DD297763A5 - INSECTICIDES AND METHODS OF CONTROLLING INSECTS - Google Patents

Abstract

The present invention is directed to a method for producing an insecticidal proteinaceous substance including the discovery and identification of the entire DNA sequence coding for the insecticidal protein MGE 1 of said proteinaceous substance, to said proteinaceous substance, to a DNA fragment characterized by the nucleotide sequence given in table 2, said fragment coding for the protein MGE 1, to the protein MGE 1 itself, to a DNA fragment originating from Bacillus thuringiensis var.kurstaki from Hpal (O) to Pstl (4355) coding for an insecticidal proteinaceous substance including truncated portions thereof, said truncated DNA portions being subject to the proviso that insecticidal activity has not been lost, to the method of constructing cloning vehicles and expression vehicles comprising the DNA fragment given in table 2 and also to said vehicles themselves.

Description

Field of application of the invention

The present invention relates to insecticidal agents and to methods of controlling insects.

Characteristic of the known state of the art

A comparable prior art can not be specified at present.

Object of the invention

The aim of the invention is to enrich the state of the art with novel substances having insecticidal activity, which are obtained by genetic engineering.

-3- 297 763 Presentation of the Essence of the Invention

The invention has for its object to provide insecticidal agents and methods for controlling insects available.

Bacillus thuringiensis (B. thuringiensis) is a Gram-positive bacterium that is usually pathogenic to insects (S. Chang ¹ ).

Reference is made to the attached bibliography, which is thus incorporated, and the publications and other material cited therein in more detail are incorporated herein by reference.

The different strains of B. thuringiensis differ considerably in terms of their toxicity and their spectrum of activity for insects. The insecticidal activity of B. thurigiensis derives, essentially or completely, from a proteinaceous parasporal crystal body formed at the time of sporulation in the growth phase. The gene (s) coding for the toxic proteins (polypeptides) of said crystal body were found on plasmid DNA and / or chromosomal DNA of B. thuringiensis.

In order to avoid any disadvantages that might arise due to the presence of other components produced by B. thuringiensis, and to obtain the insecticidally-effective polypeptide in large quantity, it is advantageous to use the corresponding gene, i. H. the corresponding DNA sequence encoding the desired insecticidally active protein, regardless of

B. to express thuringiensis.

Nevertheless, it is also possible and in some circumstances advantageous to transform B. thuringiensis itself with the mentioned DNA sequence.

In this way it is possible to obtain a protein which is analogous in its structure and properties to the natural product. The protein (polypeptides which are obtained in the context of the present invention is referred to below as MGE 1.

The present invention relates to a process for the preparation of an insecticidally active proteinaceous substance including the discovery and identification of the complete DNA sequence coding for the insecticidally active protein MGE 1 of said proteinaceous substance and which differs from the already known B. thuringiensis genes (HE Schnepf et al ⁴ ', MJ Adang et al ³¹ and Shibano et al ³⁰¹ and WO 86/01536).

The present invention furthermore relates to a DNA fragment which is characterized by the nucleotide sequence shown in Table 2 which codes for the protein MGE 1 as well as the protein MGEI itself; In addition, the present invention relates to a DNA fragment of Bacillus thuringiensis var. kurstaki from the Hpal region (O) to PstI (4355) which encodes an insecticidally active proteinaceous substance including selected portions (truncated portions) thereof provided that the insecticidal activity of said selected parts is maintained.

By the term "proteinaceous substance" is meant both the insecticidally active protein MGE 1 itself and in vitro derivatives and modifications thereof, such as the protein MGE 1 in conjunction with other protein fragments, primarily those of others cloned and for other pesticidal, preferably insecticidal activities encoding DNA, said protein combinations are classified as "fusion proteins"

 In addition to insecticidal activity, pesticidal activity also includes, for example, bacterial, viricidal, fungicidal and herbicidal activity, preferably against phytopathogenic organisms. Of the proteinaceous substances, the insecticidally active protein MGE 1 is preferred on its own.

Another object of the present invention relates to a method for the construction of cloning and expression vehicles containing the DNA fragment shown in Table 2 and said vehicle itself. Suitable DNA vectors are, for example, plasmin such as pBR322 and pUC8 or phages such as M13.

