AU714456B2

AU714456B2 - Biological insect control agent

Info

Publication number: AU714456B2
Application number: AU26472/97A
Authority: AU
Inventors: Peter Daniel Christian; Annette Claudianos; Allan Daly; Rachael Elizabeth Duncan; Marie-Marthe Suner; John David Windass
Original assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Current assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Priority date: 1996-05-22
Filing date: 1997-05-01
Publication date: 2000-01-06
Anticipated expiration: 2017-05-01
Also published as: AU2647297A; WO1997044355A1; CO4600752A1; ID16968A

Description

WO 97/44355 PCT/GB97/01205 -1- BIOLOGICAL INSECT CONTROL AGENT The present invention relates, inter alia, to the biological control of insect pests, to genetically engineered insect pathogens capable of exerting such control, and to the polynucleotide sequences (and vectors containing them) engineered into such pathogens which enable them to exert such control.

The venoms of many social wasps have been extensively studied and are known to contain a potent array of biologically active amines, pain-producing neuropeptides, allergens and neurotoxins. Much less understood are the venoms of solitary wasps, especially those which lead a parasitic lifestyle. Many solitary parasitic wasps prey upon insects and more than 250 species have been observed to paralyse their host. Many of these species are in the family Braconidae. The majority of braconid wasps are primary parasites. Adults lay their eggs almost exclusively in or on other insects and, after hatching, the wasp larvae feed upon their host. One braconid species that has attracted attention is Bracon hebetor (Bracon Microbracon Habrobracon). Bracon hebetor hebetor) is a small (3 mm) parasite of Lepidopteran larvae which have a cryptic, or cocooning, lifestyle. Adult female wasps deposit eggs on the outside of host larvae while simultaneously injecting a paralysing venom. Within minutes, the host larvae become uncoordinated and eventually suffer complete paralysis.

Although not directly fatal, this paralysis is permanent and immobilises the insect until the wasp larvae emerge to feed upon their host. The venom of B. hebetor possesses an extremely potent paralysing activity. In larvae of the greater waxmoth, Galleria mellonella (G.

mellonella), it has been estimated that complete and permanent paralysis occurs at levels of 1 part venom to 200,000,000 parts host haemolypmph. Furthermore, the venom shows selective toxicity towards insects and between insect orders. Spider, crayfish, frog, rat and guinea-pig neuromuscular preparations all appear to be insensitive to the venom.

The paralysing component of B. hebetor venom is thought to act by presynaptically blocking excitatory glutamatergic transmission at neuromuscular junctions, possibly by inhibiting the release of synaptic vesicles.

Venoms from many arthropods that prey on insects have been found to contain toxins which selectively act on insects. However, since the published information on proteinaceous toxins from B. hebetor suggests that multiple toxins may exist it was first necessary to purify and characterise a toxin that had high neurotoxic activity to Lepidopteran larvae. We purified

I

WO 97/44355 PCT/GB97/01205 -2and characterised two neurotoxic proteins, which for ease of reference have been designated bracon toxin 1 and 2 (hereinafter BrhTX-1, and BrhTX-2). Inter alia, the present invention provides polynucleotide sequences which encode combinations of the subunits comprised by BrhTX-1.

According to the present invention there is provided a polynucleotide comprising a region encoding at least two of the insecticidal toxin subunits selected from those comprised in SEQ ID Nos. 2, 4, 6, 8, and 10 and that encoded by the spliced RNA derived from the genomic clone depicted in SEQ ID No. 19, the said spliced RNA being capable of hybridising with the extension products of the primers depicted in SEQ ID Nos 20 and 21, or SEQ ID Nos 22 and 23, or SEQ ID Nos 24 and 25 using the sequence depicted in SEQ ID No. 1 as a template, with the proviso that the polynucleotide does not encode only the combination of the subunits comprised in SEQ ID Nos. 2 and 8.

"SEQ ID No. 2" includes a protein which is identical to that depicted in SEQ ID No. 2 with the proviso that the Proline residue at position 93 in the amino acid sequence is replaced by Leucine.

By "capable of hybridising" is meant hybridisation with the sequence depicted in SEQ ID No. 1 following incubation of the extension products with the SEQ ID No. 1 sequence at a temperature of between 60 and 65*C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS.

In a preferred embodiment of the polynucleotide the said subunits are the proteins represented by amino acids 17 to 158 in SEQ ID No. 2; amino acids 22 to 182 in SEQ ID No.

4; amino acids 32 to 176 in SEQ ID No.6; amino acids 23 to 275 in SEQ ID No. 8; and amino acids 22 to 184 in SEQ ID No. 10. At least one of the said proteins may optionally comprise a heterologous N-terminal extension in the form of a signal or secretory peptide.

The invention also provides a polynucleotide comprising a region encoding at least one of the insectidal toxin subunits selected from those comprised in SEQ ID Nos. 2, 4, 6, 8 and and that encoded by the spliced RNA derived from the genomic clone depicted in SEQ ID No. 19, the said spliced RNA being capable of hybridising with the extension products of the primers depicted in SEQ ID Nos 20 and 21, or SEQ ID Nos 22 and 23, or SEQ ID Nos 24 and 25, using the sequence depicted in SEQ ID No. 1 as a template, wherein the region has WO 97/44355 PCT/GB97/01205 -3been modified in that mRNA instability motifs and/or fortuitous splice regions are removed, or insect-pest preferred codons are used so that expression of the thus modified polynucleotide in the said insect yields substantially similar protein having a substantially similar activity/function to that obtained by expression of the unmodified polynucleotide in the organism in which the protein encoding regions of the unmodified polynucleotide are endogenous.

It is preferred that the modified region encodes subunits which are the proteins represented by amino acids 17 to 158 in SEQ ID No. 2; amino acids 22 to 182 in SEQ ID No.

4; amino acids 32 to 176 in SEQ ID No.6; amino acids 23 to 275 in SEQ ID No. 8; and amino acids 22 to 184 in SEQ ID No. 10. At least one of the said proteins may optionally comprise a heterologous N-terminal extension in the form of a signal or secretory peptide. The insect pest may be Lepidopteran, and the said organism may be an Hymenopteran of the superfamily Ichneumonoidea, in particular a wasp of the family Braconidae. The skilled man is aware that the family Braconidae includes the genera Apanteles, Microbracon and Stenobracon.

At least one of the protein encoding sequences in the region may be under expression control of a viral promoter, or insect strong promoter, which is not down regulated or otherwise silenced when the polynucleotide is introduced into the cells of an insect which is or becomes infected by an insect virus. Suitable promoters are known to the skilled man and include, for example, the baculovirus pl0 promoter and the polyhedrin promoter.

In one embodiment of the invention, the said region of the polynucleotide preferably encodes not less than two and not more than four of the toxin subunits selected from the proteins represented by amino acids 17 to 158 in SEQ ID No. 2; amino acids 22 to 182 in SEQ ID No. 4; amino acids 32 to 176 in SEQ ID No.6; amino acids 23 to 275 in SEQ ID No.

8; and amino acids 22 to 184 in SEQ ID No. 10. In a further embodiment, the region likewise encodes not less than two and not more than three of the toxin subunits, and in a still further embodiment of the invention the region encodes two of the toxin subunits selected from the proteins represented by amino acids 17 to 158 in SEQ ID No. 2; amino acids 22 to 182 in SEQ ID No. 4; amino acids 32 to 176 in SEQ ID No.6; amino acids 23 to 275 in SEQ ID No. 8; and amino acids 22 to 184 in SEQ ID No. 10, with the proviso that the region does not encode only the combination of the toxin subunits comprised in SEQ ID Nos. 2 and 8.

In a particularly preferred embodiment, the polynucleotide comprises a region encoding the three proteins represented by amino acids 22 to 182 in SEQ ID No. 4 or amino WO 97/44355 PCT/GB97/01205 -4acids 22 to 184 in SEQ ID No. 10; amino acids 32 to 176 in SEQ ID No.6 and amino acids 23 to 275 in SEQ ID No. 8.

Where the region has been modified as indicated above by the provision of insect pest preferred codons, or removal of mRNA instability motifs or splice regions the region may encode only one of the toxin subunits selected from the proteins represented by amino acids 17 to 158 in SEQ ID No. 2; amino acids 22 to 182 in SEQ ID No. 4; amino acids 32 to 176 in SEQ ID No.6; amino acids 23 to 275 in SEQ ID No. 8; and amino acids 22 to 184 in SEQ ID No. The invention also provides a nucleotide sequence, encoding an insecticidal toxin subunit, which is complementary to one which when incubated at a temperature of between and 55°C in single strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with single strength citrate buffered saline containing 0.1% SDS still hybridises with the sequence depicted in SEQ ID Nos. 1, 3, 5, 7, 9, or 19. "SEQ ID No. 1" includes a polynucleotide which is identical to that depicted in SEQ ID No. 1, with the proviso that the triplet CCA at positions 289-291 is replaced with the triplet CCA. It is preferred that the nucleotide sequence, encodes an insecticidal toxin sub-unit, which is complementary to one which when incubated at a temperature of between 55 and 60"C in single strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.5 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequence depicted in SEQ ID Nos. 1, 3, 5, 7, 9, or 19. It is more preferred that the nucleotide sequence, encodes an insecticidal toxin sub-unit, which is complementary to one which when incubated at a temperature of between 60 and 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequence depicted in SEQ ID Nos. 1, 3, 5, 7, 9, or 19.

The invention also provides a nucleotide sequence, encoding an insecticidal toxin subunit, which is complementary to one which when incubated at a temperature of between and 60*C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequence represented by nucleotides 61 to 486 in SEQ ID No. 1; nucleotides 285 to 766 in SEQ ID No. 3; nucleotides 147 to 584 in SEQ ID No. WO 97/44355 PCT/GB97/01205 nucleotides 161 to 219 in SEQ ID No. 7; or nucleotides 85 to 573 in SEQ ID No. 9. It is more preferred that the nucleotide sequence encodes an insecticidal toxin sub-unit, which is complementary to one which when incubated at a temperature of between 60 and 65 0 C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequence represented by nucleotides 61 to 486 in SEQ ID No. 1; nucleotides 285 to 766 in SEQ ID No. 3; nucleotides 147 to 584 in SEQ ID No. 5; nucleotides 161 to 219 in SEQ ID No. 7; or nucleotides 85 to 573 in SEQ ID No. 9.

It will be appreciated that the said nucleotide sequence can constitute a whole or part 0o of the said region of the polynucleotide.

The invention still further provides a cell transformed with the polynucleotide or complementary sequence of the invention, as well as an organism regenerated from the transformed cell.

The invention still further provides an insect pathogen, or other biological control agent, comprising the polynucleotide or complementary sequence of the invention. Included within the term biological control agent are viral, prokaryotic or eukaryotic organisms which when brought into association with an insect are capable of infecting the insect and/or interfering with the normal biochemical, physiological or electrophysiological processes and ultimately leading to the death of the insect. Suitable biological control agents within the scope of the invention include those based on bacterial, viral and fungal pathogens of insects. Bacterial pathogens include for example Bacillus species such as B. thuringiensis, B. cereus and the like. Fungal pathogens of insects include for example Beauvaria species such as B. bassiana.

Viruses are particularly preferred. Suitable insect viral pathogens include baculoviruses, entomopoxviruses, reoviruses, iridoviruses, parvoviruses, rhabdoviruses, picornaviruses and picora-like viruses, nodaviruses, ascoviruses and retroviruses, with baculoviruses being preferred. Baculoviruses can be classified as a nuclear polyhedrosis virus (NPV) (includes NPVs with singly enveloped nucleocapsids (SNPV)) and NPVs with multiply enveloped nucleocapsids (MNPV)), granulosis virus and non-occluded baculovirus.

Examples of baculoviruses include: Autographa califomica MNPV (AcMNPV), Anagrapha falcifera MNPV (AfMNPV), Bombyx mori MNPV (BmMNPV), Anticarsia gemmatalis MNPV (AgMNPV), Cydia pomonella GV (CpGV), Helicoverpa armigera SNPV (HaSNPV), WO 97/44355 PCT/GB97/01205 -6- Helicoverpa zea SNPV (HzSNPV), Lymantria dispar MNPV (LdMNPV), Choristoneura fumiferana MNPV (CfMNPV), Mamestra brassicae MNPV (MbMNPV), Neodiprion sertifer SNPV (NsSMNPV), Orgyia pseudotsugata MNPV (OpMNPV), Pieris rapae GV (PrGV) and Spodoptera exigua MNPV (SeMNPV).

The recombinant baculoviruses may be mixed or otherwise treated with known fluorescent brighteners, in particular those of the stilbene disulphonic acid group. Such brighteners may enhance the intrinsic activity of baculoviruses, and provide enhanced levels of protection of the virus against ultra-violet light, which helps to retain the activity of the virus during exposure to uv light.

Alternatively the biological control agent can be a genetically modified plant endophyte in which the genome has been altered to incorporate the polynucleotide or complementary nucleotide sequence of the present invention. When such an endophyte is brought into association with a plant the toxin subunits encoded by the introduced DNA may be produced by the endophyte within the plant and exert toxic effects on insects feeding on or dwelling within the plant.

In a further variation the biological control agents can be a plant itself, particularly a crop plant being grown for food or fibre products, in which the plant genome has been modified by incorporation into it of the polynucleotide or complementary nucleotide sequence of the invention. Accordingly, plant tissue may be transformed, by means (electroporation, micro projectille mediated transformation, Agrobacterium mediated transformation, protoplast transformation, etc.) known to the skilled man, with the polynucleotide or complementary sequence and regenerated, again by known means, into intact fertile whole plants.

The invention still further provides insecticidal compositions comprising the following combinations of proteins: the proteins represented by amino acids 17 to 158 in SEQ ID No. 2 and 22 to 182 in SEQ ID No. 4; (ii) the proteins represented by amino acids 17 to 158 in SEQ ID No. 2 and 32 to 176 in SEQ ID No. 6; (iii) the proteins represented by amino acids 22 to 182 in SEQ ID No. 4 and 32 to 176 in SEQ ID No. 6; WO 97/44355 PCT/GB97/01205 -7- (iv) the proteins represented by amino acids 22 to 182 in SEQ ID No. 4 and 23 to 275 in SEQ ID No. 8; the proteins represented by amino acids 32 to 176 in SEQ ID No. 6 and 23 to 275 in SEQ ID No. 8; (vi) the proteins represented by amino acids 17 to 158 in SEQ ID No. 2, amino acids 32 to 176 in SEQ ID No. 6, and amino acids 23 to 275 in SEQ ID No. 8; (vii) the proteins represented by amino acids 17 to 158 in SEQ ID No. 2, amino acids 22 to 182 in SEQ ID No. 4, and amino acids 32 to 176 in SEQ ID No. 6; (viii) the proteins represented by amino acids 17 to 158 in SEQ ID No. 2, amino acids 22 to 182 in SEQ ID No. 4, and amino acids 23 to 275 in SEQ ID No. 8; (ix) the proteins represented by amino acids 22 to 182 in SEQ ID No. 4, amino acids 32 to 176 in SEQ ID No. 6, and amino acids 23 to 275 in SEQ ID No. 8; the proteins represented by amino acids 17 to 158 in SEQ ID No. 2; amino acids 22 to 182 in SEQ ID No. 4, amino acids 32 to 176 in SEQ ID No. 6, and amino acids 23 to 275 in SEQ ID No. 8.

Each of the insecticidal compositions given above may be augmented by or at least one of the proteins in the composition replaced by protein represented by amino acids 22 to 184 in SEQ ID No. 10, and/or that encoded by the spliced RNA derived from the genomic clone depicted in SEQ ID No. 19, the said spliced RNA being capable of hybridising with the extension products of the primers depicted in SEQ ID Nos 20 and 21, or SEQ ID Nos 22 and 23, or SEQ ID Nos 24 and 25, using the sequence depicted in SEQ ID No. 1 as a template.

The invention still further provides a method of controlling insects, comprising exposing them or their habitat to one or more of the following: cells transformed with the polynucleotide or complementary sequence of the invention; (ii) organisms regenerated from the transformed cells; (iii) recombinant insect pathogens comprising the polynucleotide or sequence of the invention; and (iv) the insecticidal composition disclosed in the two immediately preceding paragraphs.

The invention still further provides the use of the inventive polynucleotide or sequence in the preparation of a recombinant insect-pathogen, particularly a baculovirus for the biological control of insect pests.

_i WO 97/44355 PCT/GB97/01205 -8- The invention still further provides a polynucleotide comprising a region encoding the insecticidal toxin subunit encoded by the spliced RNA derived from the genomic clone depicted in SEQ ID No. 19, the said spliced RNA being capable of hybridising with the extension products of the primers depicted in SEQ ID Nos 20 and 21, or SEQ ID Nos 22 and 23, or SEQ ID Nos 24 and 25 using the sequence depicted in SEQ ID No. 1 as a template, the translation product of the polynucleotide and recombinant insect-enteropathogenic viruses comprising the said polynucleotide or a nucleotide encoding the spliced variant thereof.

The invention will be further apparent from the following description taken in conjunction with the associated Figures and Sequence listings.

Figure 1 is a composite sequence of the genomic region encoding the BrhTX- l(a) cDNA, putative exons are indicated in bold and translation of the putative message is shown below the coding sequence.

Figure 2 is an alignment of homologous regions of the genomic clones pBH(a)X3.4- Pst.2 and pBH(a)X3.5-Pst.5, the former contains the sequences that encode BrhTX-1(a) (see Figure 1).

Figure 3 is a plasmid map of pMMS 1.

Figure 4 is a plasmid map of pAcUW21.

SEQ ID No. 1 shows a first nucleotide sequence comprising a region encoding a first insecticidal toxin subunit (BrhTX-la) having a molecular weight of about 17kDa. SEQ ID No. 2 shows the translational product of the ORF identified in SEQ ID No. 1. Amino acids 1- 16 of the sequence shown in SEQ ID No. 2 constitute a putative leader or signal sequence.

Amino acids 17 to 158 of the sequence constitute the mature toxin-subunit.

SEQ ID No. 3 shows a second nucleotide sequence comprising a region encoding a second insecticidal toxin subunit (BrhTX-lb) having a molecular weight of about 18kDa.

SEQ ID No. 4 shows the translational product of the ORF identified in SEQ ID No. 3.

Amino acids 1 to 21 of the sequence shown in SEQ ID No. 4 constitute a putative leader or signal sequence. Amino acids 22 to 182 of the sequence constitute the mature toxin-subunit.

SEQ ID No. 5 shows a third nucleotide sequence comprising a region encoding a third insecticidal toxin subunit (BrhTX-lc) having a molecular weight of about 2 kDa as judged by SDS electrophoresis.

WO 97/44355 PCT/GB97/01205 -9- SEQ ID No. 6 shows the translational product of the ORF identified in SEQ ID No. together with a putative signal peptide.

