WO1997044355A1

WO1997044355A1 - Biological insect control agent

Info

Publication number: WO1997044355A1
Application number: PCT/GB1997/001205
Authority: WO
Inventors: Rachael Elizabeth Duncan; Marie-Marthe Suner; Allan Daly; Peter Daniel Christian; John David Windass; Annette Claudianos
Original assignee: Commonwealth Scientific & Industrial Research Organisation
Priority date: 1996-05-22
Filing date: 1997-05-01
Publication date: 1997-11-27
Also published as: AU2647297A; ID16968A; AU714456B2; CO4600752A1

Abstract

The present invention provides, inter alia, 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. The invention also provides an insect pathogen, particularly a recombinant baculovirus comprising the said polynucleotide, and various modifications thereof.

Description

BIOLOGICAL INSECT CONTROL AGENT

The present invention relates, inter alia, to die 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 die 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 (B. 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 and 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 LD 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 LD No.6; amino acids 23 to 275 in SEQ ID No. 8; and a-αύno 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 10 and that encoded by the spliced RNA derived from the genomic clone depicted in SEQ LD 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 LD Nos 22 and 23, or SEQ LD Nos 24 and 25, using the sequence depicted in SEQ ID No. 1 as a template, wherein the region has been modified in that mRNA instability motifs and/or fortuitous splice regions arc 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 LD No. 8; and amino acids 22 to 184 in SEQ LD 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 p 10 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 LD No. 4; amino acids 32 to 176 in SEQ LD 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 LD No. 8; and amino acids 22 to 184 in SEQ LD No. 10, with the proviso that the region does not encode only the combination of the toxin subunits comprised in SEQ LD 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 LD No. 4 or amino acids 22 to 184 in SEQ LD No. 10; amino acids 32 to 176 in SEQ LD No.6 and amino acids 23 to 275 in SEQ LD 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 LD No. 2; amino acids 22 to 182 in SEQ LD No. 4; amino acids 32 to 176 in SEQ LD No.6; amino acids 23 to 275 in SEQ ID No. 8; and amino acids 22 to 184 in SEQ LD No. 10.

The invention also provides a nucleotide sequence, encoding an insecticidal toxin sub- unit, which is complementary to one which when incubated at a temperature of between 50 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 LD No. 1" includes a polynucleotide which is identical to that depicted in SEQ LD 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 LD 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 sub- unit, which is complementary to one which when incubated at a temperature of between 50 and 60°C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at die same temperature witii 0.3 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequence represented by nucleotides 61 to 486 in SEQ LD No. 1; nucleotides 285 to 766 in SEQ LD 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 LD 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°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 LD No. 1; nucleotides 285 to 766 in SEQ LD No. 3; nucleotides 147 to 584 in SEQ LD No. 5; nucleotides 161 to 219 in SEQ LD 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 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 witiiin 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 Beauvaήa species such as B. bassiana.

Viruses are particularly preferred. Suitable insect viral pathogens include bacuioviruses, entomopoxviruses, reoviruses, iridoviruses, parvoviruses, rhabdoviruses, picomaviruses and picoma-like viruses, nodaviruses, ascoviruses and retroviruses, with bacuioviruses being preferred. Bacuioviruses 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 (GV), and non-occluded baculovirus. Examples of bacuioviruses include: Autographa californica MNPV (AcMNPV), Anagrapha falcifera MNPV (AfMNPV), Bombyx mori MNPV (BmMNPV), Anticarsia gemmatalis

MNPV (AgMNPV), Cydia pomonella GV (CpGV), Helicoverpa armigera SNPV (HaSNPV), 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 bacuioviruses 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 bacuioviruses, 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: (i) the proteins represented by amino acids 17 to 158 in SEQ ID No. 2 and 22 to 182 in SEQ LD No. 4; (ii) the proteins represented by amino acids 17 to 158 in SEQ LD No. 2 and 32 to 176 in

SEQ LD No. 6; (iii) the proteins represented by amino acids 22 to 182 in SEQ LD No. 4 and 32 to 176 in SEQ LD No. 6; (iv) the proteins represented by amino acids 22 to 182 in SEQ LD No. 4 and 23 to 275 in

SEQ ID No. 8; (v) the proteins represented by amino acids 32 to 176 in SEQ LD No. 6 and 23 to 275 in SEQ ID No. 8; (vi) the proteins represented by amino acids 17 to 158 in SEQ LD No. 2, amino acids 32 to 176 in SEQ LD No. 6, and amino acids 23 to 275 in SEQ LD No. 8; (vii) the proteins represented by amino acids 17 to 158 in SEQ LD No. 2, amino acids 22 to

182 in SEQ LD No.4, and amino acids 32 to 176 in SEQ LD No. 6; (vϋi) 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 LD No. 8;

(ix) the proteins represented by amino acids 22 to 182 in SEQ ID No.4, amino acids 32 to

176 in SEQ LD No. 6, and amino acids 23 to 275 in SEQ LD No. 8; (x) the proteins represented by amino acids 17 to 158 in SEQ LD No. 2; amino acids 22 to 182 in SEQ ID No.4, amino acids 32 to 176 in SEQ LD No. 6, and amino acids 23 to 275 in SEQ LD 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 LD 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: (i) 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 die 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. 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 LD No. 19, the said spliced RNA being capable of hybridising with the extension products of the primers depicted in SEQ LD Nos 20 and 21, or SEQ LD Nos 22 and 23, or SEQ LD Nos 24 and 25 using the sequence depicted in SEQ LD No. 1 as a template, the translation product of die 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 die putative message is shown below the coding sequence.

Figure 2 is an alignment of homologous regions of the genomic clones pBH(a)λ3.4- Pst.2 and pBH(a)λ3.5-Pst.5, the former contains the sequences that encode BrhTX-l(a) (see Figure 1).

Figure 3 is a plasmid map of pMMS 1. Figure 4 is a plasmid map of pAcUW21. SEQ LD 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 LD No. 1. Amino acids 1- 16 of the sequence shown in SEQ LD No. 2 constitute a putative leader or signal sequence. Amino acids 17 to 158 of die 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 LD No.4 constitute a putative leader or signal sequence. Amino acids 22 to 182 of die 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 21kDa as judged by SDS electrophoresis. SEQ LD No. 6 shows the translational product of the ORF identified in SEQ LD No. 5, together with a putative signal peptide.

SEQ ID No.7 shows a fourth nucleotide sequence comprising a region encoding a fourtii insecticidal toxin subunit ((BrhTX- Id) having a molecular weight of about 32kDa. SEQ ID No. 8 shows the translational product of the ORF identified in SEQ LD No. 7. Amino acids 1 to 22 of the sequence shown in SEQ LD No. 8 constitute a putative leader or signal sequence. Amino acids 23 to 275 of me sequence constitute die mature toxin-subunit. SEQ ID No. 9 shows a fifth nucleotide sequence - substantially similar to that depicted in SEQ LD No. 3 - comprising a region encoding a fifth insecticidal toxin subunit ((BrhTX- le) having a molecular weight of about lδkDa. SEQ LD No. 10 shows the translational product of die ORF identified in SEQ LD No. 9. Amino acids 1 to 21 of the sequence shown in SEQ LD No. 10 constitute a putative leader or signal sequence. Amino acids 22 to 184 of die sequence constitute die mature toxin-subunit.

SEQ LD No. 11 shows a genomic sequence which comprises part of a spliced RNA that is related to die cDN A comprised by pBrhTX- 1 (a) 1.1 , 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 LD No. 13 shows the sequence of pBrhTX-l(a)GBsρ2 and pBrhTX-l(a)GBsp4; SEQ ID No. 14 shows the sequence of the 700bp Spe I fragment isolated from me λ(a)G- 2.1.1 genomic clone.

SEQ LD Nos. 15-18 and 20 - 44 show the sequences of the various PCR primers. SEQ LD No. 19 shows a contiguous genomic sequence which encodes part of a spliced RNA tiiat is related to the cDNA comprised by pBrhTX- 1(a) 1.1, and which encodes a protein which is substantially similar to die protein depicted in SEQ ID No. 2 SEQ ID No. 45 discloses a putative toxin-encoding-sequence derived from me genomic sequence depicted in SEQ LD No. 19 and SEQ ID No. 46 discloses the translation product of die nucleotide sequence depicted in SEQ LD. No.45.

International Patent Apphcation 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 die cloning of die sequences encoding the subunits of this toxin. These descriptions are incorporated by reference into die present application. Unless specifically indicated to die contrary, all amino acid and nucleotide sequences which are depicted in, or are referred to in die 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 die sequence listing appended to tiiis apphcation.

Example 1 -Identification of a BrhTX- 1(a) coding sequence in addition to tiiat disclosed in

WO96/16171

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 dirough MOPS/formaldehyde, 1% agarose gels. Blots for hybridisation are made widi 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 arc carried out in buffers containing 50% formamide at 42°C.

³²P-labelled probes are syndiesised 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, BrhTX- l(a)1.2 digested with Not I is used as template. Primers for the 5' end arc BH(a)F5 and BH(a)R5 (SEQ LD Nos 20, 21 ), middle BH(a)F3 and BH(a)R6 (SEQ LD Nos 22, 23) and for the 3' end BH(a)F6 and BH(a)R4 (SEQ ID Nos 24, 25)

Reaction conditions for generation of ³²P-labelled probes using die above primers are as follows:

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

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

Blots are then hybridised with one of die 3 probes (5', middle or 3') as detailed above. Results from tiiis analysis are shown in Table 1: The minimum size of the message (as deduced from die cDNA clone) is about 560 bases - with all of die 3' untranslated accounted for i.e. the clone contains die polyA tail. It therefore seems likely tiiat the 706 base band identified above corresponds to die message encoding BrhTX- 1(a). The presence of die 1694 base band however suggests tiiat anomer RNA species is produced dial contains sequences related to regions of the BrhTX- 1 (a) 1.1 cDNA clone. Genomic Southern Analysis

Genomic DNA is prepared as described for the Examples in WO-99/16171, and 5- lOmg aliquots are digested witii die restriction enzymes Aat 13, Ace I, Apa L Bam HI, Bel I, Bsp HL Cia L Eco RL Eco RV, Hind ILL Kpn I, Mbo U, Nco I, Nde L Not I, Pst I, Sac I, Sac IL Sal I, Sma L Spe I, Xba I and Xho I. Fragments arc then separated by electrophoresis dirough 0.7% agarose gels in TAE buffer and ώe DNA blotted onto nitrocellulose membranes (Nitro-pure, supported nitrocellulose, MSI, MA, USA) in a 2-20x SSC gradient and bonded to die membrane by baking at 80°C for 2 hours. ³² P-labelled probes are syntiiesised by PCR using the primers BH(a)F2 and BH(a)R4 (SEQ LD Nos 26, 25) and pBrhTX- 1(a) 1.2 plasmid DNA as target DNA under die following conditions: 5 minutes at 95°C

30 seconds at 95°C, 30 seconds at 45°C, 1 minutes at 72°C (5 cycles); then 30 seconds at 95°C, 30 seconds at 50°C, 1 minute 72°C (30 cycles) men, 5 minutes at 72°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 widi BrhTX- 1(a) cDNA

Region-Specific Probes

hybridising fragment is observed - with one of die fragments hybridising more strongly than the odier. The sizes and relative hybridisation intensity of die fragments from diese digests arc summarised below.

Major Minor Xba l 5.7Kbp 2.5Kbp

Spe I >16Kbp 0.7Kbp

Nde I 7.0Kbp 0.9Kbp

Bsp HI 3.8Kbp 1.2Kbp

Mbo ll l.lKbp Pst I 3.4Kbp

Bel I 1.4Kbp

Ace I 2.9Kbp and

1.5Kbp Initial analysis of die above data suggests tiiat tiiere is a single BrhTX- 1(a) gene in die Bracon genome, and that die gene contains an Ace 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 die three probes designed to be homologous to regions at 5' end, middle and 3' end of BrhTX- 1(a) cDNA gives the results presented in Table 2. In contrast to die results obtained using a probe generated from die cDNA using the primers BH(a)F2 and BH(a)R4 (SEQ LD Nos 26, 25) the results using the much smaller region-specific probes indicates tiiat there is a second sequence in die Bracon hebetor genome with sequence similarity to mat of the BrhTX- 1(a) cDNA. This latter observation is in agreement witii the Northern hybridisation results that indicate a second mRNA widi sequence homology to die pBrhTX-l(a) cDNA clone. Table 2. Results of Southern Analysis Performed on 17Kda cDNA using Region- Specific Probes.

