide sequences coding for equivalent toxins) having the same or similar biological activity of 86Q3(a). These equivalent toxins vwill 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 WO 92/20802 PCT/US92/04316 23 Example 3 Cloning of Novel Toxin Genes and Transformation into Escherichia coli Total cellular DNA was prepared by growing the cells B.t PS17 to a low optical density (OD6o o 1.0) and recovering the cells by centrifugation. The cells were protoplasted in TES buffer (30 mM Tris-Cl, 10 mM EDTA, 50 mM NaCI, pH 8.0) containing 20 sucrose and 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 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% Agarose-TAE (50 mM Tris-HCl, 20 mM NaOAc, 2.5 mM EDTA, buffered gel. A Southern blot of the gel was hybridized with a 32 P]-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 (GCAATITTAAATGAATATATCC) (SEQ ID NO. 23). Results showed that the hybridizing EcoRI fragments of PS17 are 5.0 kb, 4.5 kb, 2.7 kb and 1.8 kb in size, presumptively identifying ,t 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 Elutip

T

I ion exchange column (Schleicher and Schuel, Keene NH). The isolated Sau3A fragments were ligated into LambdaGEM-11T (PROMEGA). The packaged phage were plated on KW251 E. coli 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 density.

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-TAI 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 (tc 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 Sall-digested and dephosphorylated pBClac, an E. coli/B.t. 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 Ii WO 92/20802 PCT/US92/04316 24 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 pMYC1628.

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] FEMS MicrobioL Lett 60:211-218) using standard methods for expression in B.t. Briefly, Sall 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 SalIcleaved and dephosphorylated pHT3101. The ligation mixtures were used separately to transform frozen, competent E. coli NM522. Plasmids from each respective recombinant E. coli strain were prepared by alkaline lysis and analyzed by agarose gel electrophoresis. The resulting subclones, pMYC2311 and pMYC2309, 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 B.t. 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 B.t. proteins.

Example 4 Molecular Cloning of a Gene Encoding a Novel Toxin from Bacillus thuringiensis Strain PS63B Example 2 shows the aminoterminal 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 codon frequency table assembled from B.t. 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-INT) was complementary to the inverse of the internal predicted DNA sequence: 63B-INT 5' TTC ATC TAA AAT TCT TTG A/TAC 3' (SEQ ID NO. 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 HindUl fragment, and an approximately 6.4 kbp Spel fragment.

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 WO 92/20802 PCT/US92/04316 Total cellular DNA was prepared from Bacillus thuringiensis 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-HCl, 25 mM EDTA, pH 8.0) and lysozyme at a concentration of 20 mg/mL The protoplasts were ruptured by addition of ten volumes of 0.1 M NaCI, 0.1 M Tris-HCl pH 8.0, and 0.1% SDS. The cellular material was quickly frozen at and thawed to 37"C twice. The supernatant was extracted twice with phenol/chloroform The nucleic acids were precipitated with ethanol. To remove as much RNA as possible from the DNA preparation, RNase at final concentration of 200 pg/ml was added. After incubation at 37 0 C for 1 hour, the solution was extracted once with phenol/chloroform and precipitated with ethanol.

A gene library was constructed from 63B 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 NdeII fragments were ligated into BamHI-digested LambdaGEM-11 (PROMEGA). The packaged phage were plated on E. 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 KW251 cells in liquid culture for preparation of phage for DNA isolation. DNA was isolated by standard procedures (Maniatis, E.F. Fritsch, J. Sambrook [1982] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Preparative amounts of DNA were digested with SalI (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 Sal/-digested, dephosphorylated pHTBlueII (anE. coli/B.t.

shuttle vector comprised of pBlueScript S/K [Stratagene, San Diego, CA] and the replication origin from a resident B.t. plasmid [Lereclus, D. et aL (1989) FEMS MicrobioL Lett. 60:211-218]).

The ligation mix was introduced by transformation into competent E. coli NM522 cells (ATCC 47000) and plated on LB agar containing ampicillin (100 pg/ml), IPTG and XGAL White colonie,, with putative restriction fragment insertions in the (Beta)-galactosidase gene of pHTBlueII, were subjected to standard rapid plasmid purification procedures (Maniatis et aL, supra). Plasmids ere analyzed by Sail digestion and agarose gel electrophoresis. The desired plasmid construct, pMYC1641, contains an approximately 14 kb Sail insert.

For subcloning, preparative amounts of DNA were digested with XbaI and electrophoresed on an agarose gel. The approximately 4.4 kbp band containing the toxin gene was excised from the gel, electroeluted from the gel slice, and purified by ion exchange chromatography as above. This fragment was ligated into XbaI cut pHTBlueII and the resultant plasmid was designated pMYC1642.

i A A further method for identifying the toxins and genes of the subject invention is througn 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 PCT/US92/04316 26 Example 5 Cloning of a Novel Toxin Gene From B.t 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 1.0. Cells were pelleted by centrifugation and resuspended in protoplast buffer mg/ml lysozyme in 0.3 M sucrose, 25 mM Tris-Cl [pH 25 mM EDTA). After incubation at 37C 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 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 pg/ml. After incubation at 37 0 C for 1 hour, the solution was extracted once each with phenol:chloroform and TEsaturated 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, 0.1% SDS, pH=8.0. SDS was added to 1% final concentration to complete lysis. Onehalf 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 3 2 P-labelled oligonucleotide probes 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. 26) Probe 33F2B: 5' AAT GAA GTA/T TAT CCA/T 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: GCAAGCGGCCGCTATGGAATAAATTCAATT 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 EcoRI. 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). TheEcoRI inserts were ligated into EcoRI-digested pHTBlueII (an E. coli/B. thuringiensis shuttle vector comprised of pBluescript S/K [Stratagene] and the replication origin from a resident B.t plasmid (Lereclus, D. et at [1989] FEMS Microbial Let. 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-chloroii r.uuw. SL lIL.IflL lU!U DULLL 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 art. These variants WO 92/20802 PCT/US92/04316 27 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-) B.t. host, HD-1 CryB 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 puriLfed on NaBr gradients (M.A Pfannenstiel et at [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 B.t. Isolate PS86Q3 Total cellular DNA was prepared from Bacillus thuringiensis 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-HCl, 25 mM EDTA, pH 8.0) containing lysozyme at a concentration of 20 mg/ml. The protoplasts were ruptured by addition of ten volumes of 0.1 M NaCI, 0.1% SDS, 0.1 M Tris-Cl, pH 8.0. The cleared lysate was quickly frozen at -70 0 C and thawed to 37*C twice. The supernate was extracted twice with phenol:chloroform The pellet was resuspended in 10 mM Tris-Cl, 1 mM EDTA, pH and RNase was added to a final concentration of 50 pg/ml. After incubation at 37 0 C for one hour, the solution was extracted once with phenol:chloroform and then with TEsaturated chloroform. DNA was precipitated from the aqueous phase by the addition of one-tenth volume of 3M NaOAc and two volumes of ethanol. DNA was pelleted by centrifugation, washed with 70% ethanol, dried, and resuspended in TE.

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.

can be used as the reverse 3' primer. The sequence of this oligonucleotide can be: TAACGTGTAT(A or T)CG(C or G)TITAATIT(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'-TGGAATAAATCAATT(C or T)(T or G)(A or G)TC(T or (SEQ ID NO. 33).

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, more than one coding WO 92/20802 PCT/US92/043 6 28 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 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 32 P 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 kbp hybridizing fragments.

A gene library was constructed from PS86Q3 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 Ndell fragments were ligated into BamHIdigested LambdaGEM-11 (PROMEGA). The packaged phage were plated on E. coli 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 at, supra). Preparative amounts of DNA were digested with SalI (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 SalI-digested, dephosphorylated pHTBlueII (an E. coli/B.t shuttle vector comprised of pBluescript S/K [Stratagene, San Diego, CA]) and the replication origin from a resident B.t plasmid (Lereclus et at [1989], supra). The ligation mix was introduced by transformation into competent E. coli NM522 cells (ATCC 47000) and plated on LB agar containing ampicillin, IPTG, and XGAL.

White colonies, with putative restriction fragment insertions in the (Beta)-galactosidase gene of pHTBlueII, were subjected to standard rapid plasmid purification procedures (Maniatis et at, supra). Plasmid 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-) B.t., HD-1 CryB Aronson, Purdue University) host to yield MR515, a recombinant B.t 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 B.t. Toxin Protein and Gene Product Against Ants Broths were tested for the presence of f-exotoxin by a larval house fly bioassay (Campbell, Dieball, Bracket, J.M. [1987] "Rapid HPLC assay for the fi-exotoxin of 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 under 37 CFR 1.14 and 35 USC I

I

1 WO 92/20802 PCU/US92/04316 Bacillus thuringiensis," J. Agric. Food Chem. 35:156-158). Only isolates which tested free of fexotoxin 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. thuringiensis Isolates to the Pharaoh Ant (Monomorium pharaonis) B.t Isolate Percent Mortality PS140E2 91 PS 86Q3 84 Control 11 PS211B2 I90.0 Control 3.8 Example 8 Activity Against Pharaoh Ants Mint apple jelly containing 10% B.t (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 MR515 100000 40.1 86Q3 100000 29.2 211B2 100000 58.5 MAJ Blank 25.0 Control Blank 14.4 MR515 a recombinant B.t clone of 86Q3(a) gene, 10% in MAJ (Example 6) 86Q3 spray dried powder of B.t PS86Q3, 10% in MAJ 211B2 spray dried power of B.t. PS211B2, 10% in MAJ MAJ Mint apple jelly, Crosse Blackwell Control rearing diet of water, frozen flies, mealworms/honey agar mctuae treatment with aldehydes, such as formaldehyde and glutaralaenyae; anu-mecuves, sucn as zephiran chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Bouin's fixative and Helly's fixative (See: Humason, Gretchen. L,Animal Tissue Techniques, W.H.

II'

WO 92/20802 PCT/US92/04316 Table 8. Three week mortality on pharaoh ant workers.

Sample Rate ppm Percent Mortality 50000 100.0 140E2 86Q3 50000 99.6 211B2 50000 100.0 MAJ Blank 75.3 Control Blank 39.0

I

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 B.t. can be obtained from DNA of the strain by performing the standard polymerase chain reaction procedure as in Example 6 using the oligonucleotides 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 three Nterminal 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. As yet another alternative, the ITSED (SEQ ID NO 37) reverse primer (TCTCCATCTTCTGA(G or A)G(T or A)AAT) (SEQ ID NO. 37) can be used with the Nterminal forward primers (SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 27, and SEQ ID NO.

SUBSTITUTE

SHEET

microorganisms.

A wide variety of ways are available for introducing the B.t. gene expressing the toxin into the microorganism host under conditions which allow for stable maintenance and expression of ,i WO 92/20802 PCT/US92/04316 31 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. 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. coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants. The vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC184, etc.

Accordingly, the sequence coding for the B.t toxin can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli. The E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered.

Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the 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.

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 Alblasserdam, Chapter 5; Fraley et aL, Crit. Rev. Plant Sci 4:1-46; and An et aL (1985) EMBO J. 4:277-287.

Once the inserted DNA has been integrated in the genome, it is relatively stable there and, as a rule, does not come out again. It normally contains a selection marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as kanamycin, G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. The individually employed marker should accordingly permit the selection of transformed cells rather than cells that do not contain the inserted DNA.

A large number of techniques are available for inserting DNA into a plant host cell.

Those techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacteriun rhizogenes as transformation agent, fusion, injection, or electroporation as well as other possible methods. If agrol Aeria 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

L

,r au a a ul wumpeuuve aavantage against other wild-type microorgaisms m m e neiu. rur example, genes expressing metal chelating agents, siderophores, may be introduced into the host along with the structural gene expressing the toxin. In this manner, the enhanced expression WO 92/20802 PCT/US92/04316 32 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. col and in agrobacteria. They comprise a selection marker gene and a linker or polylinker which are framed by the right and left T-DNA border regions. They can be transformed directly into agrobacteria (Holsters et aL [1978] MoL Gen. Genet. 163:181-187).

The agrobacterium used as host cell is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained. The bacterium so transformed is used for the transformation of plant cells. Plant explants can advantageously be cultivated with Agrobacterium rumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant cell. Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension-cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection. The plants so obtained can then be tested for the presence of the inserted DNA No special demands are made of the plasmids in the case of injection and electroporation.

It is possible to use ordinary plasmids, such as, for example, pUC derivatives.

The transformed cells grow inside the plants in the usual manner. They can form germ cells and transmit the transformed trait(s) to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.

Example 11 Cloning of Novel B. thuringiensis Genes Into Insect Viruses A number of viruses are known to infect insects. These viruses include, for example, i 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 B.t toxin genes are well known and readily practiced by those skilled in the art. These procedures are described, for example, in Merryweather et at (Merryweather, AT., U. Weyer, M.P.G. Harris, M. Hirst, T.

Booth, R.D. Possee (1990) J. Gen. ViroL 71:1535-1544) and Martens et aL (Martens, G, Honee, D. Zuidema, J.W.M. van Lent, B. Visser, J.M. Vlak (1990) AppL Environmental MicrobioL 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.

;I

L

ii WO 92/20802 PCT/US92/04316 32 -1 BUDAPEST TeAT2 ON THE INTMW7.?ZONAL RE.COGNIION OV THE DWOSZ? OF MxICc3rAjIzS FOR TILE PUMPSES OF PA?!~4T PROCZ1UH8 fITERHATtomAL ?0W4 RECEIPT IN THlE CASE OP AN~ ORIGINAL DEPOSIT Issued pursuant to Rule 7.1 by the INTERNIATItONAL DEP'OSITARY AUTH~ORITYf identified at the bottom of this page FTEO Jewel Payne Mycogen Ccaeporation 5451 Oberlin Drive San Diego? CA 92121 WJJ4E AMD Afl'RESS OF DEPOSITRo 1. ZrENTrrmCATION Or TH.Z KXCROORCANISM Identification reorence given by the Accession number given by the DEPOSITOR; INTS"ATIONA. DEPOSTARYf AUTHORITY a B3clI.Ius thur~jj lnsi9 P586Q3 NRRL B-18763 11. SCIENTIFIC DZSCRIPTION ANI/OR PROPOSED TAXONOMIC DESIGIIATION The microorganism Identified under I above varn acco "mn1*d by: a scientific description r71a proposed taxonomic designation (Mark iith a cross where applicb..) 111. RECEIPT AMD ACCEPTW~NE This International Depositary Authority accepts the microorganism Id" ntified under I above, which was received by It on Feb.6,1991 (date of the original depoest) IV. RECEIPT OF mEumS FOR CONVERSION IThie microorganism Identiliod under I above was received by this international rDep.sitary Authority on (date of t he o rIi&% deposit) and arequest to convert the original deposit to a deposit Under the Bludapest Treaty asa received by It on (dots of receipt of request tot conversion) V. INTERNATrIONlAL DEPOSITAnY AUTHORITY Hm:AeTicul4tural Research Culture slgnature(s) of person(s) having the power Collection (NRRL) jto repmesent the\International DeposLt.ary International Depositary 4uthorit, Author I of #uthoriated off icial Adrs.Peoria,.,Illinois 61604 U.S.A.

I

Wher Rul 6.4d) pplIes, such data Is tht date on wihich the status of International depositary authority wa se acured. SUBSTITUTE SH E 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.

WO 92/20802 PCT/US92/043),6 RZCOGNXTZOtI Of THE DEPOSIT OF XZCWRC~&AMISM4 FOn THE PURPOSS Or PATfL*? PREDURi~E XNTERNATXINA FORM4 RECEIPT IN T119 CASE OF All ORICIN)AL DEP~OSIT issued pursuant to flule 7.1 by the ZNR.NATZONAI. DEPOSITAAY AUTHORITY Identified at the bottom of this page

MO

Dr. Jewvl Payne Mycogen Corporation 5451. Oberlin Drive San Diego, CA 92121 NAMEa AM ADDRESS OF DEPOSITOR 1. IDVElIPICATIOtl Or THE~ MICROORGANISM identification rolorence given by the Accession nuaibor given by the D EJO3 X MR fINTRWATXONA 4 DI'OS XTMf MUHOrIvY1 Bacillus thuringiensis PS140E2 NRR~L B-18812 11- SCIIPXIc DESCRIPTZOil AND/OR PROOSI TA-XONOIG DXSX(IMTZON The wicroorganiamt identified Under I ab~ove was accowpanied bya a 3Ciantlflo description r-Ia propo *4t b.xonocic designation (Mark with a cross where applicable) III. AECEIPT AIM ACCErFTANZ4C This International Depositary Authorit Accepts the micrroorgani;: Ident ified Under I iibov., which was received by It onApr.23,1991 (date of the originJ I pos t) IV. FUXEIPT Or RIEOREST FOn C0ItznsIOu The Miicroorganism ldentitled under I aibove was received by this international Depositary Authority on (date of the original deposit) and arequaot to convert the original deposit h.o a deposit under the B~udapest Treaty was received by It on (date of receipt of request for conversion) V. INTERATI0tt1i DEYOSIAY, AUTH~ORITY Nae Agricultural Research Culture FSignature(s) of person(s) having the power Collection (HRlUL) jto represent the International Depositary Addreaai 18 N UnlivesIity Street Dtl0 Peoria, Illinois G1604 U.S.A. Where Rule 6.4(d) applies# such date 13 the date on which the status of'Internationat depolitary thority SCJB3STITUTE SHEET Salts Solution (100 ml) MgSO 4 *7H 2 0 MnSO 4

*H

2 0 2.46 g 0.04 g WO 92/20802 rf~r. Jewel1 Payne mycogen Corporation 5451 Oberlin Drive San Diego, CA 92121 t4N AND ADDRESS OF DEPOSITOR PCT/US92/04316 32 -3 fLt!APL!S? T"1A!Y On Till! INTMM4AONAL RECONITXOl OF THR DEPOSIT or mxcnoRRANwS ?Vt THZ PPRJOSES Or PATDI? PnLW3MUR Z)NEZWATZONAL POPJ4 RECEIPT IN THlE CASE 0? AN onrXNfL4L DEPOSIT Issued pursuAnh to Rule 7.1. by the INTERN1ATXOHAL DEPOSITARY AUTHORI1TY Identified at the bottom of this pagq 1. IDWHTIFICATION Or TILE MIC11OORGAI4ISI Identification reference given by the Accession number given by the OrsOS ITOR I I"TW!UTXONAL DM$XSZAflT AUTHORtITY: Bacillus thuringiensis PS211B2 14RRL B-10921 11,* SCIENTIFIC DSCRX'TION AID/Oll PROPOSED~ TAXONOMIC DESIGNATION The muicroorganism identified under I above lias acccmpanied by; a scientific~ description a proposed t~xonoohic designation (Mark with a cross where applicable) I*X RECEIPT AND~ ACCEMANCE This International Depositary Authority accepts the microorganism Identified Under I above, which was received .y it onNDV, 15,1991 (date of the original deposit)l IV. RECEIPT or flEQUEST "OR COk4V!s3oN IThe microorganism Identified under I above wias received by this international Depositary Authority on (date of the original dep-omit) And a request to convert tiha original deposit to a deposit Under the i3udapuut T'rat was received by It on (dsto of roceipt of request for conversion) INTRNfATIONAL DEPOSITARlY AUTHORITY "".Agricultural Research Culture Signature(s) Of Pet3oA(3) having the power Collection (NRRL) to represent the International Depositary Adrs,1815 N. University Street Da to I AdrslPeoria, Illinois 61604 U.S.A.