SEQ ID No.7 shows a fourth nucleotide sequence comprising a region encoding a fourth insecticidal toxin subunit ((BrhTX-ld) having a molecular weight of about 32kDa. SEQ ID No. 8 shows the translational product of the ORF identified in SEQ ID No. 7. Amino acids 1 to 22 of the sequence shown in SEQ ID No. 8 constitute a putative leader or signal sequence.

Amino acids 23 to 275 of the sequence constitute the mature toxin-subunit.

SEQ ID No. 9 shows a fifth nucleotide sequence substantially similar to that depicted in SEQ ID No. 3 comprising a region encoding a fifth insecticidal toxin subunit ((BrhTX-le) having a molecular weight of about 18kDa. SEQ ID No. 10 shows the translational product of the ORF identified in SEQ ID No. 9. Amino acids 1 to 21 of the sequence shown in SEQ ID No. 10 constitute a putative leader or signal sequence. Amino acids 22 to 184 of the sequence constitute the mature toxin-subunit.

SEQ ID No. 11 shows a genomic sequence which comprises part of a spliced RNA that is related to the cDNA comprised by pBrhTX-l(a)l.1, and which encodes a toxin subunit which is substantially similar to the protein depicted in SEQ ID No. 2.

SEQ ID No. 12 shows the sequence of pBrhTX-l(a)GBspl and pBrhTX-l(a)GBsp3; SEQ ID No. 13 shows the sequence of pBrhTX-l(a)GBsp2 and pBrhTX-l(a)GBsp4; SEQ ID No. 14 shows the sequence of the 700bp Spe I fragment isolated from the X(a)G- 2.1.1 genomic clone.

SEQ ID Nos. 15-18 and 20 44 show the sequences of the various PCR primers.

SEQ ID No. 19 shows a contiguous genomic sequence which encodes part of a spliced RNA that is related to the cDNA comprised by pBrhTX-l(a)l.1, and which encodes a protein which is substantially similar to the protein depicted in SEQ ID No. 2 SEQ ID No. 45 discloses a putative toxin-encoding-sequence derived from the genomic sequence depicted in SEQ ID No. 19 and SEQ ID No. 46 discloses the translation product of the nucleotide sequence depicted in SEQ ID. No. International Patent Application No. PCT/GB95/02720 (Publication No. WO 96/16171) describes the isolation and characterisation of BrhTX-1 and BrhTX-2. The application also describes, inter alia, the sequence analysis of BrhTX-1 and the cloning of the sequences encoding the subunits of this toxin. These descriptions are incorporated by WO 97/44355 PCT/GB97/01205 reference into the present application. Unless specifically indicated to the contrary, all amino acid and nucleotide sequences which are depicted in, or are referred to in the following Examples have already been published in International Patent Application No.

PCT/GB95/02720 (Publication No. WO 96/16171). Accordingly, such known sequences are not included in the sequence listing appended to this application.

Example 1 -Identification of a BrhTX-l(a) coding sequence in addition to that disclosed in W096/16171 Northern Analysis of 17kDa cDNA Sequence Batches of mRNA for Northern analysis are isolated from approximately 300 female Bracon hebetor wasps using a "QuickPrep" Total RNA Extraction Kit (Pharmacia, Uppsala, Sweden). Integrity and estimates of concentration of isolated RNA from each preparation are made by electrophoresis through MOPS/formaldehyde, 1% agarose gels. Blots for hybridisation are made with a total of 20 female equivalents per lane from 1% agarose gels containing MOPS/formaldehyde, Transfers to hybridisation membrane are made using 20-2x SSC gradients and hybridisations are carried out in buffers containing 50% formamide at 42 0

C.

32 P-labelled probes are synthesised by PCR from short regions at the 5' end in the middle and at the 3' end of the BrhTX-1 cDNA. For all probes the cDNA clone, BrhTXl(a)1.2 digested with Not I is used as template. Primers for the 5' end are BH(a)F5 and BH(a)R5 (SEQ ID Nos 20, 21), middle BH(a)F3 and BH(a)R6 (SEQ ID Nos 22, 23) and for the 3' end BH(a)F6 and BH(a)R4 (SEQ ID Nos 24, Reaction conditions for generation of 32 P-labelled probes using the above primers are as follows: minutes at 95 0

C

30 seconds at 95 0 C, 30 seconds at 45 0 C, 1 minutes at 72 0 C (5 cycles); then seconds at 95 0 C, 30 seconds at 50 0 C, 1 minute 72 0 C (30 cycles) then, minutes at 72 0

C

Blots are then hybridised with one of the 3 probes middle or as detailed above.

Results from this analysis are shown in Table 1: WO 97/44355 PCT/GB97/01205 11 The minimum size of the message (as deduced from the cDNA clone) is about 560 bases with all of the 3' untranslated accounted for i.e. the clone contains the polyA tail. It therefore seems likely that the 706 base band identified above corresponds to the message encoding BrhTX-l(a). The presence of the 1694 base band however suggests that another RNA species is produced that contains sequences related to regions of the BrhTX-l(a)1.1 cDNA clone.

Genomic Southern Analysis Genomic DNA is prepared as described for the Examples in WO-99/16171, and aliquots are digested with the restriction enzymes Aat II, Acc I, Apa L Barm HI, Bcl I, Bsp HI, Cla I, Eco RI, Eco RV, Hind III, Kpn I, Mbo I, Nco I, Nde I, Not I, Pst I, Sac I, Sac IL Sal I, Sma I Spe I, Xba I and Xho I. Fragments are then separated by electrophoresis through 0.7% agarose gels in TAE buffer and the DNA blotted onto nitrocellulose membranes (Nitro-pure, supported nitrocellulose, MSI, MA, USA) in a 2-20x SSC gradient and bonded to the membrane by baking at 80 0 C for 2 hours. 32 P-labelled probes are synthesised by PCR using the primers BH(a)F2 and BH(a)R4 (SEQ ID Nos 26, 25) and pBrhTX-l(a)l.2 plasmid DNA as target DNA under the following conditions: minutes at 95 0

C

seconds at 95 0 C, 30 seconds at 45 0 C, 1 minutes at 72 0 C (5 cycles); then seconds at 95 0 C, 30 seconds at 50 0 C, 1 minute 72 0 C (30 cycles) then, 5 minutes at 72 0

C

Pre-hybridisation and hybridisation are performed as previously described.

The majority of restriction digests reveal a single hybridising fragment of over 10Kb. Eight enzymes however, generate fragments of under 10 Kbp. In some instances more than one Table 1 Results of Northern Analysis Carried out with BrhTX- cDNA Region-Specific Probes Probe Major Hybridising Minor Hybridising Band Band 706b and 1694b Middle 706b 1694b 3' 1694b 706b

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WO 97/44355 PCT/GB97/01205 -12hybridising fragment is observed with one of the fragments hybridising more strongly than the other. The sizes and relative hybridisation intensity of the fragments from these digests are summarised below.

Major Minor Xba I 5.7Kbp Spe I >16Kbp 0.7Kbp Nde I 7.0Kbp 0.9Kbp Bsp HI 3.8Kbp 1.2Kbp Mbo I l.lKbp Pst I 3.4Kbp Bcl I 1.4Kbp Acc I 2.9Kbp and Initial analysis of the above data suggests that there is a single BrhTX-l(a) gene in the Bracon genome, and that the gene contains an Acc I, Xba I, Spe I, Nde I and Bsp HI site.

However, further Southern analysis using the restriction enzymes Pst I, Xho I and Nde I and the three probes designed to be homologous to regions at 5' end, middle and 3' end of BrhTX-l(a) cDNA gives the results presented in Table 2. In contrast to the results obtained using a probe generated from the cDNA using the primers BH(a)F2 and BH(a)R4 (SEQ ID Nos 26, 25) the results using the much smaller region-specific probes indicates that there is a second sequence in the Bracon hebetor genome with sequence similarity to that of the BrhTX-l(a) cDNA. This latter observation is in agreement with the Northern hybridisation results that indicate a second mRNA with sequence homology to the pBrhTX-1(a) cDNA clone.

WO 97/44355 PCT/GB97/01205 -13- Table 2. Results of Southern Analysis Performed on 17Kda cDNA using Region- Specific Probes.

Probe Enzyme Major Minor Hybridising Hybridising Band Band Pst I 3.7Kb 4.7Kb Xho I >23Kb Nde 6.1 Kb Middle Pst I 3.7Kb 4.7Kb Xho I >23 Kb Vde I 6.1Kb and 0.9Kb 3' Pst I 3.7Kb Xho I >23Kb Nde I 0.9Kb Generation of a Genomic Library from Bracon hebetor Genomic DNA was prepared from female wasps as previously described and subjected to partial digestion with Sau3A I. The digested DNA is then ligated into XGEM-11 Xho I half site arms under conditions specified by the supplier (Promega, Madison, WI). After packaging the phage are plated into E. coli Strain LE392. Subsequent titration and amplification of the library was carried out using this strain.

Isolation and Sub-cloning of Genomic Sequences The XGEM-11 genomic library is plated and plaque lifts carried out as previously described for cDNA libraries. 32 P-labelled primers are synthesised by PCR as described above using the BH(a)F2 and BH(a)R4 primers (SEQ ID Nos 26,25). Pre-hybridisation and hybridisation of filters from plaque-lifts are performed under the conditions described for genomic Southern analysis. The primary screen identifies over a hundred plaques that hybridise strongly to the probes. Six of these are selected and taken through a further three rounds of plaque purification. DNA is then purified from the phage as previously described and restriction digests performed to estimate the size of the inserts. These insert sizes and designation of phage are shown below: WO 97/44355 PCT/GB97/01205 -14- Approx insert size ABH(A)G-2.1.1 21.0 Kbp XBH(a)G-3.1.1 19.1 Kbp ,BH(a)G-4.1.1 15.0 Kbp XBH(a)G-5.1.1 18.9 Kpb )BH(a)G-6.1.1 19.3 Kbp ABH(a)G-6.2.1 20.5 Kbp DNA is isolated from these phage and digested with the enzymes Eco RI, Spe I and Xho I and subjected to Southern blot analyses using the same probe and conditions as outlined above for their isolation. This analyses demonstrate that the clones all share a number of identical bands and are thus related to each other. Because of the close relationship between the clones further analyses are therefore concentrated upon one of the phage namely, XBH(a)G-2.1.1 Southern analysis of the genomic XBH(a)G-2.1.1 which includes the restriction enzymes Bsp HI Spe I and Pst I reveals fragments of 400bp, 700bp and 3.7kbp and 4.7kbp respectively that hybridise strongly to the 344bp probe generated by PCR from the cDNA clone, pBrhTX-l(a)1.2 (WO-99/16171) and the primers BH(a)F2 and BH(a)R4 (SEQ ID 26, Consequently, these fragments are isolated after electrophoresis on 0.8% agarose gels and cloned into the Nco I and Spe I sites of pGEM5z(f)+ (Promega) and pBluescript SK- for the Bsp HI and Spe I fragments respectively and into the Pst I site of pGEM for Pst I fragments.

Sequencing of the Lambda Genomic Clone and Subclones Sub-cloned Bsp HI fragments clones (pBrhTX-1(a)GBsp1, pBrhTX-l(a)GBsp2, pBrhTX-l(a)GBsp3 and pBrhTX-l(a)GBsp4) and Spe I fragments (pBrhTX-l(a)GSpel pBrhTX-l(a)GSpe2, pBrhTX-1(a)GSpe3, pBrhTX-1(a)GSpe4, pBrhTX-l(a)GSpe6) are sequenced using the ABI Prism Dye terminator cycle sequencing kit with AmpliTaq DNA Polymerase, FS and analysed using the ABI 373 Automated Sequencer.

Two distinct sequences are obtained from the four Bsp HI sub-clones analysed.

pBrhTX-l(a)GBspl and pBrhTX-l(a)GBsp3 are identical (SEQ ID No 12) while pBrhTXl(a)GBsp2 and pBrhTX-l(a)GBsp4 are also identical to each other (SEQ ID No 13).

WO 97/44355 PCT/GB97/01205 However, an alignment of these two sequences reveals that although similar they are not identical. Analysis of the six Spe I subclones reveals a single sequence (SEQ ID No 14).

Initial sequence analysis is also carried out by direct sequencing from XBH(a)G-2.1.1 using the primers BH(a)F2, BH(a)F3, BH(a)F6, BH(a)R6, BH(a)R4, BH(a)R7, BH(a)R8, (SEQ ID Nos 26, 22, 24, 23, 25, 27, 28)and the ABI Prism dye-terminator cycle sequencing kit with AmpliTaq DNA Polymerase, FS. Samples from cycle sequencing reactions are analysed using the ABI 373 Automated Sequencer. No sequence is obtained from the primers BH(a)F2 and BH(a)F3 and the sequencing signal from BH(a)R7 gives dual peaks at some bases. These dual peaks can in most instances be resolved by reference to the graphic summary of the sequencing analysis. Using these sequence data an initial sequence is obtained spanning the whole of the region containing all of the putative cDNA sequence represented by the pBrTX-1(a)1.2.

Alignment of this initial sequence is then carried out with the pBrhTX-1(a)GBspl/3 sequence which showed a high degree of homology. In contrast alignments performed with the pBrhTX-l(a)GBsp2/4 sequence shows considerable differences from the putative genomic sequence (including two deletions/insertions of 2 and 3 bp). As all of these fragments are isolated and cloned from a single X genomic clone, the implication from the above data is that there is more than one sequence in that X clone with relatively high homology to the 5' end of the BrhTX-l(a) cDNA.

The two Pst I sub-clones (termed pBH(a)X3.5-Pst.2 and pBH(a)X3.5-Pst.5) are sequenced using the primers BH(a)F2, BH(a)F3, BH(a)F6, BH(a)R6, BH(a)R4, BH(a)R7, BH(a)R8 (SEQ ID Nos 26, 22, 24, 23, 25, 27,28) and the ABI Prism dye-terminator cycle sequencing kit with AmpliTaq DNA Polymerase, FS. A number of Hinc II, Xba I and Acc I fragments are also subcloned from the pBH(a).3.5-Pst.2 and pBH(a)23.5-Pst.5 subclones and sequenced using the automated dye-terminator method described above.

From all of the sequence data available a consensus of the genomic region encoding the mRNA from which the BrhTX-l(a) mRNA is derived (SEQ ID 19). This sequence is shown in Figure 1 with the putative intron/exon structure and the translation of the deduced mRNA. This sequence is contained completely within the pBH(a)53.5-Pst.2 subclone.

Sequence analysis of the pBH(a).3.5-Pst.5 subclone and the subclones derived from it produce a single contiguous sequence that contains all of the pBrhTX-l(a)GBsp2/4 sequence WO 97/44355 PCT/GB97/01205 -16- (SEQ ID 13) and the 700bp Spe I subclones (SEQ ID 14) derived from XBH(a)G-2.1.1. This contiguous sequence is shown in SEQ ID 19. An alignment of this sequence with the genomic sequence that encodes BrhTX-l(a) is shown in Figure 2.

While the genomic sequence that encodes BrhTX-l(a) and the sequence (SEQ ID 19) that shows homology to it, are both located in the 21.0 kbp fragment of genomic DNA contained in XBH(a)G-2.1.1, the arrangement of the sequences relative to each other is not known. To determine this four primers are designed as shown below and in SEQ ID Nos 29, 31, 32.

BH(a)PST2R 5'-GTA ACC AGC TAA GCA TAA CG- 3' BH(a)PST2L 5'-GTT ATA CAC AGA GGA TCA GGG AG-3' 5'-AAT ACA GTC CCT ATA TAC CC-3' 5'-GGG ATG GGA ATA ATG ATG TCA-3' These primers are designed from sequences at the termini of the two Pst I subclones (BH(a),3.5-Pst.2 and pBH(a)3.5-Pst.5) with their orientation being away from the middle of those clones. All of the pairwise combinations of these primers are then used in PCR experiments under the following conditions using XBH(a)G-2.1.1 DNA as a target.

minutes at 95 0

C

Only the BH(a)PST2L and BH(a)PST5R combination produces an amplicon ie. a fragment of 305 bp. This indicates that the two fragments are virtually adjacent to each other within the XBH(a)G-2.1.1 clone. Sequencing with the primers BH(a)PST2L and directly from the XBH(a)G-2.1.1 DNA confirms that the Pst I fragments are adjacent in the genomic clone. In relation to the putative transcriptional orientation of the sequence encoding BrhTX-l(a), the homologous sequence is located in the fragment situated upstream.

Example 2 Clonin of subunit of BrhTX-1 This is performed as described in W096/16171. Briefly, from the N-terminal sequence given in Sequence ID No. 2 of WO96/16171, two multi-species primers are designed and are indicated below: BH(b)A 5'-AC(TCA) TTG TT(TC) AC(TCA) GA(TC) CG(TC) AA-3' WO 97/44355 PCT/GB97/01205 -17- BH(b)B 5'-GG (ATG)CC (AG)AA (AGT)GT (TC)TT (AG)TC-3' These primers are used in a PCR experiment to generate a PCR product from cDNA synthesised from female mRNA as previously described. The PCR reaction conditions for a 0.2 pl Taq DNA Polymerase/50 tl reaction are as follows: 5 minutes at 950C; then seconds at 95C; 1.5 minutes at 50 0 C; 1 minute at 72 0 C (5 cycles); then seconds at 95C; 1.5 minutes at 55°C; 1 minute at 72°C (30 cycles); then minutes at 72 0

C.

This PCR product are of approximately 54 bp, as estimated by PAGE in 15% gels in TBE buffer. Amplification products are visualised in the gels by staining with ethidium bromide. The fragment is then cloned into the EcoRV site of the commercially available plasmid pBluescript SK- that has been tailed with a thymine residue (T-tailing) using Taq DNA Polymerase. The cloned PCR product is sequenced using the following dye primers which are available from Applied Biosystems Inc: Universal M13-20 dye-primer: 5'-CAG GAA ACA GCT ATG ACC-3' M13 reverse dye-primer: 5'-TGT AAA ACG ACG GCC AGT-3' Sequencing is carried out using an ABI 370 A DNA analysis system (Applied Biosystems). The sequence for the PCR product is given below.

TTG TTT ACA GAC CGC AAG TGG TGT GGA CGT GCC GAT AAG ACT TTC GGC CC-3' (ii) Screening of library and Isolation of Clones Library plating is performed as described in W096/16171. Plaque lifts are performed in duplicate onto Nitropure nitrocellulose membranes, 137 mm 0.45 micron, (Microb Separations, Westboro, MA, USA). In the primary screen 3.6x10 5 plaques per filter (4 filters in total) are screened, in secondary and tertiary screens 100-250 plaques per filter are screened.

32 P-labelled probes are made by PCR. PCR is carried out in the presence of 2 P-dATP using the oligonucleotides BH(b)A and BH(b)B shown in Sequence ID Nos. 38 and 39 of W096/16171, and the cloned PCR product carrying the sequence shown in Sequence ID No.

40 (W096/16171) as the target.

The following are the PCR conditions: WO 97/44355 PCT/GB97/01205 18- 950C for 5 minutes prior to the addition of Taq polymerase; then for 30 seconds, 45°C for 1.5 minutes and 72 0 C for 1 minute (5 cycles); then for 30 seconds, 50°C for 1.30 minutes and 72°C for 1 minute (30 cycles); then 72°C for 5 minutes (1 cycle).