Furthermore, did present invention relates to living or dead microorganisms containing a DNA fragment by the in Tabel '. 2 nucleotide sequence is characterized, preferably on a microorganism of the species Sa ^ iiaromyces cerevisiae.

The invention further relates to microorganisms, in particular to microorganisms of the species Saccharomyces cerevisiae, which contain a DNA fragment which originates from the region Hpal (0) to PstI (4355) of Bacillus thuringiensis var. Kurstaki and which is an insecticidally active proteinaceous Substance including selected parts thereof, provided that the insecticidal activity of said selected parts is retained. Said microorganisms are, for example, yeasts, preferably Saccharomyces cerevisiae, bacteria and fungi on leaf surfaces, with the proviso that said microorganism, if belonging to the group of Bacillus thuringiensis, has been previously transformed with a DNA fragment, such as in Table 2. By "transformation" in this context also conjugation-like mechanisms should be understood.

The invention also includes a bioencapsulation system consisting of a first material fully embedded in a second material of biological origin, the first material being a DNA fragment as described in Table 2, the second material being a complete microorganism with the proviso that said microorganism, if it belongs to the group of Bacillus thuringiensis, has previously been transformed with the DNA fragment shown in Table 2.

The DNA material may ebonso be a DNA fragment derived from the region Hpal (0) to PstI (4355) of Bacillus thuringiensis var. Kurstaki and which encodes an insecticidal proteinaceous compound including selected portions thereof, provided that the insecticidal activity of said selected parts is maintained. Particularly suitable microorganisms are yeasts, preferably Saccharomyces cerevisiae, such as Saccharomyces cerevisiae GRF18.

Another part of the present invention relates to an agent and a method for Bekämp'ung of insects, preferably lepidopteran insects (insects of the order Lepidoptera), primarily members of the genera Pieris, Heliothis, Spodoptera and Plutella, such. B. Pieris brassicae, Heliothis virescens, Heliothis zea, Spodoptera littoralis, Plutella xylosteila and related species, using the proteinaceous substance containing the protein MGE 1, which is encoded by the DNA sequence shown in Table 2.

The method is characterized by the application of an insecticidally effective amount of a proteinaceous substance which is at least partially encoded by the DNA fragment coding for the MGE 1 protein, directly on the insects or their area of distribution. The agent includes an insecticidally effective amount of a proteinaceous substance that is at least partially encoded by the DNA fragment encoding the MGE 1 protein.

Moreover, the present invention relates to an application form consisting of transformed living or dead yeast cells containing an insecticidally effective amount of a proteinaceous substance which is at least partially encoded by the DNA fragment coding for the MGE 1 protein, said application form suitable for applying the active substance in a protected form.

It is therefore a long-lasting insecticidal activity achievable when the transformed yeast cells are applied in a conventional manner, such as. B. by spraying on the field (soil and air application). The active compound is well protected against premature degradation due to adverse conditions such as solar radiation or adverse conditions on the leaf surfaces.

The cloned gene can be placed under the control of a yeast promoter and expressed, the insecticidal activity being detectable both a) for the extract and in b) for whole cells.

Suitable yeast promoters are described in European Patent Application 100561. Particularly suitable is the PH05 promoter.

At least some of the Bacillus thuringiensis genes encoding insecticidal proteins are known to be linked to a promoter that can be recognized by the Escherichia coli (E. coli) RNA polymerase, with said promoter located in front of the respective gene is (HC Wong et al., ² ').

The process for the preparation of an insecticidally active proteinaceous substance within the scope of the present invention

involves the transcription and translation of a gene of identified DNA sequence according to the present invention

Invention, into the corresponding protein with the aid of E. coli or yeast. The method of obtaining starting material described here is carried out using Bacillus thuringiensis var.

kurstaki HDI, strain ETHZ 4449. This strain may be from the microbiological department of the Federal

Technical University in Zurich, Switzerland, and is free for anyone without any restriction

accessible.