Where Rule 6.4(d) applies# such date is the date on lihich the status of international dOP0asitar authority was acquired. SUBSTITUTE

SHEET

WO 92/20802 PCT/US92/04316 32 4 BP/A/II/12 page 14 BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL FORM Dr. Jewel Payne Entomology Mycogen Corporation 5457 Oberlin Dr.

San Diego, CA 92121 NAMZ AND ADDRESS L OF DEPOSITOR RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page I. IDENTIFICATION OF THE MICROORGANISM Identification reference given by the Accession number given by the DEPOSITOR: INTERNATIONAL DEPOSITARYX AUTHORITY: Bacillus thuringiensis PS17 NRRL B-18243 II. SCINT=FIC DESCRIPTION AND/OR PROPOSED MT=NOMIC DESIGNATION The microorganism identified under I above was accompanied by: a scientific description a proposed taxonomic designation (Mark with a croas where applicable) III. RECEIPT AND ACCEPTANCE Thil' International Depositary Authority actpts the mierorganism identified under I above, whvbich wa, received by it on July 28 ,1987(date of the original deposit) 1 IV. INTERNATIONAL DEPOSITARI AUTHORITY .,et Agricultural Research Culture Signature(s) of p-erson(s) having the power Collection (NRL) to represent the ternational Depositary International Depostary Authority Authority or of a thorized of icial(s): International Deposigr ourhCrfY 2e Addre.s, 1815 N. University Street Peoria, Illinois 61604 U.S.A. Date: Where Rule 6,4(d) applies, such date is the date on which the status of international depositary authority was acquiredi where a deposit made outside the Budapest Treaty after the acquiLition 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 tne internat.ional depositary authority.

ro= DP/4 (sole page) SUBSTITUTE SHEET ccli cells and plated on LB agar containing amnpicillin, isopropyl-(Beta)-D-thiogalactoside (IPTG) and 5-Bromo-4-Chloro-3-indoyl-(Beta).D-galactoside (XGAL). White colonies, with putative insertions in the (Beta)-galactosidase gene of pBClac, were subjected to standard rapid IWO 92/20802 PCT/US92/04316 32 BP/A/II/12 page 14 BUDAPEST TREATY ON TM INTERNATIONAL RECOGNITION OF THE DEPOSIT OF 2MICP-,ORC.XNXSMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL FORM Dr. Jewel Payne Entom~ology Mycogen Corporation 5457 Obrlin Dr.

San Diego, CA 92121 NAME AND ADDRESS L Or DEPOSITOR RECEIPT IN THE CASE OF AN ORIGINA.L DEPOSIT issued pursuant to Rule 7.1 by the INTRNATI OIAL DEPOSITARY AUTHOR.ITY identified at the bottom of this page I. IEN~TIICATION Or THE MICROORGA.NISM Ident.Lfication r~eterence given by the Accession number given by the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY: -Bacillus thuringiensis ps33F2 NRRL B-1824 4 El1. SCIEFIC DESCRIFTION MID/OR PROPOSED ILXONOMI1C DESIGNATION The microorganism identified under I above was accompanied byi Sa scientific description Sa proposed taxonomic designation (ILark with a cross where appl .czable) III. RECZIPT AC='ANCZ This International Depositary Authority accepts the xmiczvorga.Dism identified under I above, vbir-h Was received by it on July 28 ,1987(date of! rhe original deposit~l TV. n4TERNAIONAL DEPOSITARX AUTHORITY namp: Agricul.tural Research CU-lture SignatureCs of! -merson~s) having the power Collection (NRR.) to represent the international Depositary International Depositary Authority Authority or of uhrzdoficial Adrs:1815 N. tiniversity Street Penoria, Illinois 61604 U.S.A. Date: ftoie Where Rule applies, such date is the date on which the status of international depositary authority was acquired-, where a deposit made outside the Budapest Treaty alter the acquisition ot 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 tne inter-narional depositary authority.

Form BP/4 (sole page) SLUBSTITUTE

SHET

1' plumu. rlyuiLzig uinuJs LuLaiuouU an apprxIXu~Lty KOP Avai JIdgIUDI, UE approximately 2.0 kbp Hindml fragment, and an approximately 6.4 kbp SpeI fragment.

WO 92/20802 PCr/US92/043,16 32 -6 BP/A/II/12 page 14 BDAPEST TREATY~ ON THE INTERNATIONAL RECOCNITION OF THE DEPOSIT OF HIC2ROORGANISMS FOR THE PURPOSES OF PATENT PROCEDUR.E INTERNATIONAL FOR.4 Dr. Jewel Payne Entomoloegy Mycogen Corporation 5457 Oberlin Dr.

San Diego, CA 92121 NAMt AND ADDRESS L OF DEPOSITOR RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page I. IDENTI:FICATION OF T= HICMOORGANISN Ida.ntfication reference given by the Accession number gjiven by the DEPOSITOR: INTERNATIONAL DEPOSITARY A=TORITY: -Bacillus thuringiensis PS63B WRRL B-18246 :ii. scxzN,'TIFic DEscnnTxPoN AND/OR PROPOSED TXONom2c DESIGNTION The micrcorgamism identified under I above was accomanied by: a scientific deacription a proposed taxonamid designiatin (Ha~rk with a cross where appli±cable) IllE. RECMI PT MM ACCETANCE Thi.s International Depositary Authority accempts the micoorganism identified unde~r I above, which wan received by it on July 28 ,1987(date of the original deposit) I IV. ?TMNATXONAL DEPOSITARY AUTHORMT n~e: Agricultural Research Culture Signature of person(s) having-the power Collection (NTM.) to represent the 1I ter ional Depositary Int ernational Depositary Authority Authority or of -a~ria.d officialCs): Ad-as: 1815 N. University Street Peoria, Illinois 61604 U.S.A. Dae 1Where Rule 6.*4 applies, such date is the date on which the status of international depositary authority wex acquired; where a deposit made outside the Budapest Treaty after the acquisition 01 the status of internationa4 depositary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by tr-e international depositary authority.

Fr P4 (sole page) SUBSTITUTE SHEETE-a cnromatograpy as above. 'Ibis tragment was ligated into Xbai cut pk-ibiueu ana tne resuiant plasmid was designated pMYC1642.

,WO 92/20802 PCT/US92/043 16 32 -7 BUDAPEST TREATY ON THE INTERNATIONAL ECOGNITION OF THE DEPOSIT OF KICR( "AISMS FOR THE PUR~POSES OF PATENT PROC MRE INTERN4ATIONAL FORM RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT Issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page A.Lenore Linda R. Nyr Mycogen Corporation 5451 Oberlin Dr.

San Diego, CA 92121 NAME AND ADDRESS OF DEPOSITOR IDMNTIFICATION OF TUE MICROORGANISM identification reference given by the Accession number given by the DEPOSITOR: INTERLNATIONAL DEPOSITARY AUTHORITY: Escherichia coli N1,522/pKYC2316 MR608 NRRL B-18785 11. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION The microorganism identified under I above wasn accompanied bye a scientific description a proposed taxonomic designatioi (Mark with a cross where applicable) III. RECEIPT AND ACCEPTANCE This International Depositary Autppsr{b 1 ccsptsi the microorganism Identified under I above, which was received by it on tar... (date of the original deposit) 1 IV. RECEIPT OF REQUEKST 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 undeV the Bludapest Treaty was received by it on (date of receipt of request for conversion) V. INTERNATIONAL DEPOSITARY AUTHORITY IName: Agricultural Research Culture Signature(s) of psirson(s) having the power *Collection (NRRL) to represent the I-ternational Depositary International Depositary Authoritt, Authori 2 07y.au kbx-ized off Ic al(s): L-dde3:1815 N. University Street Date: e Adra:Peoria, Illinois 61604 Where Rule 6.4(d) applies, such date Is the dateo hc h ttuo nentoa eoia autortyi~s cqird. SUBSTITUTE SHEET k4,~xiu, ui. et a. 11989J FEMS Microbial Lett 60:211-218]). lie ligation mixture was transformed into frozen, competent NM522 cells (ATCC 47000). Transformants were plated on LB agar corntaining ampicillin, isopropyl-(Beta)-D-thiogalactoside (IPTG), and 5-broio-4-chloro- Iv-, WO 92/20802 PCT/US92/0431,6 32 -8 BUDAPEST TRE.ATY ON TflE INTERIMTIOHJ.L RPCOQUTZOM OF THE DEPOSIT 0F MICR-qGMIS4S k~i At't PURPOSES OF PATENT PRC IURE INITERN{ATIONAL FORM F4s. Lenre ind R. Ny~gad RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT Lenore ~issued pursuant to Rule 7.1 by the Mycogen Corporation UMERNATIONAL DEPOSITARY AUTHORITY 5451 Oberlin Dr. identified at the bottom of this page Sa2n Diego, CA 92121 NAM4E AND ADDRESS L. 01F DEPOSITOR 1. II)ENTIFICATION OF THE MICROORGANISM4 Identification reference given by the Accession number given by the DEPOSITOR: yC22 I67 INTERWATIONAL DEPOSITARY AUTHORITY: Escherichia coli NM,522/pM 21 R0 NRRL B-18770 11-* SCIEN!TIFIC DESCIPTION AMD/OR PROPOSED TAXONOMIC DESIGNATION The microorganism Identified under I above varn accompanied bys a scientific description a proposed taxonomic designation (Mark with a cross where appficable) 111. 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 REQUEST FOR CONVERSION The microorganism Identified under I above was received by this International Depositary Authority an (date of the original deposit) and arequest to convert the original deposit to a deposit under th3 Budapest Treaty Waas received by it on (date of receipt of request for conversion) V. INTERNATIONAL DEPOSITARY AUTHORITY Name Aricultural Research Culture signature(s) of person(s) having the power Collection (NRRL) to repr@3*nt the International Depositary International Depositary Authorit:,tho IAddress: 1815 N. University Street Datei t ~Peoria, Illinois 61604 U.S.A.3 9q Where Rule 6.4(d) appl.ies. such date is the date on which the status of International depositary authority was acquired. S B TT T H E Form RP/4 (sole page) S B TT T H E uw wiJ ULI iALS u LLFCULIVO nICIEnuonea Dprimer. libe sequence of the reverse primer can be: "5'-TGGAATAAAjrrCAA'rr(C or 71)(T or G)(A or G)TC(T or (SEQ ID NO. 33).

16 PCr/US92/04316 WO 92/2=02 32 -9 BUDA3'ST TREZATY ON TIME INTERNATIONAL R.coamUTZom or T1~K Dzpos= or xzc~ooacZsxs FOR THE5 FtWPOaS OF PPOCURZ INTERATIONAL FORM 7 Nyjaard 14s. Lenore Lindia R.

tmycogen Corporation 5451 Oberlin Dr.

San Diego, CA 92121 NAM4E AND ADDRESS OF DEPWZTOR RECEIPT IN THlE CASE OF AN ORIGINAL Issued pursuant to Rule 7.1 by the fIERNATIONAL DEP'OSITARY AUTHORITY identified at the bottom of this page I. zN~irzcxxoN or TR9 MICROORGANISM identification reference given by the Accession numba*r given by the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY: Estherichia coli 121522/pMiYC2317 MR609 NRRL B-18816 11, SCITFIC DESCRIPTION AND/OR PROPOSED TAXONOM4IC DESZCNATXOH The microorganism identifiLed under I above was accompaniad by: a scientific description a proposed taxonomic designation (Mark with a cross where applicable) 111. RECE17T AND ACC=P'FARCZ This International Depositary Authority accepts the microorganism identified under I bezss which was received by It on Apr.24,1993. (date of the original depoit)l IV. R=XPTOF RZOOEST I-OR CONVERSION The;, microorganism Identified under I above was received by this International Depositary Authority on (date ot the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty Iwas received by It on (date ofa cito eiee o ovrin V- INTERN~ATIONAL DEPOSITARY AUTHORITY NaHmes Aricultural Research Culture Signature(s) of person(s) having th~e power Adrs:Collection (NR.RL) to represent the International Depositary International Depositary Authorit: Authority or oa uhori dglcial(s), Adez 1815 V. University Street Da: Peorta, Illi.nois G1604 U.S.A.

Where Ruls 6.4(d) applies# such date, is the date On whica the status of International dep ositar: aunoivas acquired. SUBSTITUTE

SHEET

were test~d for the presence of fl-exotoxin by a larval house fly bioassay (Campbell, Dieball, Bracket, J.M. [19871 "Rapid HPLC assay for the P-exotoxin of p WO 92/20802 PCTP/US92/0431,6 32 BP/A/II/12 page 14 BUTDAPEST TREATY ON THE IN~TERNUATIONAL RECOGNITION OF THEt DEPOSIT OF MICROORGCANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTER.NATIONAL FORM~ Ms [7enore Linda R. Nygaard7 Mycogen Corporation 5451 Oberlin Dr.

San %Diego, CA 92121 RECEIPT IN TUE CASE OF AN ORIGIlNAL DEPOSIT issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page.

NA~t AND ADDRESS OF DEPOSITOR I IDENTIrICATION OF THE MICRORGANISM Identitication reference given by the Accession numtmr given by the DEPOSITOR: INlTERNATIONAL DEPOSIEXEY AM~ORITy: Escherichia coli NM522/pMYC1627 Mk398 NRRL B-18651 II SCTEN~rFC DESCRI1'TION AND/OR PROPOSED TAXONOMIC DESIGNATION The microorganism identified under I above was accompanied by: F7 a scientific description jrnv- a proposed taxonomic dsgnto (Mark with a cross where applicable) III RECEIPT ANDl XCCETANCE 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) 1 IV. iTEPNA~jiONAL DEPOSITAR~Y AUTrORITY nas Agricultural Rese arch Culture Signature(s) o! person~s) having the power Collection (NMR) to represent the Irr ernational Depositary International Depositary 4.uchority Authority 03Z At aut rized official(s): Address: 1815 N. University Street a Penria, Ilinhois 61604 U.S.A. Date: 4 1 w~eAR WeeRule 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 Bludapest Treaty after the acquisition Of the status of international depositary authority in converted into a deposit under the Budapest Tra aty, such date is the date on which the microorganism was received by the international depositary-.authority.

Form BP/4 (sole page) SUBSTITUTE

SHEET

WO 92/20802 PCT/US92/04316 32 11 page 14 BUDlAPEST TREATY ON THE !MERNATIONA1 RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES Of PATENT PROCEDURE INTERNATIONAL FORM MSFI-Tenore Linda R.

Mycogen Corporation 5451 Oberlin Dr.

San Diego, CA 92121 Nygaard 7 RECEIPT IN THE CASE OF AN ORIGINAL DE170SIT Issued pursuant to Rule 7.1 by the INTERNATIONA.L DEPOSITARY AUTHORITY identified at the bottom of this page.

NAM AND ADDRESS OF DEPOSITOR I IDENTIFICATION OF THE MICRORGANISM Identification reference given by the IAccession number given by the DEPOSITOR: IINTERNATIONAL DEPOSIVRY AUTHORITY; Escherichia coli. NM522/pXYCI628 M3R99 jNP.RL B-1865 2 II SC=IFIC DESCRIPTON AND/OR PROPOSED TAXONOMIC DESIGNATION The microorganism identified under I above wasn accompanied by: ma scientific description r Va proposed taxonomic designation (M.nxk with a cross where applicable) III RECEIPT AmD ACPTANCE This international Depositary Authority accepts the micoorganism identified under I above, which warn received by it on Itay 11,1990 (date of the original deposit)l IV. nTRNATIONAL DEPOSITARY AUTHORITY namet Agricultural Research Culture Sicgnature of person having the power Collection (NTRRL) to represent the I ternational Depositary Intermational Depositary Authorit-y Authorityf or Of au IVorized official(s): Address: 1815 N. University Street Peoria, Illinois 61604 U.S.A. Datet Where Rule 6.4td) applies, such date is the date on which the status of international depositary aultnority was acquiradl where a deposit made outside the Budapest Treaty attar the acquisition ot the status of international depositary authority is converted into a deposit undor the Budapest Treaty, such date Is the date on which the microorganism was received by tne Lnternational depositary ~authority, Form BP/4 (sole page) SUBSTITUTE SHEET

L

WO 92/20802 PCT/US92/04316 32 12 BUDAPEST TREATY ON THU XHTKIWATXOKAL RECOGNITION OF THU DEPOSIT Or NZCROO~AIJISX FOR THU PURPOSES Or pATENT pR0CZMME INTERNATIONAL roRx TO N~]rd RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT F s. Leor Lnd issued pursuant to Rule 7.1 by the Mycogen Corporation INTERNHATION4AL DEPOSITARY AUTHORITY 5451 Oberlin Drive Identified at the bottom of this page San Diego, CA 92121 NA)E AND ADDRESS OF DEPOSITOR 1. xDENTIrICATION or THE MICROORGANISM Identification reference given by the Accession number g Ivan by the DEPOSITORs INTERNATIONAL DEPSITARY AUTHORITY: Escherichia coli NM 522/pMYC 1642 MR626 NRRL B-18961 11. SCIENTIFIC DESCRIPTION AND/OR PRtOPOSED TAXONOMIC DESIGNATXON The microorganism Identified under I above was accoepnied by: a scientific description a proposed taxonomic designation (mark with a cross Where applicable) III. REZCEIPT AND ACCEPTANCE This International Depositary Authority Accepts the microo rganism Identified under I above# which was received by I t on 4-.10-92 (date of the original deposit)' xv. RrEiPT or REQuEsT FOR cOHvE.sxON 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) IV. INTE RNA TIONAL DEPOSITARY AUTHORITY N~a Agricultural Research Culture SignatureOs of person(s) having the power Collection (NRPRL) to represent the International Depositary International Depositary Authorit: Author ity or officialr s)a Address: 1815 N. University Street Date: Peoria, Illinois 61604 U.S.A.L 1 Where Rule 6.44d) applies, such date Is the date on which the status of International depositary authority was acquired.