Filters are probed with the 32 P-labelled probe described above under the same prehybridising and hybridising conditions as described for the Genomic Southern Blot of subunit BrhTX-l(a). Ten plaques that hybridise to this probe are initially identified. Three of these are purified through second and third round screens to homogeneity. Plaque purified phage are picked into Iml of SM buffer plus 10 pl of chloroform.

Characterisation of Clones The plaque-purified phage are screened for the presence and size of a cDNA insert by PCR analysis using the oligonucleotides Xgt 11 forward and Xgt 11 reverse. Lambda DNA for PCR is prepared from purified stocks by boiling 10 pl. of the stock for 5 minutes. Two tl of this DNA is then used in PCR experiments. The amplified fragments are sized by electrophoresis through 0.8% agarose gels in TAE buffer and sized against markers of known size. Of the three purified phage, an insert of approximately 500 bp is found in one phage, which phage is designated XBrhTX-1 (b)1.

Subcloning and Sequencing of Clones Phage DNA is purified by isopycnic centrifugation in CsC1 as described in Sambrook et al (1989).

The cDNA insert from XBrhTX-1 (b)l is removed by digestion with Not I and ligated into pBluescript SK- that is digested with Not I and phosphatase treated. The insert of the clone is sequenced using the ABI dye-primer sequencing kit (as described above). The sequence of the plasmid clone, pBrhTX-1(b)1, insert is given in Sequence ID No. 41 of W096/16171. The amino acids 27-44 match those of the N-terminal sequence shown in Sequence ID No. 2 of W096/16171, but the very short apparent size of the ORF encoded by the sequence in Sequence ID No. 41 suggests that the clone is severely truncated. Two primers to the ORF at the 5' end of the clone, namely BH(b)C and BH(b)D as shown in Sequence ID Nos. 42 and 43 respectively of WO96/16171 are designed. Using these primers and pBrhTX-1 1.1 as a target a 32 P-labelled probe by PCR is generated under the following conditions: WO 97/44355 PCT/GB97/01205 -19minutes at 950C; seconds at 950C; 1.5 minutes at 450C; 1 minute at 72C( (5 cycles); then seconds at 95C; 1.5 minutes at 50°C; 1 minute at 72°C (5 cycles); then minutes at 720C.

The library is then re-screened using the conditions described above. Six strongly hybridising plaques are purified through to homogeneity in three rounds of plaque purification. cDNA inserts are removed from the phage by digestion with Not I and ligated into pBluescript SK- that has been digested with Not I and phosphatase treated. The sizes of the inserts are estimated by digestion with Not I and separation of restriction fragments through 0.8% agarose gels in TAE buffer.

The following cDNA insert sizes are estimated: %BrhTX-l(b)2 about 500 bp; and LBrhTX-1 XBrhTX-l(b)4, XBrhTX-l(b)5 and LBrhTX-1 (b)6 all at about 1200 bp.

The plasmid designated pBrhTX-l(b)6 is sequenced using the ABI 370 A DNA analysis system and the previously described dye primers. The sequencing is completed using the ABI dye-terminator system in conjunction with the BH(b)C, BH(b)D primers and two additional primers as shown below.

BH(b)E

GTTGTCAATACACCCTG

BH(b)F

AGAACGAGATGTTATTGTAT

The nucleotide sequence obtained is shown in SEQ ID No. 3 Translation of the nucleotide sequence is shown in Sequence ID No. 4, and gives a protein of either 182 or 165 amino acids depending upon the initiation codon used. The alternative proteins have hydrophobic leader sequences of either 21 or 4 amino acids respectively, giving a mature peptide of 161 amino acids.

Analysis of Clones Peptide Digestion/Sequencing As in W096/16171, the alignment of the sequences obtained against the deduced amino acid translations is shown below: Peptide Sequence MIKPGETYGDVTNK

EWVHDNAGTLLPR

cDNA Sequence MIKPGETYGDVTNK EWVHDNA

LLPR

Peptide Sequence PHTVYDKHESLQ

DVHDNAGTLLPR

Peptide Sequence PHTVYDKHESLY WVHDNA LLPQ WO 97/44355 PCT/GB97/01205 It is not unusual for the terminal amino acids to be subject to mis-sequencing; however, one of the peptide sequences sequenced on two separate occasions contains a characteristic GT amino acid pair. These GT pair is missing in the ORF predicted for the sequenced clone.

It can therefore be deduced that the clone pBrhTX-1(b)6 contains a sequencing artefact or that the cDNA from which it is generated contains an error, i.e. a six bp deletion generated during synthesis/cloning. The region containing the apparent "GT" pair/deletion is recloned by PCR. Primers BH(b)E and BH(b)F are used in the PCR experiments with cDNA as the target, to generate an amplification product. cDNA is synthesised from female B. hebetor mRNA as previously described for the construction of the cDNA library.

The amplification products are then cloned into the EcoRV site of pBluescript that are T-tailed as previously described. These clones are then sequenced and all are found to contain the same sequence as pBrhTX-l(b)6 flanking the apparent GT anomaly, but have an additional six bp corresponding to the codons for G and T, i.e. GGAACT.

From these data it appears that the GT anomaly arises during the cDNA synthesis/cloning of the original X clone. The consensus sequence for the pBrhTX-1(b) cDNA and the putative ORF are shown in SEQ ID No. 9.

Genomic Southern Blot Southern blots of male and female B. hebetor genomic DNA digested with Pst I, EcoRV, Acc I and Xho I are generated as previously described in W096/16171. A 3 P-labelled probe is generated by PCR from the 5' region of the pBrhTXl(b)6 clone using the primers BH(b)C and BH(b)D under the following conditions: minutes at seconds at 95 0 C; 1.5 minutes at 45°C; 1 minute at 72°C (5 cycles); then seconds at 950C; 1.5 minutes at 50°C; 1 minute at 72°C (5 cycles); then minutes at 72C.

Hybridisations is carried out with the probe as previously described. Results are identical for male and female DNA. In the Pst I digest two hybridising bands are observed of 13.0 kpb and 4.1 kbp. This result is consistent with the presence of a Pst I site in the probe hybridisation region of the cDNA sequence. The EcoRV, Ace I and Xho I digests produce single hybridising bands of 3.3 kbp, 13.0 kbp and 7.0 kbp respectively.

In a second experiment, 32 P-labelled probes are made from the 3' region of the clone using the primers BH(b)E and BH(b)F under the following conditions: WO 97/44355 PCT/GB97/01205 -21minutes at 950C; seconds at 95°C; 1.5 minutes at 45°C; 1 minute at 72°C (5 cycles); then seconds at 95°C; 1.5 minutes at 500C; 1 minute at 72°C (5 cycles); then minutes at 720C.

These probes hybridise to Southern blots prepared as previously described using male and female genomic DNA that has been digested with Bcl I, Nde I, Bgl II and Pst I. Results are identical for male and female DNA with the Bcl I and Bgl II digests single hybridising bands of 1.7 kbp and 9.1 kbp are generated. Although these results do not fully agree with the presence of both these sites within the probe hybridising region, i.e. one would normally expect to see two hybridising bands, both sites are relatively close to the ends of the probe region and the fragment containing the smaller portion of the hybridising region can evade detection with the large probes that are used in this experiment. In the Nde I and Pst I digests, single hybridising bands of 2.4 and 22.2 kbp are detected.

Recovery of an alternative form of the BrhTX-l(b) subunit The aim of recovering an alternative form of the BrhTX-1(b) subunit is to obtain a cDNA without the long 5' untranslated end and encoding the GT amino acid pair which peptide sequencing suggests might be present in some BrhTX-l(b) proteins but which is not encoded by clone pBrhTX-l(b)6. Such results might be explained if there are polymorphic alleles of the BrhTX-l(b) gene, alternatively, the two forms of BrhTX-l(b) may be encoded by mRNA molecules generated by differential processing of the primary gene transcript.

The strategy chosen to access DNA sequences encoding the candidate alternative form of BrhTX- is to use PCR to obtain products from the Bracon genomic and cDNA libraries. The PCR products can then be cloned and sequenced to check if the correct editing had taken place and whether the GT amino acids are encoded.

The PCR primers (KED(b)F and KED(b)R see below) are designed for this purpose so that just the coding sequence of the gene is obtained, i.e. no 5' nor 3' untranslated regions.

Suitable restriction sites for cloning and an optimised Kozak sequence are included in the primers.

KED(b)F TTTAGATCGCGGCCGCCACCAATGTCAATCATATGT AT-3' BglII NotI Kozak Start WO 97/44355 PCT/GB97/01205 -22- KED(b)R CCGGAATCGCGGCCGCTCATTATCAGTGGAAATCTAAAG -3' EcoRI NotI Stop Stop The PCRs are performed using 0.5lP (approximately 5x10 4 pfus) of genomic or cDNA library as template, 1 00pmol of each primer, 161 Ultrapure dNTPs (Pharmacia) [1.25mM of each], 10pl 10x buffer [500mM KC1, 100mM Tris, pH8.3, 15mM MgCl 2 0.1% gelatin] and 0.8P (4 units) Taq DNA polymerase (Perkin/Elmer Cetus). The reactions are carried out in a Hybaid OmniGene programmable dri-block and subjected to the following temperature regime: 5 minutes at 95 OC; seconds at 95 oC; seconds at 47 oC; 1.2 minutes at 72 oC; cycles followed by a final incubation period of 7 minutes at 72 OC.

No products are obtained from the genomic library. However a product of the expected size is obtained from the cDNA library. This product is digested with BgII and EcoR. It is then cloned into Bgi and EcoR-digested, phosphatase-treated pMMS 1.

(pMMS1 is a derivative of pUC19, the polylinker of which has been modified so that it contains Bgm and EcoRI restriction endonuclease sites. pMMS 1 still confers the ability to metabolise X-gal. A map of pMMS 1 is shown in Figure 3.

The PCR product is sequenced using a Sequenase kit (USB, Cleveland, Ohio) and primers shown below: BH(b)F1: CGATGGTATCGTCAACAT -3' BH(b)F2: CGAATAAAGGATITICAA -3' BH(b)F3: CACCCCATACTGTATATG -3' BH(b)R1: CCAGCTCTGCGGGAGAA -3' BH(b)R2: ATGCAATATTCCACAAC -3' BH(b)R3 TCTGCAGCAATCACCAA -3' The sequencing data shows that the PCR product encodes a BrhTX1-(b) subunit including the GT amino acid pair..

Eame 3 Production of recombinant polyhedrin positive AcMNPV derivatives WO 97/44355 PCT/GB97/01205 -23expressing the BrhTX-1(a) subunit of the toxin from B. hebetor Subcloning the cDNA into pAcUW21 pAcUW21 is a commercially available baculovirus transfer vector (R&D Systems Europe Ltd., Abingdon, Oxon, UK) which can be conveniently used to generate recombinant polyhedrin positive (pol AcMNPV derivatives.

It contains the polyhedrin gene under the control of the natural polyhedrin promoter whilst foreign genes can be inserted (in either EcoRI or Bgl II sites) so that they can be transcribed from the pl0 promoter. A plasmid map of pAcUW21 is shown in Figure 4.

The cDNA encoding the BrhTX-l(a) subunit is excised from clone pBrhTX-1(a)l.1 by EcoRI digestion. The released insert is then cloned into EcoRI digested, phosphatase-treated pAcUW21 with T4 DNA ligase in appropriate buffers (Sambrook 1989). Ligation progeny are recovered by transformation of E.coli DH5a cells under standard conditions (Sambrook 1989). Transformants are selected on LB agar plates containing 100Ig/ml ampicillin.

Transformant colonies are grown overnight in L-broth containing 100g/ml ampicillin at 37°C and candidate pAcUW21/BrhTX-l(a) recombinant plasmids are recovered using a Wizard minipreparation kit (Promega Corporation, Madison, WI). These plasmids are then subjected to sequence analysis across the insert/vector junctions using internal primers BH(a)F4 and BH(a)R4 (shown below and in SEQ ID No. 15 and 25 respectively) and using a Sequenase (USB, Cleveland, Ohio) kit.

BH(a)F4: 5'-TTATATGAAGTTCTTAGA-3' BH(a)R4: 5'-TrAAATTGATTCCAACGC-3' Two transformants, designated pACBH(a). and pACBH(a).2, have inserts in the correct orientation and are selected for recombinant baculovirus construction. A caesium chloride plasmid preparation is used for this purpose.

Cotransfection of Spodoptera frugiperda (Sf21) insect cells Cotransfection is carried-out using the BacPAK6 kit (Clontech Laboratories, Palo Alto, CA, USA). Cotransfection of Sf21 insect cells with Bsu36 I-digested BacPAK6 DNA and each transfer vector containing the BrhTX-1(a) subunit is carried according to the manufacturer's recommendations.

Isolation of recombinant AcMNPV A plaque assay is performed using standard methods (King, L.A. and Possee, R.D. (1992) The Baculovirus Expression System A Laboratory Guide. Chapman and Hall, London.) with cotransfection supernatant at dilutions of 10 10' 3 as well as neat supernatant. Individual polyhedrin positive plaques, which fail to stain WO 97/44355 PCT/GB97/01205 -24blue when exposed to the chromogenic substrate X-Gal, are picked into sterile microcentrifuge tubes containing 0.5ml TC100/10% FCS medium.

Mini-amplification of the recombinant viruses Mini-amplifications are carried-out using standard methods (King and Possee, 1992). Tissue culture flat flasks (25m 3 (Corning, New York, USA) are seeded with 1x10 6 Sf21 cells and incubated overnight at 280C to form a monolayer. Next day they are infected with 0.25ml of the virus stock obtained from the isolated plug. The flasks are then incubated at 28°C for 6 days. The supernatants are then collected and cell debris and polyhedrin inclusion bodies (hereinafter referred to as PIBs) pelleted by centrifugation in Falcon 2097 tubes at 3000rpm in a Sorvall RT6000B bench top centrifuge. Supernatants are stored at 4 0 C. Pellets are resuspended in 4ml sterile distilled water. PIBs are released by sonication in an MSE Soniprep 150, 20 seconds on, 20 seconds off for 2 cycles. The number of PIBs per ml is ascertained using a counting chamber (Weber Scientific International Ltd.) and a Nikon Labophot light microscope. PIB solutions are diluted to lx10' PIB/mi to be used in biological assessment of the virus activity against first instar Heliothis virescens larvae.

Example 4 Production of recombinant polvhedrin positive AcMNPV derivatives expressing the BrhTX-l(b) subunit of the toxin from B. hebetor Subcloning the BrhTX-l b) cDNA into pAcUW21 The cDNA encoding the Brh-TX-l(b) subunit is excised from clone pBrhTX-l(b)6 by NotI digestion. The insert is blunt-ended by filling in using the Klenow fragment of DNA polymerase using standard methods (Sambrook 1989). The insert is then cloned into EcoRI-digested, Klenow polymerase blunt-ended, phosphatase-treated pAcUW21 with T4 DNA ligase in appropriate conditions for blunt end ligations (Sambrook 1989). Ligation progeny are recovered by transformation of E.coli cells under standard conditions (Sambrook 1989). Transformants are selected on LB agar plates containing 100.tg/ml ampicillin. Transformant colonies are grown overnight in Lbroth containing 100tg/ml ampicillin at 37°C. Candidate pAcUW21/BrhTX-l(b) recombinant plasmids are recovered using a Wizard minipreparation kit (Promega Corporation, Madison, WI). These plasmids are then subjected to sequence analysis across the vector/insert junctions using PACF1 and PACR1 primers (see below and SEQ ID Nos. 17 and 18) and a Sequenase kit (USB, Cleveland, Ohio). One transformant, designated pACBH(b). 1, with the insert in the p: WO 97/44355 PCT/GB97/01205 correct orientation is selected for recombinant baculovirus construction. A caesium chloride plasmid preparation is used for this purpose.

PACF1: TTCCTTACGCGAAATACG -3' PACR1: GAATTATTATCAAATCAT -3' Co-transfection of Sf21 cells, isolation and miniamplification of recombinant AcMNPV is as described in Example 3.

Example 5 Production of recombinant polyhedrin positive AcMNPV derivatives expressing an alternative form of the BrhTX- subunit of the toxin from B. hebetor Subcloning the cDNA into pAcUW21 The insert encoding the edited BrhTX-l(b) subunit is as described in Example X is excised from pMMSedl8 by BglI and EcoRI digestion. This insert is then cloned into Bgll, EcoRI-digested, phosphatase-treated pAcUW21 with T4 DNA ligase under appropriate conditions and ligation progeny are recovered by transformation of E.coli DH5a cells under standard conditions (Sambrook 1989). Transformants are selected on LB agar plates containing 100g/ml ampicillin.

Transformant colonies are grown overnight in L-broth containing 100g/ml ampicillin at 37 0 C. Candidate pAcUW21/BrhTX-l(b) recombinant plasmids are recovered using a Wizard minipreparation kit (Promega Corporation, Madison, WI). These plasmids are then subjected to sequence analysis across the vector/insert junctions performed using PACF1 and PACR1 primers shown in Example 3 and a Sequenase kit (USB, Cleveland, Ohio). One transformant, designated pACBHED(b).2, with the insert is selected for recombinant baculovirus construction. A caesium chloride plasmid preparation is used for this purpose. Cotransfection of Sf21 cells, isolation and miniamplification of recombinant AcMNPV is described in Example 3.

Example 6 Production of recombinant polyhedrin positive AcMNPV derivatives expressing the BrhTX-l(c) subunit of the toxin from B. hebetor Subcloning the cDNA into pAcUW21 The cDNA encoding the BrhTX-1(c) subunit is excised from clone XBrhTX-1(c)5 by NotI digestion. The insert is blunt-ended by filling in using the Klenow fragment of DNA polymerase using standard methods (Sambrook 1989). It is then cloned into EcoRI-digested, Klenow polymerase blunt-ended, phosphatase-treated pAcUW21 with T4 DNA ligase under appropriate conditions for blunt end ligations (Sambrook 1989). Ligation progeny are recovered by transformation of E. coli DH5ca cells WO 97/44355 PCT/GB97/01205 -26under standard conditions (Sambrook 1989). Transformants are selected on LB agar plates containing 100g/ml ampicillin. Transformant colonies are grown overnight in L-broth containing 100gpg/ml ampicillin at 37°C. Candidate pAcUW21/BrhTX-l(c) recombinant plasmids are recovered using a Wizard minipreparation kit (Promega Corporation, Madison, WI). These plasmids are then subjected to sequence analysis across the vector/insert junctions using PACF1 and PACR1 primers and a Sequenase kit (USB, Cleveland, Ohio). One transformant, designated pACBH(c).1, with the insert in the correct orientation to be expressed from the vector p10 promoter is selected for recombinant baculovirus construction.

A caesium chloride plasmid preparation is used for this purpose. Cotransfection of Sf21 cells and isolation and mini-amplification of recombinant AcMNPV is as described in Example 3.