Generation of a Genomic Library from Bracon hebetor

Genomic DNA was prepared from female wasps as previously described and subjected to partial digestion witii SauiA I. The digested DNA is tiien ligated into λGEM-11 Xho I half site arms under conditions specified by die supplier (Promega, Madison, WT). After packaging die phage are plated into E. coli Strain LE392. Subsequent titration and amplification of the library was carried out using tiiis strain. Isolation and Sub-cloning of Genomic Sequences

The λGEM-11 genomic Ubrary is plated and plaque lifts carried out as previously described for cDNA libraries. ³²P-labelled primers are syntiiesised by PCR as described above using die BH(a)F2 and BH(a)R4 primers (SEQ LD Nos 26, 25). Pre-hybridisation and hybridisation of filters from plaque-lifts are performed under die 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 tiirough a further three rounds of plaque purification. DNA is then purified from the phage as previously described and restriction digests performed to estimate die size of die inserts. These insert sizes and designation of phage are shown below: Approx insert size λBH(a)G-2.1.1 21.0 Kbp λBH(a)G-3.1.1 19.1 Kbp λBH(a)G-4.1.1 15.0 Kbp λBH(a)G-5.1.1 18.9 Kpb λBH(a)G-6.1.1 19.3 Kbp λBH(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 die same probe and conditions as outlined above for their isolation. This analyses demonstrate that die clones all share a number of identical bands and are thus related to each other. Because of the close relationship between die clones further analyses are tiierefore concentrated upon one of the phage namely, λBH(a)G-2.1.1 Southern analysis of the genomic λBH(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 tiiat 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 LD 26, 25). Consequently, tiiese fragments are isolated after electrophoresis on 0.8% agarose gels and cloned into die Nco I and Spe I sites of pGEM5z(f)+ (Promega) and pBluescript SK- for die 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-l(a)GBspl, pBrhTX-l(a)GBsp2, pBrhTX- 1 (a)GBsp3 and pBrhTX- 1 (a)GBsp4) and Spe I fragments (pBrhTX- 1 (a)GSpe 1 pBrhTX-l(a)GSpe2, pBrhTX-l(a)GSpe3, pBrhTX-l(a)GSpe4, pBrhTX-l(a)GSpe5, pBrhTX-l(a)GSpe6) are sequenced using the ABI Prism Dye terminator cycle sequencing kit widi AmpliTaq DNA Polymerase, FS and analysed using die ABI 373 Automated Sequencer. Two distinct sequences are obtained from me four Bsp HI sub-clones analysed. pBrhTX-l(a)GBspl and pBrhTX-l(a)GBsp3 are identical (SEQ ID No 12) while pBrhTX- l(a)GBsp2 and pBrhTX-l(a)GBsp4 are also identical to each other (SEQ LD No 13). However, an alignment of these two sequences reveals that although similar they are not identical. Analysis of die six Spe I subclones reveals a single sequence (SEQ LD No 14).

Initial sequence analysis is also carried out by direct sequencing from λBH(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 witii AmpliTaq DNA Polymerase, FS. Samples from cycle sequencing reactions are analysed using die ABI 373 Automated Sequencer. No sequence is obtained from die 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 die graphic summary of die sequencing analysis. Using these sequence data an initial sequence is obtained spanning die whole of die region containing all of die putative cDNA sequence represented by the pBrTX- 1(a) 1.2.

Alignment of this initial sequence is then carried out witii the pBrhTX-l(a)GBspl/3 sequence which showed a high degree of homology. In contrast alignments performed witii die 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 λ genomic clone, die implication from me above data is tiiat tiiere is more man one sequence in that λ clone with relatively high homology to die 5' end of the BrhTX-l(a) cDNA. The two Pst I sub-clones (termed pBH(a)λ3.5-Pst.2 and pBH(a)λ3.5-Pst.5) arc 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 LD 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 LT, Xba I and Ace I fragments are also subcloned from the pBH(a)λ3.5-Pst.2 and pBH(a)λ3.5-Pst.5 subclones and sequenced using die automated dye-terminator method described above.

From all of the sequence data available a consensus of die genomic region encoding die mRNA from which the BrhTX-l(a) mRNA is derived (SEQ ID 19). This sequence is shown in Figure 1 witii the putative intron/exon structure and die translation of the deduced mRNA. This sequence is contained completely witiiin die pBH(a)λ3.5-Pst.2 subclone. Sequence analysis of die pBH(a)λ3.5-Pst.5 subclone and die subclones derived from it produce a single contiguous sequence that contains all of the pBrhTX-l(a)GBsp2/4 sequence (SEQ ID 13) and the 700bp Spe I subclones (SEQ ID 14) derived from λBH(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- 1(a) is shown in Figure 2.

While the genomic sequence that encodes BrhTX- 1(a) and the sequence (SEQ ID 19) mat shows homology to it, are both located in the 21.0 kbp fragment of genomic DNA contained in λBH(a)G-2.1.1, die 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, 30, 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' BH(a)PST5R 5'-AAT ACA GTC CCT ATA TAC CC-3' BH(a)PST5L 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)λ3.5-Pst.2 and pBH(a)λ3.5-Pst.5) with tiieir 5'-3' orientation being away from die middle of those clones. All of the pairwise combinations of these primers are then used in PCR experiments under the following conditions using λBH(a)G-2.1.1 DNA as a target. 5 minutes at 95°C

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

Only the BH(a)PST2L and BH(a)PST5R combination produces an amplicon ie. a fragment of 305 bp. This indicates that die two fragments are virtually adjacent to each other within the λBH(a)G-2.1.1 clone. Sequencing with the primers BH(a)PST2L and BH(a)PST5R directly from the λBH(a)G-2.1.1 DNA confirms that the Pst I fragments are adjacent in die genomic clone. In relation to the putative transcriptional orientation of the sequence encoding BrhTX- 1(a), die homologous sequence is located in the fragment situated upstream. Example 2 - Cloning of subunit (b) of BrhTX- 1

This is performed as described in WO96716171. 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' BH(b)B 5'-GG (ATG)CC (AG)AA (AGT)GT (TC)TT (AG)TC-3'

These primers arc 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 μl Taq DNA Polymerase/50 μl reaction are as follows: 5 minutes at 95°C; then

30 seconds at 95°C; 1.5 minutes at 50°C; 1 minute at 72°C (5 cycles); then 30 seconds at 95°C; 1.5 minutes at 55°C; 1 minute at 72°C (30 cycles); then 5 minutes at 72°C.

This PCR product are of approximately 54 bp, as estimated by PAGE in 15% gels in TBE buffer. Amphfication products are visualised in the gels by staining with etiiidium bromide. The fragment is then cloned into die EcoRV site of die commercially available plasmid pBluescript SK- that has been tailed with a tiiymine residue (T-tailing) using Taq DNA Polymerase. The cloned PCR product is sequenced using the following dye primers which are available from Applied Biosystems Ine: Universal M13-20 dye-primer: 5'-CAG GAA ACA GCT ATG ACC-3¹ 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 me PCR product is given below.

5'-ACC 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 WO96/16171. Plaque lifts are performed in duplicate onto Nitropure nitrocellulose membranes, 137 mm 0.45 micron, (Microb Separations, Westboro, MA, USA). In the primary screen 3.6xl0⁵ plaques per filter (4 filters in total) arc screened, in secondary and tertiary screens 100-250 plaques per filter are screened.

³²P-labelled probes are made by PCR. PCR is carried out in die presence of ³²P-dATP using the oligonucleotides BH(b)A and BH(b)B shown in Sequence ID Nos. 38 and 39 of WO96/16171, and the cloned PCR product carrying the sequence shown in Sequence ID No. 40 (WO96/16171) as the target.

The following are the PCR conditions: 95°C for 5 minutes prior to the addition of Taq polymerase; men 95°C for 30 seconds, 45°C for 1.5 minutes and 72°C for 1 minute (5 cycles); then 95°C 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 witii the ³²P-labelied probe described above under die same pre- hybridising and hybridising conditions as described for the Genomic Southern Blot of subunit BrhTX- 1(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 1ml of SM buffer plus 10 μl of chloroform. Characterisation of Clones

The plaque-purified phage are screened for die presence and size of a cDNA insert by PCR analysis using the oligonucleotides λgt 11 forward and λgt 11 reverse. Lambda DNA for PCR is prepared from purified stocks by boiling 10 μl of die stock for 5 minutes. Two μl 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 tiiree purified phage, an insert of approximately 500 bp is found in one phage, which phage is designated λBrhTX-1 (b)l. Subcloning and Sequencing of Clones

Phage DNA is purified by isopycnic centrifugation in CsCl as described in Sambrook et al (1989).

The cDNA insert from λBrhTX-1 (b)l is removed by digestion with Not I and ligated into pBluescript SK- that is digested witii Not I and phosphatase treated. The insert of die clone is sequenced using the ABI dye-primer sequencing kit (as described above). The sequence of the plasmid clone, pBrhTX-l(b)l, insert is given in Sequence ID No.41 of WO96/16171. The amino acids 27-44 match those of die N-terminal sequence shown in

Sequence ID No. 2 of WO96/16171, 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 die ORF at the 5' end of die clone, namely BH(b)C and BH(b)D as shown in Sequence ID Nos.42 and 43 respectively of WO96716171 are designed. Using these primers and pBrhTX- 1 (b) 1.1 as a target a ³²P-labelled probe by PCR is generated under die following conditions: 5 minutes at 95°C;

30 seconds at 95°C; 1.5 minutes at 45°C; 1 minute at 72°C (5 cycles); men 30 seconds at 95°C; 1.5 minutes at 50°C; 1 minute at 72°C (5 cycles); then 5 minutes at 72°C. The library is then re-screened using die conditions described above. Six strongly hybridising plaques are purified tiirough to homogeneity in three rounds of plaque purification. cDNA inserts are removed from die phage by digestion witii Not I and ligated into pBluescript SK- tiiat has been digested witii Nor I and phosphatase treated. The sizes of die inserts are estimated by digestion widi Not I and separation of restriction fragments tiirough 0.8% agarose gels in TAE buffer.

The following cDΝA insert sizes are estimated: λBrhTX-l(b)2 - about 500 bp; and λBrhTX-1 (b)3, λBrhTX-l(b)4, λBrhTX-l(b)5 and λBrhTX-1 (b)6 all at about 1200 bp.

The plasmid designated pBrhTX-l(b)6 is sequenced using the ABI 370 A DΝA analysis system and die 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 eidier 182 or 165 amino acids depending upon die 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 WO96/16171, the ahgnment of die 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 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 die ORF predicted for die sequenced clone. It can therefore be deduced tiiat the clone pBrbTX-l(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 amphfication product. cDNA is synthesised from female fl. hebetor mRNA as previously described for die construction of the cDNA library. The amphfication products are then cloned into die EcoRV site of pBluescript that are

T-tailed as previously described. These clones are tiien sequenced and all are found to contain die same sequence as pBrhTX-l(b)6 flanking me apparent GT anomaly, but have an additional six bp corresponding to die codons for G and T, i.e. GGAACT.

From these data it appears that the GT anomaly arises during die cDN A syntiiesis/cloning of die original λ clone. The consensus sequence for die pBrhTX-l(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 witii Pst I, EcoRV, Ace I and Xho I are generated as previously described in WO96/16171. A ³²P-labelled probe is generated by PCR from the 5' region of the pBrhTX- l(b)6 clone using the primers BH(b)C and BH(b)D under the following conditions: 5 minutes at 95°C;

30 seconds at 95°C; 1.5 minutes at 45°C; 1 minute at 72°C (5 cycles); then 30 seconds at 95°C; 1.5 minutes at 50°C; 1 minute at 72°C (5 cycles); then 5 minutes at 72°C. 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 die 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, ³²P-labelled probes are made from die 3' region of the pBrhTX-l(b)5 clone using die primers BH(b)E and BH(b)F under the following conditions: minutes at 95°C;

30 seconds at 95°C; 1.5 minutes at 45°C; 1 minute at 72°C (5 cycles); then 30 seconds at 95°C; 1.5 minutes at 50°C; 1 minute at 72°C (5 cycles); then 5 minutes at 72°C. These probes hybridise to Soudiern blots prepared as previously described using male and female genomic DNA mat has been digested witii Bel I, Nde I, Bgl H and Pst I. Results are identical for male and female DNA witii die Bel I and Bgl U digests single hybridising bands of 1.7 kbp and 9.1 kbp are generated. Altiiough these results do not fully agree with the presence of botii tiiese sites within the probe hybridising region, i.e. one would normally expect to see two hybridising bands, botii sites are relatively close to the ends of die probe region and die fragment containing die smaller portion of the hybridising region can evade detection with the large probes that arc used in tiiis 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- 1(b) subunit The aim of recovering an alternative form of die BrhTX- 1(b) subunit is to obtain a cDNA without the long 5' untranslated end and encoding die GT amino acid pair which peptide sequencing suggests might be present in some BrhTX- 1(b) proteins but which is not encoded by clone pBrhTX-l(b)6. Such results might be explained if tiiere are polymorphic alleles of the BrhTX- 1(b) gene, alternatively, the two forms of BrhTX- 1(b) may be encoded by mRNA molecules generated by differential processing of the primary gene transcript. The strategy chosen to access DNA sequences encoding die candidate alternative form of BrhTX- 1 (b) is to use PCR to obtain products from the Bracon genomic and cDN A 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 die primers. KED(b)F 5'- TTTAGATCTGCGGCCGCCACCAATGTCAATCATATGTAAAAT -3'