Form BP/4 (sole page) SUBSTITUTE SHEET O LWIUILU4UUU IL, 1UlgU 1U 1L&.IL9LII.jVA L% -U other possible methods. If agroL..Aeria 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 WO 92/20802 PCT/US92/04316 33 SEQUENCE LISTING GENERAL INFORMATION: 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-Acti.ve Toxins (iii) NUMBER OF SEQUENCES: 38 (iv) CORRESPONDENCE ADDRESS: A ADDRESSEE: David R. Saliwanchik B STREET: 2421 N.W. 41st Street, Suite A-i C CITY: Gainesville D STATE: FL E COUNTRY: USA FZIP: 32606 COMPUTER READABLE FORM: A) MEDIUM TYPE: Floppy disk B COMPUTER: IBM PC compatible C)OPERATING SYSTEM: PC-DOS/MS-DOS D) SOFTWARE: PatentIn Release Version #1.25 (Vi) CURRENT APPLICATION DATA: SAPPLICATION NUMBER: US B FILING DATE: C CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: NAME: Saliwanchik, David R.

REGISTRATION NUMBER: 31,794 REFERENCE/DOCKET NUMBER: M/SCJ 104 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 904-375-8100 TELEFAX: 904-372-5800 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 4155 base pairs TYPE: nucleic acid STRANDEDNESS: double D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bacillus thuringiensis B STRAIN: PS17 C INDIVIDUAL ISOLATE: PS17a (vii) IMMEDIATE SOURCE: CLONE: E. coli NM522(pMYCi627) NRRL B-18651 (xi) SEQUENCE DESCRIPTIOMi SEQ ID NO:1: ATGGCAATTT TAAATGAATT ATATCCATCT GTACCTTATA ATGTATTGGC GTATACGCCA CCCTCTTTTT TACCTGATGC GGGTACACAA GCTACACCTG CTGACTTAAC AGCTTATGAA 120 CAATTGTTGA AAAATTTAGA AAAAGGGATA AATGCTGGAA CTTATTCGAA AGCAATAGCT 180 GATGTACTTA AAGGTATTTT TATAGATGAT ACAATAAATT ATCAAACATA TGTAAATATT 240 GGTTTAAGTT TAATTACATT AGCTGTACCG GAAATTGGTA TTTTTACACC TTTCATCGGT 300 TTGTTTTTTG CTGCATTGAA TAAACATGAT GCTCCACCTC CTCCTAATGC AAAAGATATA 360 TTTGAGGCTA TGAAACCAGC GATTCAAGAG ATGATTGATA GAACTTTAAC TGCGGATGAG 420 CAAACATTTT TAAATGGGGA AATAAGTGGT TTACAAAATT TAGCAGCAAG ATACCAGTCT 480 SUBSTITUTE

SHET

WO 92/20802 WO 9220802PCr/ US92/043 16

ACAATGGATG

AAGTTTACAG

ATTACAGATA

AGCATGCATC

ATTAATTTCA

CTTTACTCTA

TCTGATTTAG

TTAGATTTTG

GATATAAGTT

GATGGGTTAA

GGGAATGGCG

AGTTGGAGAG

CAAGATTCTG

CCTAATTATG

AAAACACCAC

GGGTTAAGTT

GCTGATACAA

TATCAAACTT

CAAGAGGCTA

ATGGGATTTC

GGTGCGAATG

ACAAGTGGAG

TTTAATGTAG

GTAGATAATA

ACAACTGATA

CAAGGTTCTT

ATATATCATG

AATATGAATT

TCTATGCATT

GAAACCTTCT

GGCCTTCCAG

AATAATGGTG

TCTTTAAGCG

GTACAAGGTA

AATACTGTAG

GCTGATAGCC

GCAACAGTAA

CAA1AATATCA

CATAATGTAA

GAAGTATTTG

AGTAGAGCAA

GGAAGAAATG

CCACCACCAG

CCAAATACAC

ATATTCAAAG

ATGAGGTACT

ATACAGCGGA

TTATGTTATT

CACCAGATGC

AAACTATTTA

AGTCCTTTGC

CAjAGATTGTT

TACAAAAAAC

CATTAAATAA

CGTTTCCAAA

CGGGACAGTA

TAGAGACTCG

TTTCCATAGA

CACAAGGTGC

TTTTACAACG

TATATAGTCT

CTGATAACTA

CTCTTCCTAA

CGTTITGAAAA

CGATGAPAGCT

AATATCAAAT

ATACTGGTGG

ATACGGGAGT

ATTCTTTTAC

CTGATOTCTT

GAAGTTATAA

ACTACGATAT

TGCTTAATAA

TTGCTACGTT

AAGTTAGTGG

GTGGTGATGG

GATCTGATCA

ATTATACCTA

TATCAAGCAT

TTGAGCTTGA

hiAAGTCCTAA

CAACACAAGT

GTGATCATGA

GAGATGAGAA

GAAATCTTCT

TAGTAACTGT

GATTGTCTCC

GTTATATTGT

CCATGGAGGA

ATCTTTAAAT

TCGAACTTTG

AAGAGATATC

AATTGATTCOC

TGACGTATTT

AAAAAAACAA

TCCTACTTTT

ACGTAGAATT

TACTTCAATT

CCCAAAAGAA

CGGTGGGCTT

TTTGTATGGG

TTCTTCTAAT

GAGTGGGTGG

AGATGGTACG

CCCTGCAACT

TTCTGGTCAC

TATTATAGGT

AGCTTCTTAT

TTCTCCTGGG

TCGTTGTCGT

AGCAAATCCA

ACAAGGAGCA

AGAAATTCCT

TTTAGACCGT

TACTTCATCA

AATAGTAAAT

AGGAAAAGTG

CCCAGTTCCA

AAATATTACJ

TGGTGGTAAT

TACGACTATT

TACAGGTACT

TCCAGTATAT

ACTACAACCT

TGTAIATTAC

AAATGCATTA

TATTGAMGAA

GAAGGCTTTA

GATAGGTGGG

ATOTGATCAT

ATCTTATATT

TTCTGGATTC

TTTAATAAGG

AGTTTTTATA

TTAGGTCTTC

ATTACTAAGG

TTTAAAACCG

CAGAAGGGAC

AAATATATTG

GATCCAGATC

CTTTCTCCTT

GATACTTCAA

AGAATATTAA

TTACAACCTT

CAGCTTCCAG

CCAATMATAC

AATACAAATT

AGACTTAGTG

CATTATCTTT

GTTGGTGCAT

CAACCAGATG

GGAGGTACAG

CAATCTATAG

TATGCAAGTA

ATTTTCCAAC

AATGGTGTCT

GCGAAGACGA

ATTGAATTTA

GGTGCAGATG

GGTCAGGCCA

ATAAAAACAA

GAAGGATTTA

GTACAATCTA

GGTGGWGGTG

TATCATGGAA

CCCGTATTAA

TCTCCTTTTG

AGATATGGTT

GATAGATCAT

TTCGCATCTG

GTTGTATTAA

CGTAAATTGG

AGTTTTGAAA

GAACTATTTA

TTCCAAAAAG

ATCGCACATG

TAGATTCTGG

CAGATCGTTT

CTTATTATGC

GTCCGACATG

ATATTAAAAA

TTGCTTCAI%

AAATTATGAC

TTTATCCAAC

TTATCCCTAT

ATTGGCCTAA

AACAATTCAA

ATTTATGGGC

CTGTAGATCC

AAATAAATAT

TAATGAGAGG

CTQGTATGGG

CTTA'PCTCTA

TGGTP.GGTGT

AACM

4

GGAAA

TTGTTAAAGA

GTATTCCTAT

ATGATAATAC

AGATAAACTT

ATGTAGTCAA

TTAATGTTCA

TACCTTTTTC

ATGTTTTATG

ATAGTAGTAG

TTGATATTCC

ATGAAGTTAG

ATAATCCGCC

ATGGTGGTCA

AACTTGAAAC

TACTGAATGC

ATATAACTAT

TTGCCACAGT

TTAhACTCCC

GAACACAAAA

AAGTGGATGC

TGAATCAAGC

ATTGGGATOC

AGAGTGATCA

TGGAGGAATC

GAAAAGACCT

ATTAATTAAA

ACCTGTATTT

TATACTTGCG

GGATTCTAAA

TAATATAAAG

CGGAACGCCT

AACACATTGT

AGq=TCACT ;iCGTACTGCA

TTATGAAAAT

ACTGTATCCT

AATAGAAGTC

ACAGGCAGGG

GGATACTTGG

AAGTGTAAGC

TGGTGGTTTT

TGGAACTCCT

GAGTACGCCT

TGTATCTACA

ATGGTTAAAT

TACAAATGTA

TAACGTTTTC

TGCATCTACT

ATCTATTGCT

TTTAACCAAC

TCTACCTCTT

GTCTTCTTCA

TATCGCTAGT

AGGGCATTCG

AATTCTTGCT

TCAACCTAGT

ATACAATTTT

TGGGATTCAT

TTACAGAAAT

ACAGACAGAA

GAATGGTACT

AATAGACTTA

TATGCTTGCT

CTTATCAGAT

AAAACGTTTG

ATOGTATAAA

TGTATTATTA

TAAATTAAAA

AGA2AATTGTT 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2 f2 0 22 2340 2400 2460 2520 2580 2640 2700 760 2820 2880 2940 3000 3060 3120

I

WO 92/20802 WO 9220802PCT/US92/04316

GTTTCACGTT

CCGTTAACAT

GGAGATCCAC

CCTGGTATTG

TTGGAAATTC

GCAAGAAATT

CCTGTTATCA

TCAGATATTT

CATTGGTTTA

GCATTAAATC

ACAAAAGATG

GGTAGACGTG

GAGAATTTTA

GTTACGTTGG

AATTTTACAA

ATTTCTTCAG

CCTACAGATG

ATGAACAACA

ATGGGCAAGA

CAAATGGACC

ATTTCTTTAG

AATTTGGTCT

GTGAAGATCG

GGAGAACCGA

ATCGAATCAA

CGTATCAGAA

TGTCAGATAG

GTGCGTATGC

CAGCTAATTG

TATTGCGACT

ATCCAGATAA

AGCATGGAGA

CTTCTCAACG

AAGATGGAGA

ACCAAAATTC

ATCAA

AGTGCAAAAG

AGTTTGTTGT

TTACAGTATC

TCGTATTGTA

TCCATTAGCA

GTATGAGAAA

CGGATTGTAT

TATAGACGCG

ATTCAGTGAA

ACAACTGGAA

GACAATAGAA

TCCAGATTGG

AGAATACAAC

AGAAACAAAA

TCAAGGACTC

ATTCTTAGTG

TGAGGGAAAT

GTCGTGCAAG

CCCCCACGTT

GATGTAGGTG

AATCCAACTG

GCAAATGAAA

GAACGTGCGG

GAAAATGGAA

ATTGTATTAC

CAAGGAGATA

CAAAGTACGC

GGCGATGCAC

TCTTCGAGTG

TTAGTATTCC

TATATAGAAA

ACGTTTGAAT

GATAATATTG

ACGGCTTCCA

TTCCTTATGG

CTACAAGTAA

CACTAGATTT

GAATGGCACG

TACGACAAGT

AAGTAACAAG

ATTGGAACGG

CAACGTTACC

TAATGGCTAA

TTCTGCATAA

ATCAGATAAC

TATCTCAAAT

ATGGGCAAGG

CGCATACACA

CAAATAAAGT

CGCTTGTGGA

GTACGAATAG

AGAAGCATTC

TGGAACCTTA

ACAAGCAAAC

CGTAAGCAAT

ACAACGTGTC

TTTAATTCAA

TTCTATTCGT

AAAGTTACGC

ATTCCAAGGT

TGGTCATTTT

ACTAGAAGAT

GATTGAAATC

AGAAGGAACG

TCATTTTGCG

GACAGTGACC

AGCTCCTCTT

CGATACAAGT

3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4155 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: BA LYET: 1 amino acid s 11LTYP: 1 amino acid s CSTRANDEDNESS: single DTOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES3 (ivr) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: I ORGANISM: BACILLUS THURINGIENSIS BSTRAIN: PS17 CINDIVIDUAL ISOLATE: PS17a (vii) IMMEDIATE SOURCE: CLONE: E. cali NM522(pMYC1627) N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Ala Ile Leu Asn Glu Leu Tyr Pro Ser V: 1 5 10 Ala Tyr Thr Pro Pro Ser Phe Leu Pro Asp AJ 25 Pro Ala Asp Leu Thr Ala Tyr Glu Gln Leu L 40 Gly Ile Asn Ala Gly Thr T r Ser Lys Ala I

S

Gly Ile Pho Ile Asp Asp Thr Ile Ann Tyr G: 70 7! Gly Leu Ser Lou Ile Thr Lou 41la Val Pro G 90 PrRhal 1 Lou Phe Phe Ala Ala Lou A: Pro Pro Pro Aen Ala Lys Asp Ile Phe Glu A: 115 120 Gln Glu Met Ile Asp Arg Thr Lau Thr Ala A: 130 135 M.R B-18651 il Pro Tyr Ann Val Letu La Gly Thr Gbn Ala Thr au Lys Asn Lou Glu Lys Ie Ala Asp Val Leu Lys In Thr Tyr Val Ann Ile 5 s0 Lu Ile Gly Ile Phe Thtr 3fl Lys His AnAla Pro la Met Lrs Pro Ala Ile sp Glu Gln Thr Phe Lou 140 Addrtant o181 N. University Street Date Peoria, Illinois 61604 U.S.A. I L I .LI I I Where Aule 6.4(d) appLies, such date I the date on which the statu4 of* internationa deoo:It.ry authority Wan acqired. S nm 14 1.^1 0 Io* SUIASTITUTEE SHEET t\ zi'

I:

t~ WO 92/20802 PCT/US92/04316 Asn 145 Thr Gly Tyr Thr Met 225 Ile Asn Gly Lye Arg 305 Asp Ile Ser Lye 38K Gln Pro Ile dly Lou 465 Ala Tyr Ala Ile Phe 545 Gly lie Ser Gly Glu Met Asp Leu Ile Thr As 193 Lou Lou 210 Lou Leu Asn Pho Aen Ile Lou Ala 275 Gin Lye 290 Leu Phe Ile Ser Arg Thr Asn Tr 3,53 Giu Arg 370 Gin Tyr Asp Ser Gin Ala Gin Ile 435 Gin Arg Asp Thr Gly Thr Leu Val 515 W G ln S30 Glu Lye Ala Asn Thr Aen Ann A.9 293 Ile Asp Lys 1 0 Arg Gly Arg Thr Lme 2 00 Ser Tyr Pro Leu Ala 340 Pro Ile Gly Val Glv Asn Thr Asp Ile Pro 500 Gly Pro Ala Ala Val 580 Aen Ser Ile 165 Lye Leu Leu Asp Pro 245 Leu Tyr Ile Thr Gin 325 Asp Aen Leu Gly Glu 405 Pro Met Asn Gly U r Tyr Val Asp Ser Met 565 Thr Thr G8y 150 Gin Phe Pro Pro Ile 230 Asp Tyr Gly Glu Phe 310 Lye Gly Tyr Lye Leu 390 Thr Aen Asp Lou Thr 470 Ser Gln Ser GiU S-0I Lye Ser Asn Leu Gin Ser His Thr Asp Val Phe 200 Tyr Tyr Ile Thr Ala Ile Ser Lye Thr Pro 280 lie Met 295 Asp Pro Thr Arg Leu Thr Giu Asn 360 Gin Phe 375 Lou Gin Arg Lou Tyr Val Thr Tr 449 Met Arg 455 Arg Lou Lou Pro Thr Ser Thr Pro 520 Gin Gly 535 f'j.lY Gly Lou Ser Gly GiU Val Phe 600 Aen Lou Aia Ala Arg Tyr Gin Ser 155 160 Gly Glu 185 Ile Ala Lye Asp Thr 265 Ser Thr Asp Arg Lou 345 Gly Lye Pro Tyr Ser 425 Lye Gly Ser Ala A a 509 Gln Aen Thr Pro gHe Phe Gl 170 Val Thr Ile Gly Ser 250 Ile Asp Thr Lou Ile 330 Aen Aen Lou Tyr Glu 4M0 Ile Thr Ser Ala Thr 490 Aen Glu Vai Val Gin 57A ln Asn Phe Leu Asp Lou Pro 235 Phe Tyr Lou His LOu Aen Gly Tyr Lou 395 Gin Asp Pro Vai G1 47K His Tyr Ala Ser Vai 555 Gln le Val Asn Ser Aen Ala 220 Thr Lye Asp Glu CgG Pro Ser Thr Ala Pro 380 Trp Lou Ser Pro Ser 460 Met Tyr Ser Thr Thr 540 Lye Sor Arg Asp Lye Lou Thr 205 Ser Trp Thr Val Ser 285 Lou Thr Pro 5cr Phe 365 Ser Ala Pro Ser Gln 445 Gly Giy Lou Gly Lou 525 Met Glu lie Cy3 Thr 605 Val Asn 190 Ala Met Asp Asp Phe 270 Phe Asp Gly Phe Ile 350 Pro Trp Ile Ala Asn 430 Gly Lou Gly Ser His 510 Pro Gly Trp Gly Gly Ser Asp His Ser Ile 255 Gln Ala Phe Ser Ile 335 Asp Aen Arg Glu Val 415 Pro Ala Ser Gly Val Amn Phe LOu Zie 575 Tyr Gly Ser Phe Arg Leu L s 2XO Lys Lys Lye Ala GIy 32 Pro Thr Pro Ala Val 400 Asp lie Ser Phe Phe 480 LOu Gly Ile Pro Aen 560 Pro Ala Ala 1 WO 92/20802 WO92/2802PCT/US92/04316 Asn Pro Ile 610 Thr Gly Val 625 Thr Thr Asp His Leu Thr Phe Ile Pro 675 Ser Ser Gly 690 Tg Asp Ile Ser Met His Pro Giy His Phe Asn Giu 755 Ile Thr Val 770 7 S Asp Gly Ser Lou Thr Gly 11.