Example 7 Production of recombinant polyhedrin positive AcMNPV derivatives expressing the BrhTX 1(d) subunit of the toxin from B. hebetor Subcloning the cDNA into pAcUW21 The cDNA encoding the BrhTX-1(d) subunit is excised from clone pBrhTX-l(d)1.2 by EcoRI digestion. The released insert is cloned into EcoRI-digested, phosphatase treated pAcUW21 with T4 DNA ligase in appropriate buffer conditions (Sambrook 1989). Ligation progeny are recovered by transformation of E.coli cells under standard conditions (Sambrook 1989). Transformants are selected on LB agar plates containing 100.g/ml ampicillin. Transformant colonies are grown overnight in Lbroth containing 100gg/ml ampicillin at 37°C. Candidate pAcUW21/BrhTX-l(d) recombinant plasmids are recovered using a Wizard minipreparation DNA purification kit (Promega Corporation, Madison, WI). These plasmids are then subjected to sequence analysis across the insert/vector junctions performed using vector specific primers PACFI and PACR1 and using a Sequenase kit (USB, Cleveland, Ohio). One transformant, pACBH(d). with the insert in the correct orientation is selected for recombinant baculovirus construction. A caesium chloride plasmid preparation is used for this purpose. Cotransfection of Sf21 cells and isolation and mini-amplification of recombinant AcMNPV is as described in Example 3.

Example 8 Physical characterisation of recombinant AcMNPV by Southern Blot Analysis These analyses are undertaken to check that the physical environment and structure of the recombinant baculoviruses is the same as that of the relevant transfer vector.

Preparation of recombinant AcMNPV DNA xl 06 Sf21 cells are used to seed 35mm Nunc Petri dishes (Gibco BRL, Paisley, Scotland). The plates are incubated at room temperature WO 97/44355 PCT/GB97/01205 -27for 15 minutes to allow cells to attach and form a monolayer. The media is then removed and Iml (approximately 1x10 Non-occluded virions [NOVs]) of mini-amplification supernatant is added. The plates are left 1 hour at room temperature for infection to occur. The inoculum is then removed and 1.4ml TC100/10%FCS medium is added. The plates are incubated at 28 0 C overnight. Next morning the infected cell monolayers are harvested by scraping them off the plate, transferring to a 1.5ml microcentrifuge tube and centrifugation at 4000rpm for 2 minutes. The supernatant is then removed and the cell pellet resuspended in 250Wl TE. Next 250g1 lysis buffer (50mM Tris HC1, pH8], 5% P-mercaptoethanol, 0.4% w/v SDS, EDTA) is added. The solution is mixed gently to obtain a viscous cell lysate. 12.51 proteinase K (10mg/ml in TE, predigested at 37 0 C for 30minutes) and 2.5pl RNase A in H 2 0) are added and the tube incubated at 37 OC for 30minutes. The solution is extracted twice with 500gl (50:50) TE-saturated phenol/chloroform:isoamyl alcohol [24:1] and once with 5001, chloroform:isoamyl alcohol Infected cell DNA is precipitated by adding 50gl 3M Na acetate and 2 volumes absolute ethanol. Precipitation is for 5 minutes at room temperature and the DNA pelleted by centrifugation at 14 000rpm for 15 minutes. The DNA pellet is washed twice in 70% ethanol, air-dried for 5 minutes at room temperature and resuspended in 100l TE, pH8 overnight at 4 0 C Digestion of the baculovirus infected cell DNA and separation by agarose gel electrophoresis of each infected cell DNA preparation is digested using EcoRI (EcoRI and BglII for BrhTX-1(c) and edited BrhTX-l(b) subunits). The digests are run on a 1xTBE agarose gel and the DNA tranferred to nylon membranes (Hybond-N, Amersham, UK) using standard "Southern Blot" methods (Sambrook 1989).

Hybridisation of the subunit cDNA probe to the recombinant baculovirus DNA Preparation of the probe The subunit cDNA is excised from the appropriate vector using EcoRI for BrhTX-l(a), BrhTX-l(d) subunits, NotI for BrhTX-l(b) and EcoRIIBglI for BrhTX-l(c) and edited BrhTX-l(b) subunits. 25ng of the excised subunit DNA is denatured (by boiling for 10min) and then labelled by random priming with c 3 P dCTP (Amersham International, Amersham, UK) and Klenow polymerase (NBL Gene Sciences Ltd.,Cramlington, Northumberland, UK) using standard methods (Sambrook 1989). This probe is denatured by incubation in a boiling water bath immediately before use.

A

WO 97/44355 PCT/GB97/01205 -28- Hybridisation The membrane is UV cross-linked in a Stratalinker (Stratagene, USA) and prehybridised at 65 0 C for 2 hours in 5 x SSPE, 5 x Denhardt's Reagent, 0.5% SDS and 2 0 0.g/ml salmon sperm DNA in a Techne hybridisation oven. Hybridisation is carried-out in 5 x SSPE, x Denhardt's reagent, 0.5% SDS plus labelled probe at 650C for 16 hours. Membranes are washed in 3 x SSC (20 x SSC: 3.0M NaCl, 0.3M trisodium citrate); 0.1%SDS at 650C for 4 x minutes and then exposed to Kodak X-AR film at -80°C with intensifying screens. If there is a high background, another series of washes are performed at 0.1 x SSC; 0.1% SDS, 4 x 15 minutes and the filters re-exposed to film.

Example 9 Construction of recombinant AcMNPV derivatives capable of co-expression of BrhTX-1 subunits.

This assembly, which is intended to provide for efficient, co-ordinated, high level expression of combinations of the various toxin subunits (BrhTX-1(a), BrhTX-1(b), BrhTXl(c) and BrhTX-1(d)), can be achieved by use of AcMNPV transfer vectors which have been constructed to allow introduction of three or four heterologous genes downstream of independent polyhedrin or p10 very late promoters. Such transfer vectors can be custom built, in which case they could be designed so that they also carry an intact functional polyhedrin gene and can therefore be used to generate polyhedrin positive (pol+) progeny by in vivo recombination with one of several convenient intermediate AcMNPV viral vector DNA preparations, including: Baculogold T M (Pharmingen), BacPAK6 T (Clontech) or AcRP23.1acZ (Pharmingen).

Alternatively, commercially available co-expression transfer vectors can be used. In the case that three subunits are selected, pAcAB3 (Pharmingen) may be used. This can accommodate up to three heterologous genes under the transcriptional control of either two pl0 promoters or one polyhedrin promoter. In addition, pAcAB4 (Pharmingen)) is also available. This expression vector can accommodate up to four heterologous genes under the transcriptional control of two p10 promoters and two polyhedrin promoters. However, if this transfer vector is used to construct recombinant AcMNPV derivatives by recombination with conventional intermediate AcMNPV viral vectors, such as Baculogold T M BacPAK6 T or AcRP23.1acZ, only polyhedrin negative AcMNPV derivatives are made. These can be bioassayed by injection into suitable lepidopteran hosts e.g. 3rd/4th instar H.virescens.

However in a preferred embodiment they are used to construct polyhedrin positive (Pol+) WO 97/44355 PCT/GB97/01205 -29- AcMNPV derivatives directly by in vivo recombination with Bsu36 I linearised AcUW1-PH DNA (Weyer et al.(1990) J.Gen.Virol. 71 1525-1534) since this AcMNPV derivative has a functional polyhedrin gene in place of its non-essential p10 gene and has a replaceable lacZ gene within the region homologous and hence exchangeable with the above mentioned transfer vectors (see also PCT/GB95/00677). Pol+ recombinant baculoviruses can be readily employed in a variety of bioassays, including that described in Example 10 and model plant protection studies, when delivered per os.

The skilled worker will appreciate that the above options provide various permutations for construction of recombinant baculovirus derivatives capable of coexpression of the various subunits of the toxin.

Example 9(a) Construction of recombinant AcMNPV derivatives capable of co-expression of four subunits of BrhTX-1 Concerning the combination of BrhTX-l(a), BrhTX-l(b), BrhTX-l(c) and BrhTX-1(d) one may initially insert the BrhTX-l(d) gene, isolated from pBrhTX-1(d)1.2 as an EcoRI fragment and blunt ended by a fill-in reaction with Klenow DNA polymerase performed under standard conditions (Sambrook 1989), into a similarly filled BamHI site in the transfer vector pAcAB4. DNA sequence analysis is then used with likely recombinant plasmids to select those with an intact BrhTX-l(d) gene correctly orientated for transcription from one of the two polyhedrin promoters in pAcAB4. Next an insert encoding the BrhTX-l(c) gene, isolated from pBrhTX-l(c)5 as a NotI fragment and blunt ended by a fill-in reaction with Klenow DNA polymerase, is introduced into a similarly filled Spel site in an authenticated preparation of the intermediate pAcAB4/BrhTX-1(d) transfer vector. DNA sequence analysis is then again used to select those recombinant plasmids with an intact BrhTX-l(c) gene correctly orientated for functional transcription from one of the two p10 promoters carried by pAcAB4. One such authenticated pAcAB4/BrhTX-1(c)/BrhTX-1(d) recombinant transfer vector preparation is selected for further work and subject to Bgl digestion to linearise it downstream of the other pl0 promoter and blunt ended by Klenow fill in reaction. NotI digestion is then used to isolate a BrhTX-l(b) gene from pBrhTX-l(b)6. This fragment is again blunt ended by Klenow fill-in before ligation with the BglII pAcAB4/BrhTXl(c)/BrhTX-l(d) preparation under appropriate conditions to promote blunt end fragment joining. Again recombinant transfer vectors containing the correctly orientated BrhTX-l(b) WO 97/44355 PCT/GB97/01205 gene are recognised by DNA sequence analysis. Finally, the selected pAcAB4/BrhTX- 1(b)/BrhTX-l(c)/BrhTX-1(d) recombinant transfer vector is subject to StuI digestion to linearise it just downstream of the remaining polyhedrin promoter. EcoRI digestion is then used to isolate a BrhTX-l(a) gene from a suitable pUC19/BrhTX-l(a) recombinant. This fragment is again blunt ended by Klenow fill-in before ligation with the StuI pAcAB4/BrhTX-1(b)/BrhTX-(c)/BrhTX- preparation under appropriate conditions to promote blunt end fragment joining. The target recombinant transfer vectors containing the correctly orientated BrhTX-l(a) gene are then recognised by DNA sequence analysis. An ethidium bromide/CsCI purified preparation of the selected pAcAB4/BrhTX-l(a)/BrhTXl(b)/BrhTX-l(c)/BrhTX-l(d) recombinant transfer vector is then prepared for use in assembly of recombinant pol+ AcMNPV derivatives by co-tranfection of Sf21 cells with Bsu36 I linearised AcUW1-PH DNA as described in Example 3.

Alternatively, initially four independent linkers are inserted into the EcoRI/HindIII sites of pAlter-1 vector (Promega) to assemble four independent intermediate cloning vectors to facilitate cloning of the four subunits into pAcAB4. Then an insert encoding the BrhTXl(d) gene, isolated from pBrhTX-l(d)1.2 as an EcoRI fragment, is introduced into an EcoRI site in one of the pAlter-1 intermediate cloning vectors which carries a pair of Spe9 sites flanking the EcoRl site. DNA sequence analysis is then used with likely recombinant plasmids to select those with an intact BrhTX-l(d) gene. Spel digestion is then utilised to isolate the BrhTX-l(d) gene from the pAlter- intermediate vector. This fragment is subsequently introduced into an Xbal digested pAcAB4. DNA sequence analysis is then performed on recombinant plasmids to select those with an intact BrhTX-l(d) gene correctly orientated for transcription from one of the two polyhedrin promoters in pAcAB4. An insert encoding the BrhTX-l(b) gene, isolated from pBrhTX-1(b)6 as a Notl fragment, is then introduced into a NotI site in one of the pAlter-1 intermediate cloning vectors which carries a pair of BglII sites flanking the NotI site Again, DNA sequence analysis is used to select recombinant vectors with an intact BrhTX-1(b) gene. This BrhTX-1(b) gene is then released from the pAlter-1 vector with the Bglll restriction enzyme to clone it into the BamHI site in an authenticated preparation of the intermediate pAcAB4/BrhTX-l(d) transfer vector. DNA sequence analysis is then again performed on the product in order to select those recombinant plasmids with an intact BrhTX-1(b) gene correctly orientated for functional transcription WO 97/44355 PCT/GB97/01205 -31from the remaining polyhedrin promoter in pAcAB4. EcoRI digestion is then used to isolate a BrhTX-l(a) gene from pUC19/BrhTX- to clone it into an EcoRI site in one of the pAlter-1 intermediate cloning vectors which carries a pair of flanking Xba sites flanking the EcoR1 site. Once again, recombinant vectors containing the intact BrhTX-1(a) gene are recognised by DNA sequence analysis. One authenticated pAcAB4/BrhTX-l(d)/BrhTX-l(b) recombinant transfer vector preparation is selected for further work and subject to Spel digestion to linearise it downstream of one of the pl0 promoters. Xbal digestion is then used to release the BrhTX-l(a) gene from pAlter-1 before ligation with the Spel digested pAcAB4/BrhTX-1(d)/BrhTX-1(b) preparation. Recombinant transfer vectors containing the correctly orientated BrhTX-l(a) gene are recognised by DNA sequence analysis. Finally, NotI digestion is used to isolate a BrhTX-l(c) gene from a suitable recombinant and introduce it into the last pAlter-1 cloning vector which carries a pair of BamH1 sites flanking the EcoR1 site. Recombinant progeny of this cloning step are again analysed by sequencing, to isolate a plasmid with an intact BrhTX-l(c) gene. The selected pAcAB4/BrhTX-1(d)/BrhTX-l(b)/BrhTX-I(a) recombinant transfer vector is then subjected to BglI digestion to linearise it just downstream of the remaining pl0 promoter. Finally, the BrhTX-l(c) gene, isolated from pAlter-1 by BamHI digestion, is ligated to the Bglll pAcAB4/BrhTX- l(d)/BrhTX- (b)/BrhTX- preparation. The target recombinant transfer vectors containing the correctly orientated BrhTX-1(c) gene are then recognised by DNA sequence analysis. An ethidium bromide/CsCl purified preparation of the selected pAcAB4/BrhTX- 1(a)/BrhTX-l (b)/BrhTX- 1 (c)/BrhTX- 1(d) recombinant transfer vector is then prepared for use in assembly of recombinant pol+ AcMNPV derivatives by cotransfection of Sf21 cells with Bsu36 I linearised AcUW1-PH

DNA.

The skilled man will appreciate that adapters may be used to introduce an EcoRI or a NotI digested subunit into pAcAB4. Initially the BrhTX-1(d) gene, isolated from pBrhTX- 1(d)1.2 may be inserted as an EcoRI fragment, into an Xbal digested pAcAB4 vector using an Xbal-EcoRI adapter. DNA sequence analysis is then used with likely recombinant plasmids to select those with an intact BrhTX-1(d) gene correctly orientated from transcription from one of the two polyhedrin promoters in pAcAB4. Next an insert encoding the BrhTX-1(b) gene, isolated from pBrhTX-1(b)6 as a NotI fragment, is introduced into the BamHI site of an authenticated preparation of the intermediate pAcAB4/BrhTX-l(d) transfer vector utilising a WO 97/44355 PCT/GB97/01205 -32- BamHL/Bgll-Notl adapter. DNA sequence analysis is then again used to select those recombinant plasmids with an intact BrhTX-l(b) gene correctly orientated for functional transcription from the second polyhedrin promoter carried by pAcAB4. One such authenticated.pAcAB4/BrhTX- (d)/BrhTX-l(b) recombinant transfer vector preparation is selected for further work and subject to Spel digestion to linearise it downstream of one of the two pl0 promoters. EcoRI digestion is then used to isolate a BrhTX-l(a) gene from pUCI9/BrhTX-l(a). An Spel-EcoRI adapter is again utilised to allow ligation of the BrhTX- 1(a) gene with the Spel pAcAB4/BrhTX-1(d)/BrhTX-1(b) preparation under appropriate conditions. Again recombinant transfer vectors containing the correctly orientated BrhTX- 1(a) gene are recognised by DNA sequence analysis. Finally, the selected pAcAB4/BrhTXl(d)/BrhTX- (b)/BrhTX-l(a) recombinant transfer vector is subjected to Bglll digestion to linearise it just downstream of the remaining pl0 promoter. Notl digestion is then used to isolate a BrhTX-l(c) gene from a suitable pBrhTX-l(c)5 recombinant. A BamHI/Bgill.NotI adapter is again utilised to allow ligation of the BrhTX-l(c) gene with the BglII pAcAB4/BrhTX- (d)/BrhTX-l(b)/BrhTX-1(a) preparation under appropriate conditions.

The target recombinant transfer vectors containing the correctly orientated BrhTX-l(c) gene are then recognised by DNA sequence analysis and are purified as indicated above.

Alternatively, the two methods described above can also be combined to produce a pAcAB4/BrhTX- (a)/BrhTX- (b)/BrhTX- 1(c)/BrhTX- recombinant transfer vector.

Eample 9(b) Construction o recombinant AcMNPV derivatives capable of co-expression f three subunits of BrhTX-l Concerning the production of recombinant baculovirus derivatives capable of providing for co-expression of BrhTX-l(a), BrhTX-1(c) and BrhTX-1(d) a pACAB4/BrhTX-l(c)/BrhTX- 1(d) recombinant transfer vector is selected as indicated above, and subjected to Stu I digestion to linearise it downstream of the other polyhedrin promoter. Eco RI digestion is then used to isolate a BrhTX-l(a) gene from a suitable pUC19/BrhTX-1(a) recombinant plasmid. This fragment is again blunt ended by Klenow fill-in before ligation with the Stu I pAcAB4/BrhTX-1(c)/BrhTX-1(d) preparation under appropriate conditions to promote blunt end fragment joining. Again recombinant transfer vectors containing the correctly orientated BrhTX-l(a) gene are recognised by DNA sequence analysis. An ethidium bromide/CsCI purified preparation of the selected pAcAB4/BrhTX- (a)/BrhTX- 1(c)/BrhTX- 1(d) WO 97/44355 PCT/GB97/01205 -33recombinant transfer vector is then prepared for use in assembly of recombinant pol+ AcMNPV derivatives by co-tranfection of Sf21 cells with Bsu36 I linearised AcUWI-PH DNA as described above for the four sub-unit assembly.