The origin of this strain is the H. Dulmage Collection at the Cotton Insects Research Unit, US. Department of Agriculture, Agriculture Research Center, in Brownsville, Texas, where it is also freely accessible to everyone. According to the present invention, the DNA coding for the protein MGE 1 can be obtained by

a) Isolation and lysis of B. thuringiensis var. kurstaki HDI cells, as well as separation of the plasmids from the material obtained in this way, by means of methods known per se. The resulting plasmid material is then purified and dialyzed;

b) preparation of a DNA library of B. thuringiensis var. kurstaki HDI plasmid DNA;

c) cloning the fragmented plasmid DNA obtained according to point b) in a suitable vector, preferably a plasmid;

d) screening for the presence of an MGE 1 protein, which can be carried out according to one of the methods mentioned under points e) to g);

e) screening the clones for the presence of an antigen using appropriate antibodies prepared using B. thuringiensis var. kurstaki crystal body protein (screening for expression of the corresponding polypeptides);

f) reading the clones that react specifically with goat antiserum; and

g) testing the extracts of the clones obtained according to point f) for insecticidal activity.

The identification of the DNA can be carried out according to methods known per se, as described, for example, in US Pat Points h) and i) are listed:

h) Mapping of the DNA of positive clones by digestion with restriction endonucleases and hybridization of the thus obtained

Fragments with radioactively labeled RNA; and

i) sequencing of DNA fragments encoding the respective proteins.

Meaning of the abbreviations used in the following: Bp: base pairs BSA: bovine serum albumin (bovine serum albumin) DEAE: diethylaminoethyl DFP: diisopropylfluorophosphate DTT: 1,4-dithiothreitol (1,4-dimercapto-2,3-butanediol) EDTA: ethylenediaminetetraacetic acid IPTG: isopropyl-β-thiogalactopyranoside (Serva) Kb: kilobases PSB ¹ : 0.01 M phosphate buffer, pH 7.4 and 0.8% NaCl PSB ² : 1 mM sodium phosphate, pH 7.8 and 0.14% NaCl PEG 6,000: 6,000 molecular weight polyethylene glycol PMSF: phenylmethylsulfonyl fluoride (Fluka) RT: room temperature SDS: sodium dodecyl sulfate STE: see TNE TBS: 10 mM Tris-HCl, pH 7.5 and 0.14 M NaCI TE: solution containing 10 mM Tris · HCl (pH 7.5) and 1 mM EDTA TES: 0.5 M Tri * pH 8.0, 0.005 M NaCl and 0.005 M EDTA TNE: Solution containing 100 mM NaCl, 10 mM Tris · HCl (pH 7.5) and 1 mM EDTA Tris · HCl: tris- (hydroxymethyl) -aminomethane, pH adjustment with HCl X-GAL: 5-Bromo-4-chloro-3-indoxyl-β-D-galactoside

2 x YT: 16gBactoTrypton,

10 g of yeast extract (Bacto), BgNaCl

The following used media, buffers: md solutions:

20x SSC Dissolve 175.3 g NaCl and 88.2 g sodium citrate in 800 ml H ₂ O. Adjust the pH to pH 7.0 with a few drops of a 10 N NaOH solution. Adjust the volume to 1 liter; Splitting the solution into aliquots;

Sterilization by autoclaving

 2xSSC 10% from20xSSC

6xSSC 33% of 20x SSC

Denhardt's Ficoll 70 [molecular weight approx. 700,000; Solution (5Ox) Pharmacia) 5g

polyvinylpyrrolidone

(Calbiochem-Behring Corp.) 5 g

DSA (Sigma) 5g

H ₂ O to 500 ml

Filtration through a Nalgene ^e -Einmalfilter (· Nalgene, Nalge Co. Inc., Rochester, NY, USA). Separate into 25 ml aliquots and store at -2O ⁰ C.

solutions

M 9 medium: per liter:

a) Na ₂ HPO ₄ 6 g KH ₂ PO ₄ 3 g NaCl 0.5 g NH ₄ Cl 1 g

Adjust the pH to 7.4, autoclave, cool and then add from:

b) 1mMgSO ₄ 2ml 20% glucose 10ml 1MCaCl ₂ 0.1ml

Vitamin B1 (40 mg / 1 OmI) 5 ml The abovementioned solutions a) and b) should be sterilized separately by filtration (glucose, Vit. B1) or

by autoclaving.