Leu Ile Leu 835 Val T rSer Giu Lou Giu 865 Ala Thr Val Pro Ile Asp Ser Gly Thr 915 Glu GlU Val 930 AsGiu Lys Ser Arg Ala Ala Trp Tyr Phe Lys Ser 995 Tyr Ile Phe 1010 Ty'r Ile Val Val Ser Axg Phe Gin Gin Ile Asn Phe 615 Gin Giy Ala Asn Gly Val 630 Ann Ser Phe Thr Giu Ile 645 Asn Gin Gly Ser Ser Asp 660 665 Phe Ser Leu Pro Lou Ile 680 Ala Asp Asp Vai Leu Trp 695 Ile Val Ass Giy Gin Ala 710 Leu Leu Asn Lys Giy Lys 725 SAr Glu Thr Phe Phe Ala 7, 745 Vai Arg Ile Leu Ala Gly 760 Gin Ser Asn Ass Pro Pro 775 Gly Gly Asn Gly Gly Giy 790 Gly Ser Asp His Thr Thr 805 ,i:s Val Gin Gly Asn Tr Asn Ala Tyr Arg Ass Ass 840 Pro Phe Asp Ile Thr Ile 855 Leu Gin Pro Arg Tyr Gly 870 Lys Ser Pro Ass Vai Ann 885 Lou Gin Asn Ile Thr Thr 900 905 Gin Asn Met Lou Ala His 920 Val Leu Lys Val Asp Ala 935 Lys Aia Lou Arg Lys Lou 950 Arg Asn Leu Lou Ile Gly 965 Lys Gly Arg Asn Val Vai 9 0 985 Asp His Val Lou Leu Pro 1000 Gin Lys Val Giu Glu Ser 1015 Ser Gly Phe Ile Ala His 1030 TyrgSGin Giu Val Gin Ala Ser Thr Val Asp Ass Ann 620 Tyr Val Vai Lys Ser Ile Ala 635 640 Pro Ala Lys Thr Ile Ass Vai 650 655 Val Phe Leu Asp Ar Ile Giu Tyr His Giy Ser Tyr Asn Thr 685 Ser Ser Ser Asn Met Ass Tyr 700 Asn Ser Ser Ser Ile Ala Ser 715 720 Val Ile Lye Thr Ile As p Ile 730 735 Thr Phe Pro Val Pro Giu Gly 750 Leu Pro Giu Val Ser Giy Ass 765 Gin Pro Ser Ann Ass Gly Gly 780 Apg Gly Gin Tyr Ass Phe 79 800 Ile Tyr His Giy Lye Leu Glu 810 815 Thr Tyr Thr Giy Thr Pro Val 830 Thr Val Val Sor Ser Ile Pro 845 Gin Thr Giu Ala Asp Ser Lou 860 Phe Ala Thr Val Ass Gly Thr 875 880 Tgr Asp Arg Ser Phe Lps Lou Gin Val Ass Ala Leu Phe Ala 910 Ass Val Ser An His Asp Ile Lou Ser As Glu Vai Pho Gly Val Ass Gin Ala Lys Arg Lou 955 960 Gl1 Ser Phe Glu Ass Tr~ s Thr Val Ser Asp His Giu Lou 990 Pro Pro Gly Lou Ser Pro Ser 1005 Lys Lou Lye Pro Ann Thr Arg Giy L% Asp Lou Giu le Val M's 1040 98VlVal Gin Val Pro Tyr I BO l05s Gl lUAaPhe Pro Lou Thr Ser Ass Gly Pro Val cys a p pro Gi lUAa1060 1065 %7 WO 92/20802 WO 9220802PCr/US92/04316 38 Arg Ser Thr Ser Asn Gly Thr Lou Gly Asp Pro His Phe Phe Ser Tyr 1075 1080 1085 Ser Ile Asp Val Gly Ala Leu Asp Lou Gin Ala Asn Pro Gly Ile Glu 1090 1095 1100 Phe Gly Lou Arg Ile Val Asn Pro Thr Giy Met Ala Arg Val Ser Asn 1105 1110 ills 1120 Leu Glu Ile Arg Giu Asp Arg Pro Lou Ala Ala Aan Glu Ile Arg Gln 1125 1130 1135 ValGlnArgVal Ala Arg Asn Trp Ar Thr lTyGuL GuAr Va in g1140 M15 Gl Tr iu 0 Gi r Ala Giu Val Thr Ser Leu Ile Gin Pro Val Ile Asn Arg Ile Asn Gly 1155 1160 11 Leu T r Glu Asn Gly Asn Tr~ Asn Gly Ser Ile Arg Ser Asp Ile Ser TyrGlnAsnIleAspAla Ile Val Leu Pro Thr Leu Pro Lys Leu Arq 1 85 Anli s 1190 1195 120 His Trp Phe Met Ser Asp Arg Phe Ser glu Gin Gly Asp Ile Met Ala 1205 1210 1215 Lys Phe Gin Gly Ala Leu Asn Arg Ala.Tyr Ala Gin Leu Giu Gin Ser 1220 1225 1230 Thr Leu Leu His Asn Gly His Phe Thr Lys Asp Ala Ala Asn Trp Thr.

1235 1240 1245 Ile Glu Gly Asp Ala His Gin Ile Thr Leu Giu AglyArg rVa 1250 1255 A1gVa Leu Arg Leu Pro Asp Trp Ser Ser Ser Val Ser Gin Met Ile Giu Ile 1265 1270 1275 1280 Giu Asn Phe Asn Pro Asp Lys Giu Tyr Asn Lou Val Phe His Gly Gin hr1285 1290 12 Gl GuGl TrVal Thr Leu Giu His Gly Glu Glu Thr L y le Gl iuGy1300 1305 M1s0y Glu Thr His Thr His His Phe Ala As= Phe Thr Thr Ser Gin Arg Gin 1315 1320 1325 Gly Lou Thr Phe Glu Ser Asn Lys Val Thr Val Thr Ile Scr Ser Glu 1330 1335 1340 Asp Gly Giu Phe Lou Val Asp Aen Ile Ala Leu Val Giu Ala Pro Lou 1345 1350 1355 1360 Pro Thr Asp Asp Gin Asn Ser Glu Gly Asn Thr Ala Ser Ser Thr Asn 1365 1370 1375 Ser Asp Thr 5cr Met Asn Asn Asn Gin 1380 1385 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: IA) LENGTH: 3867 base pairs BTYPE: nucleic acid CSTRANDEDNESS: double DTOPOLOGY: linear (ii) MO0LECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: SORGANISM: Bacillus thuringionois BSTRAIN: PS17 CINDIVIDUAL ISOLATE: PSl7b (vii) IMMEDIATE SOURCEi CLONE: E. ccli NH522(VHYC1628) NRRL B-18652 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATGOCAATTT TAAATOAATT ATATCCATCT GTACCTTATA ATGTATTGGC OTATACOCCA -Where Rule 6.41d) 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 inzernat-ional depositaty authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by tne international depositary authority.

Form BP/4 (sale page) SUBSTITUTE SHEPET -WO 92/20802 PCT/US92/04316

CCCTCTTTTT

CAATTGTTGA

GATGTACTTA

G-TTTAAGTT

TTGTTTTTTG

TTTGAGGCTA

CAAACATTTT

ACAATGGATG

AAGTTTACAG

ATTACAGATA

AGCATGCATC

ATTAATTTCA

CTTTACTCTA

TCTGATTTAG

STAGATTTTG

GATATAAGTT

GATGGGTTAA

GGGAATGGCG

AGTTGGAGAG

CAAGATTCTG

CCTAATTATG

AAAACACCAC

GGGTTAAGTI

GCTGATACAA

TATCAAACTT

CAAGAGGCTA

ATGGGATTTC

GGTGCGAATG

ACAAGTGGAG

TTTAATGTAG

GTAGATAATA

ACAACTGATA

AACCAAGGTT

AATACTGTAA

ATAGCTCCTC

GGTCGAACTA

ACACAAACCA

GACATAGTTT

AAATTAGATT

CAAAATGATT

GATGCACTTG

GCATTATCTG

GCGAAGCGCT

GCTTGGTATA

TACCTGATGC

AAAATTTAGA

AAGGTATTTT

TAATTACATT

CTGCATTGAA

TGAAACCAGC

TAAATGGGGA

ATATTCAAAG

ATGAGGTACT

ATACAGCGGA

TTATOTTATT

CACCAGATGC

AAACTATTTA

AGTCCTTTGC

CAAGATTGTT

TACAAAAAAC

CATTAAATAA

CGTTTCCAAA

CGGCACAGTA

TAGAGACTCG

TTTCCATAGA

'CAAGGTGC

.CTTTACAACG

TATATAGTCT

CTGATAACTA

CTCTTCCTAA

CGTTTGAAAA

CGATGAAGCT

AATATCAAAT

ATACTGGTGG

ATACGGGAGT

ATTCTTTTAC

CTTCTGATGT

CTATATTCAA

TTTGGAGTAC

CCCCTAACAG

TTCCTATTCC

CTATTGATAT

TTACCAATAA

TAGAGAATAT

CAACAGATGT

ATGAAGTGTT

TAAGCAAGGC

GAGGAAGAAA

GGGTACACAA

AAAAGGGATA

TATAGATGAT

AGCTGTACCG

TAAACATGAT

GATTCAAGAG

AATAAGTGGT

CCATGGAGGA

ATCTTTAAAT

TCGAACTTTG

AAGAGATATC

AATTGATTCC

TGACGTATTT

AAAAAAACAA

TCCTACTTTT

ACGTAGAATT

TACTTCAATT

CCCAAAAGAA

CGGTGGGCTT

TTTGTATGGG

TTCTTCTAAT

GAGTGGGTGG

AGATGGTACG

CCCTGCAACT

TTCTGGTCAC

TATTATAGGT

AGCTTCTTAT

TTCTCCTGGG

TCGTTGTCGT

AGCAAATCCA

ACAAGGAGCA

AGTAAAAATT

CTTTTTAGAT

CAATTCATAT

TAGTTCAGAT

TGATGATGCT

GGGTTCCGGA

TTTTGTCGGA

TAATAGTGGT

CACAACACAA

GAGTGATCAT

TGGAAAAGAG

GCGTAATCTC

TGTAGTAAAC

GCTACACCTG

AATGCTGGAA

ACAATAAATT

GAAATTGGTA

GCTCCACCTC

ATGATTGATA

TTACAAAATT

TTTAATAAGG

AGTTTTTATA

TTAGGTCTTC

ATTACTAAGG

TTTAAAACCG

CAGAAGGGAC

AAATATATTG

GATCCAGATC

CTTTCTCCTT

GATACTTCAA

AGAATATTAA

TTACAACCTT

CAGCTTCCAG

CCAATCATAC

AATACAAATT

AGACTTAGTG

CATTATCTTT

GTTGGTGCAT

CAACCAGATG

GGAGGTACAG

CAATCTATAG

TATGCAAGTA

ATTTTCCAAC

AATGGTGTCT

CCTGCGAAGA

CGTATTGAGT

ACTACAGGTT

AAAGCCCTTA

TTGCTTCGAT

AAAGATTTTA

TCTGGTCTAC

AGTGGTGGCT

GTGAATGCTC

GATATTGAAG

AAAAAAACAT

CTGGTAGGAG

GTATCTAATC

CTGACTTAAC

CTTATTCG7AA

ATCAAACATA

TTTTTACACC

CTCCTAATGC

GAACTTTAAC

TAGCAGCAAG

TAGATTCTGG

CAGATCGTTT

CTTATTATGC

GTCCGACATG

ATATTAAAAA

TTGCTTCATA

AAATTATGAC

TTTATCCAAC

TTATCCCTAT

ATTGGCCTAA

AACAATTCAA

ATTTATGGGO

CTGTAGATCC

AAATAAATAT

TAATGAGAGI

CTGGTATGGG

CTTATCTCTA

TGGTAGGTGT

AACAGGGAAA

TTGTTAAAGA

GTATTCOT3AT

ATGATAATAC

AGATAAACTT

ATGTAGTCAA

CGATTAATGT

TTGTTCCAAT

CAGCAAATCT

CAGGTTCTAT

TTTTTAAAAC

CAAATACTCT

ATGGATCCGA

CTCCAAAGAG

TATTCACATC

AAGTGGTTCT

TGCGTAAATT

GLAATTTTGA

ACGAACTGTT

AGCTTATGAA

AGCAATAGCT

TGTAAATATT

TTTCATCGGT

AAAAGATATA

TGCGGATGAG

ATACCAGTCT

ATTAATTAAA

ACCTGTATTT

TATACTTGCG

GGATTCTAAA

TAATATAAA(,'

CGGAACGCCT

AACACATTGT

AGGATCAGGT

ACGTXACTGCA

TTATGAAAAT

ACTGTATCCT

AATAGAAGTC

ACAGGCAGGG

GQ~ATACTTGG

AAGTGTAAGC

TGGTGGTTTT

TGGAACTCCT

GAGTACGCCT

TGTATCTACA

ATGGTTAAAT

TACAAATGTA

TAACGTTTTC

TGCATCTACT

ATCTATTGCT

TCATTTAACC

TCTAGAATCA

TATACCAGCA

GTCAATAACA

TAATTATGAT

AGAAATACAA

TGGATCTATA

TTTCACCGAG

TAATACACAA

AAAAGTAGAT

TGTAAATCAA

TAACTTGGAT

GAAGAGTGAT

120 180 240 300 360 420 480 540 600 660 72 0 780 840 900 960 1020 1080 1,140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 ""erC M~ule 6.4(d) applies, such date is the date On which the status of international depositary autzlori-y 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, suc 'h date is the date on which the microarganis= was received by tne inlternati.onal depositary authority.

P'O= BP/4 (sale page) SUB3STITUTE SHEE WO 92/20802 PCr/US92/0431,6

CATGTATTAT

TCTAAATTAA

TTAGAAATTG

GGAGAAGCAT

AATGGAACTT

GTAGACACAA

CGCGTAAGCA

GTACAACGTG

AGTTTAATTC

GGTTCTATTC

CCAAAGTTAC

AAATTCCAAG

AATGGTCATT

GTATTAGAAG

ACGATTGAAA

GGAGAAGGAA

CATCATTTTG

GTGACAGTGA

GAAGCTCCTC

AGCGATACAAN

TACCACCACC

AACGAAATAC

TGGTTTCTCG

TCCCATTAAC

TAGGCAATCC

ACCCTGGTAT

ATTTGGAAAT

TCGCAAGAAA

AACCTGTTAT

GTTCAGATAT

GCCATTGGTT

GTGCATTAAA

TTACAAAAGA

ATGGTAAACG

TCGAGAATTT

CGGTTACGTT

CGAATTTTAC

CCATTTCTTC

TTCCTACAGA

GTATGAACAA

AGGATTGTCT

ACGTTATACG

TTATGGGCAA

ATCAAGTGGA

ACATTTCTTT

TGAATTCGGT

TCGTGAAGAT

TTGGAGAACC

CAATCGAATC

TTCGTATCAG

TATGTCAGAT

TCGTGCGTAT

TGCAGCCAAT

TGTATTACGA

TGATCCAGAT

GGAGCATGGA

AACTTCTCAA

AGAAGATGGA

TGACCAAAAT

CAATCAA

CCATCTTATA

GTTTCTGGAT

GAAATAAAGA

CCAGTTTGTT

AGTTACAGTA

CTTCGTATTG

CGTCCATTAG

GAGTATGAGA

AATGGATTGT

AATATAGACG

AGATTTAGTG

GCACAACTGG

TGGACGGTAG

TTGCCAGATT

AAAGAATATC

GAAGAAACAA

CGTCAAGGAC

GAATTCTTAG

TCTGAGGGAA

TTTTCCAAA.A

TTATTGCGCA

AAGTGGTGCA

GTATCCCACA

TTGATGTAGG

TAAATCCAAC

CAGCAAATGA

AAGAACGTGC

ATGACAATGG

CGATTGTATT

AACJAAGGAGA

AACAAAATAC

AAGGCGATGC

GGTCTTCGAG

AATTAGTATT

AATATATAGA

TCACGTTTGA

TGGATAATAT

ATACGGCTTC

AGTGGAGGAA

TGCAACAGAT

AGTTCCTTAT

TTCTACAAGT

TGCATTAGAT

TGGAATGGCA

AATACGACAA

GGAAGTAACA

AAATTGGAAC

ACCAACGTTA

TATCATGGCT

GCTTCTGCAT

ACATCAGGTA

TGTGTCTCAA

TCATGGGCAA.