Bglll Notl Kozak Start KED(b)R

5'- CCC<JAATTCGCGGCCGCTCATTATTCAGTTGGAAATCTAAAG -3'

EcoRI Norl Stop Stop

The PCRs arc performed using 0.5μl (approximately 5xl0⁴ pfus) of genomic or cDΝA Ubrary as template, 1 OOpmol of each primer, 16μl Ultrapure dΝTPs (Pharmacia) [ 1.25mM of each], lOμl lOx buffer [500mM KCl, lOOmM Tris, pH8.3, 15mM MgCl₂, 0.1% (w/v) gelatin] and 0.8μl (4 units) Taq DΝA polymerase (Perkin/Elmer Cetus). The reactions are carried out in a Hybaid OmniGene programmable dri-block and subjected to die foUowing temperature regime: 5 minutes at 95 °C;

30 seconds at 95 °C; 30 seconds at 47 °C; 1.2 minutes at 72 °C;

35 cycles followed by a final incubation period of 7 minutes at 72 °C. No products are obtained from the genomic library. However a product of the expected size is obtained from die cDNA library. This product is digested with BgiTL and EcoRI. It is tiien cloned into BglΩ. and EcoRI-digested, phosphatase-treated pMMS 1. (pMMSl is a derivative of pUC19, the polyUnker of which has been modified so that it contains BglR and EcoRI restriction endonuclease sites. pMMSl 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)Fl 5'- CGATGGTATCGTCAACAT -3' BH(b)F2 5^*- CGAATAAAGGATTTTCAA -3' BH(b)F3 5'- CACCCCATACTGTATATG -3'

BH(b)Rl 5'- CCAGCTCTTGCGGGAGAA -3' BH(b)R2 5'- ATGCAATATTTCCACAAC -3'

BH(b)R3 5'- TTCTGCAGCAATCACCAA -3'

The sequencing data shows that die PCR product encodes a BrhTX 1 -(b) subunit including the GT amino acid pair..

Example 3 - Production of recombinant polyhedrin positive AcMNPV derivatives expressing die BrhTX- 1(a) subunit of the toxin from B. hebetor Subcloning the cDNA into pAcUW21 pAcUW21 is a commerciaUy available baculovirus transfer vector (R&D Systems Europe Ltd., Abingdon, Oxon, UK) which can be convenientiy used to generate recombinant polyhedrin positive (pot) 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 U sites) so that they can be transcribed from the p 10 promoter. A plasmid map of pAcUW21 is shown in Figure 4.

The cDNA encoding die BrhTX- 1(a) subunit is excised from clone pBrhTX- 1(a) 1.1 by EcoRI digestion. The released insert is then cloned into EcoRI digested, phosphatase-treated pAcUW21 witii T4 DNA Ugase in appropriate buffers (Sambrook 1989). Ligation progeny are recovered by transformation of E.coli DH5α ceUs under standard conditions (Sambrook 1989). Transformants are selected on LB agar plates containing lOOμg/ml ampicillin. Transformant colonies are grown overnight in L-broth containing lOOμg/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 tiien subjected to sequence analysis across die 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'-TTAAATTGATTCCAACGC-3'

Two transformants, designated pACBH(a).l 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 ceUs Cotransfection is carried-out using the BacPAK6 kit (Clontech Laboratories, Palo Alto, CA, USA). Cotransfection of Sβ\ insect cells with J9.ϊw361-digested BacPAKό DNA and each transfer vector containing the BrhTX- 1(a) subunit is carried according to die 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 IO^'1, 10^' ², 10^"3 as weU as neat supernatant. Individual polyhedrin positive plaques, which fail to stain blue when exposed to the chromogenic substrate X-Gal, are picked into sterile 1.5ml microcentrifuge tubes containing 0.5ml TC 100/10% FCS medium. Mini-amphfication of the recombinant viruses Mini-amphfications arc carried-out using standard methods (King and Possee, 1992). Tissue culture flat flasks (25m³ ) (Corning, New York, USA) are seeded with lxlO⁶ S/21 cells and incubated overnight at 28°C to form a monolayer. Next day tiiey are infected witii 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 PBBs) pelleted by centrifugation in Falcon 2097 tubes at 3000rpm in a Sorvall RT6000B bench top centrifuge. Supernatants are stored at 4°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 lxlO⁷ PTJB/ml to be used in biological assessment of die virus activity against first instar Heliothis virescens larvae.

Example 4 - Production of recombinant polyhedrin positive AcMNPV derivatives expressing the BrhTX- 1 (h) suhunit of the toxin from B. hebetor

Subcloning the BrhTX- 1(b) cDNA into pAcUW21 The cDNA encoding the Brh-TX- 1 (b) subunit is excised from clone pBrhTX-l(b)6 by Nσfl digestion. The insert is blunt-ended by filling in using the Klenow fragment of DΝA polymerase using standard methods (Sambrook 1989). The insert is then cloned into EcoRI-digested, Klenow polymerase blunt-ended , phosphatase-treated pAcUW21 with T4 DΝA hgase in appropriate conditions for blunt end Ugations (Sambrook 1989). Ligation progeny are recovered by transformation oi E.coli DH5α cells under standard conditions (Sambrook 1989). Transformants are selected on LB agar plates containing lOOμg/ml ampicilhn. Transformant colonies are grown overnight in L- brodi containing lOOμg/ml ampicillin at 37°C. Candidate pAcUW21 /BrhTX- 1(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 Νos. 17 and 18) and a Sequenase kit (USB, Cleveland, Ohio). One transformant, designated pACBH(b).1 , with die insert in the correct orientation is selected for recombinant baculovirus construction. A caesium chloride plasmid preparation is used for this purpose.

PACF1: 5'- TTCCTTACGCGAAATACG -3'

PACR1: 5'- GAATTATTATCAAATCAT -3' Co-transfection of 5/21 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- 1(b) subunit of the toxin from B. hebetor Subcloning die cDNA into pAcUW21 The insert encoding the edited BrhTX- 1 (b) subunit is as described in Example X is excised from pMMSedlδ by Bglϋ. and EcoRI digestion. This insert is then cloned into BgtLλ, EcoRI-digested, phosphatase-treated pAcUW21 witii T4 DNA ligase under appropriate conditions and ligation progeny arc recovered by transformation oiEcoli DH5α cells under standard conditions (Sambrook 1989). Transformants arc selected on LB agar plates containing lOOμg/ml ampicillin. Transformant colonies are grown overnight in L-brotii containing lOOμg/ml ampicillin at

37°C. Candidate pAcUW21/BrhTX-l(b) recombinant plasmids arc recovered using a Wizard minipreparation kit (Promega Coφoration, 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, witii the insert is selected for recombinant baculovirus construction. A caesium chloride plasmid preparation is used for this purpose. Cotransfection of 5/21 cells, isolation and miniamphfication of recombinant AcMNPV is described in Example 3. Example 6 - Production of recombinant polyhedrin positive AcMNPV derivatives expressing the BrhTX- 1(c) subunit of the toxin from B. hebetor

Subcloning the cDNA into pAcUW21 The cDN A encoding the BrhTX- 1 (c) subunit is excised from clone λBrhTX-l(c)5 by Nofl digestion. The insert is blunt-ended by filling in using the Klenow fragment of DΝA polymerase using standard methods (Sambrook 1989). It is tiien cloned into EcoRI-digested, Klenow polymerase blunt-ended, phosphatase-treated pAcUW21 with T4 DΝA ligase under appropriate conditions for blunt end ligations

(Sambrook 1989). Ligation progeny are recovered by transformation of E. coli DH5α ceUs under standard conditions (Sambrook 1989). Transformants are selected on LB agar plates containing lOOμg/ml ampicillin. Transformant colonies are grown overnight in L-brotii containing lOOμg/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).l, with the insert in the correct orientation to be expressed from the vector plO promoter is selected for recombinant baculovirus construction. A caesium chloride plasmid preparation is used for this purpose. Cotransfection of 5/21 cells and isolation and mini-amphfication of recombinant AcMNPV is as described in Example 3. Example 7 - Production of recombinant polvhedrin positive AcMNPV derivatives expressing the BrhTX Kd) 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- 1(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 DH5α cells under standard conditions (Sambrook 1989). Transformants are selected on LB agar plates containing lOOμg/ml ampicillin. Transformant colonies are grown overnight in L- broth containing lOOμg/ml ampicillin at 37°C. Candidate pAcUW21/BrhTX- 1(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 PACF1 and PACR1 and using a Sequenase kit (USB, Cleveland, Ohio). One transformant, pACBH(d).l with the insert in die correct orientation is selected for recombinant baculovirus construction. A caesium chloride plasmid preparation is used for this purpose. Cotransfection of 5/21 ceUs and isolation and mini-amphfication of recombinant AcMNPV is as described in Example 3. Example 8 - Physical characterisation of recombinant AcMNPV bv Southern Blot Analysis These analyses are undertaken to check tiiat the physical environment and structure of the recombinant bacuioviruses is the same as that of the relevant transfer vector. Preparation of recombinant AcMNPV DNA lxlO⁶5/21 ceUs are used to seed 35mm Nunc Petri dishes (Gibco BRL, Paisley, Scotland). The plates are incubated at room temperature for 15 minutes to allow cells to attach and form a monolayer. The media is then removed and lml (approximately 1x10⁷ Non-occluded virions [NOVs]) of iriini-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°C overnight. Next morning the infected ceU 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 ceU pellet resuspended in 250μl TE. Next 250μl lysis buffer (50mM Tris HCI, [ pH8], 5% β-mercaptoethanol, 0.4% w/v SDS, lOmM EDTA) is added. The solution is mixed gently to obtain a viscous ceU lysate. 12.5μl proteinase K ( 1 Omg/ml in TE, predigested at 37°C for 30minutes) and 2.5 μl RNase A

(lOmg/ml in H₂O) are added and the tube incubated at 37 °C for 30minutes. The solution is extracted twice with 500μl (50:50) TE-saturated phenol/chloroform:isoamyl alcohol [24:1] and once with 500μl chloroformtisoamyl alcohol [24: 1]. Infected cell DNA is precipitated by adding 50μl 3M Na acetate and 2 volumes absolute ethanol. Precipitation is for 5 minutes at room temperature and die DNA pelleted by centrifugation at 14 OOQrpm for 15 minutes. The DNA pellet is washed twice in 70% ethanol, air-dried for 5 minutes at room temperature and resuspended in lOOμl TE, pH8 overnight at 4°C .

Digestion of the baculovirus infected cell DNA and separation bv agarose gel electrophoresis 15μl of each infected cell DNA preparation is digested using EcoRI (EcoRI and BglD. for BrhTX- 1 (c) and edited BrhTX- 1 (b) subunits). The digests are run on a 0.8%, 1 xTBE agarose gel and the DNA tranferrcd 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- 1 (a), BrhTX- 1 (d) subunits, Notl for BrhTX- 1 (b) and EcoRVBgiH for

BrhTX- 1(c) and edited BrhTX- 1(b) subunits. 25ng of the excised subunit DΝA is denatured (by boiling for lOmin) and tiien labelled by random priming with α ³²P dCTP (Amersham International, Amersham, UK) and Klenow polymerase (ΝBL Gene Sciences Ltd.,Cramiington, Northumberland, UK) using standard methods (Sambrook 1989). This probe is denatured by incubation in a boiting water bath immediately before use. Hybridisation The membrane is UV cross-linked in a StrataUnker (Stratagene, USA) and pre- hybridised at 65°C for 2 hours in 5 x SSPE, 5 x Denhardt's Reagent, 0.5% SDS and 200μg/ml salmon sperm DNA in a Techne hybridisation oven. Hybridisation is caπied-out in 5 x SSPE, 5 x Denhardt's reagent, 0.5% SDS plus labelled probe at 65°C for 16 hours. Membranes are washed in 3 x SSC (20 x SSC: 3.0M NaCl, 0.3M tiisodium citrate); 0.1 %SDS at 65°C for 4 x 15 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, 65°C 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), BrhTX- 1(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 p 10 very late promoters. Such transfer vectors can be custom built, in which case they could be designed so tiiat 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™ (Pharmingen), BacPAKό™ (Clontech) or AcRP23.1acZ (Pharmingen).