LBILuria-

Bertani) Per liter: Medium Bacto-tryptone 10g Bacto yeast extract 5 g

NaCl 10g

Adjust the pH to 7.5 with sodium hydroxide. L-Broth see LB medium

For the induction of sporulation in B. thuringiensis var. Kurstaki, a GYS medium is used according to the data of Yousten and Rogoff (AA Yousten and MH Rogoff ⁶¹ ) [1969] (g / Γ ¹ ):

glucose 1 Yeast extract (Difco) 2 (NH ₄ I ₂ SO ₄ 2 K ₂ HPO ₄ 0.5 MgSO ₄ 7 H ₂ O 0.2 CaCl ₂ -2H ₂ O 0.08 MnSO ₄ -H ₂ O 0.05

Before autoclaving, the pH is adjusted to 7.3 with potassium hydroxide. Characterization of the microorganisms used in the context of the present invention:

1. HDI-ETHZ 4449 is a Bacillus thuringiensis strain of the subspecies kurstaki, characterized on the one hand by its immunological reaction against its flagellum antigen - HDI-ETHZ 4449 belongs to the 3a, 3b scrotype (A. Krieg ³ ") - to the other by its specific Southern blot pattern, which is obtained by carrying out the so-called ether blot experiments, as described in Section III 5.b.

The total DNA of this strain is isolated and digested to completion with the restriction enzyme Hind III; the resulting fragments are then separated according to their size on an agarose gel and then transferred to a nitrocellulose paper. The radioactively labeled EcoRI fragment Pos.423 to Pos. 1149 (Table 2) hybridizes specifically with 3 fragments having a size of 6.6 Kb, 5.3 Kb and 4.5 Kb, respectively.

2. HB 101 is a hybrid between Escherichia coli K12 χ Escherichia coli B. This strain is well suited for large-scale DNA purification. It is used for transformation experiments, and CaCI ₂ -competent cells are commercially available eg from Gibco AG, Basel, Switzerland, Catalog No. 5303260SA.

3. JM103 is an Escherichia coli K-12 which is e.g. commercially available from Pharmacia P-L Biochemicals, catalog no. 27-1545-xx (1984).

The E. coli strains HB101 and JM103 are described in Maniatis et al. (T. Maniatis et al., ⁶ '): Strain genotype HB101F ", hsdSjo (ri, mi),

recAi3, ara-14, proA ₂ , lacYi, galK ₂ , rpsL ₂ o (Sm ¹ ), xyl-5, mt 1 -1, supE ₄₄ , λ "JM103 Δ (lac per), thi, strA, supE, endA , sbcB, hsdR, F'traD36, proAB, lad ", Z ΔΜ15

In the following example, the term "yeast" is synonymous with Saccharomyces cerevisiae. Examples I. Plasmid DNA Processing: The workup of the plasmid DNA is carried out as described below as described in White and Nester (F. F. White

and EW nests ⁷¹ ).

1.1. B. thurlglensl plasmids

E. coli HB 101 cells are cultured in 1 liter LB medium with shaking for 12-14 hours at 37 ^° C. and worked up as described in White and Nester (FF White and EW Nester ") resuspended in alkaline lysing buffer and incubated for 20-30 minutes at a temperature of 37 ^° C. A clear lysate is obtained which is neutralized by the addition of 2 M Tris HCl (pH 8) The chromosomal DNA is then purified by addition of SDS and NaCl precipitated. the lysate is packed on ice, and the chromosomal DNA were removed by centrifugation. the plasmid DNA, which is now in the supernatant is precipitated with 10% PEG 6000. After on the plasmid DNA reinforcement overnight at 4 ⁰ C resuspension is carried out in 7-8 ml of TE The plasmid DNA obtained from 1 liter of culture solution is then further purified by means of 2 CsCl gradients, Festos CsCl being added to the solution (8.3 g of CsCl to 8.7 ml of supernatant) and) After adding ethidium bromide (Sigma; Final concentration 1 mg / ml supernatant), the solution is transferred to 13.5 ml Quick Seal polyallomer tubes (Beckmann) and centrifuged in a Beckman Ti50 rotor for 40 hours at 40000 rpm. Under long-wave UV light (366nm) two fluorescent bands are visualized. The bottom band contains overspiral plasmid DNA, which is collected by side-puncturing the centrifuge tube with a 2 ml syringe (18G needle). Ethidium bromide is removed by washing 5 times with equal volumes of isopropanol (saturated with CsCl) and then transferring the product to 30ml Corex tubes. 2.5 volumes of TE are added and then the DNA is precipitated with ethanol. This solution is then kept for 12-15 hours at -20 ^0C. The precipitated DNA is then collected by centrifugation in a Sorvall HB-4 rotor over a period of 30 min at 12000rpm and a temperature of 0 ° C and dissolved in 200 μΙ TE. (E. coli JM 103 can also be extracted and treated in an analogous manner.)