AACGCATACA

ATCAAATAAA

TGCGCTTGTG

CAGTACGAAT

2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3867 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: B LYET: 1 amino acid s LTYP 1 amino acid s CSTRANDEDNESS: single (DTOPOLOGY: linear (i)MOLECULE TYPE: protein (i)HYPOTHETICAL: YES (iv) ANTI-SENSE: WO (Vi) ORIGINAL SOURCE: SORGANISM: BACILLUS THURINGIENSIS BSTRAIN: PS17 CINDIVIDUAL ISOLATE: PS17b (Vii) IMMEDIATE SOURCE: (B)-CLONE: E. coli NM522(pMYC1628) N~ (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Ala Ile Leu Aen Glu Leu Tyr Pro Ser V~ 1 5 10 UIRL B-18652 il Pro Tyr Asn Val Leu Ala Tyr Thr Pro Pro Ser Phe Leu Pro Asp 25 Pro Ala Asp Leu Thr Ala Tyr Glu Gln Leu 40 Gly Ile Asn Ala Gly Thr Tyr Ser Lye Ala 55 Ala Gly Thr Gln Ala Thr 30 Leu Lys Aen Leu Glb Lys Ile Ala Asp Val LeU Lys Gly Ile Phe Ile Asp Asp Thr Ile Aen Tyr Gin Thr Tyr Val. Asn 70 75 Gly Leu ser Leu Ile Thr Leu Ala Val Pro Giu Ile Gly Ile 9 Phe Thr Pro Phe ig1 Leu Phe Phe Ala Ala Leu Pro Pro Pro Asn Ala Lye Asp Ile Phe Giu 115 120 Asn Lys His As~ Ala Pro Ala Met L s Pro Ala Ile SUBSTITUTE SHEET name: -6J.4U.LLUraJ. ±esearcn Luuirure Signature(s) of p ron(s) having the powar Collection (NRRL) to represent the ternational Depositary International Depositary Authorit:' Authori ,~joyau b-affi zed oftic al(s): Peoria, Illinois 61604 U.SA. ''2z where Rule 6.4(d) applies, such date is the date on which the status of international depositar authority wa cquird SUBSTITUTE

SHEET

rsolp nace) WO 92/20802 PCr/IJ592/04316 Gin Asn 145 Thr Gly Tyr Thr Met 225 Ile Asn Gly Lys Asp Ile Ser Lys Ala 385 Gln Pro Ile Gly Leu 465 Ala Tyr Ala Ile Phe 545 Gly Ile Ile Ile Asp Arg Gly Arg Thr Ser Tyr Pro Leu Ala 340 Pro Ile Gly Val Asn Thr Asp I le Pro 500 Gly Pro Ala Ala Val 580 Arg Gin Phe Pro Pro Ile 230 Asp Tyr Gly Giu Phe 310 Lys Gly Tyr Lys Leu 390 Thr Asn Asp Leu Thr 470 Ser Gin ser Giu Lys Ser Thr Asn Gly Giu 185 Ile Ala Lys Asp Thr 265 Ser Thr Asp Arg Leu 345 Gly Lye Pro Tyr Ser 425 Lys Giy Ser Ala As Gin Asn Thr Pro Asp Ala 155 Phe Leu.

Asp Leu Pro 235 Phe Tyr Leu His Leu Asn Gly Tyr Leu 395 Oin A sp Pro Val His Tyr Aia Ser Val 555 Gin le Giu 140 Ala Asn Ser Asn Ala 220 Thr Lys Asp Giu Pro Ser Thr Ala Pro 380 Trp Leu Ser Pro Ser 460 Met Tyr Ser Thr Thr 540 Lys Ser Arg Gin Thr Arg Tyr Lys Val Leu Asn 190 Thr Ala 205 Ser Met Trp Asp Thr Asp Val Phe 270 Ser Phe 285 Leu Asp Thr Gly Pro Phe Ser Ile 350 Phe Pro 365 Ser Trp Ala Ile Pro Ala Ser Asn 430 Gin Gly 445 Gly Leu Gly Gly Leu Ser Gly His 510 Leu Pro 525 Met Gly Giu Trp Ile Gly Cys A Y~ I a WO 92/20802 PCT/US92/O4316 Ser An As 599 Asn Pro Ile 610 Thr Gly Val 625 Thr Thr Asp Val. His Leu Glu Phe Vai 675 Ser Ur Thr Tr~ Ser Thr Gly Arg Thr Thr Asn Tyr Phe Thr Asn 755 Vai Gil Ser 77 Thr Asn Asn 785 Gin Asn Asp Ser Asn Thr Glu Giu Val 835 Lys Giu Lys 850 Ser Lys Ala 865 Ala Trp Tyr Leu Lys Ser Tyr Ile Phe 915 Tyr Thr Vai 930 Vai Ser Arg 945 Gly Giu Ala His Ser Thr Ser Ile Asp 999 Phe G Leu Leu Giu Ile 1025 Asn Thr Phe Gin Gin Gly Asn Ser 645 Thr Asn 660 Pro Ile Thr Gly Ser Ser Thr Pro 725 AsE Thr 740 Thr Leu Gly Leu Asn Ser Leu Glu 805 Gin Asp 820 Vai Leu Lys Thr Arg Asn Arg Gl 885 As His Gin Lys Ser Gly Tyr Gly Phe Pro 965 Ser Aen 980 Val Gly Arg Ile Arg Glu Ass Gin Ala 630 Phe Gin Leu Ser Asn Gin Glu His Gl 790 Ass Ala Lys Leu Leu 870 Arg Val Val Phe Gin 950 Leu Gly Ala Val As Val Phe Phe 600 Ile Asn Phe 615 Asn Giy Vai Thr Val Lys Gly Ser Ser 665 Glu Ser Ass 680 Ala Ass Leu 695 Lys Aia Leu Ser Asp Asp Thr Ile Pro 745 Ile Gin Asp 760 Gl Ser Asp 775 Ser Giy Gly Ile Thr Thr Leu Aia Thr 825 Vai Asp Ala 840 Arg Lys Phe Leu Val Gly Ass Vai Val Leu Leu Pro 905 Giu Giu Ser 920 Ile Aia His 935 Glu Ile Lys Thr Ser Ser Thr Leu GIy 985 Leu As Ass Pro Thr 1015 Arg Pro Leu Ass Ala Tyr Ile 650 Asp Thr Ile Thr Ala 730 Ile Ile Gly Ser Gin 810 Asp Leu Vai Gly Asn 890 Pro Lys Ala Lys GIy 97 Ass Asp Gly Ala Val Asp Thr Gly 605 Ser Thr Val Asp 620 Val Vai Lys Ser 635 Pro Aia Lys Thr Val Phe Leu Asp 670 Vai Thr Ile Phe 685 Pro Ala Ile Ala 700 G1 Ser Met Ser 715 Leu Leu Arg Phe Pro Giy Ser Gl 75 Vai Ser Ile Asp 765 Ser Ile Lys Leu 780 Pro Lys Ser Phe 795 Val Asn Aia Leu Val Ser Asp His 830 Ser Asp Giu Vai 845 Ass Gin Ala Lys 860 Ass Phe Asp Aen 875 Val Ser Ass His Pro Gly Leu Ser 910 Leu Lys Arg Ass 925 Thr Asg Leu Glu 940 Val Val Gin Vai 955 Pro Vai Cys Cys Pro His Phe Phe 990 Thr Asn Pro Gly 1005 Met Ala Arg Vai 1020 Ala Ass Giu Ile 1035 Gly Ass Ile Ile 655 Arg Ass Pro Ile Phe 735 Lys Ile Asp Thr Phe 815 Asp Phe Arg Leu Glu 895 Pro Thr Ile Pro Ile 975 Ser Ile Ser Arg Ala Ass Ala 640 Ass Ile Ass Leu Thr 720 Lys Asp Phe Phe Glu 800 Thr Ile Gly Leu Asp Leu Srr ArV Val Pro Tyr Glu Ass Gin 1040

I

Val Gin Arg Val Ala Arg Ass Trp Arg Thr Giu Tyr Giu Lys GIu Arg 1045 1050 1055 i i WO 92/20802 PCT/US92/04316 Ala Glu Val Thr Ser Leu Ile Gin Pro Val Ile Asn Arg Ile Asn Gly 1060 1065 1070 Leu Tyr Asp Asn Gly Asn Trp Asn Gly Ser Ile Arg Ser Asp 'le Ser 1075 1080 1085 Tyr Gin Asn Ile Asp Ala Ile Val Leu Pro Thr Leu Pro Lys Leu Arg 1090 1095 1100 His Trp Phe Met Ser Asp Arg Phe Ser Glu Gin Gly Asp Ile Met Ala 1105 1110 1115 1120 Lys Phe Gin Gly Ala Leu Asn Arg Ala Tyr Ala Gln Leu Glu Gin Asn 1125 1130 1135 Thr Leu Leu His Asn Gly His Phe Thr Lys Asp Ala Ala Asn Trp Thr 1140 1145 1150 Val Giu Gly Asp Ala His Gin Val Val Leu Glu Asp GlyLys Arg Val 1155 1160 1165 Leu Ar Leu Pro Asp Trp Ser Ser Ser Val Ser Gin Thr Ile Glu Ile 11 0 1175 1180 Glu Asn Phe Asp Pro Asp Lys Glu Tyr Gin Leu Val Phe His Gly Gin 1185 1190 1195 1200 Gly Glu Gly Thr Val Thr Leu Glu His GlyGlu Glu Thr Lys Tr Ile 1205 12 0 1 5 Glu Thr His Thr His His Phe Ala Asn Phe Thr Thr Ser Gin Arg Gin 1220 1225 1230 Gly Leu Thr Phe Glu Ser Asn Ly s Val Thr Val Thr Ile Ser Ser Glu 1235 1240 1245 Asp Gl Glu Phe Leu Val Ap Asn Ile Ala Leu Val Glu Ala Pro Leu 12 Lu 125 1260 Pro Thr Asp Asp Gin Asn Ser Glu Gly Asn Thr Ala Ser Ser Thr Asn 1265 1270 1275 1280 Ser Asp Thr Ser Met Asn Asn Asn Gin 1285 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 3771 base pairs TYPE: nucleic acid STRANDEDNESS: double DTOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: A ORGANISM: Bacillus thuringiensis INDIVIDUAL ISOLATE: 33F2 (vii) IMMEDIATE SOURCE: CLONE: E. coli NM522(pMYC2316) B-18785 (ix) FEATURE: NAME/KEY: misc feature B LOCATION: 4..24 OTHER INFORMATION: /function= "oligonucleotide ybridization probe" product= "GCA7T ACA/T TTA AAT GAA GTA/T TAT" /standard name= "probe a"ll /note= "Probe A" (ix) FEATURE: NAME/KEY: misc feature SLOCATION: 13..33 OTHER INFORMATION: /function= "oligonucleotide ybridization probe" roduct= "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 L WO 92/20802 PCT/US92/04316

ATGGCTACAC

CAACAATTAG

AAAAAATGGA

ACTACA(;GAG

ATTCCTGAAG

AAAATATTTG

GAAGCATTPA

GACAGTCTTC

ACAGCAAAAA

ATTCCAGAAG

ATATTATTGT

GTAGACACAC

AAAGCATTCT

AAAGCAAATT

ACTTTCGATC

TCTCCAATTT

AGCGATCTAT

AACGATGGTC

ACTCATGAAA

AGAGGCTCTT

AGAAATTCAT

ACTCAAGGGT

GAATCTGATG

ACATTAAGAG

TCAACAGAAA

ATGAAATATT

CAGCAAACAT

CGTTATGCCA

ACGCTTAATA

GATTTATATA

CATAATGATA

CAAGATTCAC

TCATCTCCAA

TCATATACAA

GACCCTAACA

AAAGATTCTG

GCTTGGTATA

ATAACTCCTA

TTATTCGCAT

CAGGTCGTTA

TTACGTAAAT

GGTAATTTTG

CATGAATTAT

ATTTTCCAAA

TTAATGAAGT

ATACAACAGG

AAAAAGGGGC

AAATTGACCC

TTGGTACTGT

GAGATAAACC

TTCAACAAGA

AGAAAACAAT

CGCAACTCGA

GATATGAAAC

TAAGAGACGC

ATAAAAAATA

TAAATGGACT

ATATTAAAGG

CAGATCATTA

ACCAACCTGT

TTCACTATCA

TTGCAAAAAT

CATACCATGT

CAAATCCGAT

TTTATAAGGC

ATGCATTTGC

GTGCCCCAGA

ATTTCATCAA

AAATCAAAGG

ACGGTAAACC

TAATATTCGA

GTACCCAAGG

TACCTACTTC

CAATAGGTTC

AAAATGGAAT

CTCAAGATTC

CTATATGGTC

GTCAGGGAAG

GAAATCATAC

TAGCCGATGG

GCGGTACTAT

AATTTGAACT

CTAGTGCACA

TGAAAGTCGA

TGGTAAATCA

ACAATTTAGT

TTAAAAGTGA

AGGTGGAAGA

ATATCCTGTG

TTTTAAAAGT

AAAAGGAAAA

TTTAAATGTA

GGCCTCTGCA

AAATGCAAAA

TATAACAAP.C

TAATCTATAT

AAATCTAAAT

TGGAGGTTTA

TATAGTTAAT

TATCAAAATG

TGACAAATTT

TATGACAGAA

TCAAAAAGAA

ACCTAAAAAC

AGGAGATCTT

TTTTACTGGT

AGATTTTAGT

TCCAATTGAT

AATAGCGGGA

CCAAGCACCA

AGGGCATAAA

TGTATATACT

CTTTCCTGCG

AGAATATATT

ATTTCATGCT

AACAAAAGGT

ACACAACGGT

ATATACAATT

GGTTTTAGAT

ACCTCCAGAA

TTCTAACAAA

TTATCCACAC

TATTCATGTT

GTTAAATTTT

TACTTCTATG

TTCTAATGAA

AGATACTCTC

TGCCTTATCA

AGCAAAACGT

CGCTTGGTAT

TCATGTCTTA

ATCAAAACTA

AATTATAATG

AAATATGATG

GACCTTTTAG

ATTAAAGGTG

GCAAGTACTA

AATATATTTG

TATCAAGATG

ACAGTAGCTA

TCTATACTTA

CCTTATTATG

GCAGAGAAAT

ACAATACACA

AAGAGTTTAG

ATGGTTCTTG

GTAGAAATTG

ATGCAAAATA

GTAAAATTAG

ATTCGAAACA

TATAATACCC

CTTAATAATC

TCTTCTGTTT

ACAGGAGGTG

TTAAACTATA

CTTATAAGTA

GAAAAAGGAT

AATGGAGCTC

TCAAAAACAG

TATTTTCGTT

TATGTAACCG

ACAGAAGGTA

CGTATTGAAT

GTTCACGAAT

CACTCATATA

AATTTATTAA

AACAATGGTG

AATAAAATAA

CACTTATTTA

TTAGAAAACA

GCAAGTAATG

GATGAAGTAT

CTCAGTAAAA

ATGGGAAAAG

CTACCTCCCC

AAACCAAATA

TATTATCTTC TGATGCTTTT AAATGATAAA AGCATTCGAA ATGTTGCATG GACTTATATA

TTTTATCTGTATTAACTTTA

TTGTAAGTTT !TA.TTTGGCCT AAGAGCTCAA GCCTCAAATT CAATTAATCA AAAAAAATTT TAGATAACAA TGATTACGTA CCTCAGATAT CTCCATATTT CTATGGTTGC TAATGCTCAT TAGGCTTTAG TGATAAAGAA ATCATACTGA AGCAGTAATA ATGTAAATAG CTATAATAAA ATCTAGTTGC TCTATGGCCA AATTTACAAG AACTATTTCT CCTCTAGCTC TATTGTACCT AATTTTCTAC AAGAACGGAC CATTCTACAA ATCGCCTAAT AATCTAGTGG TAATATTTCA CCAT-TATTTC AACTTGTATT TAGTTAATTT TAAAGATGGC CCTGGGACCA TTCTTTTATT TTTATACTTC TCCAGGTGAT CTCCAACTAT AAATGAACTA ATATCAAAAA TCAAGGGATC AACCAGTTAA TCTGGAAAAC CTCAATATAC CATTCGTATA TAGATAATCA GGAACTGCAA GTAATATTGG TGAAAATTAT ACCATACTCT TCAAATCCAA TTGTTCCTAA AGATTCACTT CAACAATTAT TTTTGATAAA GCCATATACA TTTAGAAGGA TTAATTTATT TCATCCTACA ATATGAATGT TGATTATGGA CTGCTACGAT ACCAAGTGAT ATGATAATAA TTTTAAAACA TCACAACTCA AGTAAATGCT TAAGTGATTA CTGGATTGAA TTGGAAAAGA GP.AAAAAGCA TACGAAATCT TCTCATAGGT ATGTAGTAAA AGAATCGGAT CAACATTCCA TCCTTCTTAT CACGTTATAC TATTTCTGGT 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 Budapest Treaty, such date is the date on which the microorganism was received by tne 3.nzernat~icnal depositary .authority.

Form BP/, (sole page) SUBSTITUTE

SHEET

WO 92/20802 PCI'/US92/04316

TTTATCGCAC

AAAGTGATGC

TGTTGTGTTC

AGCATCGATG

ATTGTCAAAC

TTAACAGCAA

GAACAAGAAC

TTATACGAAA

GAGCAAATTA

CCAGCTTTTT

RCTATTTTAG

AATCTCCTGC

GCCCATCATA

AATGCAACTC

ATTCATGCAA

GAAACACATA

AAAGGAAATC

ATTACAGTAA

ATCAATACAA

ATGGAGAAGA

AAGTGCCATA

CAAATTTAAA

TTGGTTCTCT

CAACAGGTAT

AAGAAATTCG

GAACAGAGAT

ATGAAGATTG

TGCTTCCTAC

TATTAAAAGT

CACGTTTCCA

ATAACGGTCA

CAATCTTAGA

AAACAATTGA

AAGGAAAAGG

CTCATCATAC

AAATTGAAGT

TAGAAGTTTC

GTATGAATAG

TGTAGAGCTT

TGAAGAAGCA

TATAAATGAA

GGAAATGGAA

GGCACGTGTA

TCAAGTACAA

CACAGCTATA

GAATGGTTCT

TTTATTAAAA

ATATCATTGG

AGAAGCATTA

TTTTACAACT

AGATGGTAGA

AATTGAAGAT

TTCCATTACT

AAATGATTTT

CCATATTACT

TAAAACAGAC

TAATGTAAGA

GTTGTCTCTC

CTTCCTCTTA

ACACTAGCTG

GCGAATCCTG

AGTAATTTAG

CGTGCAGCAA

ATTCAACCTG

ATTCGTTCAA

ACTGAGGAAA

TTTATGACAG

GATCGTGCAT

GATACAGCGA

CGTGTGTTAC

TTTGACTTAG

TTACAACATG

ATAACATCCC

TCAGAAGATG

ACAAATACAA

GTAGATATAC

GTTATGGGCA

CATCTGAATC

ATCCACATTT

GTATTGAATT

AAATTCGAGA

GAGATTGGAA

TTCTTAATCA

ATGTTTCCTA

TAAATTGTAA

ATCGTATAGG

ATACACAATT

ATTGGACAAT

GTTTACCAGA

ATCAAGAATA

GAGAAGAAAA

AAAATATTCC

GAGAGTTTTT

ATATTATTGA

CAAGAAGTCT

AGAAATACAA

TAATTCTAGT

CTTTAGTTAT

TGGTCTCCGT

AGACCGTCCA

ACAAAACTAT

AATTAATGCG

TCATGATCTA

TTATGATCAT

AGAACATGGT

AGAAAGTCGT

AGAAGGAGAT

TTGGTCTTCT

CCAATTGCTC

CGAATATGTG

TTTCACTTTT

AATCGATCAC

AAATTCACCA

C

2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3771 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: B LYET: 1 amino acid s IILTYP: 1 amino acid s CSTRANDEDNESS: single DTOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: A? ORGANISM: Bacillus thuringiensis CINDIVIDUAL ISOLATE: PS33 F (vii) IMMEDIATE SOURCE: CLONE: E. ccli NM522(pMYC2316) B-18785 (ix) M~ATUJRE: A NAME/KEY: Protein B? LOCATION: 1. .1257 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Ala Thr Leu Asn Glu Val Tyr Pro Val Asn Tyr Asn 1 5 10 Ser Asp Ala Phe Gln Gln Leu Asp Thr Thr Gly Phe Lys 25 ApGuMet Ile Lys Ala Phe Glu Lys Lys Trp LysL AspG 5 40 eK Gly Lys Asp Leu Leu Asp Val Ala Trp Thr Tyr Ile Thr 55 Ile Asp Pro Leu Asn Val Ile Lys Gly Val. Leu Ser Val.