In the case that the recombinant baculoviruses should comprise genes encoding BrhTX-l(a), BrhTX-l(b) and BrhTX-l(d), a pACAB4/BrhTX-l(d) recombinant transfer vector is constructed as indicated above for either the three or four subunit assembly. Next an insert encoding the BrhTX-l(b) gene, isolated from pBrhTX-l(b)6 as a Not I fragment and blunt ended by a fill-in reaction with Klenow DNA polymerase, is introduced into a similarly filled Bgl II site in an authenticated preparation of the intermediate pAcAB4/BrhTX- (d) transfer vector. DNA sequence analysis is then used to select those recombinant plasmids with an intact BrhTX-l(b) gene correctly orientated for functional transcription from one of the two pl0 promoters carried by pAcAB4. One such authenticated pAcAB4/BrhTXl(b)/BrhTX-1(d) recombinant transfer vector preparation is selected for further work and subject to Stu I digestion to linearise it downstream of the other polyhedrin promoter. Eco RI digestion is then used to isolate a BrhTX-l(a) gene from a suitable pUC19/BrhTX-1(a) recombinant plasmid. This fragment is again blunt ended by Klenow fill-in before ligation with the Stu I pAcAB4/BrhTX-l(b)/BrhTX-l(d) preparation under appropriate conditions to promote blunt end fragment joining. Again recombinant transfer vectors containing the correctly orientated BrhTX-l(a) gene are recognised by DNA sequence analysis. An ethidium bromide/CsCI purified preparation of the selected pAcAB4/BrhTX- (a)/BrhTX-l(b)/BrhTX- 1(d) recombinant transfer vector is then prepared for use in assembly of recombinant pol+ AcMNPV derivatives by co-tranfection of Sf21 cells with Bsu36 I linearised AcUWI-PH

DNA.

In the case that the recombinant baculoviruse comprise genes encoding the BrhTX- BrhTX-l(c) and BrhTX-l(d) subunits, an authenticated pAcAB4/BrhTX-l(c)/BrhTX- 1(d) recombinant transfer vector preparation is produced as indicated above and subjected to BglI digestion to linearise it downstream of the other p10 promoter. NotI digestion is then used to isolate a BrhTX-l(b) gene from pBrhTX-l(b)6. This fragment is again blunt ended by Klenow fill-in before ligation with the Bgl II pAcAB4/BrhTX-l(c)/BrhTX-l(d) preparation under appropriate conditions to promote blunt end fragment joining. Again recombinant transfer vectors containing the correctly orientated BrhTX-l(b) gene are WO 97/44355 PCT/GB97/01205 -34recognised by DNA sequence analysis. An ethidium bromide/CsCl purified preparation of the selected pAcAB4/BrhTX- (b)/BrhTX-l(c)/BrhTX- recombinant transfer vector is then again prepared for use in assembly of recombinant pol+ AcMNPV derivatives by cotranfection of Sf21 cells with Bsu36 I linearised AcUWl-PH DNA.

Concerning the construction of recombinant baculovirus derivatives capable of coexpression of BrhTX-l(a), BrhTX-1(b) and BrhTX-1(c), one may initially insert the BrhTX- 1(c) gene, isolated from pBrhTX-l(c)5 as a Not I fragment and blunt ended by a fill-in reaction with Klenow DNA polymerase performed under standard conditions (Sambrook 1989), into a similarly filled Spe I site in the transfer vector pAcAB4. DNA sequence analysis is then used with likely recombinant plasmids to select those with an intact BrhTXl(c) gene correctly orientated for transcription from one of the two p10 promoters in pAcAB4. Next an insert encoding the BrhTX-l(b) gene, isolated from pBrhTX-l(b)6 as a Not I fragment and blunt ended by a fill in reaction with Klenow DNA polymerase, is introduced into a similarly filled Bgl II site in an authenticated preparation of the intermediate pAcAB4/BrhTX-l(c) transfer vector. DNA sequence analysis is then used to select those recombinant plasmids with an intact BrhTX-l(b) gene correctly orientated for functional transcription from the other plO promoter carried by pAcAB4. One such authenticated pAcAB4/BrhTX- (b)/BrhTX- recombinant transfer vector preparation is selected for further work and subject to Stu I digestion to linearise it downstream of one of the polyhedrin promoters. Eco RI digestion is then used to isolate a BrhTX-l(a) gene from a suitable pUC19/BrhTX-l(a) recombinant plasmid. This fragment is again blunt ended by Klenow fill in before ligation with the Stu I pAcAB4/BrhTX-l(b)/BrhTX-1(c) preparation under appropriate conditions to promote blunt end fragment joining. Again recombinant transfer vectors containing the correctly orientated BrhTX-l(a) gene are recognised by DNA sequence analysis. An ethidium bromide/CsCl purified preparation of the selected pAcAB4/BrhTX-l(a)/BrhTX-l(b)/BrhTX-l(c) recombinant transfer vector is then prepared for use in assembly of recombinant pol+ AcMNPV derivatives by co-tranfection of Sf21 cells with Bsu36 I linearised AcUW1-PH

DNA.

Example Constrnction of recombinant AcMNPV derivatives capable of co-expression of two subunits of BrhTX-1 WO 97/44355 PCT/GB97/01205 In addition to the AcMNPV transfer vectors used in the production recombinant baculoviruses comprising genes providing for expression of three or four toxin subunits, suitable commercially available co-expression transfer vectors can be used for the production of viruses comprising genes encoding only two subunits. One suitable example, pAcUW51 (Pharmingen), can accommodate up to two heterologous genes under the transcriptional control of one plO promoter or one polyhedrin promoter.

Recombinant baculovirus derivatives capable of co-expression of BrhTX-1(a) and BrhlX- 1(b) can be made by a method which involves the insertion, into an EcoRi digested phosphatase-treated pAcUW51 plasmid, of a BrhTX-1(a) gene isolated from a suitable pUC19/BrhTX-1(a) recombinant plasmid as an EcoRI fragment. DNA sequence analysis is then used with likely recombinant plasmids to select those with an intact BrhTX-l(a) gene correctly orientated for transcription from the pl0 promoter. An insert encoding the BrhTXl(b) gene is isolated from pBrhTX-l(b)6 as a Not fragment and blunt-ended by a fill-in reaction with Klenow DNA polymerase. This is introduced into a similarly filled Bar HI site in an authenticated preparation of the intermediate pAcUW51/BrhTX- 1(a) transfer vector.

DNA sequence analysis is then used with likely recombinant plasmids to select those with an intact BrhTX- gene correctly orientated for transcription from the polyhedrin promoter.

One such authenticated pAcUW51/BrhTX- (a)/BrhTX- recombinant transfer vector preparation is selected for further work. A caesium chloride purified preparation of the selected pAcUW51/BrhTX-l(a)/BrhTX-1(b) recombinant transfer vector is prepared for use in assembly of recombinant pol+ AcMNPV derivatives by co-tranfection of Sf21 cells with Bsu36 I linearised AcUWI-PH DNA as described in PCT/GB95/00677. Pol+ AcMNPV derivatives capable of co-expressing BrhTX-1(a) and BrhTX-l(b) are then isolated by the method described above in Example 3.

This method can be repeated for the production of recombinant baculoviruses comprising sequences encoding other binary combinations of subunits, for example, (i) BrhTX-l(a) and BrhTX-l(c); (ii) BrhTX-l(b) and BrhTX-l(c); (iii) BrhTX-l(b) and BrhTX- (iv) BrhTX-l(c) and BrhTX-l(d).

Whatever the combination of subunit encoding sequences the recombinant baculoviruses comprise, Pol+ AcMNPV derivatives capable of co-expressing the various combinations of sub-units are isolated by:- WO 97/44355 PCT/GB97/01205 -36- Plaque purification of the co-transfection supernatants on Sf21 monolayers by selecting for pol+/lacZ- plaques by standard procedures (King Possee 1992).

Restriction digestion/Southern blot analysis of mini-amplifications of candidate recombinant viral DNA to confirm purity and the appropriate diagnostic restriction pattern in the vicinity of the introduced combinations of genes, for example, BrhTX-1 BrhTX- (b)/BrhTX- (c)/BrhTX- (ii) BrhTX- (a)/BrhTX- (c)/BrhTX- (iii) BrhTX- 1(b)/BrhTX- (c)/BrhTX- (iv) BrhTX-1(a)/BrhTX- (b)/BrhTX-l(c); (v) In vitro protein expression studies to demonstrate that the viruses have the capacity to express immunologically detectable BrhTX-l(a), BrhTX-l(b), BrhTX-l(c) and BrhTXl(d).

The selected AcMNPV/BrhTX- (a)/BrhTX- 1(b)/BrhTX- (c)/BrhTX-l(d); AcMNPVBrhTX- (a)/BrhTX- (c)/BrhTX- AcMNPVBrhTX- (b)/BrhTX- 1 (c)/BrhTX- AcMNPVBrhTX- (a)/BrhTX- l(b)/BrhTX- etc. recombinant baculovirus isolates are then subject to diet based bio-assays on first instar H.virescens larvae as described in Example Example 10 Diet surface dosing assay for evaluation of viruses vs.

Heliothis virescens larvae Petri dishes (Falcon model 1006, 50 x 9mm, tight fit lid) are filled with a thin layer of a freshly made, pinto bean based, lepidopteran diet (velvetbean caterpillar diet Green et al 1976 Louisiana State University) sufficient to evenly cover the floor of the dish and labelled as appropriate for the test treatment list (one to two dishes per treatment). Twenty 1/2" clear 'minipots' (Ashwood Plastics, London cat. no. 4161) per virus treatment, are similarly prepared.

The virus stock solution concentrations are measured by using a counting chamber (Weber Scientific International Ltd.) and diluted to a concentration of 1x106 PIB/ml using deionised water. In Primary tests two tenfold serial dilutions are made to give a dose range, for each virus tested, of 1x10 6 1xl0 5 and 1x10 4 PIB/ml. In follow up tests on viruses of interest the dose range consists of five rates in four-fold serial dilution i.e. Ixl0 6 2.5x105, 6.3x10 4 1.6x10 4 3.9x10 3 PIB/ml.

Using a sterile syringe, 0. 1ml of the appropriate treatment solution is deposited on the surface of the diet in each of the pre-labelled Falcon dishes. The solution is spread evenly over WO 97/44355 PCT/GB97/01205 -37the surface of the diet using a sterile inoculating loop. Control dishes are treated in the same way with an equivalent volume of deionised water. The dishes are left to dry thoroughly prior to infestation.

Approximately 30 first instar Heliothis virescens larvae of uniform size are transferred to the surface of the diet in each dish using a fine artists paint brush. The dishes are tightly sealed and stored in trays in a holding room at 27 0 C and 60% relative humidity.

After 24 hours on the treated diet surface, twenty larvae per treatment are individually transferred to the prepared minipots of clean diet using a fine artists paint brush (one larva per minipot). Each minipot is labelled with the appropriate treatment and replicate number. The minipots are held under the same conditions as the treated Falcon dishes (see above) for the duration of the test.

Assessments of mortality and symptomology are made at three, four, and seven days after exposure to the treated diet. The numbers of dead and affected larvae are recorded and used for calculating percentage mortality/affected.

AcMNPV/BrhTX-l(c) recombinants Table 3 summarises data generated comparing the BrhTX-l(c) clones with wild type and Tox 34#4 AcMNPV. The three compare three clones over a comprehensive dose range.

Whilst the insecticidal activity at 3DAT appears to be inconsistent between tests, the three selected clones all show good intrinsic viral activity compared to the wild type.

WO 97/44355 PCT/GB97/01205 -38- Rate Mortait (Jfected (PIB/ml) 3 DAT 4 DAT 7 DAT AcNPV wild type 1.0 x 10 6 0 65 100 x 10' 40 6.3 x10 4 5 1.6 x 10 4 3.9 x 10' AcNPV 1.0x 106 70301 100 100 pAc Tox34#4 2.5 x IC 15"' 75 6.3 x 1W4 50 35 1.6 x 10 4 0 0 3.9 x 10' 0 Bracon 21#j 1.0 x 106 0 65(10) x 105 35(15) 6.3 x 104 10 00) 1.6 x 104 15 3.9 x 10 3 0 0 Bracon 21#x 1.0 x 106 10 45('0) 100 x I 10) 95 100 6.3 x 10 4 0(5) 50 1.6 x 10 4 25 3.9 x 10 3 0 Bracon 21#p 1.0 x 106 0 70(20) 100.

x jQS 75(10) 6.3 x 10 4 20 1.6 x 10 4 0 3.9x IW I 0 Controll 0 0 0 Control2 0 10 10 Table 3 AcMNPVBrhTX- recombinants Table 4 summarises data generated in a diet surface dosing assays comparing the edited BrhTX-I(b) (SEQ ID No. 9) clones with wild type and Tox 34#4 AcMNPV.

WO 97/44355 WO 9744355PCT/GB97/01205 39 Virus Rate Mortaity Afected) 3 DAT 4 DAT 7 DAT AcNPV wild type 1.0OX 106 15 95 100 (Ac9) 1.0Ox W 10 42 x10 4 15 AcNPV 1.0OX 106 95 95 pAc Tox34#4 1.0x Xi0 5 5(5 15 5 5 Bracon 18#a 1.0 X10 6 5 11 53 10, 104 Bracon 18#c 1.0 x10 6 10 58 100 1.0OX10 15(10) X10) 4 20 Bracon 18#d 1.0OX 106 205 45 x1W 0 9 24 4 Bracon 18#e 1.0 X 106 30 65(15) 100 10, 10 15 X10 4 Bracon 18#f 1.0 X 106 1021 95 100 X10 4 0 0 0 Braconl18#g 1.0Ox 106 10 47 x10 5 19 38 X10 4 -10 Contral 1 0 5 5 Control 2 00 0 Table 4 Table 5 shows the insecticidal activity of recombinant baculovirus based on he AcUTW1-PN/pAcAB4 system comprising genes encoding the BrhTX-1(a) (17KDa -SEQ I]) No. BrhTX-1I(b) (1l8kDa SEQ ID No. Brh-TX- 1(c) (2 1kDa -SEQ ID No. 6) or BrhTfX-1(d) (32kDa SEQ ID No. 8) toxin subunits.

WO 97/44355 PCT/GB97/01205 Virus Rate Mortality Affected) (PIB/mI) 3 DAT 4 DAT 5 DAT 7 DAT AcNPV wild type 1.0 x 106 0(5) 40 100 100 (Ac9) 1.0 x I0, 20 80 x 104 0 35 AcUWl-PHpV 1392 1.0 x 107 0 20 100 100 x 106 5 90 100 5 15- 15 AcNPV 1.0 x 106 30(M) 65(3 95 pAc Tox34#4 1.0 x 101 000) 15 20 x 10 4 10 20 Bracon BH4#67 1.0 x 107 0 5 45(50) 100 SEQ ID No. 2 1.0 x 106 20(10) x 10 5 5 Bracon BH4#68 1.0 x 107 0 5(10) 75(20) 100 SEQ ID No. 2 1.0 x 106 0 2e) x 105 0010) Bracon BH469 1.0 x 10 10 15(15) 65(30) 100 SEQ ID No. 2 1.0 x 106 10(5 30(40) 100 x 10 5 5 5 Bracon BH4#59 1.0 x 107 0 000) 55(30) 100 SEQ ID No. 10 1.0 x 10 6 15 x 10 5 5 Bracon BH4#39 1.0 x 10' 11 26 70(30) 100 SEQ ID No. 6 1.0 x 10 6 016) 4 84 x 105 0 32 Bracon BH4#51 1.0 x 107 5 5) 20('5) SEQ ID No. 6 1.0 x 106 5 21 x 10 5 0 0 Bracon BH4#52 1.0 x 10' 0 5(20) 50 SEQ ID No. 8 1.Ox 106 10 60 x 101 5 Bracon BH4#87 1.0 x 10' 0 0 0 SEQ ID No. 8 1.0 x 10 6 0 0 x 10 5 0 Bracon BH4#88 1.0 x 107 0 0 0 0 SEQ ID No. 8 1.0 x 106 0 0 x 10 5 0 Control 1 0 0 0 0 0 Control 2 0 0 0 0 57 Table Table 6 shows the insecticidal activity of recombinant baculovirus comprising various combinations of the genes encoding the Brh TX-1I(a) (I7kDa SEQ ID No. BrhTX-I(b) WO 97/44355 WO 9744355PCT/GB97/01205 -41- (18kDa SEQ ID No. 10), BrhTX-1(c) (2lkDa SEQ ID No. 6) or BrhTX-1(d) (32kDa SEQ ID No. 8) toxin subunits.

Virus Rate Mortality Affected) (PIB/mi) 3 DAT 4 DAT 5 DAT 7 DAT AcNPV wild type 1.0Ox 106 0 15 75 (Ac9) 1.0Ox 10 5 60 1.0x 1W 15 AcUTW1-PH~pVL1392 1.0 x 10' 5 100 100 106 1(5) 85 1.0x i0W 0 10 AcNPV 1.0Ox 106 5(10) 65 75 pAc Tox34#4 1.0Ox 1W~ 0 10 10 1.O 0~ 10 0 Bracon BH4#L 1.0 x 10 5 40 95 100 SEQ ID Nos 6and 8 1.0OX 106 0 6625) 100 .Ox 1W0I 15(10) Bracon BH4#50 1.0Ox 10 0 11011) 585 SEQ ID Nos 6and 8 1.0 x1 -c 5 20 105 0 Bracon BH44*13 1.0 x i0, 0 20(10) 95 100 SEQ ID Nos. 2,10,6 &8 1.0 x 106 5 60(' 105 205 Bracon BH4#15 1.0x X 0 5 1615 75(15) 100 SEQ ID Nos 2, 10 &8 1.0 X 106 0(5 80010) 100 X 0 35 Bracon BH4#19 1.0OX10 0 0(20 80 SEQ ID Nos 2, 10 &8 1.0OX 106 0' x1 0 0 Bracon BH4#12 1.0 x 1W~ 0 25(10) 100 100 SEQ ID Nos 2 10 1.0 X 106 15 75(10) 100 X10 5 5 15 Bracon BH4#17 1.0 x 1, 0 0(5) 4020 SEQED Nos 2& 8 1.0 X10 6 15('0) X 10 Bracon BH4#22 1.0Ox i0, 0 95 100 SEQ ID Nos 2 8 1.0 X 106 10(1 35(10) 1.0 X10 5 1 5 30 Controll1 0 0 0 0 0 Control 2 0 0 0 0 0 Table 6 Whilst the present invention has been particularly described with reference to the production of recombinant baculoviruses comprising nucleotide sequences depicted in various of SEQ ED Nos. 1.3,5,7 and 9, the skilled man will appreciate that the baculoviruses may be engineered WO 97/44355 PCT/GB97/01205 -42to contain other sequences which are also part of the present invention. For example, the baculoviruses may comprise a polynucleotide comprising a region encoding a protein encoded by the spliced RNA derived from the genomic clone depicted in SEQ ID No. 19, the said spliced RNA being capable of hybridising with the extension products of the primers depicted in SEQ ID Nos 20 and 21, or SEQ ID Nos 22 and 23, or SEQ ID Nos 24 and 25 using the sequence depicted in SEQ ID No. 1 as a template. SEQ ID No. 45 discloses a toxin encoding sequence derived from the genomic clone depicted in SEQ ID No. 19 and SEQ ID No. 46 discloses the translation product of the nucleotide sequence depicted in SEQ ID. No. 45. The present invention thus includes Baculoviruses which are engineered to comprise the SEQ ID No. 45 sequence, alone or in combination with one or more of the other toxin encoding sequences disclosed herein.