1.2. E. coli plasmids

The cells of a 100 ml culture (LB medium) are collected by centrifugation (Sorvall, GSA rotor, 10 min at 6000 rpm, 4 ° C), resuspended in 10 μMITE (10 mM Tris · HCl, 1 mM EDTA, pH 8.0) and centrifuged again under the same conditions. The cell pellet is then resuspended in 3 ml Tsuc (50 mM Tris HCl, pH 7.5.25% [w / v] sucrose) and transferred to SS-34 polypropylene Sorvall tubes. All subsequent steps are carried out on ice: first, 0.3 ml of a lysozyme solution (10 mg / ml, supplied by Worthington, 11000 U / mg) after 5 min 1.2 ml EDTA (50 mM, pH 8.0) and after a further 5 min 4, 8 ml of detergent (0.1% Triton X-100 [Merck], 50 mM EÜTA, 50 mM Tris · HCl, pH 8.0). After 5 minutes, the lysate is centrifuged in a pre-cooled SS-34 rotor for 40 minutes at 4 ° C. The supernatant is gently removed and purified on a CsCl gradient after addition of solid CaCl 2 according to the information given for the B. thuringiensis plasmid DNA

 From 100 ml of culture solution 50-100pg hybrid plasmid DNA are obtained.

II. Δ-endotoxin antigen and goat antibodies

11.1. Preparation of δ-endotoxin crumbbone specimens:

Bacillus thuringiensis (var kurstaki HD 1, strain ETHZ 4449) is in a Fernbach bottle on a medium to Yousten

and Rogoff, (AA Yousten and MH Rogoff ⁵ ') as previously described, but with an increased level of glucose (0.3% rather than 0.1%).

The incubation period is 4-5 days at a temperature of 3O ⁰ C. The colonies are unmitteiuar after sporulation

(B.Trümpi ⁸¹ ) harvested. For the separation of spores and parasporal bodies, the method of Delafield et al. (FP Delafield et al. ⁸¹ ).

a) Separation of spores and crystal bodies:

Suspend η autolysed cultures in 1M NaCl / 0.02M potassium phosphate buffer (pH 7.0) with 0.01% Triton-X-100 (Merck).

- Filter the suspension to remove particulate components, such as agar residues u.a.

- Centrifuge.

- Repeated washing of the sediment with the solution described above, until only traces of components that absorb at 260nm are present in the supernatant.

Washing the particulate components in 0.2M NaCl 0.004M phosphate buffer (pH 7.0) / 0.01% Triton-X-100.

Repeat washing with 0.01% Triton-X-100.

- Resuspending the particles in water.

- Remove the remaining cells from the suspension.

- Centrifuge 3 times and then wash the remaining spores and crystals in 0.02 M phosphate buffer IpH 7.0) / 0.01% Triton-X-100.

Transfer the suspension, in 182 ml of the same buffer, into a cylindrical separatory funnel containing 105 g of a 20% (w / w) aqueous sodium dextran sulfate 500 solution (Sigma), 13.2 g of solid polyethylene glycol 6000 (Merck), 3.3 ml 3M phosphate buffer (pH 7.0) and 7.5g NaCl.

- Shake to dissolve the solid ingredients.

- Set the volume to 600 ml by adding a well-mixed solution of the same composition, but ο'in bacterial components.

- Shake vigorously.

- Leave at 5 ⁰ C for 30 minutes.

- After phase separation, remove the upper phase (containing most of the spores but very few crystals).

- Centrifuge the upper phase.

Transferring the supernatant into the separating funnel to the lower phase remaining there (containing a mixture of spores and crystals).

- Repeat the extraction process.