70 75 Val. Leu 3er 15 Ser Lys Tyr 30 Gly Ala Lys Thr Gly Glu Leu Thr Leu Ile Pro Glu Val. Gly Thr Val. Ala Scqr Ala Ala Ser Thr Ile Val. Ser 90 Phe Ile Trp Pro Lys Ile Phe G2.y As~ y r s l v s i 100 10 0o b, .i f WO 92/20802 PCT/US92/043 16 Phe Thr 1 5 Thr Ile Tyr Val Lys Ser Leu Lys Gin 305 Ser Thr Asn Phe Asn 385 Arg Phe Gly His Phe 465 Ser Asn Ala Hiis Thr 545 Thr Glu Asn 130 Thr Ala Ser Ala Asn 210 Lys Ala Tyr Asp Glu 290 Pro Asp Arg Thr Ser 370 Pro Aen Lys Al IL Lyn 4 0 Ile Thr Gln Gin Ala 530 Gin Leu Lys Asp Leu Gin 165 Ile Ala Lys Lys Asn 245 Lys Ala Ile Lys His 325 Asn Lys Thr Ile Thr His Tyr Tyr Ile 485 Met Asn Thr Lys Pro 565 Pro Ala Leu Pro Asn Leu Met 230 Gly Ala Leu Glu Aen 310 Tyr Asp Ser Gin As 39E Lys Gin Ser Ile Thr 470 Lys Lys Leu Ala Gl Thr Gin Ile 135 Thr Glu Glu Ala Gly 215 Thr Leu Asn Trp Phe 295 Met Gin Gly Pro Ser 375 Leu Ala Gly Phe T~r Leu Gly Tyr Glu Gln 535 Tyr Ser Asp Leu Tyr Ser 175 Pro Ala Thr Val Val 255 Met Tyr Ile Val Phe 335 Ile Val Ser Cys Val 415 Thr Glu Arg Glu Ile 495 Asn Glu Ala Leu Asn 575 Ile Gln Val 160 Asp Tyr Ile His Ile 240 Asn Val Gin Tyr Pro 320 Ser Arg Asp Ser Ile 400 Asn Gly Gly Asp Leu 480 Lys Gly Phe Ser Gln 560 Ile )i b. ~iiin 92/20802 PCT/US92/04316 Gly Gly Leu Gin 625 Ser His Leu His Ala 705 Ala Asn Asn Thr Lys 785 LIeu Leu Lye Val Val 865 I Phe Gin Leu Asn Gly 945 Ile Gltu Ala Ala Glu Asn Asn His 595 As Arg 630 Asp Ser Ser Pro Leu Glu Ile Asn 675 Val Asn 690 Asp Gly Trp Tyr Phe Lys Ile Thr 755 Leu Ala 770 Val Asp Arg Lys Leu Ile Asp Val 835 Leu Leu 850 Glu Glu 114 Ala Glu Ile Thr Ser 915 Glu Thr 930 Ser Leu Val Lye Asp Arg Arg As 995 Ile Ile 1010 Leu Gin Phe Glu 630 Trp Tyr His Asp Phe 710 Thr Thr Val Val Ser 790 Asn Asn Glu Pro Leu 870 Glu Val Asn Asp Glu 950 Gly Thr Gin Val Tyr Thr Ile 585 Ile Gin His 600 Val Pro Lys 615 Val His Glu Ser Ser Asn Thr Ser Gin 665 Pro Thr Asp 680 Met Asn Val 695 Asn Lys Ile Ile Thr Ser Pro Lye Phe 745 Asn Ala Leu 760 Ser Asp Tyr 775 Asp Glu Val Gin Ala Lye Phe Asp Asn 825 Ser Asp His 840 Thr Phe His 855 Lys Pro Asn Asp Val Glu Met Gin Val 905 Ser Ser Cya 920 Pro His Phe 935 Ala Asn Pro Met Ala Arg Ala Lye Glu 985 Asn Tyr Glu 1900 Let Asn Gin 1015 Gly Asn Asp Ser Lys 650 Gly Pro Asp Thr Met 730 Glu Phe Trp Phe Arg 81 Leu Glu Pro Thr Leu 890 Pro Cys Phe Gly Val 970 Ile Gin Ile Tyr Thr Ile 590 Lye Asn Gly 605 Leu Gin Asp 620 Ile Ile Phe Ser Tyr Ser Tyr Pro His 670 Arg Aen His 685 Gly Lys Asp 700 Thr Ile Pro Leu Phe Asn Ser Asn Glu 750 Ser Ser Ala 765 Glu Gin Val 780 Lys Glu Lye Ser Lye Ile Ala Trp Tr 8 0 Phe Lye Ser 845 Tyr Ile Phe 800 Tyr Thr Ile Val Ser Arg Glu Glu Ala 910 Pro Asn Leu 925 Tyr Ser Ile 940 Glu Phe Gly Asn Leu Glu Gin Val Gin 990 Arg Thr Glu 1005 Ala Leu Tyr 1020 Thr Met Ser Asp His 655 Asn Thr Ser Ser As 735 Leu Gin Val Lye Arg 81 Met Asp Gin Ser Tr 895 Leu Aen Asp Leu Ile 975 Arg Ile Glu Glu Val Pro Lys 640 Ile Leu Ile Val 72B Asn Glu Asp Met Ala 800 Asn Gly His Lye Gly 880 Gly Pro Ile Val Arg 960 Arg Ala Thr Aen Glu Asp Trp Aen Gly Ser lie Arg Ser Asn Val Ser Tyr His Asp Leu 1025 1030 1035 1040

L

WO 92/20802 WO 9220802PCI'/U592/0431 6 48 GlU Gn Ile Met Leu Pro Thr Leu Leu W~s Thr GlU GlU Ile Asfl Cys 1045 1 01055 Asn Tyr Asp His Pro Ala Phe Leu Leu Lys Val Tyr His Trp Phe Met 1060 1065 1070 Thr Asp Ar le Gly GiU His Gl Thr Ile Leu Ala Arg Phe Gin Glu 1075 1 Ala Leu Asp Arg Ala Tyr Thr Gin Leu Glu ser Arq As n Leu Leu His 1090 1095 110 Asn Gly His Phe Thr Thr Asp Thr Ala Asn Trp Thr Ile Glu Gly Asp0 1105 1110 11 5 112 Ala His His Thr Ile Leu, Giu Asp Gly ArArVaLeAgLePo 1125 1135 Asp Trp ser Ser Asfl Ala Thr Gin Thr Ile Giu Ile Giu AsPhAs 1140 1145 go1 h As Leu Asp Gin Giu Tyr Gin Leu Leu Ile His Ala Lys Gi 1155 1160 jlyy Giy Ser Ile Thr Leu Gin His Gly GlU Giu Asn Giu Tyr Val Giu Thr His Thr 1170 1175 1180 His His Thr Asn Asp Phe Ile Thr Ser Gin Asn Ile Pro Phe Thr Phe 1185 1190 1195 1200 Lys Gly Asn Gin Ile Giu Val His Ile Thr Ser Glu Asp Gly Giu Phe 1205 1210 1215 Leu Ile Asp His Ile Thr Val Ile Giu Val Ser Lys Thr Asp Thr Asn 1220 1225 12 .0 Thr Asn Ile Ile Giu Asn Ser Pro Ile Asn Thr 5cr Met Asn Ser Asn 1235 1240 1245 Val ArgoVai Asp Ile Pro Ar~ Ser Leu INFORM4ATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS:.

LENGTH: 3738 base pai~rs JB) TYPE: nucleic wcid C)STRANDEDNESS: double D) TOPOLOGY: linear (i MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (Vi) ORIGINAL SOURCE: ORGANISM: Bacillus thuringiensis C) INDIVIDUAL ISOLATE: PS86Q3 (vii) IMMEDIATE SOURCE: LIBRARY: Laxnbdagem (TM) 11 LIBRARY CLONE: 86Q3a (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

ATGGCAACAA

GAAGTCGATG

GAATGGGGAA

CTTTATAAAA

GGATTTATTC

TGGCCTAAAT

ATGGATGCAG

AATAAAACAA

GCCATTTGTC

AATCCAAATC

AATTCTCAAT

TTAATGAGTT GTATCCAGTT ATCCTTATTC TTGGTCAAAT AAACAGGACA AAAAAA.ACTT CAGGAAA.ATT AGATTATTTC CAGGGGCAGA AGCAGCAGTT TATTTGGTGC GAATACAGAA TTAATAAAAT GGTAGATAAT TTGAAGGACT TCAAGGTAAT AAGGCAGTAC ACCAGAAAGA AACCTTGTAA AGATGATTTG TCACACAGCA TTTACCAGAA

CCTTATAATG

TTATTAAAGG

TTTGAAGACC

GCTTTGACAA

CCCTTTATTA

GGAAAAGATC

AAGTTCTTAA

TTAGGCCTAT

GTAAATTTTG

GATAGAGTTG

TTTAAAAATC

TGCTAGCTCA TCCAATTAAA

GTATACAAGA

ATCTTACGAT

AAGCATCAAT

ATATGTTTGT

AACAGTTGTT

GTTATAATCT

TTCAAAATGC

ATCAAAATTG

CTTCACGTTT

CTTGGTCGGA

AGGTTGGGAA

TGCATGGAAT

ATCATTGATT

AGACTTTGTT

TAATGCTATC

TAGTACACTT

TATACAAGTA

TACACCATGT

TGATACGGCT

TGAAAACTCT

SUBSTITUTE

SH-EET

e. WO 92/20802PC/U9/41 PCr/US92/04316 49 ACTCAGGAAT TTAAAAGAAC ATCTGTTGAA TTAACTTTAC CAATGTATAC AACAGTAGCT

ACGTTACATC

AIATGAACAAT

GAAACTGTTC

TCCGTAAATG

GCTA.CATGGC

CGTATTAT'Lt

TCAGGACCTG

TATCAGCACT

TTAGACAThC4

CTTTCATATA

TCCAATAACA

CATTTATATA

AATGGTGGAA

ACAAGTAAAA

ACTGTAAATA

CTTGTACCAG

TTAAAGGGCT

GTTCGCGAAT

ATACAAATTA

GGAGATAATA

TCTTTTGGAT

TTGAP.ATCAA

CAAGGTTCTT

TTCTGTGATA

TTTTGTTGCG

TGTACGCTAT

CAAAATATTA

ACAGACGTGA

GAAGTGTTTG

AGCAAAGCGC

GGCCGAAATG

CCACCACCAA

GCGAPITACAC

GTGTCTCGTT

CCATTGACAT

GCTGATCCAC

CCTGGTATCG

TTAGAAATTC

GCAAGAAATT

CCTGTATTAA

TCTGGAGTTT

CATTGGTTTA

GCATTAGATC

TTTTATTATA

ATTTAAATAA

GTACAAGTTT

CTTATAACCG

CTACATTTGA

TTTCAGATAC

AACATAGTAA

CATTTGTATC

CTACTTATAG

CAGATGGAAG

ATTATTGTCA

ATGCAAAAGG

GTGGTTCATG

ATGAATCACG

AGGGGACTGCv

AAAACGTTAT

TTCCATTTGA

GGATAAATGG

CGAATCAAAC

ATGTTTATTT

CTACTGAAAG

TCACAACGGT

CAGATCTCTT

ATAATAATCT

TTTGCACTAG

GTTGTCAGGT

CGACACAAGT

GTGATTATGA

GAAAAGAGAA

GTAACCTCTT

TAGTAAACGT

CACTGTACTC

GTTATACTGT

ATGG;GCAAGA

CGAGGGGAGC

ATTTCTTTAG

AATTGGGTCT

GTGAAGATCG

GGAGAACAGC

ATCAhATCAA

CTTATCATGA

TGTCTGATAT

GTGCGTATAC

TGAAGGATAT

TTTAAAGGTA

CCTTCAATTT

TTATGTCCGC

TACACATAAT

AGCAGGACCA

CATTACACCA

TGTTGATTCT

TACAAATAAT

CAGATATGAT

TAATAGCTAT

CTCTTTACAA

CATTTGTGAT

TCCTGATCAA

AGGAAATTTA

TQGAGATGTT

AAAATATGGT

TAACAATGCA

CAAACAAAAA

TAATGTGGAT

TTCTGTTGTA

AGAAATACCT

TTTAGATCGT

TCACTGTGAT

TCTTACTGAT

AGAAAATCAG

AAATGCATTA

GATTGAAGAA

AAAAGCATTG

GATAGGAGGA

ATCTGATCAT

ATCTTATATG

GTCTGGTTTT

AGTGAAGAAA

GATTTGTTGC

TTACAGTATT

TCGTATTGTA

TCCATTAAAG

ATATGACCAA

TGCGTTGTAT

CTTAGAAGCA

GTTAGGGGAA

GCAACTCGAA

ATAGAATTTA

GAATTACAAC

TTACCTACCT

AATATGACTG

TATCATCAAG

ATAGAAGAAT

AATAATATTC

ATTGTATATT

AGTAATAATT

TATGGAGATA

ACTGCCCCTA

AATGTAGAAT

GCATGGATTA

AAAATTAATG

GGAGTTATTT

AATGCTGATA

TCTGAGTATA

GTTAAACTTT

TATGAAATAC

TTAAGTGAAA

GGAGTACAAG

GCTGGAAGTT

ATTGAGTTTG

TGTAATAACC

TGTGATTGTA

CTACCTTCTT

GTTGCATCGA

GTTGTACTGA

CGTAAATTGG

AATTTTGATA

GAACTATTTA

TTCCZAAAAG

ATTGCACATG

GTGGTTCAAG

CCTCCACGTT

GATGTGGGAA

GAACGAACTG

AAAAATGAAC

GAACGTGCAG

GAAAATGAAG

ATTGTTTTAC

CAAGGTTCCA

GAAAGTACAA

TGACAAAATG

AATTGATACA

TGAATZAATCG

TTAACTGTTT

GTGGTAAATT

ATACTACTGG

TAGATACACC

CTAGAAAAGA

GTCACCCTTA

ATCAACCTGA

TTACACTTGT

CTTTAGTGGT

ATTATTTACG

TTTTGTATCC

CTGCCTATGT

CTAAATTGCC

ATAATCGG

CTAATAGTC4,

GTTGCCGTTA

ATCCATTTAG

GTGAAAATGG

TCTATGTTCA

TTCCAAAAAT

CTGTTGACAC

ATAACCCTCG

TTGTGACACT

GCGAACATGA

AAGTAGATGC

TAAATCACAC

ACTTGGATGC

AGAGTGATCA

TAGAGGAATC

CAGAAGATTT

TTCCATATGG

CTACAAGTAA

CATTAGATGT

GAATGGCACG

TCCGCAATGT

AAGTAACGGC

ATTGGAATGG

CAACATTACC

TTTTAGCTCA

TTCTGCATAA

GAATTTTCAC

CTCATATTCA

TTCAAAATCA

AGATATTGCT

AGATTTAACT

CGACAAAACT

ATCTCCAACA

ATTACAACAA

TGGATTACGA

TTTTACTACT

GAATGCACGA

TAGTACTGTA

TCCTCCTCAA

AATAACAGAA

TCCAATGGAA

ACTTACACAA

TATCTCTCTT

ATCTGTTGGC

TGCGAGTAAA

AAATTCCATT

AAAGTATATA

TATAACAAAC

CCAATTCCAA

CGATTGTACA

TGGCCTAGAT

TACAGATTTA

TACACTTGCA

ATTATCTGGT

AAAACGTTTA

TTGGTACAGA

TGTATTATTG

GAAATTAAAA

AGAAATTGTT

AGAAGCATTC

TGGAAAACCT

AGAAGCAAAC

TGTAAGTAAT

ACAACGTGCA

CTTGATTCAA

AGCAATTCGT

AAAATTAAAT

ATTTCAAGAA

TGGTCATTTC

780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 WO 92/20802 WO 9220802PCI'/US92/04316 ACAACAGATG CAGCAAATTG GGTAGACGCG TATTACGTCT GAAAATTTTG ATCCAGATAA GTCTCCCTTC AACATGGTGA AATTTTACAA CTTCACACCG ATTACCTCAG AAGATGGAGA CCTACAGATG ACCAAAGTTC ATGAATAATA ATCAATAA