Alternatively, commercially available co-expression transfer vectors can be used. In die case that three subunits are selected, pAcAB3 (Pharmingen) may be used. This can accommodate up to three heterologous genes under die transcriptional control of either two plO 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 plO 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™ , BacPAKό™ or AcRP23.1acZ, only polyhedrin negative AcMNPV derivatives are made. These can be bioassayed by injection into suitable lepidopteran hosts e.g. 3rd/4tiι instar H.virescens. However in a preferred embodiment they are used to construct polyhedrin positive (Pol+) AcMNPV derivatives directly by in vivo recombination with Bsu361 linearised AcUWl-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 plO gene and has a replaceable lacZ gene witiiin the region homologous and hence exchangeable witii the above mentioned transfer vectors (see also PCT/GB95/00677). Pol+ recombinant bacuioviruses 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 wiU appreciate that the above options provide various permutations for construction of recombinant baculovirus derivatives capable of co- expression of the various subunits of die 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-l(d) one may initially insert the BrhTX- 1(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 BαmHI site in die transfer vector pAcAB4. DNA sequence analysis is then used witii likely recombinant plasmids to select those with an intact BrhTX- 1(d) gene correctly orientated for transcription from one of the two polyhedrin promoters in pAcAB4. Next an insert encoding die BrhTX- 1(c) gene, isolated from pBrhTX-l(c)5 as a Notl fragment and blunt ended by a fύl-in reaction with Klenow DNA polymerase, is introduced mto a similarly filled Spel site in an authenticated preparation of the intermediate pAcAB4/BrhTX-l(d) transfer vector. DNA sequence analysis is then again used to select those recombinant plasmids with an intact BrhTX- 1(c) gene correctly orientated for functional transcription from one of the two plO promoters carried by pAcAB4. One such authenticated pAc AB4/BrhTX- 1 (c)/BrhTX- 1 (d) recombinant transfer vector preparation is selected for further work and subject to BglΩ. digestion to linearise it downstream of the other plO promoter and blunt ended by Klenow fiU in reaction. Notl digestion is then used to isolate a BrhTX- 1(b) gene from pBrhTX-l(b)6. This fragment is again blunt ended by Klenow fill-in before ligation with the BglΩ. 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- 1(b) gene are recognised by DNA sequence analysis. Finally, the selected pAcAB4/BrhTX- l(b)/BrhTX-l(c)/BrhTX-l(d) recombinant transfer vector is subject to 5/uI digestion to linearise it just downstream of the remaining polyhedrin promoter. EcoRI digestion is ώen used to isolate a BrhTX- 1(a) gene from a suitable pUC19/BrhTX-l(a) recombinant. This fragment is again blunt ended by Klenow fill-in before Ugation with the Stul pAcAB4/BrhTX-l(b)/BrhTX-l(c)/BrhTX-l(d) preparation under appropriate conditions to promote blunt end fragment joining. The target recombinant transfer vectors containing the correctly orientated BrhTX- 1(a) gene are men recognised by DNA sequence analysis. An ethidium bromide/CsCl purified preparation of the selected pAcAB4/BrhTX-l(a)/BrhTX- 1 (b)/BrhTX- 1 (c)/BrhTX- 1 (d) recombinant transfer vector is then prepared for use in assembly of recombinant pol+ AcMNPV derivatives by co-tranfection of 5/21 cells with Bsuiβ I linearised AcUWl-PH DNA as described in Example 3.

Alternatively, initially four independent linkers are inserted into the EcoRI/Hindlll sites of pAlter-1 vector (Promega) to assemble four independent intermediate cloning vectors to facihtate cloning of die four subunits into pAcAB4. Then an insert encoding die BrhTX- 1(d) gene, isolated from pBrhTX- 1(d) 1.2 as an EcoRI fragment, is introduced into an EcoRI site in one of die pAlter-1 intermediate cloning vectors which carries a pair of 5pe9 sites flanking die EcoRI site. DNA sequence analysis is then used with likely recombinant plasmids to select those with an intact BrhTX- 1(d) gene. Spel digestion is then utilised to isolate the BrhTX- 1(d) gene from the pAlter-1 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- 1(d) gene correcdy orientated for transcription from one of the two polyhedrin promoters in pAcAB4. An insert encoding the BrhTX- 1(b) gene, isolated from pBrhTX-l(b)6 as a Notl fragment, is then introduced into a Notl site in one of the p Alter- 1 intermediate cloning vectors which carries a pair of BglΩ. sites flanking die Notl 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 die intermediate pAcAB4/BrhTX-l(d) transfer vector. DNA sequence analysis is then again performed on die product in order to select those recombinant plasmids whh an intact BrhTX- 1(b) gene correcdy orientated for functional transcription from the remaining polyhedrin promoter in pAcAB4. EcoRI digestion is then used to isolate a BrhTX-l(a) gene from pUC19/BrhTX-l(a), to clone it into an EcoRI site in one of the pAlter-1 intermediate cloning vectors which carries a pair of flanking Xbal sites flanking the EcoRI site. Once again, recombinant vectors containing the intact BrhTX- 1(a) gene arc recognised by DNA sequence analysis. One authenticated p Ac AB4/BrhTX- 1 (d)/BrhTX- 1 (b) recombinant transfer vector preparation is selected for further work and subject to Spel digestion to linearise it downstream of one of die p 10 promoters. Xbal digestion is then used to release the BrhTX- 1(a) gene from p Alter- 1 before ligation with the Spel digested pAcAB4/BrhTX-l(d)/BrhTX-l(b) preparation. Recombinant transfer vectors containing the correctly orientated BrhTX-l(a) gene are recognised by DNA sequence analysis. Finally, Notl digestion is used to isolate a BrhTX- 1(c) gene from a suitable pBrhTX-l(c)5 recombinant and introduce it into the last pAlter- 1 cloning vector which carries a pair of BamHI sites flanking the EcoRI site. Recombinant progeny of this cloning step are again analysed by sequencing, to isolate a plasmid with an intact BrhTX- 1(c) gene. The selected pAc AB4/BrhTX- 1 (d)/BrhTX- 1 (b)/BrhTX- 1 (a) recombinant transfer vector is then subjected to Bglll digestion to linearise it just downstream of die remaining plO promoter. Finally, the BrhTX- 1(c) gene, isolated from pAlter-1 by BamHI digestion, is ligated to the Bglll pAcAB4/BrhTX-l(d)/BrhTX-l(b)/BrhTX-l(a) 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 pAc AB4/BrhTX- 1 (a)/BrhTX- 1 (b)/BrhTX- 1 (c)/BrhTX- 1 (d) recombinant transfer vector is then prepared for use in assembly of recombinant pol+ AcMNPV derivatives by co¬ transfection of 5/21 ceUs with Bsu36 1 linearised AcUWl-PH DNA.

The skilled man will appreciate mat adapters may be used to introduce an EcoRI or a Notl 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 witii likely recombinant plasmids to select tiiose with an intact BrhTX- 1(d) gene correcdy orientated from transcription from one of the two polyhedrin promoters in pAcAB4. Next an insert encoding the BrhTX- 1(b) gene, isolated from pBrhTX- l(b)6 as a Notl fragment, is introduced into die BamHI site of an authenticated preparation of the intermediate pAcAB4/BrhTX-l(d) transfer vector utilising a BamHI/BgUI-Notl adapter. DNA sequence analysis is dien again used to select those recombinant plasmids witii an intact BrhTX- 1(b) gene correctly orientated for functional transcription from the second polyhedrin promoter carried by pAcAB4. One such 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 two plO promoters. EcoRI digestion is then used to isolate a BrhTX- 1(a) gene from pUC19/BrhTX-l(a). An Spel-EcoRI adapter is again utilised to allow Ugation of the BrhTX- 1(a) gene with the Spel pAcAB4/BrhTX-l(d)/BrhTX-l(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/BrhTX- l(d)/BrhTX-l(b)/BrhTX-l(a) recombinant transfer vector is subjected to Bglll digestion to linearise it just downstream of die remaining plO promoter. Notl digestion is then used to isolate a BrhTX- 1(c) gene from a suitable pBrhTX-l(c)5 recombinant. A BamHI/Bglll-Notl adapter is again utiUsed to allow ligation of the BrhTX- 1(c) gene with the Bglll pAcAB4/BrhTX- 1 (d)/BrhTX- 1 (b)/BrhTX- 1 (a) preparation under appropriate conditions. The target recombinant transfer vectors containing the correctly orientated BrhTX- 1(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-l(a)/BrhTX-l(b)/BrhTX-l(c)/BrhTX-l(d) recombinant transfer vector. Example 9(b) Construction of recombinant AcMNPV derivatives capable of co-expression of three subunits of BrhTX-1

Concerning the production of recombinant baculovirus derivatives capable of providing for co-expression of BrhTX-l(a), BrhTX-l(c) and BrhTX-l(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 odier polyhedrin promoter. Eco RI digestion is tiien 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 Ugation witii die Stu I pAcAB4/BrhTX-l(c)/BrhTX-l(d) preparation under appropriate conditions to promote blunt end fragment joining. Again recombinant transfer vectors containing die correcdy orientated BrhTX- 1(a) gene are recognised by DNA sequence analysis. An ethidium bromide/CsCl purified preparation of the selected pAcAB4/BrhTX-l(a)/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 5/21 cells with Bsu361 linearised AcUWl-PH DNA as described above for die four sub-unit assembly.

In the case that the recombinant bacuioviruses 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- 1(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 TI site in an authenticated preparation of the intermediate pAcAB4/BrhTX-l(d) transfer vector. DNA sequence analysis is then used to select those recombinant plasmids with an intact BrhTX- 1(b) gene correcdy orientated for functional transcription from one of die two plO promoters carried by pAcAB4. One such authenticated pAcAB4/BrhTX- l(b)/BrhTX-l(d) recombinant transfer vector preparation is selected for further work and subject to Stu I digestion to linearise it downstream of die odier polyhedrin promoter. Eco RI digestion is then used to isolate a BrhTX- 1 (a) gene from a suitable pUC 19/BrhTX- 1 (a) recombinant plasmid. This fragment is again blunt ended by Klenow fill-in before Ugation 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- 1(a) gene are recognised by DNA sequence analysis. An etiiidium bromide/CsCl purified preparation of the selected pAc AB4/BrhTX- 1 (a)/BrhTX- 1 (b)/BrhTX- 1(d) recombinant transfer vector is then prepared for use in assembly of recombinant pol+ AcMNPV derivatives by co-tranfection of 5/21 ceUs with Bsu361 linearised AcUWl-PH DNA .

In die case that the recombinant baculoviruse comprise genes encoding the BrhTX- 1 (b), BrhTX- 1 (c) and BrhTX- 1 (d) subunits, an authenticated pAc AB4/BrhTX- 1 (c)/BrhTX- 1(d) recombinant transfer vector preparation is produced as indicated above and subjected to Bglll digestion to linearise it downstream of the other plO promoter. Norl digestion is then used to isolate a BrhTX- 1(b) gene from pBrhTX-l(b)6. This fragment is again blunt ended by Klenow fill-in before ligation with the Bgl H 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- 1(b) gene are recognised by DNA sequence analysis. An ethidium bromide/CsCl purified preparation of the selected pAcAB4/BrhTX-l(b)/BrhTX-l(c)/BrhTX-l(d) recombinant transfer vector is then again prepared for use in assembly of recombinant pol+ AcMNPV derivatives by co- tranfection of 5/21 cells with B.w361 linearised AcUWl-PH DNA . Concerning the construction of recombinant baculovirus derivatives capable of co- expression of BrhTX- 1(a), BrhTX- 1(b) and BrhTX- 1(c), one may initiaUy insert the BrhTX- 1(c) gene, isolated from pBrhTX-l(c)5 as a Not I fragment and blunt ended by a fiU-in reaction with Klenow DNA polymerase performed under standard conditions (Sambrook 1989), mto a similarly filled Spe I site in the transfer vector p Ac AB4. DNA sequence analysis is then used witii likely recombinant plasmids to select those witii an intact BrhTX- 1(c) gene correctly orientated for transcription from one of the two plO promoters in pAcAB4. Next an insert encoding the BrhTX- 1(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 E 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- 1(b) gene correctly orientated for functional transcription from the other p 10 promoter carried by pAcAB4. One such autiienticated pAcAB4/BrhTX-l(b)/BrhTX-l(c) 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- 1(a) gene from a suitable pUC19/BrhTX- 1(a) recombinant plasmid. This fragment is again blunt ended by Klenow fill in before Ugation with the Stu I pAcAB4/BrhTX-l(b)/BrhTX-l(c) preparation under appropriate conditions to promote blunt end fragment joining. Again recombinant transfer vectors containing die correcdy orientated BrhTX- 1(a) gene are recognised by DNA sequence analysis. An etiiidium bromide/CsCl purified preparation of the selected pAc AB4/BrhTX- 1 (a)/BrhTX- 1 (b)/BrhTX- 1 (c) recombinant transfer vector is then prepared for use in assembly of recombinant pol+ AcMNPV derivatives by co-tranfection of 5/21 ceUs with Bsu361 linearised AcUWl-PH DNA . Example 9(c) Construction of recombinant AcMNPV derivatives capable of co-expression of two subunits of BrhTX-1 In addition to the AcMNPV transfer vectors used in the production recombinant bacuioviruses comprising genes providing for expression of tiiree 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 me transcriptional control of one plO promoter or one polyhedrin promoter.