I

WO 97/44355 PCT/GB97/01205 -43- SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: ZENECA Ltd STREET: 15 Stanhope Gate CITY: London COUNTRY: UK POSTAL CODE (ZIP): W1Y 6LN NAME: Commonwealth Scientific and Industrial Research Organisation STREET: 407 Royal Parade CITY: Parkville STATE: Victoria COUNTRY: Aus-ralia POSTAL CODE (ZIP): 3052 (ii) TITLE OF INVENTION: Biological Insect Control Agent (iii) NUMBER OF SEQUENCES: 46 (iv) COMPUTER READABLE

FORM:

MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 564 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bracon Hebetor (ix) FEATURE: NAME/KEY:

CDS

LOCATION:13..487 5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TTTGGATAAA TC ATG AAA TTT TTA TAT CTA ATA CTC CTT TTA ATT GCA 48 Met Lys Phe Leu Tyr Leu Ile Leu Leu Leu Ile Ala 1 5 GGA GTA GTA TCA TTC AAT CCG GAG ACA CAT CGT GAA TGT AAG AAT TAT 96 Gly Val Val Ser Phe Asn Pro Glu Thr His Arg Glu Cys Lys Asn Tyr 20 WO 97/44355 PCT/GB97/01205 -44- TGC GCC AAA GAG CAC GGC Cys Ala Lys Glu His Gly

GAA

Glu

ATG

Met

CAT

His

CCA

Pro

TGT

Cys cc Pro 125

OTT

Val

TCA

CTT GGT GAT ATT Leu Gly Asp Ile GGA AGO GAG AAT Gly Ser Glu Asn GGA GCG GGA GGC Oly Ala Oly Gly 80 OCA TCA TOG GCA Ala Ser Trp Ala ATA CAT GOT TGC Ile His Ala Cys 110 ATA GTT ATO AAA Ile Val Met Lys GAC AGO TAT TGT Asp Arg Tyr Cys 145 ATT T AACAATGATC

TTT

Phe 50

TAT

Tyr

TTT

Phe

TGC

Cys

TCA

Ser

AAT

Asn 130

GAA

GAG GAA TAT CGT Glu Giu Tyr Arg 35 AAA TGT OTT TGC Lys Cys Val Cys GGT AAO TOT AGA Gly Lys Cys Arg 70 AAA TAT GCC TTT Lys Tyr Ala Phe 85 ATA TGO ACT CAG le Cys Thr Gin 100 OAA ATT CAT CAC Olu Ile His His 115 GGA CAA TGC TAC Gly Gin Cys Tyr OTT TAT ATO AAO

ACG

Thr

ACT

Thr 55

OAA

Olu

CCC

Pro

GAG

Olu

AAG

Lys

TAC

Tyr 135

TTC

TGG

Trp

CAC

His

GCA

Ala

ATA

Ile

AAA

Lys 0CC Ala 120

CAA

Gin

TTA

TCT TTC Ser Phe OGA AAG Gly Lys TGT ATT Cys Ile TAC AGC Tyr Ser AAT AAG Asn Lys 105 CCA CCT Pro Pro OAT CAC Asp His GAT GCG

CGT

Arg

AAT

Asn

CAA

Gin

GAA

Glu

ACA

Thr

AAG

Lys

AGO

Arg

TTG

TAC

Tyr

OTT

Leu

AAT

Asn

GTA

Val

TTT

Phe

AAT

Asn

GGT

Gly 140

GAA

144 192 240 288 384 432 480 537 Glu Val Tyr Met Lys Phe Leu Asp Ala Leu Glu 150 155 AAATTCATGT TATCAATOAA GGAAGAATAA TGAATTAATA Ser Ile ATAATTATCA AAAATCAAAA AAAAAAA 564 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 158 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Lys Phe Leu Tyr Leu Ile Leu Leu Leu Ile Ala Gly Val Val 1 5 10 is Phe Asn Pro Glu Thr His Arg Giu Cys Lys Asn Tyr Cys Ala Lys 25 His Gly Glu Olu Tyr Arg Thr rrp Ser Phe Arg Tyr Giu Leu Gly 40 Ile Phe Lys Cys Val Cys Thr His Oly Lys Asn Leu Met Gly Ser 50 55 Asn Tyr Oly Lys Cys Arg Glu Ala Cys Ile Gin Asn His Gly Ala 70 75 Ser Glu Asp Glu Oly WO 97/44355 WO 9744355PCT/GB97/01205 Gly Phe Lys Tyr Phe Pro Ile Tyr Ser Giu Val Pro Ala Ser Trp Ala Cys Ile Cys Ser Glu 115 Lys Asn Gly 130 Cys 100 Ile Gin Gin Glu Lys Lys Thr Phe Cys Ile His Ala 110 Ile Val Met His His Lys Cys Tyr Tyr 135 Ala 120 Gin Pro Lys Asn Pro 125 Asp His Arg Gly Val Asp 140 Glu Ser Ile Arg Tyr Cys 145 (2) Giu Val Tyr Met Lys 150 Phe Leu Asp Ala Leu 155 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 1197 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bracon Hebetor (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CACGCCTACT TAGATAATTT CTCAATTCTT

GTATGACACC

TGTACAAAAG

CCCTGAAAAA

TAATCTTGTT

GAAAGTGGTG

TTGGTGATTG

ATGGAGATGT

TTTTTCAATG

TTGACGTTGT

ATACTGTATA

AGGAGTGGGT

TCAATCTGAG

AAAACTACCT

CTGAGTAAGG

GGTGCTACTG

TGGACGTGCC

CTGCAGAAGT

TACGAATAAA

TCTTCAGCGT

CAATACACCC

TGATAAACAC

GCATGATAAT

TTTGTTCATA

CATATAAATC

CAATAATTTT

AGTTGGACAT

GATAAGACTT

CATGACAGCT

GGATTTTCAA

TCCAkI'GGTA

TGTTACTTCA

GAATCACTCT

GCTCTTCTCC

TGCAAATTAT

ATTCGAGAGG

TTGCATTTTT

NCCTCATAAC

CGATGGTATC

TTGGTCCTTC

GTGGCCGCAT

ATATTTGGGA

AAATGAAAAA

TGAAAGATGG

ATCAACTTAT

CGCAAGAGCT

GAAATAAGTG

GATAAATAAG

CCGTGAGAGA

AATGTCAATC

GTCAACATTA

ACGGTCGCTA

GATTAAACCA

ATGCCGATGT

TGTTGTGGAA

CCGTGCTAAA

ACTACACAAA.

GGGGATTAAA

CAAGAGATGT

GAAGTCTCTG

GAAAAAAAAA

ATATGTAAAA

TTTACAGACC

GGAGGAGGTG

GGAGAGACTT

GACTATGCAT

ATATTGCATT

ATATCACCCC

GATAATTTTA

GATGAGCATG.

120 180 240 300 360 420 480 540 600 660 720 TGTGGGAGAC ACTGATGGCA TGGATGGACT TTAGATTTCC AACTGAATAA TAAATATTCC WO 97/44355 PCTGB97/01205 -46-

AAATACAGAT

TAATGAAAAG

ATATTTGCCT

GGAGGATTAG

ATTGATTGAT

CATAACAGTA

AAATAAAAAT

ATCCTTTTGA

ATTGATTGAA

TATTNGATAA

GATATTTTAC

CATGTATTAA

TGCACAAAAA

AGTTAGACAG

TAAAATGTCG

AATGTCTGAA

ACTTCTACCN

AAGGATTTTA

ATACAATAAC

TAATGACGGT

GTACCAAAAA

TAAACATGAT

GTAACTNNNG

TTAANAAAGG

AAAATAATTA

ATCTCGTTCT

AAATATCTAT

AAAAAAAAAA

TGTTTAGATG

GATNNGACAT

AAAAAGGAGG

AACAATTAGA

CATAGTACAA

GTATGTATGT

AAAAAAAAAA

AATGGTAAAT'

ATAATATATA

AGGNGTAGGA

TCTTCTGTAA

TGAAAAAGAA

AGAGAGAAGA

AAAAAAA

840 900 960 1020 1080 1140 1197 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 182 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bracon Hebetor (xi) Met SEQUENCE DESCRIPTION: SEQ ID NO: 4: Ser Ile Ile Cys Lys Ile Ile Leu Leu Asp Val Leu Leu Ser Trp Thr Gly Arg Ser Met Val Ala Asp Lys Asp Cys Cys Ser Ser Thr Leu Phe Thr 25 Arg Lys Trp Cys Thr Phe Gly Pro Ser 40 Arg Ser Leu Gly Gly Gly Val Gly Arg Ser His Asp 55 Ser Cys Gly Arg Met Ile Lys Pro Gly Glu Thr Tyr Gly Asp Val 70 Thr Asn Lys Gly Ser Asn Ile Trp Glu Gly Cys Arg Cys Asp Ala Phe Phe Gln Cys Leu Gin Arg Ser Lys Met Lys Pro Cys Tyr 115 Val Tyr Asp 130 Val Val Glu Ile Leu 105 His Phe Asp Val Val Asn Thr 110 Pro His Thr Met Lys Asp Arg Ala Lys Ile Ser 125 Lys His Glu Ser Leu Tyr Gln Leu Ile 135 140 Leu His Lys Asp WO 97/44355 WO 9744355PCT/GB97/01205 -47 Msn Phe Lys-Glu Trp Val His Asp Asn Ala Leu Leu Pro Gin Olu LeU 145 150 155 160 Gly Ile Lys Asp Glu His Val Trp, Glu Thr Leu Met Ala Trp Met Asp 165 170 175 Phe Arg Phe Pro Thr Olu 180 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 669 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bracon Hebetor (ix) FEATURE: NAME/KEY: CDS LOCATION:147. .581 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AAAAGACTAA AAATAAGAAA AAAAAACATA. GAAGAATGTT TACAATAATT TATATTTTTA AACTTTCATT TCTATTAGTT CCGTGCTGGA GTTTTTCAAC CTACGCTGGG TATOOTOAAT 120 ATAATCGGTC CATTACTAAG CGACAG ATG GAC OAT GOT GAG ACG TGC GAA AGG 173 Met Asp Asp Oly Glu Thr Cys Olu Arg 160 165 TOT TTO AAT CCA CTC GAA TTA GTA AAT GAC OCT GTA GAC TCG TGC ATT 221 Cys Leu Asn Pro Leu Glu Leu Val Asn Asp Ala Val Asp Ser Cys Ile 170 175 180 GAA OCT CAT GAG OAA TOT GAG GAA TTC ATT GAA GOC 000 ATG GAA ATG 269 Glu Ala His Olu Glu Cys Glu Olu Pile Ile Glu Gly Oly Met Glu Met 185 190 195 CTT CAT GTA CAC AAT CCA OGA AAC TTC CGA GTC TCC AAA TOT OTA TOC 317 Leu His Val His Asn Pro Gly Msn Phe Arg Val Ser Lys Cys Val Cys 200 205 210 215 GAC ATT 0CG CTC AAG GAO TGC CTC ACT ACT CAT CCT OAA ATO AOT TTC 365 Asp Ile Ala Leu Lys Giu Cys Leu Thr Thr His Pro Olu Met Ser Phe 220 225 230 AAA TTT OTT AAA GCA CTC TTT TTT OAT TTO CTT OCT CCA CCC TOT TTT 413 Lys Pile Val Lys Ala Leu Phe Pile Asp Leu Leu Ala Pro Pro Cys Pile 235 240 245 OAT CAG AT? OCT OAT TOG GOT AAG AAA AAA TTO AAA AAT AAG CAG OCA 461 s WO 97/44355 PCT/GB97/01205 -48- Asp Gln Ile Ala Asp Trp Gly Lys Lys Lys Leu Lys Asn Lys Gln Ala 250 255 260 TTT TCA CTG CAT GAT TTA CAA TCA GCT GCC CAC GCG CTC TGG CAA ACA Phe Ser Leu His Asp Leu Gin Ser Ala Ala His Ala Leu Trp Gin Thr 265 270 275 CTC TAT GAC GCT GTC AAG GGC ATA GCT CAG GAT GTC GGA CAT GCT GCA Leu Tyr Asp Ala Val Lys Gly Ile Ala Gln Asp Val Gly His Ala Ala 280 285 290 295 CAT TCT TTT GAA AAA ATG TTA CAG TAACAGTTAA ATATGAAAAA GGTCCATGAT His Ser Phe Glu Lys Met Leu Gin 300 AGTAGAATAC AGTTATTGTT GTATAAATAA ATAATATATT CAGAATGATA AAAAAAAA INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 176 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Met Trp Gly Leu Ser Phe Leu Leu Val Pro Cys Trp Ser Phe Ser Thr 509 557 611 669

.L

Tyr Asp Asn Phe Phe Thr Asp Lys Ala 145 Ala Asp Asp 50 Ile Arg Thr I Leu I Lys I 130 Ala I Gly Gly Ala l1u Jal iis Leu Leu His Cys Gly Gly Glu Thr Cys Val Asp Ser Gly Gly Met 70 Ser Lys Cys Pro Glu Met 100 Ala Pro Pro Lys Asn Lys Ala Leu Trp 150 Tyr Glu Cys 55 Glu Val Ser Cys Gin 135 Gin Asn Arg 40 Ile Met Cys Phe Phe 120 Ala Thr Arg Ser 25 Cys Leu Glu Ala Leu His Asp Ile 90 Lys Phe 105 Asp Gin Phe Ser Leu Tyr Ile Asn His Val 75 Ala Val Ile Leu Asp 155 Thr Pro Glu His Leu Lys Ala His 140 Ala Lys Leu Glu Asn Lys Ala Asp 125 Asp Val Arg Glu Cys Pro Glu Leu 110 Trp Leu Lys Gin Leu Glu Gly Cys Phe Gly Gin Gly Met Val Glu Asn Leu Phe Lys Ser Ile 160 Ala Gin Asp Val Gly 165 His Ala Ala His Ser Phe Glu Lys Met Leu Gin 175 WO 97/44355 WO 9744355PCT/GB97/01205 -49 INFORMATION FOR SEQ ID NO: 7: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1057 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (Vi) ORIGINAL SOURCE: ORGANISM: Bracon Hebetor (ix) FEATURE: NAME/KEY: CDS LOCATION:95. .919 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CGCGGCCGCT GTTGATATAT AACAATTTAT TAAAAATTTC AAGTGGAAAG AAAAACTATC TTGTTTTTTT TTTTGTTTTT TTTCATAATT TAAA ATG CAT TTC TTC GCC TCC Met His Phe Phe Ala Ser

ATC

Ile

ATA

Ile

TAT

Tyr Leu 200

CAC

His

AAT

Asn

TCT

Ser

TCC

Ser

GAT

CTG

Leu

ATA

Ile

TTA

Leu 185

CTA

Leu

AGA

Arg

AAG

Lys

GAG

Glu

ATA

Ile 265

ATT

GTA

Val

AAT

Asn 170

CAA

Gin

ACT

Thr

ACG

Thr

CCA

Pro

TAC

Tyr 250

CAT

His

OCT

TTC

Phe

CAT

His

TCA

Ser

AAA

Lys

AAT

Asn 220

ATG

Met

GGA

Gly

ACA

Thr

ATA

TTA

Leu

GAT

Asp

GCT

Ala

CAT

His 205

ATT

Ile

CAA

Gin

GGA

Gly

TTT

Phe

ACG

GGC

Gly

ACT

Thr 175

AAG

Lys

ATG

Met

GTA

Val

AGA

Arg

CGT

Arg 255

GGA

Oly

AGC

AAG

Lys 160

GAG

Glu

TOT

Cys

ACA

Thr

GCC

Ala

AAG

Lys 240

AAG

Lys

CCG

Pro

CAG

GCA ATT CAT GAT GTG GAA GGA Ala

GGA

Oly

TCT

Ser

GCT

Ala

CTT

Leu 225

GTT

Val

TCA

Ser

TCT

Ser

OAA

le

CAA

Gin

TAT

Tyr

OCT

Ala 210

GOT

Gly

GAA

Glu

TTA

Leu

GGG

Gly

GTA

His

TTT

Phe

OTA

Val 195

CAT

His

OTT

Val

CAT

His

AAA

Lays

GAT

Asp 275

ACA

Asp

CCC

Pro 180

TOT

Cys

TGT

Cys

ACG

Thr

ATA

Ile

AAT

Asn 260

AAA

Lys

CTA

Val 165

CAT

His

GGC

Oly

GTA

Val

GAT

Asp

AAA

Lys 245

TOG

Trp

OAA

Oiu

GGA

GiU

ATO

Met

GOT

Gly

GCA

Ala

TTT

Phe 230

GTC

Val

TAT

TAC

CCA

Gly

OCT

Ala

OCT

Ala

ATO

Met 215

CAT

His

CAT

His

CC

Arg

AAT

Asn

GTA

WO 97/44355 PCT/GB97/01205 Asp 280

GTA

Val

CAA

Gin

AAT

Asn

AAG

Lys

GCT

Ala 360

GAC

Asp

GTT

Val

TAT

Ile

AAG

Lys

GAT

Asp

GAT

Asp

GTT

Val 345

GAT

Asp

AGC

Ser

TCT

Ser

TAT

Ala

ACT

Thr

GCT

Ala

CCA

Pro 330

CTT

Leu

AAA

Lys

GGT

Gly

GTG

Val

TTG

Ile

ATT

Ile

AGA

Arg 315

ATT

Ile

CAT

His

GAA

Glu

AGT

Ser

AGT

Ser 395

GAT

Ile

AAT

Asn 300

TTG

Leu

CCT

Pro

TCA

Ser

TAT

Tyr

CCC

Pro 380

GAT

Asp

TGG

Thr 285

TTA

Leu

TCG

Ser

CCA

Pro

CGA

Arg

GGT

Gly 365

TTA

Leu

GAA

Glu

ATC

Leu

CCC

Pro

GGC

Gly

CCC

Pro

GAT

Asp 350

GAT

Asp

GTC

Val

GAA

Glu

AAG

Ser

CCA

Pro

TTT

Phe

ACT

Thr 335

GCT

Ala

GGA

Gly

AAG

Lys

CAT

His

AAA

Gin

AAG

Lys

GGC

Gly 320

ACA

Thr

ATT

Ile

ACT

Thr

GAT

Asp

ACT

Thr 400

TAT

Glu Val 290 AGC TAT Ser Tyr 305 CGA ACA Arg Thr CAT TTA His Leu GTC ACT Val Thr TGG TCT Trp Ser 370 AAT CAA Asn Gin 385 ACA CGC Thr Arg GCC GAA Thr

CGG

Arg

GTC

Val

CAA

Gin

GAT

Asp 355

AAT

Asn

GTA

Val

TTT

Phe

CTT

Leu

CTT

Leu

ATT

Ile

TGG

Trp 340

AGT

Ser

GCA

Ala

ATT

Ile

CAA

Gin

GCG

Gly Pro Val 295 CCT TTT GAT Pro Phe Asp 310 GTC AAA GAA Val Lys Glu 325 CTA GAT ATG Leu Asp Met GAA TTT CTC Glu Phe Leu GCT AAG GGA Ala Lys Gly 375 GGC GTA GCC Gly Val Ala 390 ATA GTC ACT Ile Val Thr 592 640 688 736 784 832 880 929 989 1049 1057 405