GACGATAGAA

TCCAGATTGG

AGAATATCAG

AGAAGGAGAA

TCAAGGAGTC

ATTCCTAGTC

AGATGGAAAT

GGCGATGCAC

TCTTCTAGCG

TTAGTTTTCC

TATGTGGAAA

ACATTTGAAA

GATCATATTG

ACGACTTCCA

ATCATGCGAT

TTTCACAAAC

ATGCACAAGG

CACACCCGCA

CAAATAAAGT

CTCTTGTGGA

ATACGAATAG

ATTAGAAGAT

CATTGAAATA

AGAAGGAACG

TAAGTCTGCG

AACAGTTGAA

AGCTCCTCTT

CAATACAAGT

3360 3420 3480 3540 3600 3660 37 2 0~ 3738 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 1245 amino acids STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: BACILLUS THURINGIENSIS INDIVIDUAL ISOLATE: P586Q3 (vii) IMMEDIATE SOURCE: LIBRARY: LAMBDAGEM (tin) 11 library CLONE: 86Q3A (xi) SEQUENCZ DESCRIPTION: SEQ ID NO:8: met Ala Thr Ile Asfl 1 5 His Pro Ile L S Giu Lys Gly Ile Gin GiU Lys Leu Phe Giu Asp Giu Leu Tyr Pro Pro Tyr Asn Val LeU Ala Val Asp Asp Pro Tyr 5cr Trp Ser Giy Trp Giu Giu Trp Gly Lye 40 Asn Leu Leu Gly Gin Lys Tyr Lys Thr His Leu Thr le Ala Trp Giy Lys Leu Asp Tyr Phe Ala Leu 70 Thr Lys Ala 5cr Ile Ser Leu Ile 75 Gly Phe Ile Pro Gly Ala GiU Ala Ala Val Pro Phe Ile Asn 90 Met Phe Val Asp Asp Gin Asp As Phe Val Trp Pro 100 Gin Leu Phe Asn 115 Lys Phe LeU Scr Leu Gin Gly Asn 150 Lys Leu Phe Gly Ala Asn Thr 105 Ala Ile 120 T~r Asn LeU Giy met Asp Ala Val Giu Gly Lys 110 Lys Met Val Lys Thr Ile Leu scr Thr Leu Phe Ala Ile Gin Ile CyS Gin Scr Thr Pro GiU Aen Phe ASP Gin 175 Cys Thr Pro Val Ala Ser 195 Pro GiU Phe 210 LsArg Thr Thr Leu His Aen Pro Asn Gin Phe Asp Thr Pro CyS Lys ASP Asp 185 Aen Ser Gin Phe Thr 205 ASP GiU Aen ser Thr 220 LeU Asp Arg 190 Gin His Leu Gin Giu Phe Lys Asn Pro Tr~ 5cr Val GiU 230 Leu LeU leu 245 Leu Thr Leu Tyr Glu Gly Pro met 235 T le Tyr Thr Thr Val Giu Phe Met Thr 255 Ala 240 Lys SUBSTITUTE

SHEET

L

L,

CEL7~TPL1 ,WO 92/20802 PCr/US92/04316 Trp, Gin Gln Tr Ala Leu Glu Thr Phe 385 Leu Tyr Asp Ser Ala 465 Asn Arg Aen Asn Asn 545 Leu Gly Leu Gln Val 625 Ser Gly Sar Asp Ann 705 Tyr Ser 280 Asn Met Thr Leu Thr 360 Thr Val Thr Thr Thr 440 Leu Val Ile Aen Glu 520 Tyr Ala Lye Trp

GIY

Arg Sar Ser Ile Ann 680 Lys Aen Aen Val Lys Asn Tyr Thr Pro Pro Ser Ser Aen Ala Leu 475 Ala Arg Asn Met 5 5 Se? Gly Ile Ser Pro 635 Gly Val Ser Phe Val 715 Leu Arg Ser 300 CYs His Ala Glu Thr 380 Lye Asn Arg Asn Val Trp Pro Lye Glu 540 Leu Glu Aen Th 6 0 Phe Val Glu Sal? Gln 700 Asp Lye Thr 285 Ser Leu ('1n Gly His Tyr Glu Aen Tyr His Val Ile Asp Gly 52K Leu Pro Tyr Aen Asn 605 Giy Arg Gln le Asp 685 Pha Thr Val 270 Ser Val Asp Gly Pro 350 Ser Oln Leu Cys Cys Leu Ser Aen Gin 510 Thr Val Leu Aen Ala 590 Gln Asp Aen Giy Pro 670 Lou Cye Asp Leu Leu Ala Ala 320 Lys Glu Ile Ser Gin 400 Pro Gly Asn Asn Val 480 Leu Ile Gly Glu Gln 560 Arg Lye Lye Asn ile 640 Aen Gly Lou Aen Thr 720 WO 92/20802 PCT/US92/04316 52 Phe cys Cys Val C~s Thr Ser Leu Thr As Cys Asp Cys Ann Ann Pro 7 5 739735 Arg Gly Leu Asp Cys Thr Leu Cys Cgs Gin Val Giu Ann Gin Lou Pi.,0 740 750 Ser Phe Val Thr Leu Thr Asp Leu Gin Ann Ile Thr Thr Gin Val Ann 755 760 765 Ala Leu Val Ala Ser Ser Glu His Asp Thr Leu Ala Thr Asp Val Ser 770 775 780 As? Tyr Glu Ile Glu Giu Val Val Leu Lys Val Asp Ala Leu S3er GI~ 7790 795 0 Giu Val Phe Gly L~s Glu Lys Lye Aia Leu Arg Lys Leu Val Atri His 85810 815 Thr Lys Arg Leu Ser Lye Ala Arg Ann Leu Lou Ile Gly Gly Ann Phe 820 825 830 Asp Ann Leu Asp Ala Trp Tyr Ar~ Giy Arg Ann Val Val Ann Val Ser 835 89845 Asp His Glu Leu Phe Lye Ser Asp His Val Leu Leu Pro Pro Pro Thr 850 855 860 Leu Tyr Ser Ser Tyr Met Phe Gin Lye Val Giu Glu Ser Lye Leu Lye 865 870 875 8 0 Ala Ann Thr Arg Tyr Thr Val Ser Gly Phe le Ala His Ala Glu Asp 885 890 895 Leu Giu Ile Val Val Ser Arg Tyr GlIn i a y a a 90090 Laln Val Pro Tyr Gly Glu Ala Phe Pro Lou Thr Ser Arg Gly Aia Ile 915 920 92 Cy3 Cys Pro Pro Arg Ser Thr Ser Ann Gly Lye Pro Ala Asp Pro His 930 935 940 Phe Phe Ser Tyr Ser Ile Asp Val. Gly Thr Leu Asp Val Giu Ala Ann 945 950 955 960 Pro Gly Ile Glu Leu Gly Leu Arg Ile Val Giu Arg Thr Gly Met Ala 965 970 975 Arg Val Sor Asn Leu Glu Ile Arg Giu Asp Arg Pro Leu L~a Lye Ann 980 98590 Giu Lou Arg Asn Val Gin Arg Ala Ala Arg Ann Trp Arg Thr Ala Tyr 991000 105 Asp Gin Glu Arg Ala Giu Val Thr Ala Lou Ile Gin Pro Val. Lou Ann 1010 1015 1020 Gin Ile Ann Ala Lou T~r Glu Ann Glu Asp TrAs GlAali Ser Gly Val. Sor Tyr His Asp Lou GiU Ala Ile Va. Lou Pro Thr Lou 1651050 1055 Pro Lye Lou Ann His Trp Phe Met Sor Asp Mot Lou Gly GiU Gln Gly 1060 1065 1070 Ser Ile Lou Ala Gin Phe Gin Giu Ala Lou Asp Arg Ala Tyr Thr Gin 1075 1080 1085 Lou Glu Oiu Sor Thr Ioe Lou His Asn Gly His Pho Thr Thr Asp Ala 1090 1095 1100 Ala Asn Trp Thr Ile Giu Gly Asp Ala His His Ala Ile Lou Giu As 1105 1110 111s 1130 Gly Arg Arg Val Lou Arg Lou Pro Asp Tr3Ser Sor Sor Val Sor Gin 112S 1 1135 Thr Ile GlU 11e Giu Ann Phe Asp Pro Asp Lye Giu Tyr Gin Lou Val 1140 1145 1150 Pho His Ala Gin Gly Clu Gly Thr Val Sor Lou Gin His Gly G].u GiU 1155 1160 1165 Oiy Giu Tyr Val GlU Thr His Pro His Lye Ser Ala Ann Phe Thr Thr 1170 1175 1180 WO 92/20802 Ser His Arg Gin 1185 Ile Thr Ser Glu Giu Ala Pro Leu 1220 Ser Asn Thr Asn 1235 PCr/US92/04316 Giy Val Thr Phe Glu Thr Asn Lys Val 1.190 1195 Asp Gly Glu Phe Leu Val Asp His Ile 1205 1210 Pro Thr Asp Asp Gin Ser Ser Asp Gly 1225 Ser Asn Thr Ser Met Asn Asn Asn Gin 1240 1245 Thr Val Glu 1200 Ala Leu Val 1215 Asn Thr Thr 12.30 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: BTYPE: nucleic acid CSTRANDEDNESS: double DTOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: A ORGANISM: Bacillus thuringiensis CINDIVIDUAL ISOLATE: PS63B (vii) IMMEDTATE SOURCE: CLONE: E. coli NM522(pMYC1642) NR7 L B-18961 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:S9:

ATGACTTGTC

ACTAGTAATA

ACCGTTAAAG

GCAGCTCTTG

TTAGTTCAAG

GTGGCAGTGC

CAAGAAAACC

TCTGATCAGT

CGTTTGGAAG

AGTAAATCAA

CTTGGCATGA

CTAGGCGCAA

OTTAATAAAG

CGAGCTAAAC

TTTACTGGGA

TACACTCGAG

CCAGATGACT

GTCGGGCAAA

GATTCACATC

CAGAAAGCAC

TGCTGGCCGT

GATCCAGGTC

ACTCAAACAG

CTTTGTACTC

AATAGTACTG

CTATATCCTT

AGTCATATTC

AATTACAAGC

CAGGTAGTCC

AGCTCAAGGA

AAAAGGGAT'r

CCGGCCTAGG

CTCTTATTAG

TTATTACAGT

TAATAAAGAA

AAGTAZTAAT

ATTATATGAA

GTGATTTTCT

CTATGAAACT

TTTATGATTT

AGCATATGCG

ATCTCCCTTC

CAATGGTATT

ATTCGTCTCA

GTGAGAGTAG

AACATGGATC

AACTTCGCAT

ATGGAGTGAT

TTTCAGGAGA

CCCAATATAC

TACGTGGCTA

GATATGGAGA

TTACACAAAC

CATATGACCT

GCAACCACTT

AATCGGCAAT

AGCATGGGAA

TGATGCAGCA

ATTAGTTGGT

CATGC7V2LGTT TATTG7?TAAG

ATTGAACGCA

AGATGCAACT

AGTGGATTCA

TACTGATACC

TTCAGCATAT

ATCATCAGAT

CCAAGACATA

ATTATCATCT

GAATGGCTTA

GATAAAACTG

AGATGGCAGC

CATAGGTCTC

GTATGATTAT

TTTAAACTAT

CGTTCAACTC

AGATGGAGAA

CTGTACTACA

AAGTTGCAAT

AAATGTGCTG

AAGTCCGAAC

ATTCCCTATA

GCAGGTAATC

GCGTTCCAAA

ATCGGAGGAG

ACGCTAGGCG

GGTGTTTTTT

GAAGTTCAGI.

GATTTAXTG

TTCG,'!,.TC

GCATATTTCT

TATTCAAAGC

CATAGTTATA

GAGGGAAAAA

GCATTTTATA

AATAAATATG

GATATAGTAG

GAGAAAACAC

GTAACGATTA

AATTCAATCT

AATCACAAAC

AACAAGAATA

CCAGCACCTA

AACATATGGA

AACTGTTTTC

CAATCACTTC

GGACAATCAG

AATACGATTG

ACGTACTAGC

AATTTGATCA

AA AACGGAAG

GATCCTTTGA

CCGCAATCCC

GGCCAAAGGG

GAATACTAGA

CTTTTACGGA

ACAAGCCTGT

CAACAGGAGG

TTACCTTCCC

TACAATTCGG

CAATGTCGCA

CAAGCCAAGC

CAATTAATGA

CAACATGGCC

GCGTGATCTT

AAAATATTTT

CTTATTTCCC

CTTATTGTAC

CCTTTAGATA

TGAGTGTAGT

CAGATACTG

CAGGAAGAGG

CAGGTCAAAA

GCAAACTAGG

GTGACAAAGA

AGGAGTTCCA

GTTTGAGCAA

TTTCTCATTA

TTATTTAGGT

TGGTGTTTCA

CACAAACAAC

TGAAAAGCTA

CCTAGTAACT

ACTACAAGTA

TATTCTTACT

ATTATATGTA

AAATACATGG

GGCTTTAGCA

TTTAAACATG

CTATAATGTA

TACCCTATAT

TTCAGATATG,

TGACAATACA

AGATGAGTTA

GGACTGTTTC

TGGCGATAAT

TAATGCCCAA

CCGCAGTTGG

TTGTTATAAT

AATACATGCA

ATTGCTAGCA

TACAGATTCT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 WO 92/20802 WO 9220802PCT/US92/04316

ACGAATATTG

GTTGAAATTA

TCTTGGGGAA.

GCAAGTACAA

ATTTATAACC

AACAAGATAC

TCTGTCGAAC

ATTTATTTAG

ACACAACCAA

GCAATAATAT

GGTGTTCCAG

CGTAGCAACG

GCTAGCTCGT

TCTAATCTAT

TCGrAAAAGG

'XACGAGAGTG

TGGATTTTAC

ACGATACTCC

AGATGACATT

TAGGCATAAA

TTCCATCTGG

ATCGACTTGA

TTAATTATCC

GGGAGAAATC

AAAATTCCCA

GATTTAAATT

CAAATTTAGT

AA

AATTCCAGTGW

GATAAATGGT

CAATAGCACA

AATCTTTTTT

CCCTGCTACA

AGGAATAAAT

GAAATTTCAT

GTTTCTTCCT

TATCACAAGT

AGGGAATGTT

AATATATCTT

AGTTAATTAC

AGATATTACA

GAAAAAGGGT

GCGAATGTAG

r7GTGGTCAAT

AATTTAGTGT

AAAGAGACTC

GGAAATTATT

GTTTTTTTCA

TTAGATCAAC

AGGTTACCTC

CGCGGGAATC

TCGGTGGGTG

TCACCTACTT

AGTGGTACCA

ATGCATCCAG

TTCAATTATC

'TATGGTCCG

ATGACGGGGG

CAGCTCACGA

CACTCATG?A

CAAATAATGG

CAGCAGCGCC

ATCGTTCCGG

AACTAACTAT

GCGATCGCCA

ATTCTTTCAC

TCACTGGCCA

TGGACAAAAA

TCCAGGCCAA

CTGTCGATAT

ATCGAATCCT

TTCAGTAGAT

TGTAAAAGAT

ATCATCTGCT

AACACAGTCA

AGAACCACCT

ATCGGCACA.A

AATTTTAGAC

TAACATTCAG

AGTACAAGTA

1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2412 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: B LYET: 8 amino acid s IILTYP :8 amino acid s CSTRANDEDNESS: single DTOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: A ORGArAISM: Bacillus thuringiensis CINDIVIDUAL ISOLATE: PS63B (vii) IMMEDIATE SOURCE: CLONE: E. coli NM522(pMYC1642) (xi) SEQUENCE DESCRIPTION: SEQ ID NRRL B-18961 Met 1 Thr Cys Gln Gin Ala Gin Pro Leu 10 Ile Pro Tyr Asn Val Leu Ala Gly Val Pro Thr Ser Asn Thr Giy Ser Pro Ile 25 Asn Gin Phe Trp glu Ala Asp Gin Phe Giu Gin Thr Val Lys Glu 40 Gly Asn Ala Gly Lou Lys Giu Ala Ala Ala Leu Giu Phe Gin Lys Ann Gly Ser Phe Ser Phe Asp Ala Ile Sly Gly Gly Phe Asp Tyr Lou Val Gin Ala Leu Gly Lou Pro Gly Val Phe Trp Pro Asp L 8 GiU Val Ala Val Pro Val Gly Lou Ile 105 Gin Giu Asp Giu Thr Leu Gly Ala Ala Ile Gly Thr Asn Ser Met Leu Asn Leu Ile 125 Lys Leu Ser Val Gly Val 110 Thr Val Ile Asp Gin Leu Val Gin Arg -130 Ile Lys Lys Leu An Leu Asn Ala Asp Leu Val 145 Arg Thr 160 Leu Glu Glu Ile Ile Asp Ala Thr 170 Lys Ser Ann T~r Met Lo5i y a s e Glu Ann His L s Pro Ala Tyr Val Leu Gln 190 i WO 92/20802 PCI/US92/04316 Phe Asp Met 225 Leu Gin Tyr Ser Met 305 Pro Phe Ile Gly Leu 385 Cys Tyr Pro Gly Argg Asn Lye Ser Pro Ala 545 Val Ser Gln Phe Met 625 Asn Ser Thr 210 Lye Aen Ala Thr Ser 290 Val Asp Ser Lys Leu 370 Arg Trp Gly Met Glu 450 Giy Ser Ile Gly Asn 530 Lye Glu Pro Tyr Phe 610 Thr Lys Thr 195 Tyr Leu Lys Leu Ser 275 Aen Leu Asp Asp Asn 355 Asn Met Pro Asp Ser 435 Asn Tyr Thr His Asn Gly Ile Gly Met 595 Aen Phe Ile Gly Ser Ser Val Ala 260 Gin Lye Asn Tyr Met 340 Ile Ser Tyr Tyr Asn 420 Val Ile Cys Gly Ala 500 Leu Thr Ile Ile Gin 580 Val Leu Pro Leu Leu Thr 215 His Leu Lye Asn Ile 295 Asp Gin Gin Asn Asn Ile Gly Ala As Aen Glu Pro Leu As~ Glu Trp Gly Arg As Lye Lye Thr 200 Phe Ser Ser Gin Met 280 Asn Ile Ile Ser Thr 360 Phe His Leu Leu Gin 440 Thr Cys Ser Phe Ala 520 Lye Lye Ile Met Gly Giu Gly Met Tyr Gin 235 Glu Arg Gly Aen Thr 315 Glu Arg His Glu Asn Asp Thr Ser Gl 475 Gin Thr Ile Asp Ala 555 Ala Thr Thr Asn Ala 635 Gly Ser Val 220 Phe Gly Gin Asn Val 300 Trp Lys Asp Gin Leu 380 Cys Lye Val Ala Arg Ser Aen Pro Ser Ser Aan Asn Aen Pro 620 His Aen Leu Gly Lye Asp Leu 285 Tyr Pro Thr Gly His 365 Gin Thr Asn Gin Gin 445 Leu Gly Leu Val Thr Ser Val Ser As Ile Asp Tyr Phe Gly Asn Thr Ile 270 Pro Thr Thr Arg Ser 350 Gly Lye Asp Thr Leu 430 Tyr Cys Cys Pro Leu 510 Asp Asn Gly Val Thr 590 Thr Tyr Ser Ser I WO 92/20802 PCT/US92/0431.6 Asn Val Lys Phe Thr Asn 675 Val Pro Leu Ser Val Glu Leu Ser Gly Lys Phe Gly Ser Ser Tyr Leu Asp His Val Phe 670 Leu Glu Phe Gin Pro Ile Asp Gin Pro Pro Thr Gin 690 Asn Tyr Pro Ile Thr Leu Pro His Gly Glu Pro Ala Ile Ile Trp Glu Lys Ser Gly Asn 725 Gly Asn Gin Leu Thr 735 Ile Ser Ala Gly Gly As 755 Asn Tyr Ser 770 Gin Gly 740 Val Pro Glu Asn 745 Arg Gin Ile Tyr Arg Gin Ile Leu Asp 760 Phe Ser Asn Gly Leu Ser Val 750 Lys Leu Val Ser Ser Ser Pro Thr Tyr Thr Asn Ile Asn Leu Val 785 Ser Asn Leu Asp Ile Ser Gly Thr Ile Gin Val Gin INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: SLENGTH: 8 amino acids B TYPE: amino acid C STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) Arg 1 SEQUENCE DESCRIPTION: SEQ ID NO:11: Glu Trp Ile Asn Gly Ala Asn INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: A LENGTH: 21 bases STYPE: nucleic acid SSTRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: AGARTRKWTW AATGGWGCKM A INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: A LENGTH: 20 bases B TYPE: nucleic acid SSTRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GARTGGWTAA ATGGRMSAA INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: A LENGTH: 8 amino acids BTY)E: amino acid CSTIANDEDNESS: single DTOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