Recombinant baculovirus derivatives capable of co-expression of BrhTX- 1(a) and BrhT'X- 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- 1(a) gene correctly orientated for transcription from the p 10 promoter. An insert encoding die BrtϊTX- 1(b) gene is isolated from pBrhTX-l(b)6 as a Notl fragment and blunt-ended by a fill-in reaction with Klenow DNA polymerase. This is introduced into a similarly filled Bam HI site in an autiienticated 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- 1(b) gene correctly orientated for transcription from the polyhedrin promoter. One such authenticated pAcUW51/BrhTX- 1 (a)/BrhTX- 1 (b) recombinant transfer vector preparation is selected for further work. A caesium chloride purified preparation of the selected pAcUW51/BrhTX- 1 (a)/BrhTX- 1 (b) recombinant transfer vector is prepared for use in assembly of recombinant pol+ AcMNPV derivatives by co-tranfection of 5/21 ceUs with Bsu36 1 Unearised AcUWl-PH DNA as described in PCT/GB95/00677. Pol+ AcMNPV derivatives capable of co-expressing BrhTX- 1(a) and BrhTX- 1(b) are then isolated by the method described above in Example 3. This method can be repeated for the production of recombinant bacuioviruses 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- 1(d); (iv) BrhTX-l(c) and BrhTX-l(d).

Whatever the combination of subunit encoding sequences die recombinant bacuioviruses comprise, Pol+ AcMNPV derivatives capable of co-expressing the various combinations of sub-units are isolated by:- • Plaque purification of the co-transfection supernatants on Sf21 monolayers by selecting forpol+/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 die vicinity of the introduced combinations of genes, for example, (i) B±TX- 1 (a)/

BrhTX-l(b)/BrhTX-l(c)/BrhTX-l(d): (ii) BrhTX-l(a)/BrhTX-l(c)/BrhTX-l(d); (iii) BrhTX-l(b)/BrhTX-l(c)/BrhTX-l(d); (iv) BrhTX- l(a)/BrhTX-l(b)/BrhTX- 1(c); (v)

• In vitro protein expression studies to demonstrate that the viruses have die capacity to express immunologicaUy detectable BrhTX- 1(a), BrhTX- 1(b), BrhTX- 1(c) and BrhTX- 1(d).

The selected AcMNPV/BrhTX-l(a)/BrhTX-l(byBrhTX-l(c)/BrhTX-l(d); AcMNPVBrhTX- 1 (a)/BrhTX- 1 (c)/BrhTX- 1 (d); AcMNPVBrhTX- 1 (b)/BrhTX- 1 (c)/BrhTX-

1(d); AcMNPVBrhTX- l(a)/BrhTX-l(b)/BrhTX- 1(c) etc. recombinant baculovirus isolates are then subject to diet based bio-assays on first instar H.virescens larvae as described in Example 10.

Example 10 - Diet surface dosing assay for evaluation of viruses vs.

Heliothis virescens larvae

Petri dishes (Falcon model 1006, 50 x 9mm, tight fit Ud) 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 labeUed as appropriate for the test treatment list (one to two dishes per treatment). Twenty \Yι" 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 lxlO⁶ PIB/ml using deionised water. In Primary tests two tenfold serial dilutions are made to give a dose range, for each virus tested, of lxlO⁶, 1x10^s and 1x10* PTB/rnl. In follow up tests on viruses of interest the dose range consists of five rates in four-fold serial dilution i.e. lxlO⁶, 2.5x10%

6.3xlθ\ 1.6x10*, 3.9xl0³ PTB/rnl. Using a sterile syringe, 0.1ml of the appropriate treatment solution is deposited on die surface of the diet in each of the pre-labelled Falcon dishes. The solution is spread evenly over the surface of the diet using a sterile inoculating loop. Control dishes are treated in the same way witii an equivalent volume of deionised water. The dishes are left to dry dioroughly prior to infestation.

Approximately 30 first instar Heliothis virescens larvae of uniform size are transferred to die 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°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 witii the appropriate treatment and rephcate number. The minipots are held under die same conditions as the treated Falcon dishes (see above) for the duration of the test.

Assessments of mortaUty 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 mortaUty /affected. AcMNPV/BrhTX-l(c) recombinants

Table 3 summarises data generated comparing the BrhTX- 1(c) clones witii 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 die wild type.

Table 3

AcMNPV/BrhTX-l(b) recombinants Table 4 summarises data generated in a diet surface dosing assays comparing the edited BrhTX- 1(b) (SEQ ID No.9) clones with wild type and Tox 34#4 AcMNPV.

Table 4

Table 5 shows die insecticidal activity of recombinant baculovirus based on he AcUWl-PN/pAcAB4 system comprising genes encoding die BrhTX-l(a) (17KDa -SEQ ID No. 2), BrhTX-l(b) (18kDa - SEQ ID No. 4), Brh-TX-l(c) (21kDa -SEQ ID No. 6) or BrhTX- 1(d) (32kDa - SEQ TD No. 8) toxin subunits.

Table 5

Table 6 shows die insecticidal activity of recombinant baculovirus comprising various combinations of the genes encoding the Brh TX-l(a) (17kDa - SEQ ID No. 2), BrhTX-l(b) (18kDa - SEQ TD No. 10), BrhTX-l(c) (21kDa - SEQ ID No. 6) or BrhTX-l(d) (32kDa SEQ ID No. 8) toxin subunits.

Table 6

Whilst the present invention has been particularly described with reference to the production of recombinant bacuioviruses comprising nucleotide sequences depicted in various of SEQ ID Nos. 1,3,5,7 and 9, the skilled man will appreciate that the bacuioviruses may be engineered to contain odier sequences which are also part of the present invention. For example, the bacuioviruses may comprise a polynucleotide comprising a region encoding a protein encoded by the spliced RNA derived from die genomic clone depicted in SEQ ID No. 19, die said spticed 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 TD No. 45 discloses a toxin encoding sequence derived from the genomic clone depicted in SEQ ID No. 19 and SEQ ID No. 46 discloses die translation product of the nucleotide sequence depicted in SEQ ID. No. 45. The present invention thus includes Bacuioviruses 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.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: ZENECA Ltd

(B) STREET: 15 Stanhope Gate

(C) CITY: London (E) COUNTRY: UK

(F) POSTAL CODE (ZIP) : W1Y 6LN

(A) NAME: Commonwealth Scientific and Industrial Research Organisation (B) STREET: 407 Royal Parade

(C) CITY: Parkville

(D) STATE: Victoria

(E) COUNTRY: Aus-ralia

(F) POSTAL CODE (ZIP) : 3052

(ii) TITLE OF INVENTION: Biological Insect Control Agent

(iii) NUMBER OF SEQUENCES: 46 (iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30 ⁽EPO⁾

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 564 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bracon Hebetor

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION:13..487

(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 lie Leu Leu Leu lie Ala 1 5 10 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 15 20 25 TGC GCC AAA GAG CAC GGC GAG GAA TAT CGT ACG TGG TCT TTC CGT TAC 144 Cys Ala Lys Glu His Gly Glu Glu Tyr Arg Thr Trp Ser Phe Arg Tyr 30 35 40

GAA CTT GGT GAT ATT TTT AAA TGT GTT TGC ACT CAC GGA AAG AAT CTT 192

Glu Leu Gly Asp lie Phe Lys Cys Val Cys Thr His Gly Lys Asn Leu

45 50 55 60

ATG GGA AGC GAG AAT TAT GGT AAG TGT AGA GAA GCA TGT ATT CAA AAT 240 Met Gly Ser Glu Asn Tyr Gly Lys Cys Arg Glu Ala Cys lie Gin Asn

65 70 75

CAT GGA GCG GGA GGC TTT AAA TAT GCC TTT CCC ATA TAC AGC GAA GTA 288 His Gly Ala Gly Gly Phe Lys Tyr Ala Phe Pro He Tyr Ser Glu Val 80 85 90

CCA GCA TCA TGG GCA TGC ATA TGC ACT CAG GAG AAA AAT AAG ACA TTT 336 Pro Ala Ser Trp Ala Cys He Cys Thr Gin Glu Lys Asn Lys Thr Phe 95 100 105

TGT ATA CAT GCT TGC TCA GAA ATT CAT CAC AAG GCC CCA CCT AAG AAT 384 Cys He His Ala Cys Ser Glu He His His Lys Ala Pro Pro Lys Asn 110 115 120 CCC ATA GTT ATG AAA AAT GGA CAA TGC TAC TAC CAA GAT CAC AGG GGT 432 Pro He Val Met Lys Asn Gly Gin Cys Tyr Tyr Gin Asp His Arg Gly 125 130 135 140

GTT GAC AGG TAT TGT GAA GTT TAT ATG AAG TTC TTA GAT GCG TTG GAA 480 Val Asp Arg Tyr Cys Glu Val Tyr Met Lys Phe Leu Asp Ala Leu Glu

145 150 155

TCA ATT T AACAATGATC AAATTCATGT TATCAATGAA GGAAGAATAA TGAATTAATA 537 Ser He

ATAATTATCA AAAATCAAAA AAAAAAA 564

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 158 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Lys Phe Leu Tyr Leu He Leu Leu Leu He Ala Gly Val Val Ser 1 5 10 15

Phe Asn Pro Glu Thr His Arg Glu Cys Lys Asn Tyr Cys Ala Lys Glu 20 25 30

His Gly Glu Glu Tyr Arg Thr Trp Ser Phe Arg Tyr Glu Leu Gly Asp 35 40 45

He Phe Lys Cys Val Cys Thr His Gly Lys Asn Leu Met Gly Ser Glu 50 55 60

Asn Tyr Gly Lys Cys Arg Glu Ala Cys He Gin Asn His Gly Ala Gly 65 70 75 80 Gly Phe Lys Tyr Ala Phe Pro He Tyr Ser Glu Val Pro Ala Ser Trp 85 90 95

Ala Cys He Cys Thr Gin Glu Lys Asn Lys Thr Phe Cys He His Ala 100 105 110

Cys Ser Glu He His His Lys Ala Pro Pro Lys Asn Pro He Val Met 115 120 125

Lys Asn Gly Gin Cys Tyr Tyr Gin Asp His Arg Gly Val Asp Arg Tyr

130 135 140

Cys Glu Val Tyr Met Lys Phe Leu Asp Ala Leu Glu Ser He 145 150 155

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1197 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bracon Hebetor

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

CACGCCTACT TAGATAATTT CTCAATTCTT TGCAAATTAT GAAATAAGTG CAAGAGATGT 60 GTATGACACC TCAATCTGAG TTTGTTCATA ATTCGAGAGG GATAAATAAG GAAGTCTCTG 120

TGTACAAAAG AAAACTACCT CATATAAATC TTGCATTTTT CCGTGAGAGA GAAAAAAAAA 180

CCCTGAAAAA CTGAGTAAGG CAATAATTTT NCCTCATAAC AATGTCAATC ATATGTAAAA 240

TAATCTTGTT GGTGCTACTG AGTTGGACAT CGATGGTATC GTCAACATTA TTTACAGACC 300

GAAAGTGGTG TGGACGTGCC GATAAGACTT TTGGTCCTTC ACGGTCGCTA GGAGGAGGTG 360 TTGGTGATTG CTGCAGAAGT CATGACAGCT GTGGCCGCAT GATTAAACCA GGAGAGACTT 420

ATGGAGATGT TACGAATAAA GGATTTTCAA ATATTTGGGA ATGCCGATGT GACTATGCAT 480

TTTTTCAATG TCTTCAGCGT TCCAATGGTA AAATGAAAAA TGTTGTGGAA ATATTGCATT 540

TTGACGTTGT CAATACACCC TGTTACTTCA TGAAAGATGG CCGTGCTAAA ATATCACCCC 600

ATACTGTATA TGATAAACAC GAATCACTCT ATCAACTTAT ACTACACAAA GATAATTTTA 660 AGGAGTGGGT GCATGATAAT GCTCTTCTCC CGCAAGAGCT GGGGATTAAA GATGAGCATG 720

TGTGGGAGAC ACTGATGGCA TGGATGGACT TTAGATTTCC AACTGAATAA TAAATATTCC 780 AAATACAGAT ATCCTTTTGA TAAAATGTCG TAAACATGAT TGTTTAGATG AATGGTAAAT 840

TAATGAAAAG ATTGATTGAA AATGTCTGAA GTAACTNNNG GATNNGACAT ATAATATATA 900

ATATTTGCCT TATTNGATAA ACTTCTACCN TTAANAAAGG AAAAAGGAGG AGGNGTAGGA 960

GGAGGATTAG GATATTTTAC AAGGATTTTA AAAATAATTA AACAATTAGA TCTTCTGTAA 1020

ATTGATTGAT CATGTATTAA ATACAATAAC ATCTCGTTCT CATAGTACAA TGAAAAAGAA 1080

CATAACAGTA TGCACAAAAA TAATGACGGT AAATATCTAT GTATGTATGT AGAGAGAAGA 1140

AAATAAAAAT AGTTAGACAG GTACCAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAA 1197

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 182 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

_. (A) ORGANISM: Bracon Hebetor

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Ser He He Cys Lys He He Leu Leu Val Leu Leu Ser Trp Thr 1 5 10 15 Ser Met Val Ser Ser Thr Leu Phe Thr Asp Arg Lys Trp Cys Gly Arg