TAAJAAAGAAT

Tyr Tyr Leu Asp Trp Ile Lys Lys Tyr Ala Glu Leu Ala 410 420 AAAGAGCAAA ATTGCTCAGA TGGTGAATAT ACATTTTTCC AATAAGCTCA GAAAAAATCG ATTTATATGT AATTAAAAAA TTAAAGATTG TTTTTTCTCT TTTAACAGAA GAATTTGGCG

CGTGAATT

INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 275 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Met His Phe Phe Ala Ser Ile Leu Val Ser Phe Leu Leu Gly Lys Ala 1 5 10 Ile His Asp Val Glu Gly Ile Ile Asn Gly His Asp Ala Thr Glu Gly 25 Gin Phe Pro His Met Ala Tyr Leu Gin Ala Ser Ala Gly Lys Cys Ser 35 40 Tyr Val Cys Gly Gly Ala Leu Leu Thr Lys Lys His Ile Met Thr Ala 55 ~ii_ WO 97/44355 PCT/GB97/01205 -51- Ala Gly Glu Leu Gly Val 145 Tyr Thr Leu Thr Ser 225 Gin Arg Glu His Cys Val Ala Met His Arg Thr 70 Val His Lys Asp 130 Thr Arg Val Gin Asp 210 Asn Val Phe Leu Thr Ile Asn 115 Lys Leu Leu Ile Trp 195 Ser Ala Ile Gin Ala Asp Lys 100 Trp Glu Gly Pro Val 180 Leu Glu Ala Gly Ile 260 Phe Val Tyr Tyr Pro Phe 165 Lys Asp Phe Lys Val 245 Val His His Arg Asn Val 150 Asp Glu Met Leu Gly 230 Ala Thr Asn Ser Ser Asp 135 Val Gin Asn Lys Ala 215 Asp Val Tyr Pro Tyr 105 His Ala Thr Ala Pro 185 Leu Lys Gly Val Leu 265 Gly Ser 90 Lys Arg Ile Ile Arg 170 Ile His Glu Ser Ser 250 Asp Asn 75 Met Gly Thr Ile Asn 155 Leu Pro Ser Tyr Pro 235 Asp Trp Ile Gin Gly Phe Thr 140 Leu Ser Pro Arg Gly 220 Leu Glu Ile Lys Gin Arg Thr 125 Leu Pro Gly Pro Asp 205 Asp Val Glu Lys Val Arg Arg 110 Gly Ser Pro Phe Thr 190 Ala Gly Lys His Lys 270 Ala Lys Lys Pro Gin Lys Gly 175 Thr Ile Thr Asp Thr 255 Tyr Leu Val Ser Ser Glu Ser 160 Arg His Val Trp Asn 240 Thr Ala 275 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 594 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bracon Hebetor (ix) FEATURE: NAME/KEY: CDS WO 97/44355 PCT/GB97/01205 -52- LOCATION:22..573 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: AGATCTGCGG CCGCGCCACC A ATG TCA ATC ATA TGT AAA ATA ATC TTG TTG Met Ser Ile Ile

GTG

Val

CGA

Arg

CTA

Leu

CGC

Arg

TTT

Phe 350

CTT

Leu

TTT

Phe

AAA

LyS

OTT

Leu

GGA

Gly 430

GAG

AGT

Ser

TGT

Cys 305

GGT

Gly

AAA

Lys

ATT

Ile

TCC

Ser

GTC

Val 385

CCC

Pro

CAC

His

CTT

Leu

ATG

TCG

Ser

GCC

Ala

GAT

Asp

GAG

Glu 340

TGC

Cys

AAA

Lys

CCC

Pro

GTA

Val

AAT

Asn 420

GAG

Glu

ATC

Met

GAT

Asp

TGC

Cys 325

ACT

Thr

CGA

Arg

ATG

Met

TGT

Cys

TAT

Tyr 405

TTT

Phe

CTG

Leu

GTA

Val

AAG

Lys 310

TGC

Cys

TAT

TGT

Cys

AAA

Lys

TAC

Tyr 390

GAT

Asp

AAG

Lys

GGA

Gly Cys 280

TCA

Ser

TTT

Phe

AGT

Ser

GAT

Asp

TAT

Tyr 360

GTT

Val

ATG

Met

CAC

His

TGG

Trp

AAA

Lys 440

ACA

Thr

GGT

Gly

CAT

His

OTT

Val 345

GCA

Ala

GTG

Val

AAA

Lys

GAA

Glu

GTG

Val 425

GCT

Ala

TTA

Leu

CCT

Pro

CAC

Asp 330

ACC

Thr

TTT

Phe

GAA

Glu

GAT

Asp

TCA

Ser 410

CAT

His

GAG

Clu Lys Ile Ile Leu Leu

TTT

Phe

TCA

Ser 315

AGO

Ser

AAT

Asn

TTT

Phe

ATA

Ile

GGC

Gly 395

CTC

Leu

GAT

Asp

CAT

His

ACA

Thr 300

CGG

Arg

TGT

Cys

AAA

Lys

CAA

Gin

TTC

Leu 380

CGT

Arg

TAT

Tyr

AAT

Asn

GTG

Val 99 147 195 243 291 339 387 435 483 Glu Thr Leu Met Ala Trp 450 ATG GAC TTT Met Asp Phe AGA TTT Arg Phe 455 CCA ACT GAA Pro Thr Glu TAATCAGCGG CCGCGAATTO C INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 184 amino acids TYPE: amino acid TOPOLOGY: linear WO 97/44355 PCT/GB97/01205 -53- (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Ser Ile Ile Cys Lys Ile Ile Leu Leu Val Leu Leu Ser Trp Thr Ser Met Ala Asp Asp Cys 50 Glu Thr Cys Arg Lys Met Pro Cys Val Tyr 130 Asn Phe 145 Glu Leu Met Asp Val Ser Lys Thr Cys Arg Tyr Gly Cys Asp Lys Asn 100 Tyr Phe 115 Asp Lys Lys Glu Gly Ile Phe Arg 180 Ser Phe Ser Asp Tyr Val Met His Trp Lys 165 Phe Thr Gly His Val 70 Ala Val Lys Glu Val 150 Ala Pro Leu Phe Pro Ser 40 Asp Ser 55 Thr Asn Phe Phe Glu Ile Asp Gly 120 Ser Leu 135 His Asp Glu His Thr Glu Thr 25 Arg Cys Lys Gin Leu 105 Arg Tyr Asn Val Asp Ser Gly Gly Cys 90 His Ala Gin Ala Trp 170 Arg Leu Arg Phe 75 Leu Phe Lys Leu Gly 155 Lys Gly Met Ser Gin Asp Ile Ile 140 Thr Trp Gly Ile Asn Arg Val Ser 125 Leu Leu Cys Gly Lys Ile Ser Val 110 Pro His Leu Gly Val Pro Trp Asn Asn His Lys Pro Arg Gly Gly Glu Gly Thr Thr Asp Arg 160 Glu Thr Leu Met Ala Trp 175 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 1326 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bracon Hebetor (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: TTTGGATAAA TCATGAAATT TTTATATCTA ATACTCCTTT TAATCGCAGG AGTAGTATCA TTCAATCCGG AGACACGTAA GTAAATXGGA AAATTTTTTT AATTAATTCA ATTCTATAAT WO 97/44355 WO 9744355PCT/GB9701205 54-

CAATTCAATT

AATATAAATT

TGCGCCAAAG

ATTTTTAAAT

TATTCATGAT

TCACGGAAAG

TTTTGGTTTA

AGAGA&GCAT

AGCGAAGTAC

ATTATTTCAT

GAAAAATAAG

GAATCCCATA

GTATTGTGAA

TTCATGTTAT

TTGTTTTTAA

CCTTTCTCCA

TCCAATAAAC

APATGCAAATT

TTTGTATATA

TCCCAGTCTC

TGGAAC

GTTCACAAAT

TATTTTTCGC

AGCACGGCGA

GTGTTTGCAC

TAAATCAAGA

AATCTTATGG

ATAATATTAA

GTATTCAAAA

TAGCATCATG

TTTAAATCCA

ACATTTTGTA

GTTATGAAAA

GTTTATATGA

CAATGAAGGA

TTATTAAAAA

TTACAACTAC

GTAACGAAAC

TAGGCGAGAG

TACTATTTTA

AAAGTATCCT

TTTI'GTTTTT

TTTAAATTGA

GGAATATCGT

GTAAGTAACA

TTTATTAATT

GAAGCGAGAA

TTTTCCTTTA

TCATGGAGCG

GGCATGCATA

ATATATTAAT

TACATGCTTG

ATGGACAATG

AGTTCTTAGA

AGAATAATGA

AAAAGGCTAC

CCTCTCATCT

AATTTCACTA

CACTATAATA

AGAATGACTT

TGCGTGTATA

CATTTTTATT

TAA.AATTTAG

ACGTGOGTCTT

AAATTTTCAT

AATGAATTAT

TTATGGTAAG

AATTATCCGT

GGAGGCTTTA

TGCACGTATG

AATATAATTG

CTCAGAAATT

CTACTACCAA

TGCGTTGGAA

ATCAATAATA

ATTGGATT

CAA.TTAGAAA

GCATAGAAAG

AGGGGGGGGG

TCCTTTCTGC

TATATATACG

AAAATTATAC

ATCGGGAATG

TCCGTTACGA

TACTAATATA

GTGTGACTTC

TATTTACTCA

CCTTCTTGCC

AATATGCCTT

"'AACATTAGA

TATAATGTTC

CATCACAAGG

GATCACAGGG

TCAATTTAAC

ATAATCAAAA

TAACTTGTCG

CAACCTAAAA

AGCTTAATTG

GCTCCGTTCC

ATTCCATTTC

TACGTTAGAT

CAATAATGAT

TAAGAATTAT

ACTAGGTGAT

CTTCTTTTAT

ATTATGTAAG

AAAATITTAG

AGGTAAGTGT

TCCCATATAC

TGTAATTATT

TTAGTCAGGA

CCCCACCTAA

GTGTTGACAG

AATGATCAAA

ATCAATGATT

ATATAATACC

AACTAATGAT

CCTAAAAATT

AAAAATCCAT

TTTTTCTCTC

CCTCGCAACT

180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1326 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 358 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bracon Hebetor (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CATGGAAATT TTTATATCTA ATACTCCTTT TAATTGCAGG AGTAGTATCA TTCAATCCGG WO 97/44355 WO 9744355PCT/GB97/01205 55 AGACACGTAA GTAATTGGA AAATTTTTTT AATTAATTCA ATTCTATAAT CAATTTrATT GTTCACTTAT TTTTGTT''IT CATTTTTATT AAAATTATAC CAATAATGAT

AATATAAATT

TATTTTCGC TTTATTA TAAAATTTAG ATCGTGAATG TAAGAATTAT

TGCGCCAAAG

AGCACGGCGA GGAATACCGT ACGTGGTCTT TCCGTTACGA ACTTGGTGAT ATTTTTAAAT GTGTTTGCAC GTAAGTAACA AAATTTTCAT TACTAATATA TTCTTTTATT

ATTCATGG

INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 358 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL:

NO

(iV) ANTI-SENSE:

NO

(vi) ORIGINAL SOURCE: ORGANISM: Bracon Hebetor (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 120 180 240 300 358

CATGGAAATT

CCATACGTAA

TTGTTCACTT

ATTTTTCGCT

GCACGGCGAG

TATTTGCACG

TTTATATCTA

GTAAATTGGA

TTTTTTTTTC

TTAAATTTAT

GAATATGCTA

TAAGTAACAA

ATACCCCTTT

TTTTTTTTTT

ATTTTTATTA

AAAATTTAGA

CATGGTCTTT

AATTTTCATT

TAATTGCAGG

TTTATAAATT

AAATTATACC

TCGTGAATGT

TCGTTACGAA

ACTAGTATTC

AGTAGCATCA

CAATTCTATA

AATAATGATA

AAGAATTTTT

CTGGGTGATA

TTCCTTTGTT

TTCAATGCGG

ATTAATTTTA

ATATAAATTT

GCGCCAAAGA

CGTrTAAATG

TTTCATGG

120 180 240 300 358 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 737 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: ORGANISM: Bracon Hebetor (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: WO 97/44355 WO 9744355PCT/GB97/O1205 56- ACTAGTATTC TTCCTTTGTT TGTGTTGATT TGATTATATA GTATCTACTT AGAATTATTA CTCTATTATT CTCTCTCTCT AACAGCATGG AGCGGGAGGC CATGGGCATG C.ATGTGCACG ATCAATAATT TTAATAATAC TTGTATACAT GCTTGCATGA GAAAAATGGA GAATGCTACT AATGAATAGC ATTGATTGAA AGAATAATAA ATCAATAATA AAAAAAGGCT ACATTGGATT TAAGGGCTTG TACTACT

TTTCATGATT

GTATCGGAAA

GTTTTGGTTT

CTCTCTCCCA

TTTAAATATG

TATGTAACAT

AATTGTATGA

AAGTTGAAGA

ATCAAGATCA

TAAATTCAAC

TATATCAACA

TTTAACTTCT

AAATCAAGAT

CGGCCTCATG

AATAATTTCA

ATCAAGATAA

CCTTCAGCAT

TAGATGCTAT

TGTTTTTAGT

AAAGGCCCCA

CAGGGGTGTT

AATTATCAAA

ATCAATAACT

CGACATACTA

TTATTAATTA

AGAACAGAGA

ATTTTCTTTA

GTGTAGACAA

ATACAGTGAA

TATTATTATT

CAGGTGAAAA

CCTATGAATC

GAGAGGTCGT

TTCATGTTAT

TTGTTTTTAA

CTCGAATTGT

ATGAATTATG

ATAATGGTAA

CTTTTAATA

AAATGTATTC

GTACCAGCTT

TCCTTTTTAA

ATAAGACATA

CCATAGTGAT

GTGAATTGGA

CAATGAAGGA

TCATTTAAAA

GAGGGAGAAT

120 180 240 300 360 420 480 540 600 660 720 737 INFORMATION FOR SEQ ID NO: Wi SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: DNA TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TTATATGAAG TrCTTAGA INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: DNA TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: TTAAATTGAT TCCAACGC INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: DNA TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid WO 97/44355 WO 9744355PCT/GB97/01205 57 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: TTCCTTACGC GAAATACG INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: DNA TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GAATTATTAT CAAATCAT INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 1811 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bracon Hebetor (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: TCTAGAACAA TGATATAGGA TAGCCGCATC AACGCCCCTA GAGTGCCGTT AGAGGGGCG TAACTATACA AAATTCAAAA AAAAAGATTC TGAAGCTGTT AGAAAACATT GATATAAATT ACTAGTAATG GCTTACGTGT AAATACTCGT GATTCTTAGT GAGCTATATA AGAATGAGTA TTCAGATAAA TCATGAAATT TTCA.ATGCGG CCATACGTAA ATTCAATTTT ATTGTTCACT CCGTTGTTAG GTAAAAAGCA GTGCCAATTG TCTATAGTCC

AAACACGTAA

TGTACCAATT

ATATTCTAAA

CTAAGAAATG

GTTAGGGACA

.ATTGTTTCAC

CTATTTAAAA

AGGTTTAGCA

TTTATATCTA

GTAAATTGGA

TTrTTTTTTTT

GAA.GACAAAA

TCAGAATTAT

ACTCCTCCAA

TCCCAGTTTT

ATTGAGGAGT

ACACTAGTAA

AAAATTTTCA

TAAGTATAAT

ATACCCCTTT

TTTTTTTTTT

CATTTTTATT

AGCTATAGCG

GTTATCCATC

ATAACTTAAA

GGAACGCAAA

TGTACTGGTC

TAIAATATGGA

TTTGAATATC

TCATTTCTTT

TAATTGCAGG

TTTATAAATT

AAAATTATAC

GTGATATATA

ATTCTGCATA

ACAAAA.CGAA

ATGGAGTTGT

GATATAATAA

CGGGATACAA

TCAAAGAGAG

AGTAGCATCA

CAATTCTATA

CAATAATGAT

WO 97/44355 WO 9744355PCTIGB97O 1205 58 AATATAAATT TATTTrTCGC TTTAAATTTA TAAAATTTAG ATCGTGAATG

TGCGCCAAAGA

ACGTTTAAAT G TTTI'CATGAT T AGTATCGGAA A, AGTTI'TGGTT T

TCTCTCTCCCA

CI'TAAATAT G GTATGTAACA T' CAATTGTATG A!