L

S hybridization probe" /product= "AAT GAA GTA/T TAT CCA/T GTA/T AAT" standard name= "Probe B" /label= pobe-b /note= "probe b" (xi) SEQUENCE DESCRIPTION: SEQ ID WO 92/20802 PCT/US92/04316 57 Pro Thr Phe Asp Pro Asp Leu Tyr 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: I LENGTH: 24 bases BTYPE: nucleic acid SSTRANDEDNESS: single D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID CCNACYTTTK ATCCAGATSW YTAT 24 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: A LENGTH: 24 bases TYPE: nucleic acid STRANDEDNESS: single DTOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ iD NO:16: CCWACWTTYG ATMCASATMW TTAT 24 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: (A LENGTH: 14 amino acids BTYPE: 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 1 5 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: A LENGTH: 14 amino acids B TYPE: amino acid C) STRANDEDNESS: single D) TOPOLOSY: 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 1 5 1 INFORMATION FOR SEQ ID NC:19: SEQUENCE CHARACTERISTICS: A) LENGTH: 16 amino acids B) TYPE: amino acid C) STRANDEDNESS: single DTOPOLOGY: 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 1 5 10 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICSt A) LENGTH: 14 amino acids B) TYPE: amino acid STRANDEDNESS: single SUBSTITUTE

SHEET

WO 92/20802 PCT/US92/04316 58 TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Gin Leu Gln Ala Gin Pro Leu Ile Pro Tyr Asn Val Leu Ala 1 5 1D INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: SLENGTH: 10 amino acids STYPE: amino acid CSTRANDEDNESS: 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 1 5 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid C STRANDEDNESS: single DTOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Val Gin Arg Ile Leu Asp Glu Lys Leu Ser Phe Gin Leu Ile Lys 1 5 10 INFORMATION FOR SEQ ID N0:23: SEQUENCE CHARACTERISTICS: A LENGTH: 23 bases BTYPE: nucleic acid STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GCAATTTTAA ATGAATTATA TCC 23 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: A LENGTH: 17 bases BTYPE: nucleic acid CSTRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: CAAYTACAAG CWCAACC 17 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: A) LENGTH: 21 hases B)TYPE: nucleic acid SSTRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:25: TTCATCTAAA ATTCTTTGWA C 21 L I i l-~;r-uclcun I---ra ~~nzr;un~ .WO 92/20802 PCT/US92/04316 59 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: A LENGTH: 21 bases TYPE: nucleic acid STRANDEDNESS: single STOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: GCWACWTTAA ATGAAGTWTA T 21 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: A LENGTH: 21 bases TYPE: nucleic acid STRANDEDNESS: single SD TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: AATGAAGTWT ATCCWGTWAA T 21 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: (A LENGTH: 38 bases (B TYPE: nucleic acid (C STRANDEDNESS: single (D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: GCAAGCGGCC GCTTATGGAA TAAATTCAAT TYKRTCWA 38 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: SLENGTH: 37 bases BTYPE: nucleic acid CSTRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) i (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: AGACTGGATC CATGGCWACW ATWAATGAAT TATAYCC 37 I INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids (BI TYPE: amino acid C) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Glu Ser Lys Leu Lys Pro Asn Thr Arg Tr 1 5 1F INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: AI LENGTH: 29 bases TYPE: nucleic acid C STRANDEDNESS: single DTOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) L t-^ WO 92/20802 PCT/US92/043.16 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: TAACGTGTAT WCGSTTTTAA TTTWGAYTC 29 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: SLENGTH: 9 amino acids STYPE: amino acid SSTRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: Tyr Ile Asp Lys Ile Glu Phe Ile Pro 1 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: A LENGTH: 23 bases BTYPE: nucleic acid STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: TGGAATAAAT TCAATTYKRT CWA 23 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: A LENGTH: 23 bases TYPE: nucleic acid STRANDEDNESS: single DTOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: AGGAACAAAY TCAAKWCGRT CTA 23 INFORMATION FOR SEQ ID (xi) SEQUENCE DESCRIPTION: SEQ ID TTTAGATCGT MTTGARTTTR TWCC 24 INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: A LENGTH: 5 amino acids BTYPE: amino acid CSTRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: Ile Thr Ser Glu Asp 1 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: A LENGTH: 20 bases TYPE: nucleic acid STRANDEDNESS: single D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: TCTCCATCTT CTGARGWAAT WO 92/20802 PCT/US92/04316 61 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: I A LENTH: 8 amino acids BI TYPE amino acid CSTRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Leu Asp Arg Ile Glu Phe Val Pro 1

Claims (25)

1. A substantially pure toxin protein which is toxic to ants and which has at least one characteristic selected from: the amino acid sequence of said toxin conforms to the following Generic Formula: I MOXLt.EBYP XxxxxxXJXX 101 XXBBXXBXXX BXZLUXXXXX xxxx-xXXUZX LBLXJXXLxx XLXXXXXXXJ XLXXOKIXLXZ 401 XXXXBxxxxx xxxxxxxxxx 501 XXZXXxxxxx XXXxXBXxBB 601 XxxxxxxxxB XXXBXBEXXX 701 xxLXxxxxxx BXYUBLXxxx XXBXXXXXXX XXXXXXXXKX XXXOBXXXX* XOXXLXXBXX xxxxxXIXXB xXZXXXXXXY XxxxxxXXXX xxxxXXXXXX xxxUx*xxxx xx*x*xxxxx ZXXXXXE;XYX XLUZXUXBXL xUXBXXXXXX xxxXUXXXXB xxxxXXXXXX XXLXXXXXXX xxLBXXBXXX XXXXxxxxxX xxxxxxXXXE XXJXxBXXX1K BJXBOXX*LE BXXXXXZXXX LXXXXXXXXX XXPLXXX*XJ XX(XXXXXxxX XXEXZXXXXX XXXUXBXBXB XZXXXXXXZX BLKLXBBPX XXXXXBXXxX XXLZBLZBxB BXXBBXXXBX xxxxxxxxxX XXXXXxxxXL XXLXXXLXXX BXXXXPOBEX ZXXXXXXxXX XXX*)xxXXX XxXXXXXXXX XXXXXXXLXX XXBXXXXBXx XXXXXXXYXL XXXXXXYXBX FXxxXKXXXIKX PxxxxxXXxX xxXXxXXUXX XX*xxxxxxx PXYOXBOXXH XLOBXXXBXX XXYXXxxxxx XXXBXXXXXX XxXxxxxxxx XXXXXBxXXX LYXXXXXXXJ xxXXIxxxxx IE*IUPZXXXX ZXOxxxxxxX 4 I wherein A is ala, G is gly, M is Met, S is ser, C is cys, 1-1 is his, N is asn, T thr, D is asp, I is ile, P is pro, V is val, E is glu, K is lys, Q is gin, W is trp, F is ple, L is leu, R is argY is tyr, K is K or R, J is E or D, L is L or I, B is M, L, 1, V, or F, J is K, R, E, or D, 0 is A or T, U is N or Q, Z is G or S, X is any naturally occurring amino acid, lo except C, is any naturally occurring amino acid, x is any naturally occurring amino acid, except C, or complete omission of any amino acids, the amino acid sequence of said toxin is at least 50% homologous with toxin 86Q3(a) as herein defined; the amino acid sequence of said toxin has an alignment value of at least 100 Is with toxin 86Q3(a) the DNA which codes for said toxin hybridizes with DNA which codes for all or part of toxin 86Q3(a); the DNA which codes for said toxin hybridizes with a probe selected from tile group consisting of SEQ ID NO, 12, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO, 16, SEQ ID NO. 34, SEQ ID NO, 33, SEQ ID NO. 31, SEQ ID NO, 27, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO, 29, and SEQ ID NO. 37; (1 a portion of the nucleotide sequence codinig for said toxin can be amplified from total cellular DNA from a Bacillus t/itripngiensis strain using polymerase chain reaction with a reverse, primeir selected from SEQ ID NO. 34, SEQ ID NO. 33, SEQ ID NO. 31, SEQ ID NO, 37, or the complements of SEQ ID NO. 12 or SEQ ID NO. 13; and a forward primer selected from SEQ ID NO. t\WI'\nUS R\LUtRlus317TCWlAR L _I _U 62A 12, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 27, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 335, or SEQ ID NO. 29; or said toxin is immunoreactive with an antibody which immunoreacts with a toxin selected from toxins expressed by PS86Q3, toxins expressed by PS140E2, or toxins expressed by PS211B2, as herein defined.

2. The ant toxin, according to claim 1, wherein said toxin conforms to the Generic Formula.

3. The ant toxin, according to claim 1, wherein said toxin has an alignment value i of at least 100 with toxin 86Q3(a).

4. The ant toxin, according to claim 1, wherein the DNA coding for said toxin hybridizes with DNA which codes for all or part of toxin 86Q3(a). The ant toxin, according to claim 1, wherein the DNA coding for said toxin hybridizes with a probe selected from the group consisting of SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID I *i C i t1.\\VPUS1\LD)RR]0017:rTC\Vdfl\ I WO 92/20802 PCI/US92/04316 63 3 NO. 15, SEQ ID NO. 16, SEQ ID NO. 34, SEQ ID NO. 33, SEQ ID NO. 31, SEQ ID NO. 27, 4 SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 29, and SEQ ID NO. 37. 1 6. The ant toxin, according to claim 1, wherein said toxin is immunoreactive with an 2 antibody which immunoreacts with toxin 86Q3(a). 1 7. The ant toxin, according to claim 1, wherein a portion of the nucleotide sequence 2 coding for said toxin can be amplified from total cellular DNA from a Bacillus thuringiensis strain 3 using polymerase chain reaction with a reverse primer selected from the group consisting of SEQ 4 ID NO. 34, SEQ ID NO. 33, SEQ ID NO. 37, SEQ ID NO. 31, and the complements of SEQ ID NO. 12 or SEQ ID NO. 13; and a forward primer selected from the group consisting of SEQ ID 6 NO. 12, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 27, SEQ ID NO. 23, 7 SEQ ID NO. 24, SEQ ID NO. 35, and SEQ ID NC. 29. 1 8. The ant toxin, according to claim 7, wherein said reverse primer is SEQ ID NO. 33 2 or SEQ ID NO. 34 and 3 the forward primer is SEQ ID NO. 12 or SEQ ID NO. 13, and the poiymerase 4 chain reaction fragment is approximately 330 to 600 bp; the forward primer is SEQ ID NO. 15 or $EQ ID NO. 16, and the polymerase 6 chain reaction fragment is approximately 1000 to 1400 bp; or 7 the forward primer is SEQ ID NO. 27, SEQ ID NO. 23, SEQ ID NO. 24, or 8 SEQ ID NO. 29, and the polymerase chain reaction fragment is 1800 to 2100 bp. 1 9. The ant toxin, according to claim 7, wherein said reverse primer is a complement of 2 SEQ ID NO. 12 or SEQ ID NO. 13 and 3 the forward primer is SEQ ID NO. 15 or SEQ ID NO. 16, and the polymerase 4 chain reaction fragment is approximately 650 to 1000 bp; or the forward primer is SEQ ID NO. 27, SEQ ID NO. 23, SEQ ID NO. 24, or 6 SEQ ID NO. 29, and the polymerase chain reaction fragment is approximately 7 1000 to 1400 bp. 1 10. The ant toxin, according to claim 7, wherein said reverse primer is SEQ ID NO. 31 2 and 3 the forward primer is SEQ ID NO. 27, SEQ ID NO. 23, or SEQ ID NO. 29, and 4 the polymerase chain reaction fragment is approximately 2550-3100 bp; the forward primer is SEQ ID NO. 15 or SEQ ID NO. 16, and the resulting 6 polymerasc chain reaction fragment is 1750-2150 bp; 7 the forward primer is SEQ ID NO. 12 or SEQ ID NO, 13, and the polymerase 8 chain reaction fragment is approximately 850-1400 bp; llllllP4**---" WO 92/20802 PCT/US92/04316 the forward primer is SEQ ID NO. 35, and the polymerase chain reaction fragments are approximately 550-1050 bp.

11. The ant toxin according to claim 7, wherein said reverse primer is SEQ ID NO. 37 and the forward primer is SEQ ID NO. 27, SEQ ID NO. 23, or SEQ ID NO. 29, and the polymerase chain reaction fragment is approximately 3550-4050 bp; the forward primer is SEQ ID NO. i5 or SEQ ID NO. 16, and the resulting polymerase chain reaction fragment is 2600-3100 bp; the forward primer is SEQ ID NO. 12 or SEQ ID NO. 13, and the polymerase chain reaction fragment is approximately 1800-2400 bp; the forward primer is SEQ ID NO. 35, and the polymerase chain reaction fragment is approximately 1500-2050 bp.

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

13. The toxin, according to claim 1, wherein said toxin is expressed by PS140E2.

14. The toxin, according to claim 1, wherein said toxin is expressed by PS211B2. A nucleotide sequence encoding an ant toxin as defined in claim 1.

16. The nuclcotide sequence, according to claim 15, which encodes 86Q3(a).

17. The nucleotide sequence, according to claim 15, which codes for a toxin expressed by PS140E2.

18. The nuclcotide sequence, according to claim 15, which codes for a toxin expressed by PS211B2.

19. A host comprising a nuclcotide sequence which codes for an ant toxin as defined in claim 1. The host, according to claim 19, wherein said host expresses a toxin which immunorcacts with an antibody, which antibody immunoreacts with an ant.active toxin expressed by a microbe selected from the group consisting of PS86Q3, PS140E2, and PS211132.

21. The host, according to claim 19, which is a Bacllus thuringlensls r_

22. The host, according to claim 21, wherein said host has the characteristics of PS140E2.

23. The host, according to claim 21, wherein said host has the characteristics of PS211B2.

24. The host, according to claim 21, wherein said h vt has the characteristics of Bacillus thurineiensis PS86Q3. The host, according to claim 19, where said nucleotide sequence is a heterologous sequence which has been transformed into said host and wherein said heterologous sequence is expressed at sufficient levels to result in the production of said ant toxin.

26. The host, according to claim 25, wherein said host is capable of inhabiting the phylloplane or rhizosphere of a plant or is capable of survival in a baited trap.

27. The host, according to claim 25, which is transformed with a nucleotide sequence which codes for 86Q3(a).

28. A process for controlling 'herein said process comprises contacting said ants with an ant-controlling effective amount cr a toxin as defined in claim 1.

29. A formicidal composition comprising substantially intact cells which express a toxin as defined in claim 1. The formicidal composition, according to claim 29, wherein said cells have been treated to prolong their formicidal activity.

31. A biologically pure culture of Bacillus thuringiensis PS140E2, having the identifying characteristic of activity against hymenopteran pests of NRRL B-18812, or mutants thereof.

32. A biologically pure culture of Bacillus thuringiensis PS211B2, having the identifying characteristic of activity against hymenopteran pests of NRRL B-18921, or mutants thereof.

33. A biologically pure culture of Bacillus thuringiensis PS86Q3, having the identifying characteristic of activity against hymenopteran pests of NRRL B-18765, or mutants thereof.

34. A substantially pure toxin protein which is toxic to ants, substantially as hereinbefore described with reference to any one of the Examples. A nucleotide encoding an ant toxin, substantially as hereinbefore described with reference to any one of the Examples.

36. A host comprising a nucleotide sequence which codes for an ant toxin, substantially as hereinbefore described with reference to any one of the Examples. 3s 37. A process fc- controlling ants, substantially as hereinbefore described with reference to any one of the Examples. Dated 2 March, 1995 Mycogen Corporation Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON t? 1 t [GAWPUSER\LIBRR]003 I7:TCWV:IAR L