20 25 30

Ala Asp Lys Thr Phe Gly Pro Ser Arg Ser Leu Gly Gly Gly Val Gly 35 40 45

Asp Cys Cys Arg Ser His Asp Ser Cys Gly Arg Met He Lys Pro Gly 50 55 60

Glu Thr Tyr Gly Asp Val Thr Asn Lys Gly Phe Ser Asn He Trp Glu 65 70 75 80

Cys Arg Cys Asp Tyr Ala Phe Phe Gin Cys Leu Gin Arg Ser Asn Gly 85 90 95 Lys Met Lys Asn Val Val Glu He Leu His Phe Asp Val Val Asn Thr

100 105 HO

Pro Cys Tyr Phe Met Lys Asp Gly Arg Ala Lys He Ser Pro His Thr 115 120 125

Val Tyr Asp Lys His Glu Ser Leu Tyr Gin Leu He Leu His Lys Asp 130 135 140 Asn Phe Lys Glu Trp Val His Asp Asn Ala Leu Leu Pro Gin Glu Leu 145 150 155 160

Gly He Lys Asp Glu His Val Trp Glu Thr Leu Met Ala Trp Met Asp 165 170 175

Phe Arg Phe Pro Thr Glu 180

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 669 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Bracon Hebetor

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION:147..581

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: AAAAGACTAA AAATAAGAAA AAAAAACATA GAAGAATGTT TACAATAATT TATATTTTTA 60

AACTTTCATT TCTATTAGTT CCGTGCTGGA GTTTTTCAAC CTACGCTGGG TATGGTGAAT 120

ATAATCGGTC CATTACTAAG CGACAG ATG GAC GAT GGT GAG ACG TGC GAA AGG 173 Met Asp Asp Gly Glu Thr Cys Glu Arg

160 165

TGT TTG AAT CCA CTC GAA TTA GTA AAT GAC GCT GTA GAC TCG TGC ATT 221 Cys Leu Asn Pro Leu Glu Leu Val Asn Asp Ala Val Asp Ser Cys He 170 175 180

GAA GCT CAT GAG GAA TGT GAG GAA TTC ATT GAA GGC GGG ATG GAA ATG 269 Glu Ala His Glu Glu Cys Glu Glu Phe He Glu Gly Gly Met Glu Met 1B5 190 195

CTT CAT GTA CAC AAT CCA GGA AAC TTC CGA GTC TCC AAA TGT GTA TGC 317 Leu His Val His Asn Pro Gly Asn Phe Arg Val Ser Lys Cys Val Cys 200 205 210 215 GAC ATT GCG CTC AAG GAG TGC CTC ACT ACT CAT CCT GAA ATG AGT TTC 365 Asp He Ala Leu Lys Glu Cys Leu Thr Thr His Pro Glu Met Ser Phe 220 225 230

AAA TTT GTT AAA GCA CTC TTT TTT GAT TTG CTT GCT CCA CCC TGT TTT 413 Lys Phe Val Lys Ala Leu Phe Phe Asp Leu Leu Ala Pro Pro Cys Phe 235 240 245

GAT CAG ATT GCT GAT TGG GGT AAG AAA AAA TTG AAA AAT AAG CAG GCA 461 Asp Gin He Ala Asp Trp Gly Lys Lys Lys Leu Lys Asn Lys Gin Ala 250 255 260

TTT TCA CTG CAT GAT TTA CAA TCA GCT GCC CAC GCG CTC TGG CAA ACA 509 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 557 Leu Tyr Asp Ala Val Lys Gly He Ala Gin Asp Val Gly His Ala Ala 280 285 290 295

CAT TCT TTT GAA AAA ATG TTA CAG TAACAGTTAA ATATGAAAAA GGTCCATGAT 611 His Ser Phe Glu Lys Met Leu Gin 300

AGTAGAATAC AGTTATTGTT GTATAAATAA ATAATATATT CAGAATGATA AAAAAAAA 669

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 176 amino acids

(B) TYPE: amino acid (D) 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 1 5 10 15

Tyr Ala Gly Cys Gly Gly Tyr Asn Arg Ser He Thr Lys Arg Gin Met 20 25 30

Asp Asp Gly Glu Thr Cys Glu Arg Cys Leu Asn Pro Leu Glu Leu Val 35 ' 40 45

Asn Asp Ala Val Asp Ser Cys He Glu Ala His Glu Glu Cys Glu Glu 50 55 60

Phe He Glu Gly Gly Met Glu Met Leu His Val His Asn Pro Gly Asn 65 70 75 80 Phe Arg Val Ser Lys Cys Val Cys Asp He Ala Leu Lys Glu Cys Leu

85 90 95

Thr Thr His Pro Glu Met Ser Phe Lys Phe Val Lys Ala Leu Phe Phe 100 105 110

Asp Leu Leu Ala Pro Pro Cys Phe Asp Gin He Ala Asp Trp Gly Lys

115 120 125

Lys Lys Leu Lys Asn Lys Gin Ala Phe Ser Leu His Asp Leu Gin Ser 130 135 140

Ala Ala His Ala Leu Trp Gin Thr Leu Tyr Asp Ala Val Lys Gly He 145 150 155 160 Ala Gin Asp Val Gly His Ala Ala His Ser Phe Glu Lys Met Leu Gin

165 170 175 (2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1057 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Bracon Hebetor

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION:95..919

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CGCGGCCGCT GTTGATATAT AACAATTTAT TAAAAATTTC AAGTGGAAAG AAAAACTATC 60

TTGTTTTTTT TTTTGTTTTT TTTCATAATT TAAA ATG CAT TTC TTC GCC TCC 112

Met His Phe Phe Ala Ser 150

ATC CTG GTA TGC TTC TTA CTG GGC AAG GCA ATT CAT GAT GTG GAA GGA 160 He Leu Val Cys Phe Leu Leu Gly Lys Ala He His Asp Val Glu Gly 155 160 165 ATA ATA AAT GGT CAT GAT GCT ACT GAG GGA CAA TTT CCC CAT ATG GCT 208 He He Asn Gly His Asp Ala Thr Glu Gly Gin Phe Pro His Met Ala 170 175 180

TAT TTA CAA GCA TCA GCT GGA AAG TGT TCT TAT GTA TGT GGC GGT GCT 256 Tyr Leu Gin Ala Ser Ala Gly Lys Cys Ser Tyr Val Cys Gly Gly Ala 185 190 195

CTT CTA ACT AAA AAA CAT ATT ATG ACA GCT GCT CAT TGT GTA GCA ATG 304 Leu Leu Thr Lys Lys His He Met Thr Ala Ala His Cys Val Ala Met 200 205 210 215

CAC AGA ACG GGA AAT ATT AAA GTA GCC CTT GGT GTT ACG GAT TTT CAT 352

His Arg Thr Gly Asn He Lys Val Ala Leu Gly Val Thr Asp Phe His

220 225 230

AAT AAG CCA TCA ATG CAA CAA AGA AAG GTT GAA CAT ATA AAA GTC CAT 400

Asn Lys Pro Ser Met Gin Gin Arg Lys Val Glu His He Lys Val His 235 240 245 TCT GAG TAC AAA GGA GGA AGG CGT AAG TCA TTA AAA AAT TGG TAT CGC 448 Ser Glu Tyr Lys Gly Gly Arg Arg Lys Ser Leu Lys Asn Trp Tyr Arg 250 255 260

TCC ATA CAT CGT ACA TTT ACA GGA CCG TCT GGG GAT AAA GAA TAC AAT 496 Ser He His Arg Thr Phe Thr Gly Pro Ser Gly Asp Lys Glu Tyr Asn 265 270 275

GAT ATT GCT ATT ATA ACG TTG AGC CAG GAA GTA ACA CTA GGA CCA GTA 544 Asp He Ala He He Thr Leu Ser Gin Glu Val Thr Leu Gly Pro Val 280 285 290 295

GTA AAG ACT ATT AAT TTA CCC CCA AAG AGC TAT CGG CTT CCT TTT GAT 592 Val Lys Thr He Asn Leu Pro Pro Lys Ser Tyr Arg Leu Pro Phe Asp

300 305 310

CAA GAT GCT AGA TTG TCG GGC TTT GGC CGA ACA GTC ATT GTC AAA GAA 640 Gin Asp Ala Arg Leu Ser Gly Phe Gly Arg Thr Val He Val Lys Glu 315 320 325

AAT GAT CCA ATT CCT CCA CCC ACT ACA CAT TTA CAA TGG CTA GAT ATG 688 Asn Asp Pro He Pro Pro Pro Thr Thr His Leu Gin Trp Leu Asp Met 330 335 340

AAG GTT CTT CAT TCA CGA GAT GCT ATT GTC ACT GAT AGT GAA TTT CTC 736 Lys Val Leu His Ser Arg Asp Ala He Val Thr Asp Ser Glu Phe Leu 345 350 355 GCT GAT AAA GAA TAT GGT GAT GGA ACT TGG TCT AAT GCA GCT AAG GGA 784 Ala Asp Lys Glu Tyr Gly Asp Gly Thr Trp Ser Asn Ala Ala Lys Gly 360 365 370 375

GAC AGC GGT AGT CCC TTA GTC AAG GAT AAT CAA GTA ATT GGC GTA GCC 832 Asp Ser Gly Ser Pro Leu Val Lys Asp Asn Gin Val He Gly Val Ala

380 385 390

GTT TCT GTG AGT GAT GAA GAA CAT ACT ACA CGC TTT CAA ATA GTC ACT 880 Val Ser Val Ser Asp Glu Glu His Thr Thr Arg Phe Gin He Val Thr 395 400 405

TAT TAT TTG GAT TGG ATC AAG AAA TAT GCC GAA CTT GCG TAAAAAGAAT 929 Tyr Tyr Leu Asp Trp He Lys Lys Tyr Ala Glu Leu Ala 410 415 420

AAAGAGCAAA ATTGCTCAGA TGGTGAATAT ACATTTTTCC AATAAGCTCA GAAAAAATCG 989

ATTTATATGT AATTAAAAAA TTAAAGATTG TTTTTTCTCT TTTAACAGAA GAATTTGGCG 1049 CGTGAATT 1057

(2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 275 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Met His Phe Phe Ala Ser He Leu Val Ser Phe Leu Leu Gly Lys Ala 1 5 10 15

He His Asp Val Glu Gly He He Asn Gly His Asp Ala Thr Glu Gly 20 25 30

Gin Phe Pro His Met Ala Tyr Leu Gin Ala Ser Ala Gly Lys Cys Ser 35 40 45

Tyr Val Cys Gly Gly Ala Leu Leu Thr Lys Lys His He Met Thr Ala 50 55 60 Ala His Cys Val Ala Met His Arg Thr Gly Asn He Lys Val Ala Leu 65 70 75 BO

Gly Val Thr Asp Phe His Asn Lys Pro Ser Met Gin Gin Arg Lys Val 85 90 95

Glu His He Lys Val His Ser Glu Tyr Lys Gly Gly Arg Arg Lys Ser 100 105 HO

Leu Lys Asn Trp Tyr Arg Ser He His Arg Thr Phe Thr Gly Pro Ser 115 120 125

Gly Asp Lys Glu Tyr Asn Asp He Ala He He Thr Leu Ser Gin Glu 130 135 140

Val Thr Leu Gly Pro Val Val Lys Thr He Asn Leu Pro Pro Lys Ser

145 150 155 160 Tyr Arg Leu Pro Phe Asp Gin Asp Ala Arg Leu Ser Gly Phe Gly Arg

165 170 175

Thr Val He Val Lys Glu Asn Asp Pro He Pro Pro Pro Thr Thr His

180 185 190

Leu Gin Trp Leu Asp Met Lys Val Leu His Ser Arg Asp Ala He Val 195 200 205

Thr Asp Ser Glu Phe Leu Ala Asp Lys Glu Tyr Gly Asp Gly Thr Trp 210 215 220

Ser Asn Ala Ala Lys Gly Asp Ser Gly Ser Pro Leu Val Lys Asp Asn 225 230 235 240 Gin Val He Gly Val Ala Val Ser Val Ser Asp Glu Glu His Thr Thr

245 250 255

Arg Phe Gin He Val Thr Tyr Tyr Leu Asp Trp He Lys Lys Tyr Ala 260 265 270

Glu Leu Ala 275

. (2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 594 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: Bracon Hebetor

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION : 22 . . 573

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

AGATCTGCGG CCGCGCCACC A ATG TCA ATC ATA TGT AAA ATA ATC TTG TTG 51

Met Ser He He Cys Lys He He Leu Leu 280 285 GTG CTA CTG AGT TGG ACA TCG ATG GTA TCG TCA ACA TTA TTT ACA GAC 99 Val Leu Leu Ser Trp Thr Ser Met Val Ser Ser Thr Leu Phe Thr Asp 290 295 300