After the tenth extraction, the crystal bodies in the lower phase are practically free of spores and can be isolated by centrifugation. Both the spores and the crystal bodies are then washed 5 times in cold distilled water. The crystal bodies are stored as an aqueous suspension at a temperature of -5 ° C.

b) dissolving the crystal bodies

An aliquot of the crystal-containing suspension is centrifuged for 10 minutes at 12000g. The sediment thus obtained

is resuspended in 0.05M carbonate buffer and 10 mM dithiothreitol (DTT, Sigma; the mixtures of carbonate buffer and dithiothreitol, hereinafter referred to as carbonate / DTT) at a concentration of 5 mg sediment / ml carbonate / DTT mixture.

After 30 minutes incubation at 37 ⁰ C, the insoluble particles are separated by centrifugation at 25000g lOminütige. The supernatant is dialyzed against carbonate buffer and then assayed for protein content and activity in the bioassay

examined.

For storage purposes, the protoxin solution is divided into individual portions which are deep-frozen. Protein that is free

DTT has a gelatinous consistency when thawed. Complete dissolution of the protein is achieved by addition of 1 mM DTT.

c) Inactivation of crystal-bound proteases

Serine proteases and metal proteases car- body suspension (Chestukhina et al., ¹⁰¹ ) are prepared by adding Diisopropylfluorophosphate (DFP, Serva) and EDTA inactivated as follows: The crystal bodies are dissolved in 0.01 M phosphate buffer, pH 8.0 and 1 mM EDTA in a concentration of 5-10 mg crystal body / ml Buffer / EDTA mixture, suspended. The suspension is then sonicated until a monodisperse solution

present (this is checked by means of a light microscope).

In a hood, with the usual safety precautions, 1 mM DFP is added to the suspension. The tube, the

receives the suspension is sealed airtight and shaken vigorously. After incubation overnight at room temperature, the inactivated suspension is dialyzed until equilibrium with H ₂ O and 1 mM EDTA is achieved.

11.2. Immunization of goats

The antigen is prepared from crystal bodies of B. thuringiensis serotype H-3 by dissolving the crystal bodies in carbonate / DTT, dialyzing the solution thus obtained against carbonate buffer and 1 mM DTT and purifying the antigen by Sterile filtration using a 0.45 pm Millipore filter. The antigen solution is mixed with "complete Freund's adjuvant" (Bacto) in a ratio of 1: 1 and at 4 ° C

stored.

At the experimental station of CIBA-GEIGY AG in St. Aubin (Friborg, Switzerland), 2 goats are immunized with H-3-protoxin. each

the two goats received an intracutaneous injection of 0.5 mg antigen and a subcutaneous injection of 1.5 ml pertussis (Behring), the latter to increase the immune response. The entire treatment is carried out on the Togen 0.28 and 76.

Blood samples are taken on the 35th, 40th, 84th and 89th day, with 5ml on the 35th day and 80ml on the remaining days

be removed.

After coagulation of the blood, the sera are incubated for 30 minutes at 56 ⁰ C to inactivate the complement system. The sera are stored at -2O ⁰ C.

11.3. Purification and [ ^12S l] Labeling of Goat H3 Antielodies

a) Purification of the immunoglobulin

The IgG (immunoglobulin G) fraction of the goat H ₃ antiserum is purified by ammonium sulfate precipitation, followed

from a chromatography on DEAE cellulose and an analysis on Ouchterlony immunodiffusion plates (O.Ouchterlony " ¹ ) according to the method described by Huber-Lukac (H. Huber-Lukac ¹²¹ ).

30 ml of 3.2 M ammonium sulfate are added to 15 ml of goat H ₃ antiserum in 15 ml PBS ¹ (0.01 M phosphate buffer, pH 7.4 and

0.8% NaCl). The mixture is allowed to stand for 15 minutes. After centrifugation of the mixture (10000g, 20min) is resumed, the sediment in 7.5 ml PBS ^1, three times at a temperature of 4 ° C against 1000 ml PBS ¹ dialysed, then dialyzed against 1000 ml 0.01 M phosphate buffer pH 7.8 and finally centrifuged (3000g, 20min).

The supernatant is applied to a 30 ml DEAE column and eluted at RT with 0.01 M phosphate buffer pH 7.9 (flow rate:

20-80ml / h). The fractions of the first peak are pooled and lyophilized. The average IgG yield is 180ml / 15ml serum. The purity of the IgG fraction is determined by immunodiffusion (O. Ouchterlony ¹¹ ') against anti-goat IgG.