AAAGTTGAAG

TATCAAGATCA

ATAAATTCAA C~

ATATATCAACA

TTTTAACTTC T CGAAACAGCC A TGTTGAAATT2 AGTTTAAATT G AATGTTTGAA A

GCACGGCGA

TATTTGCAC

AAATCAAGA

CGGCCTCAT

AATAAT1TTC

ATCAAGATA

CTTCAGCA

TAGATGCTA

IGTTTTTAG

khAGGCCCC

:AGGGGTGT

AATTATCAA

kTCAATAAC

CGACATACT

~TTCCACTT

GGAATATGCT

GTAAGTAACA

TTTATTAATT

GAGAACAGAG

AATTTTCTTT

AGTGTAGACA

TATACAGTGA

TTATTATTAT

TCAGGTGAAA

ACCTATGAAT

TGAGAGGTCG

ATTCATTTTA

TTTGTTTTTA

ACTCGAATTG

TT.AGAACGCA

ACATGGTCTT

AAATTTTCAT

AATGAATTAT

AATAATGGTA

ACTTTTTAAT

AAAATGTATT

AGTACCAGCT

TTCCTTTTTA

AATAAGACAT

CCCATAGTGA

TGTGAATTGG

TCAATGAAGG

ATCATTTAAA

TGAAGGAGAA

CCTCTATACA

ACATCCTCCA

ACTTTAATAA

TTCGTTACGA

TACTAGTATT

GTGTGTTGAT

AGTATCTACT

ACTCTATTAT

CAACAGCATG

TCATGGGCAT

AATCAATAAT

ATTGTATACA

TGAAAAATGG

AAATGAATAG

AAGAATAATA

AAAAAAAGGC

TTAAGGGCTT

CATACATGTG

TTAAATGCCT

TGATGGAAAG

TAAGAATTTT

ACTGGGTGAT

CTTCCTTTGT

TTGATTATAT

TAGAATTATT

TCTCTCTCTC

GAGCGGGAGG

GCATGTGCAC

TTTAATAATA

TGCTTGCATG

AGAATGCTAC

CATTGATTGA

AATCAATAAT

TACATTGGAT

GTACTAGTCA

TCCGTCCAAA

AACCGTTACA

AAAGGAAAGA

780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1811 PTGCCGAAA ATTTTTTTTC .CAACTGGT TGAAAGCCGA INFORMATION FOR SEQ ID NO: Wi SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: DNA TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ATGAAATTTT TATATCTAAT INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: DNA WO 97/44355 PCT/GB97/01205 -59- TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: GTCTCCGGAT TGAATGATAC INFORMATION FOR SEQ ID NO: 22: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: DNA TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: CATATACAGC GAAGTACC 18 18 INFORMATION FOR SEQ ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: DNA TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: CATAACTATG GGATTCTTAG G 21 INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: DNA TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: AAATGGACA ATGCTACTAC C 21 INFORMATION FOR SEQ ID NO: SEQUENCE

CHARACTERISTICS:

LENGTH: 18 base pairs TYPE: DNA TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TTAAATTGAT TCCAACGC 18 INFORMATION FOR SEQ ID NO: 26: WO 97/44355 PCT/GB97/01205 SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: DNA TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: AATGTGTTTG CACTCACG 18 18 INFORMATION FOR SEQ ID NO: 27: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: DNA TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: CCTCCCGCT CCATGATTTT G 21 INFORMATION FOR SEQ ID NO: 28: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: DNA TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: CCTCGCCGTG CTCTTTGGC 19 INFORMATION FOR SEQ ID NO: 29: SEQUENCE

CHARACTERISTICS:

LENGTH: 21 base pairs TYPE: DNA TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: GTAACCAGC TAAGCATAAC G 21 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: DNA TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: GTTATACAC AGAGGATCAG GGAG WO 97/44355 PCT/GB97/01205 -61- INFORMATION FOR SEQ ID NO: 31: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: DNA TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: AATACAGTCC CTATATACCC INFORMATION FOR SEQ ID NO: 32: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: DNA TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: GGGATGGGAA TAATGATGTC A 21 INFORMATION FOR SEQ ID NO: 33: SEQUENCE CHARACTERISTICS: LENGTH: 15 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: GACTCCTGGA GCCCG INFORMATION FOR SEQ ID NO: 34: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: TTGACACCAG ACCAACTGGT AATG 24 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid 71'L WO 97/44355 PCT/GB97/01205 -62- STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GCTGATGTGC TGCAAGGCGA TTAAG INFORMATION FOR SEQ ID NO: 36: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: TTCACAGAGG AAACAGCTAT GAC 23 INFORMATION FOR SEQ ID NO: 37: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: TCGTCAAGTG AAGAATTA 18 INFORMATION FOR SEQ ID NO: 38: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: TTATATGAAG TTCTTAGA 18 INFORMATION FOR SEQ ID NO: 39: WO 97/44355 PCT/GB97/01205 -63- SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: GGCGCAATAA TTCTTCAC 18 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TAACTATGGG ATTCTTAG, 18 INFORMATION FOR SEQ ID NO: 41: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: ATATTTAAAG CCTCCCGC 18 INFORMATION FOR SEQ ID NO: 42: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: TTYAAYCCNG ARACNCAYNG NGA 9a WO 97/44355 PCT/GB97/01205 -64- INFORMATION FOR SEQ ID NO: 43: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: GCNAAAGARC ASGGNGARGA INFORMATION FOR SEQ ID NO: 44: SEQUENCE CHARACTERISTICS: LENGTH: 59 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: TTCAACCCNG ARACNCACNG NGARNNNAAR AACTACNNNG CNAARGARCA TGGNGARGA 59 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 456 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bracon Hebetor (ix) FEATURE: NAME/KEY: CDS LOCATION:1..456 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ATG AAA TTT TTA TAT CTA ATA CCC CTT TTA ATT GCA GGA GTA GCA TCA 48 Met Lys Phe Leu Tyr Leu Ile Pro Leu Leu Ile Ala Gly Val Ala Ser 1 5 10 WO 97/44355 PCT/GB97/01205 TTC AAT GCG GCC ATA GAT CGT GAA Phe Asn Ala Ala Ile Asp Arg Glu CAC GGC His Gly ACG TTT Thr Phe AAT GAT Asn Asp TTT AAA Phe Lys TGC ATG Cys Met AAA GTT

L

y s Val GGA GAA Gly Glu 130 TTG GAA Leu Glu 145

GAG

Glu

AAA

Lys

AAG

Lys

TAT

Tyr

TGC

Cys

GAA

Glu 115

TGC

Cys

ATG

Met GAA TAT Glu Tyr TGT ATT Cys Ile TGT AGA Cys Arg GCC TTC Ala Phe 85 CAG GTG Gin Val 100 GAA AAG Glu Lys TAC TAT Tyr Tyr AAT AGC Asn Ser

GCT

Ala

TGC

Cys

CAA

Gin 70

AGC

Ser

AAA

Lys

GCC

Ala

CAA

Gin

ATT

Ile 150

ACA

Thr

ATC

Ile 55

AAA

Lys

ATA

Ile

AAT

Asn

CCA

Pro

GAT

Asp 135

GAT

Asp

TGG

Trp 40

GGA

Gly

TGT

Cys

TAC

Tyr

AAG

Lys

CCT

Pro 120 TGT AAG Cys Lys 25 TCT TTT Ser Phe AAC GGC Asn Gly ATT CAA Ile Gin AGT GAA Ser Glu 90 ACA TAT Thr Tyr 105 ATG AAT Met Asn

AAT

Asn

CGT

Arg

CTC

Leu

CAG

Gin 75

GTA

Val

TGT

Cys

CCC

Pro

TTT

Phe

TAC

Tyr

ATG

Met

CAT

His

CCA

Pro

ATA

Ile

ATA

Ile

TGC

Cys

GAA

Glu

AGA

Arg

GGA

Gly

GCT

Ala

CAT

His

GTG

Val 125

GCC

Ala

CTG

Leu

ACA

Thr

GCG

Ala

TCA

Ser

GCT

Ala 110

ATG

Met

AAA

Lys

GGT

Gly

GAG

Glu

GGA

Gly

TGG

Trp

TGC

Cys

AAA

Lys

GAG

Glu

GAT

Asp

AAT

Asn

GGC

Gly

GCA

Ala

ATG

Met

AAT

Asn 288 336 384 CAC AGG GGT GTT GAG AGG TCG TGT GAA His Arg Gly Val Glu Arg Ser Cys Glu 140 456 INFORMATION FOR SEQ ID NO: 46: SEQUENCE CHARACTERISTICS: LENGTH: 152 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: Met Lys Phe Leu Tyr Leu Ile Pro Leu Leu Ile Ala Gly Val Ala Ser 1 5 10 Phe Asn Ala Ala Ile Asp Arg Glu Cys Lys Asn Phe Cys Ala Lys Glu 25 His Gly Glu Glu Tyr Ala Thr Trp Ser Phe Arg Tyr Giu Leu Gly Asp 40 Thr Phe Lys Cys Ile Cys Ile Gly Asn Gly Leu Met Arg Thr Glu Asn 55 Asn Asp Lys Cys Arg Gin Lys Cys Ile Gin Gin His Gly Ala Gly Gly 70 75 WO 97/44355 PCT/GB97/01205 66- Phe Cys Lys Gly Leu 145 Lys Tyr Ala Phe Met Cys Gin Val 100 Val Glu Glu Lys 115 Glu Cys Tyr Tyr 130 Giu Met Asn Ser Ser Ile Tyr Ser Glu Val Pro Ala Ser Trp Ala 90 Lys Asn Lys Thr Tyr Cys Ile His Ala Cys Met 105 110 Ala Pro Pro Met Asn Pro Ile Val Met Lys Asn 120 125 Gin Asp His Arg Gly Val Glu Arg Ser Cys Glu 135 140 Ile Asp 150

Claims

1. A polynucleotide comprising a region encoding at least two of the insecticidal toxin subunits selected from those comprised in SEQ ID Nos. 2, 4, 6, 8, and 10 and that encoded by the spliced RNA derived from the genomic clone depicted in SEQ ID No. 19, the said spliced RNA being capable of hybridising with the extension products of the primers depicted in SEQ ID Nos 20 and 21, or SEQ ID Nos 22 and 23, or SEQ ID Nos 24 and using the sequence depicted in SEQ ID No. 1 as a template following incubation at a temperature of between 60 0 C and 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS, with the proviso that the polynucleotide does not encode only the combination of the subunits comprised in SEQ ID Nos. 2 and 8.

2. A polynucleotide according to claim 1, wherein the said subunits are the proteins represented by amino acids 17 to 158 in SEQ ID No. 2; amino acids 22 to 182 in SEQ ID No. 4; amino acids 32 to 176 in SEQ ID No. 6; amino acids 23 to 275 in SEQ ID No. 8; and amino acids 22 to 184 in SEQ ID 20 No.

3. A polynucleotide according to claim 2 wherein at least one of the said Sproteins comprises a heterologous N-terminal extension in the form of a signal or secretory peptide.

4. A polynucleotide comprising a region encoding at least one of the insecticidal toxin subunits selected from those comprised in SEQ ID Nos. 2, 4, 6, 8, and 10 and that encoded by the spliced RNA derived from the genomic clone depicted in SEQ ID No. 19, the said spliced RNA being capable of hybridising with the extension products of the primers depicted in SEQ ID Nos 20 and 21, or SEQ ID Nos 22 and 23, or SEQ ID Nos 24 and using the sequence depicted in SEQ ID No. 1 as a template following incubation at a temperature of between 60 0 C and 65 0 C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS, R y, ZZherein the region has been modified in that mRNA instability motifs and/or 67A fortuitous splice regions are removed, or insect-pest preferred codons are used so that expression of the thus modified polynucleotide in the said insect yields substantially similar protein having a substantially similar activity/function to that obtained by expression of the unmodified polynucleotide in the organism in which the protein encoding regions of the unmodified polynucleotide are endogenous. *SS. S S S WO 97/44355 PCT/GB97/01205 -68- A polynucleotide according to claim 4, wherein the said subunits are the proteins represented by amino acids 17 to 158 in SEQ ID No. 2; amino acids 22 to 182 in SEQ ID No. 4; amino acids 32 to 176 in SEQ ID No.6; amino acids 23 to 275 in SEQ ID No. 8; and amino acids 22 to 184 in SEQ ID No.

6. A polynucleotide according to claim 5, wherein at least one of the said proteins comprises a heterologous N-terminal extension in the form of a signal or secretary peptide.

7. A polynucleotide according to any one of claims 4-6, wherein the insect pest is Lepidopteran, and the said organism is an Hymenopteran of the superfamily Ichneumonoidea, in particular a wasp of the family Braconidae.

8. A polynucleotide according to any of the preceding claims wherein at least one of the protein encoding sequences in the region is under expression control of a viral promoter, or insect strong promoter, which is not down regulated or otherwise silenced when the polynucleotide is introduced into the cells of an insect which is or becomes infected by an insect virus.

9. A polynucleotide according to the preceding claim, wherein the promoter is selected from the group consisting of the baculovirus p10 promoter and the polyhedrin promoter.

10. A polynucleotide according to any preceding claim, wherein the region encodes not less than two and not more than four of the toxin subunits selected from the proteins represented by amino acids 17 to 158 in SEQ ID No. 2; amino acids 22 to 182 in SEQ ID No. 4; amino acids 32 to 176 in SEQ ID No.6; amino acids 23 to 275 in SEQ ID No. 8; and amino acids 22 to 184 in SEQ ID No. WO 97/44355 PCT/GB97/01205 -69-

11. A polynucleotide according to any preceding claim, wherein the region encodes not less than two and not more than three of the toxin subunits selected from the proteins represented by amino acids 17 to 158 in SEQ ID No. 2; amino acids 22 to 182 in SEQ ID No. 4; amino acids 32 to 176 in SEQ ID No.6; amino acids 23 to 275 in SEQ ID No. 8; and amino acids 22 to 184 in SEQ ID No.

12. A polynucleotide according to any preceding claim, wherein the region encodes two of the toxin subunits selected from the proteins represented by amino acids 17 to 158 in SEQ ID No. 2; amino acids 22 to 182 in SEQ ID No. 4; amino acids 32 to 176 in SEQ ID No.6; amino acids 23 to 275 in SEQ ID No. 8; and amino acids 22 to 184 in SEQ ID No.

13. A polynucleotide according to any one of claims 1 to 9, wherein the region encodes the three proteins represented by amino acids 22 to 182 in SEQ ID No. 4 or amino acids 22 to 184 in SEQ ID No. 10; amino acids 32 to 176 in SEQ ID No.6 and amino acids 23 to 275 in SEQ ID No. 8.

14. A polynucleotide according to any one of claims 4-7, wherein the said modified region encodes one of the toxin subunits selected from the proteins represented by amino acids 17 to 158 in SEQ ID No. 2; amino acids 22 to 182 in SEQ ID No. 4; amino acids 32 to 176 in SEQ ID No.6; amino acids 23 to 275 in SEQ ID No. 8; and amino acids 22 to 184 in SEQ ID No. A nucleotide sequence, encoding an insecticidal toxin sub-unit, which is complementary to one which when incubated at a temperature of between 50 and in single strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with single strength citrate buffered saline containing 0.1% SDS still hybridises with the sequence depicted in SEQ ID Nos. 1, 3, 5, 7, 9, or

19. i~ WO 97/44355 PCT/GB97/01205 16. A nucleotide sequence, encoding an insecticidal toxin sub-unit, which is complementary to one which when incubated at a temperature of between 55 and 0 C in single strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.5 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequence depicted in SEQ ID Nos. 1, 3, 5, 7, 9, or 19. 17. A nucleotide sequence, encoding an insecticidal toxin sub-unit, which is complementary to one which when incubated at a temperature of between 60 and 65 0 C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequence depicted in SEQ ID Nos. 1, 3, 5, 7, 9, or 19. 18. A nucleotide sequence, encoding an insecticidal toxin sub-unit, which is complementary to one which when incubated at a temperature of between 50 and in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequence represented by nucleotides 61 to 486 in SEQ ID No. 1; nucleotides 285 to 766 in SEQ ID No. 3; nucleotides 147 to 584 in SEQ ID No. nucleotides 161 to 219 in SEQ ID No. 7; or nucleotides 85 to 573 in SEQ ID No. 9. 19. A nucleotide sequence, encoding an insecticidal toxin sub-unit, which is complementary to one which when incubated at a temperature of between 60 and in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequence represented by nucleotides 61 to 486 in SEQ ID No. 1; nucleotides 285 to 766 in SEQ ID No. 3; nucleotides 147 to 584 in SEQ ID No. nucleotides 161 to 219 in SEQ ID No. 7; or nucleotides 85 to 573 in SEQ ID No. 9.

20. A polynucleotide according to any one of claims 1-13, wherein the said region comprises the nucleotide sequence of any one of claims 15 to 19. WO 97/44355 PCT/GB97/01205 -71-

21. A cell transformed with the polynucleotide of any one of claims 1-13 or 20, or the nucleotide sequence of any one of claims 14 to 19.

22. An organism regenerated from the cell of the preceding claim.

23. An insect pathogen comprising the polynucleotide of any one of claims 1-13 or 20, or the nucleotide sequence of any one of claims 14 to 19.

24. A pathogen according to the preceding claim, in the form of an insect virus. A pathogen according to claim 23, in the form of a recombinant baculovirus such as HaSNPV, AcMNPV or AFMNPV, a fungus or a Bacillus bacterium.

26. An insect virus according to either of claims 24 or 25, in combination with a fluorescent brightener particularly those comprising a stilbene diphosphonic acid group, other uv-stabiliser and/or an anti-oxidant.

27. An insecticidal composition comprising the following combinations of proteins: the proteins represented by amino acids 17 to 158 in SEQ ID No.2 and 22 to 182 in SEQ ID No. 4; (ii) the proteins represented by amino acids 17 to 158 in SEQ ID No.2 and 32 to 176 in SEQ ID No. 6; (iii) the proteins represented by amino acids 22 to 182 in SEQ ID No. 4 and 32 to 176 in SEQ ID No. 6; (iv) the proteins represented by amino acids 22 to 182 in SEQ ID No.4 and 23 to 275 in SEQ ID No. 8; the proteins represented by amino acids 1 to 145 in SEQ ID No. 4 and 23 to 275 in SEQ ID No. 8; (vi) the proteins represented by amino acids 17 to 158 in SEQ ID No.2, amino acids 32 to 176 in SEQ ID No. 6, and amino acids 23 to 275 in SEQ ID No. 8; 72 (vii) the proteins represented by amino acids 17 to 158 in SEQ ID No. 2, amino acids 22 to 182 in SEQ ID No. 4, and amino acids 32 to 176 in SEQ ID No. 6; (viii) the proteins represented by amino acids 17 to 158 in SEQ ID No. 2, amino acids 22 to 182 in SEQ ID No. 4, and amino acids 23 to 275 in SEQ ID No. 8; (ix) the proteins represented by amino acids 22 to 182 in SEQ ID No. 4, amino acids 32 to 176 in SEQ ID No. 6, and amino acids 23 to 275 in SEQ ID No. 8; the proteins represented by amino acids 17 to 158 in SEQ ID No. 2; amino acids 22 to 182 in SEQ ID No. 4, amino acids 32 to 176 in SEQ ID No. 6, and amino acids 23 to 275 in SEQ ID No. 8.

28. A composition according to the precedinig claim, wherein the composition is augmented by or at least one of the proteins in a :composition is replaced by the toxin subunit represented by amino acids 22 to 184 of SEQ ID No. 10 or that encoded by the spliced RNA derived from the genomic clone depicted in SEQ ID No. 19, the said spliced RNA being capable of hybridising with the extension products of the primers depicted in 20 SEQ ID Nos 20 and 21, or SEQ ID Nos 22 and 23, or SEQ ID Nos 24 and *9:using the sequence depicted in SEQ ID No. 1 as a template following incubation at a temperature of between 60 0 C and 65 0 C in 0.3 strength citrate :buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS.

29. A method of controlling insects, comprising exposing them or their habitat to the cell of claim 21, organism of claim 22, pathogen of claims 2 3-26 or composition of claims 27 or 28.

30. Use of the polynucleotide of any one of claims 1-13 and 20 or the nucleotide sequence of any one of claims 14-19, in the preparation of a recombinant baculovirus for the biological control of insect pests.

31. Use of the cell of claim 21, organism of claim 22, pathogen of claims 2 3-26 or composition of claims 27 or 28 to control insect pests. 73

32. A polynucleotide comprising a region encoding the insecticidal toxin subunit encoded by the spliced RNA derived from the genomic clone depicted in SEQ ID No. 19, the said spliced RNA being capable of hybridising with the extension products of the primers depicted in SEQ ID Nos 20 and 21, or SEQ ID Nos 22 and 23, or SEQ ID Nos 24 and 25 using the sequence depicted in SEQ ID No. 1 as a template following incubation at a temperature of between 60 0 C and 65 0 C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS, the translation product of the polynucleotide and recombinant entomopathogenic viruses comprising the said polynucleotide or a nucleotide comprising the spliced variant thereof. Dated this 4th day of November 1999 COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION Patent Attorneys for the Applicant: F B RICE CO