CGA AAG TGG TGT GGA CGT GCC GAT AAG ACT TTT GGT CCT TCA CGG TCG 147 Arg Lys Trp Cys Gly Arg Ala Asp Lys Thr Phe Gly Pro Ser Arg Ser 305 310 315

CTA GGA GGA GGT GTT GGT GAT TGC TGC AGA AGT CAT GAC AGC TGT GGC 195 Leu Gly Gly Gly Val Gly Asp Cys Cys Arg Ser His Asp Ser Cys Gly 320 325 330

CGC ATG ATT AAA CCA GGA GAG ACT TAT GGA GAT GTT ACG AAT AAA GGA 243

Arg Met He Lys Pro Gly Glu Thr Tyr Gly Asp Val Thr Asn Lys Gly

335 340 345

TTT TCA AAT ATT TGG GAA TGC CGA TGT GAC TAT GCA TTT TTT CAA TGT 291

Phe Ser Asn He Trp Glu Cys Arg Cys Asp Tyr Ala Phe Phe Gin Cys

350 355 360 365 CTT CAG CGT TCC AAT GGT AAA ATG AAA AAT GTT GTG GAA ATA TTG CAT 339 Leu Gin Arg Ser Asn Gly Lys Met Lys Asn Val Val Glu He Leu His 370 375 380

TTT GAC GTT GTC AAT ACA CCC TGT TAC TTC ATG AAA GAT GGC CGT GCT 387 Phe Asp Val Val Asn Thr Pro Cys Tyr Phe Met Lys Asp Gly Arg Ala 385 390 395

AAA ATA TCA CCC CAT ACT GTA TAT GAT AAA CAC GAA TCA CTC TAT CAA 435 Lys He Ser Pro His Thr Val Tyr Asp Lys His Glu Ser Leu Tyr Gin 400 405 410

CTT ATA CTA CAC AAA GAT AAT TTT AAG GAG TGG GTG CAT GAT AAT GCT 483

Leu He Leu His Lys Asp Asn Phe Lys Glu Trp Val His Asp Asn Ala 415 420 425

GGA ACT CTC CTT CCG CGA GAG CTG GGA ATT AAA GCT GAG CAT GTG TGG 531 Gly Thr Leu Leu Pro Arg Glu Leu Gly He Lys Ala Glu His Val Trp 430 435 440 445 GAG ACA CTG ATG GCA TGG ATG GAC TTT AGA TTT CCA ACT GAA 573

Glu Thr Leu Met Ala Trp Met Asp Phe Arg Phe Pro Thr Glu 450 455

TAATGAGCGG CCGCGAATTC C 594

(2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 184 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE : protein

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 10 :

Met Ser He He Cys Lys He He Leu Leu Val Leu Leu Ser Trp Thr

1 5 10 15

Ser Met Val Ser Ser Thr Leu Phe Thr Asp Arg Lys Trp Cys Gly Arg 20 25 30

Ala Asp Lys Thr Phe Gly Pro Ser Arg Ser Leu Gly Gly Gly Val Gly 35 40 45

Asp Cys Cys Arg Ser His Asp Ser Cys Gly Arg Met He Lys Pro Gly 50 55 60

Glu Thr Tyr Gly Asp Val Thr Asn Lys Gly Phe Ser Asn He Trp Glu 65 70 75 80 Cys Arg Cys Asp Tyr Ala Phe Phe Gin Cys Leu Gin Arg Ser Asn Gly

85 90 95

Lys Met Lys Asn Val Val Glu He Leu His Phe Asp Val Val Asn Thr 100 105 110

Pro Cys Tyr Phe Met Lys Asp Gly Arg Ala Lys He Ser Pro His Thr 115 120 125

Val Tyr Asp Lys His Glu Ser Leu Tyr Gin Leu He Leu His Lys Asp 130 135 140

Asn Phe Lys Glu Trp Val His Asp Asn Ala Gly Thr Leu Leu Pro Arg 145 150 155 160 Glu Leu Gly He Lys Ala Glu His Val Trp Glu Thr Leu Met Ala Trp

165 170 175

Met Asp Phe Arg Phe Pro Thr Glu 180

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1326 base pairs

(B) TYPE: nucleic acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: genomic DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Bracon Hebetor

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: TTTGGATAAA TCATGAAATT TTTATATCTA ATACTCCTTT TAATCGCAGG AGTAGTATCA 60

TTCAATCCGG AGACACGTAA GTAAATXGGA AAATTTTTTT AATTAATTCA ATTCTATAAT 120 CAATTCAATT GTTCACAAAT TTTTGTTTTT CATTTTTATT AAAATTATAC CAATAATGAT 180

AATATAAATT TATTTTTCGC TTTAAATTGA TAAAATTTAG ATCGGGAATG TAAGAATTAT 240

TGCGCCAAAG AGCACGGCGA GGAATATCGT ACGTGGTCTT TCCGTTACGA ACTAGGTGAT 300

ATTTTTAAAT GTGTTTGCAC GTAAGTAACA AAATTTTCAT TACTAATATA CTTCTTTTAT 360 TATTCATGAT TAAATCAAGA TTTATTAATT AATGAATTAT GTGTGACTTC ATTATGTAAG 420

TCACGGAAAG AATCTTATGG GAAGCGAGAA TTATGGTAAG TATTTACTCA AAAATTTTAG 480

TTTTGGTTTA ATAATATTAA TTTTCCTTTA AATTATCCGT CCTTCTTGCC AGGTAAGTGT 540

AGAGAAGCAT GTATTCAAAA TCATGGAGCG GGAGGCTTTA AATATGCCTT TCCCATATAC 600

AGCGAAGTAC TAGCATCATG GGCATGCATA TGCACGTATG "'AACATTAGA TGTAATTATT 660 ATTATTTCAT TTTAAATCCA ATATATTAAT AATATAATTG TATAATGTTC TTAGTCAGGA 720

GAAAAATAAG ACATTTTGTA TACATGCTTG CTCAGAAATT CATCACAAGG CCCCACCTAA 780

GAATCCCATA GTTATGAAAA ATGGACAATG CTACTACCAA GATCACAGGG GTGTTGACAG 840

GTATTGTGAA GTTTATATGA AGTTCTTAGA TGCGTTGGAA TCAATTTAAC AATGATCAAA 900

TTCATGTTAT CAATGAAGGA AGAATAATGA ATCAATAATA ATAATCAAAA ATCAATGATT 960 TTGTTTTTAA TTATTAAAAA AAAAGGCTAC ATTGGATTTT TAACTTGTCG ATATAATACC 1020

CCTTTCTCCA TTACAACTAC CCTCTCATCT CAATTAGAAA CAACCTAAAA AACTAATGAT 1080

TCCAATAAAC GTAACGAAAC AATTTCACTA GCATAGAAAG AGCTTAATTG CCTAAAAATT 1140

AATGCAAATT TAGGCGAGAG CACTATAATA AGGGGGGGGG GCTCCGTTCC AAAAATCCAT 1200

TTTGTATATA TACTATTTTA AGAATGACTT TCCTTTCTGC ATTCCATTTC TTTTTCTCTC 1260 TCCCAGTCTC AAAGTATCCT TGCGTGTATA TATATATACG TACGTTAGAT CCTCGCAACT 1320

TGGAAC 1326

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 358 base pairs

(B) TYPE: nucleic acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE:

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bracon Hebetor

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

CATGGAAATT TTTATATCTA ATACTCCTTT TAATTGCAGG AGTAGTATCA TTCAATCCGG 60 AGACACGTAA GTAAATTGGA AAATTTTTTT AATTAATTCA ATTCTATAAT CAATTTTATT 120

GTTCACTTAT TTTTGTTTTT CATTTTTATT AAAATTATAC CAATAATGAT AATATAAATT 180

TATTTTTCGC TTTAAATTTA TAAAATTTAG ATCGTGAATG TAAGAATTAT TGCGCCAAAG 240

AGCACGGCGA GGAATACCGT ACGTGGTCTT TCCGTTACGA ACTTGGTGAT ATTTTTAAAT 300 GTGTTTGCAC GTAAGTAACA AAATTTTCAT TACTAATATA TTCTTTTATT ATTCATGG 358

(2) INFORMATION FOR SEQ ID NO: 13: (I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 358 base pairs

(B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bracon Hebetor

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

CATGGAAATT TTTATATCTA ATACCCCTTT TAATTGCAGG AGTAGCATCA TTCAATGCGG 60

CCATACGTAA GTAAATTGGA TTTTTTTTTT TTTATAAATT CAATTCTATA ATTAATTTTA 120

TTGTTCACTT TTTTTTTTTC ATTTTTATTA AAATTATACC AATAATGATA ATATAAATTT 180

ATTTTTCGCT TTAAATTTAT AAAATTTAGA TCGTGAATGT AAGAATTTTT GCGCCAAAGA 240 GCACGGCGAG GAATATGCTA CATGGTCTTT TCGTTACGAA CTGGGTGATA CGTTTAAATG 300

TATTTGCACG TAAGTAACAA AATTTTCATT ACTAGTATTC TTCCTTTGTT TTTCATGG 358

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 737 base pairs

(B) TYPE: nucleic acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: genomic DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bracon Hebetor

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: ACTAGTATTC TTCCTTTGTT TTTCATGATT AAATCAAGAT TTATTAATTA ATGAATTATG 60

TGTGTTGATT TGATTATATA GTATCGGAAA CGGCCTCATG AGAACAGAGA ATAATGGTAA 120

GTATCTACTT AGAATTATTA GTTTTGGTTT AATAATTTCA ATTTTCTTTA CTTTTTAATA 180

CTCTATTATT CTCTCTCTCT CTCTCTCCCA ATCAAGATAA GTGTAGACAA AAATGTATTC 240 AACAGCATGG AGCGGGAGGC TTTAAATATG CCTTCAGCAT ATACAGTGAA GTACCAGCTT 300

CATGGGCATG CATGTGCACG TATGTAACAT TAGATGCTAT TATTATTATT TCCTTTTTAA 360

ATCAATAATT TTAATAATAC AATTGTATGA TGTTTTTAGT CAGGTGAAAA ATAAGACATA 420

TTGTATACAT GCTTGCATGA AAGTTGAAGA AAAGGCCCCA CCTATGAATC CCATAGTGAT 480

GAAAAATGGA GAATGCTACT ATCAAGATCA CAGGGGTGTT GAGAGGTCGT GTGAATTGGA 540 AATGAATAGC ATTGATTGAA TAAATTCAAC AATTATCAAA TTCATGTTAT CAATGAAGGA 600

AGAATAATAA ATCAATAATA TATATCAACA ATCAATAACT TTGTTTTTAA TCATTTAAAA 660

AAAAAAGGCT ACATTGGATT TTTAACTTCT CGACATACTA CTCGAATTGT GAGGGAGAAT 720

TAAGGGCTTG TACTAGT 737

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: DNA

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

TTATATGAAG TTCTTAGA 18

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs

(B) TYPE: DNA (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: nucleic acid (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

TTAAATTGAT TCCAACGC 18

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: DNA (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

TTCCTTACGC GAAATACG ¹⁸

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: DNA

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: nucleic acid

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

GAATTATTAT CAAATCAT 18

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1811 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

. (vi) ORIGINAL SOURCE: (A) ORGANISM: Bracon Hebetor

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

TCTAGAACAA TGATATAGGA CCGTTGTTAG GTAAAAAGCA GTGCCAATTG TCTATAGTCC 60

TAGCCGCATC AACGCCCCTA AAACACGTAA GAAGACAAAA AGCTATAGCG GTGATATATA 120 . GAGTGCCGTT AGAGGGGCGG TGTACCAATT TCAGAATTAT GTTATCCATC ATTCTGCATA 180

TAACTATACA AAATTCAAAA ATATTCTAAA ACTCCTCCAA ATAACTTAAA AAAAAAAAAA 240

AAAAAGATTC TGAAGCTGTT CTAAGAAATG TCCCAGTTTT GGAACGCAAA ACAAAACGAA 300

AGAAAACATT GATATAAATT GTTAGGGACA ATTGAGGAGT TGTACTGGTC ATGGAGTTGT 360

ACTAGTAATG GCTTACGTGT ATTGTTTCAC ACACTAGTAA TAAATATGGA GATATAATAA 420 AAATACTCGT GATTCTTAGT CTATTTAAAA AAAATTTTCA TTTGAATATC CGGGATACAA 480