Antibody and rabbit anti-goat serum antibody (Miles Laboratories). The further purification of the antibodies is carried out by Absorpti ι on a Sepharose® (Pharmacia) column, the H ₃ -Protoxin bound

contains. The binding of the protoxin to CNBr-Sepharose® (Pharmacia) is performed according to the manufacturer's instructions, which can be summarized as follows:

1 gCNBr-activated Sepharose®6MB is weighed for a gel-end volume of 3 ml. The gel is washed and placed on a

Glass frit re-swollen using 200ml 1mM HCl. The H ₃ antigen protein, which can be obtained as described under point 11.1 .c

is dissolved in 0.1 M NaHCO ₃ and 0.5 M NaCl dissolved. 1 ml of the gel then contains 5-10 mg of protein. The gel suspension and the antigen are mixed for 2 hours at room temperature. The excess protein is washed by washing with 0.1 M

NaHCO ₃ (pH 8.3), 0.5 M NaCl, and 0.5 M ethanolamine. Another wash is followed by 0.1 M NaHCO ₃ (pH

8.3) and 0.5 M NaCl, followed by 0.1 M CH ₃ COONa (pH 4) and 0.5 M NaCl. The last wash is then again with 0.1 M

NaHCO ₃ and 0.5M NaCl performed. The protein Sepharose * conjugate can then be packed in a Pharmacia K9 column (Pharmacia). The IgG can

then be cleaned very effectively on this column.

Specially bound antibodies are eluted with 3M KSCN and against PBS ² (10 mM sodium phosphate pH 7,8,0,14M NaCl).

dialyzed. The antibodies are then radiolabeled with ¹²⁸ I using the chloramine-T method (Amersham Buchler Review ¹³¹ ).

b) iodine labeling

1 ml of sodium iodide- ¹²⁵ Ii is placed in a tube with 100 μM of 0.5M phosphate buffer (pH 7.2). With continued stirring, 5 mg of the protein solution (0.5 mg / ml in TBS) and 50 mg of chloramine T in 0.05 M phosphate buffer (pH 7.2) are added. After a one-minute incubation at RT, the addition of 120 μρ Na ₂ S ₂ O _{6 takes place} . Free iodine is released from the labeled protein via a

0.9 cm x 12 cm column, packed with Sephadex «G-25, separated. In order to prevent absorption of the labeled krotein to the column, first 0.5 ml of BSA (100 mg / ml) is passed through the packed column. Thereafter, the labeled material is quantitatively transferred to the column and eluted with phosphate buffer (pH 7.2). Lie fractions (1 ml) are collected until all protein is eluted.

III. Cloneal daro-endotoxin-Qene

A partial Sau3A DNA library of B.thiringiensis var. Kurstaki HDI, strain ETHZ4449 plasmid DNA essentially as described by Maniatis et al. (T. Manialis et al. ' ⁴ ) and subcloned into the 8 amHI restriction site of pBR322, which acts as the vector DNA, as described below:

111.1. Partial digestion of high molecular weight B. turinglensis DNA

The digestion with Sau3A is carried out in such a way that the staining of the DNA fragments on the agarose gel with ethidium bromide preferably in the 2-10Kb range. This is done by using the methods described by Maniatis et al. (T. Maniatis et al., ^M) ).

The separation of the partially fragmented DNA is carried out on a preparative agarose gel as described in Section IV.2.

is described or preferably on a NaCl salt gradient. Adjust the linear salt gradient between 5 and 20% NaCl in TE buffer and centrifuge at 35Krpm for 3 hours in a SW40 Ti Beckman rotor. The collected fractions are precipitated by addition of ethanol and analyzed on an agarose gel.

10pg of the plasmid pBR322 are digested with 10 units of the endonuclease BamHI in 50μΙ 1OmM Tris-HCI, pH 7,4,10OmM NaCl and 1OmM MgCl ₂ at 37 ⁰ C 1-2h.

The phosphatase treatment of the cleaved DNA is carried out as follows:

10μg of DNA are first dissolved in 50μl Tris HCl, pH 8, followed by the addition of bovine intestinal alkaline phosphatase (Boehringer) at a concentration of 3 units / pg of DNA. After a 30 minute incubation at 37 ⁰ C, the DNA is treated twice with phenol and then extracted with chloroform. After ethanol precipitation, the DNA is resuspended in 20μΙ H ₂ O and used for the linkage reaction with DNA fragments obtained by partial Sau3A digestion. The reaction is carried out as follows: 0.1 μg of the phosphatase-treated vector is added to 0.4 μg Sau3A digested DNA in 10 μl H ₂ O.