GAGCTATATA AGAATGAGTA AGGTTTAGCA TAAGTATAAT TCATTTCTTT TCAAAGAGAG 540

TTCAGATAAA TCATGAAATT TTTATATCTA ATACCCCTTT TAATTGCAGG AGTAGCATCA 600

TTCAATGCGG CCATACGTAA GTAAATTGGA TTTTTTTTTT TTTATAAATT CAATTCTATA 660

ATTCAATTTT ATTGTTCACT TTTTTTTTTT CATTTTTATT AAAATTATAC CAATAATGAT 720 AATATAAATT TATTTTTCGC TTTAAATTTA TAAAATTTAG ATCGTGAATG TAAGAATTTT 780 TGCGCCAAAG AGCACGGCGA GGAATATGCT ACATGGTCTT TTCGTTACGA ACTGGGTGAT 840 ACGTTTAAAT GTATTTGCAC GTAAGTAACA AAATTTTCAT TACTAGTATT CTTCCTTTGT 900 TTTTCATGAT TAAATCAAGA TTTATTAATT AATGAATTAT GTGTGTTGAT TTGATTATAT 960 AGTATCGGAA ACGGCCTCAT GAGAACAGAG AATAATGGTA AGTATCTACT TAGAATTATT 1020 AGTTTTGGTT TAATAATTTC AATTTTCTTT ACTTTTTAAT ACTCTATTAT TCTCTCTCTC 1080 TCTCTCTCCC AATCAAGATA AGTGTAGACA AAAATGTATT CAACAGCATG GAGCGGGAGG 1140 CTTTAAATAT GCCTTCAGCA TATACAGTGA AGTACCAGCT TCATGGGCAT GCATGTGCAC 1200 GTATGTAACA TTAGATGCTA TTATTATTAT TTCCTTTTTA AATCAATAAT TTTAATAATA 1260 CAATTGTATG ATGTTTTTAG TCAGGTGAAA AATAAGACAT ATTGTATACA TGCTTGCATG 1320 AAAGTTGAAG AAAAGGCCCC ACCTATGAAT CCCATAGTGA TGAAAAATGG AGAATGCTAC 1380 TATCAAGATC ACAGGGGTGT TGAGAGGTCG TGTGAATTGG AAATGAATAG CATTGATTGA 1440 ATAAATTCAA CAATTATCAA ATTCATTTTA TCAATGAAGG AAGAATAATA AATCAATAAT 1500 ATATATCAAC AATCAATAAC TTTGTTTTTA ATCATTTAAA AAAAAAAGGC TACATTGGAT 1560 TTTTAACTTC TCGACATACT ACTCGAATTG TGAAGGAGAA TTAAGGGCTT GTACTAGTCA 1620 CGAAACAGCC AGTTCCACTT TTAGAACGCA CCTCTATACA CATACATGTG TCCGTCCAAA 1680 TGTTGAAATT TATGCCGAAA ATTTTTTTTC ACATCCTCCA TTAAATGCCT AACCGTTACA 1740 AGTTTAAATT GACAACTGGT TGAAAGCCGA ACTTTAATAA TGATGGAAAG AAAGGAAAGA 1800 AATGTTTGAA A 1811

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: DNA

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

ATGAAATTTT TATATCTAAT 20

(2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: DNA (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

GTCTCCGGAT TGAATGATAC ²⁰

(2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: DNA

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

CATATACAGC GAAGTACC 18

(2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs

(B) TYPE: DNA (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

CATAACTATG GGATTCTTAG G ²¹

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: DNA (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: AAATGGACA ATGCTACTAC C 21

(2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: DNA

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

TTAAATTGAT TCCAACGC ¹⁸

(2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: DNA

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

AATGTGTTTG CACTCACG 18

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs

(B) TYPE: DNA (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

CCTCCCGCT CCATGATTTT G 21

(2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: DNA (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: nucleic acid

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: CCTCGCCGTG CTCTTTGGC 19

(2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: DNA

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

GTAACCAGC TAAGCATAAC G 21

(2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: DNA

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: nucleic acid

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

GTTATACAC AGAGGATCAG GGAG 24 (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: DNA

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31;

AATACAGTCC CTATATACCC 20

(2) INFORMATION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: DNA

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

GGGATGGGAA TAATGATGTC A 21

(2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

GACTCCTGGA GCCCG 15

(2) INFORMATION FOR SEQ ID NO: 34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

TTGACACCAG ACCAACTGGT AATG 24

(2) INFORMATION FOR SEQ ID NO: 35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: GCTGATGTGC TGCAAGGCGA TTAAG 25

(2) INFORMATION FOR SEQ ID NO: 36:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:

TTCACAGAGG AAACAGCTAT GAC 23

(2) INFORMATION FOR SEQ ID NO: 37: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

TCGTCAAGTG AAGAATTA 18

(2) INFORMATION FOR SEQ ID NO: 38:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: TTATATGAAG TTCTTAGA 18

(2) INFORMATION FOR SEQ ID NO: 39: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: GGCGCAATAA TTCTTCAC 18

(2) INFORMATION FOR SEQ ID NO: 40:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: TAACTATGGG ATTCTTAG 18

(2) INFORMATION FOR SEQ ID NO: 41:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: ATATTTAAAG CCTCCCGC 18

(2) INFORMATION FOR SEQ ID NO: 42: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:

TTYAAYCCNG ARACNCAYNG NGA 23 (2) INFORMATION FOR SEQ ID NO: 43:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:

GCNAAAGARC ASGGNGARGA 20

(2) INFORMATION FOR SEQ ID NO: 44: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 59 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:

TTCAACCCNG ARACNCACNG NGARNNNAAR AACTACNNNG CNAARGARCA TGGNGARGA 59

(2) INFORMATION FOR SEQ ID NO: 45:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 456 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bracon Hebetor

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION:!..456

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 45 :

ATG AAA TTT TTA TAT CTA ATA CCC CTT TTA ATT GCA GGA GTA GCA TCA 48

Met Lys Phe Leu Tyr Leu He Pro Leu Leu He Ala Gly Val Ala Ser

1 5 10 15 TTC AAT GCG GCC ATA GAT CGT GAA TGT AAG AAT TTT TGC GCC AAA GAG 96 Phe Asn Ala Ala He Asp Arg Glu Cys Lys Asn Phe Cys Ala Lys Glu 20 25 30

CAC GGC GAG GAA TAT GCT ACA TGG TCT TTT CGT TAC GAA CTG GGT GAT 144 His Gly Glu Glu Tyr Ala Thr Trp Ser Phe Arg Tyr Glu Leu Gly Asp 35 40 45 ACG TTT AAA TGT ATT TGC ATC GGA AAC GGC CTC ATG AGA ACA GAG AAT 192 Thr Phe Lys Cys He Cys He Gly Asn Gly Leu Met Arg Thr Glu Asn 50 55 60

AAT GAT AAG TGT AGA CAA AAA TGT ATT CAA CAG CAT GGA GCG GGA GGC 240 Asn Asp Lys Cys Arg Gin Lys Cys He Gin Gin His Gly Ala Gly Gly 65 70 75 80

TTT AAA TAT GCC TTC AGC ATA TAC AGT GAA GTA CCA GCT TCA TGG GCA 288 Phe Lys Tyr Ala Phe Ser He Tyr Ser Glu Val Pro Ala Ser Trp Ala 85 90 95

TGC ATG TGC CAG GTG AAA AAT AAG ACA TAT TGT ATA CAT GCT TGC ATG 336

Cys Met Cys Gin Val Lys Asn Lys Thr Tyr Cys He His Ala Cys Met 100 105 110

AAA GTT GAA GAA AAG GCC CCA CCT ATG AAT CCC ATA GTG ATG AAA AAT 384

Lys Val Glu Glu Lys Ala Pro Pro Met Asn Pro He Val Met Lys Asn

115 120 125 GGA GAA TGC TAC TAT CAA GAT CAC AGG GGT GTT GAG AGG TCG TGT GAA 432 Gly Glu Cys Tyr Tyr Gin Asp His Arg Gly Val Glu Arg Ser Cys Glu 130 135 140

TTG GAA ATG AAT AGC ATT GAT 456 Leu Glu Met Asn Ser He Asp 145 150

(2) INFORMATION FOR SEQ ID NO: 46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 152 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:

Met Lys Phe Leu Tyr Leu He Pro Leu Leu He Ala Gly Val Ala Ser 1 5 10 15

Phe Asn Ala Ala He Asp Arg Glu Cys Lys Asn Phe Cys Ala Lys Glu 20 25 30 His Gly Glu Glu Tyr Ala Thr Trp Ser Phe Arg Tyr Glu Leu Gly Asp 35 40 45

Thr Phe Lys Cys He Cys He Gly Asn Gly Leu Met Arg Thr Glu Asn 50 55 60

Asn Asp Lys Cys Arg Gin Lys Cys He Gin Gin His Gly Ala Gly Gly 65 70 75 80 Phe Lys Tyr Ala Phe Ser He Tyr Ser Glu Val Pro Ala Ser Trp Ala 85 90 95

Cys Met Cys Gin Val Lys Asn Lys Thr Tyr Cys He His Ala Cys Met 100 105 110

Lys Val Glu Glu Lys Ala Pro Pro Met Asn Pro He Val Met Lys Asn 115 120 125 Gly Glu Cys Tyr Tyr Gin Asp His Arg Gly Val Glu Arg Ser Cys Glu 130 135 140

Leu Glu Met Asn Ser He Asp 145 150

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 tiiat encoded by the spliced RNA derived from the genomic clone depicted in SEQ TD No.

19, the said spliced RNA being capable of hybridising witii the extension products of the primers depicted in SEQ TD 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, witii the proviso that the polynucleotide does not encode oniy 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 TD No.6; amino acids 23 to 275 in SEQ ID No. 8; and amino acids 22 to 184 in SEQ ID No. 10.

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

4. 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 10 and that encoded by die spliced RNA derived from the genomic clone depicted in SEQ TD No. 19, the said spliced RNA being capable of hybridising witii the extension products of the primers depicted in SEQ ID Nos 20 and 21 , or SEQ ED Nos 22 and 23, or SEQ TD

Nos 24 and 25 using die sequence depicted in SEQ ID No. 1 as a template, wherein the region has been modified in tiiat mRNA instability motifs and/or fortuitous splice regions arc removed, or insect-pest preferred codons are used so that expression of die tiius modified polynucleotide in die 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 die unmodified polynucleotide are endogenous.

5. A polynucleotide according to claim 4, wherein the said subunits are die proteins represented by amino acids 17 to 158 in SEQ ID No. 2; amino acids 22 to 182 in SEQ TD 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.

6. A polynucleotide according to claim 5, wherein at least one of die 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 die family Braconidae.

8. A polynucleotide according to any of the preceding claims wherein at least one of the protein encoding sequences in die region is under expression control of a viral promoter, or insect strong promoter, which is not down regulated or otiierwise silenced when the polynucleotide is introduced into die 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 plO 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 die toxin subunits selected from the proteins represented by amino acids 17 to 158 in SEQ ED 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.

11. A polynucleotide according to any preceding claim, wherein the region encodes not less tiian two and not more man 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 TD 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.

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 ED No. 4; amino acids 32 to 176 in SEQ TD No.6; amino acids 23 to 275 in SEQ ED No. 8; and amino acids 22 to 184 in SEQ

ED No. 10.

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

14. A polynucleotide according to any one of claims 4-7, wherein die said modified region encodes one of the toxin subunits selected from die proteins represented by amino acids 17 to 158 in SEQ ED No. 2; amino acids 22 to 182 in SEQ ED No. 4; amino acids 32 to 176 in SEQ ED No.6; amino acids 23 to 275 in SEQ ED No. 8; and amino acids 22 to 184 in SEQ ID No. 10.

15. A nucleotide sequence, encoding an insecticidal toxin sub-unit, which is complementary to one which when incubated at a temperature of between 50 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.

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 60°C in single strengdi citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.5 strength citrate buffered sahne containing 0.1% SDS still hybridises witii 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°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 sahne 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

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 sahne containing 0.1% SDS still hybridises with die 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.

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 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at die same temperature with 0.3 strength citrate buffered sahne containing 0.1 % SDS still hybridises with die 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.

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.

21. A cell transformed witii 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 me nucleotide sequence of any one of claims 14 to 19.

24. A pathogen according to die preceding claim, in the form of an insect virus.

25. A pathogen according to claim 23, in die 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 anϋ-oxidant.

27. An insecticidal composition comprising the following combinations of proteins: (i) 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) die 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;

(v) die proteins represented by amino acids 1 to 145 in SEQ ID No. 4 and 23 to

275 in SEQ TD No. 8; (vi) die 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 TD No.2, amino acids 22 to 182 in SEQ ID No. 4, and amino acids 32 to 176 in SEQ ID No.6; (viii) die proteins represented by amino acids 17 to 158 in SEQ ED 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; (x) 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 TD No.6, and amino acids 23 to 275 in SEQ ID No. 8.

28. A composition according to die preceding 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 DD No. 19, the said spliced RNA being capable of hybridising witii 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 die sequence depicted in SEQ ID No. 1 as a template.

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 23-26 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 23-26 or composition of claims 27 or 28 to control insect pests

32. A polynucleotide comprising a region encoding die insecticidal toxin subunit encoded by die spliced RNA derived from the genomic clone depicted in SEQ ID No. 19, the said sphced 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 die sequence depicted in SEQ ID No. 1 as a template, the translation product of the polynucleotide and recombinant entomopatiiogenic viruses comprising the said polynucleotide or a nucleotide comprising the sphced variant thereof.