The linking reaction is achieved by addition of 50 mM Tris.HCl, pH 7.4, ImMATP, 10 mM MgCl ₂ and 15 mM DTT, with subsequent application of 20 units of T ₄ DNA ligase (Biolabs). After incubation at 15 ^° C. overnight, the DNA is used to transform competent E. coli HB 101 cells.

Alternatively to the preparation of a partial Sau3 Α library, a DNA library of B. thuringiensis (var kurstaki HDI, strain ETH 2 4449) can also be prepared by complete BamHI and partial CIaI digestion of the plasmid DNA. Thereafter, the K'onierung takes place in pBR322 between the CIa I and BamHI interfaces.

111.2. Transformation using the Calclumchlorld method »

Competent cells are provided by treatment of cells grown to a population density of 5x10 ⁷ cells / ml with calcium chloride (Maniatis et al., ¹⁶¹ ).

The transformation is achieved by adding DNA to these cells. The cells are then incubated at 42 ⁰ C for 3 minutes followed by dilution with 1 ml of LB medium, incubated for 60 minutes at 37 ⁰ C as well as the distribution on selective media using well known methods (T. Maniatis et al. ¹⁶¹ ).

111.3. Producing raw cell lysates

The colonies containing the δ-endotoxin gene express a protein with similar biological activity to the purified and dissolved toxin crystals (HF Schnepf et al., ¹⁶¹ ). They are therefore screened by immunological methods using goat antibodies (the H ₃ antibodies) prepared against B. thuringiensis var. Kurstaki crystal body protein. The bacterial colonies are cultured individually in 5 ml LB medium in the presence of ampicillin. Ten cultures are each pooled, harvested and washed in 10 mM NaCl; Finally, the cells are lysed in 2 ml of 40 mM NaCl, 0.1 M NaOH and 1 mM PMSF. After incubation at room temperature for 20 minutes, the lysates are neutralized by addition of 20 μl of 2 M Tris.HCl, pH 7.0. After centrifugation in a SS34 Sorvall rotor (20 min, 10000 rpm), the lysates are dialyzed extensively against TBS (10 mM Tris-HCl, pH 7.5, 0, 14 M NaCl).

111.4. Radioimmunological screening of cell extracts

The extracts are purified by the plastic cup method as described by Clarke et al. (L. Clarke et al., ¹⁷¹ ), radioimmunoassay for the presence of δ-endotoxin antigen. Individual plastic cups are incubated overnight with 150 μΙ of purified H3 goose antibodies (10 μg / ml) in 10 mM Tris.HCl, pH 9.3 coated, and stored overnight at 4 ⁰ C. the cups are washed three times with TBS / Tween (TBS + 0.5% Tween 20) filled with 150μΙ the bacterial extract and incubated for 6 hours at 37 ° C. After the Wash the cups with 150μΙ ( ¹²⁶ L) labeled rabbit anti-goat HS antibodies (60ng, 10 ⁶ cpm) in TBS containing 25% horse serum and incubate overnight at room temperature The individual plastic cups are measured in a scintillation counter.

111.5. Restriction map and localization of the δ-endotoxin gene on the recombinant plasmid pK 19 a) Restrlktionskartierung

The restriction map of the DNA clone pK19, which is obtained according to the above-described immunological screening of the Sau3 Α library of B. thuringiensis HDI, ETHZ 4449, can be derived from the results of one-time, two times and three times digestions of the plasmid DNA with various restriction enzymes. The enzyme digestions are all carried out according to the manufacturer's instructions. Briefly, the DNA (1 μρ / δΟμΙ) is first dissolved in a suitable buffer for the respective restriction endonucleases and then the digested DNA after an incubation period of 1-2 hours at 37 ⁰ C applied to an agarose gel and subjected to electrophoresis. If further treatment with a second enzyme becomes necessary under conditions incompatible with the first enzyme

(For example, due to an incorrect buffer), the DNA is first extracted with a 1: 1 mixture of phenol and chloroform, then precipitated with ethanol and finally under the previously incompatible Pedingungen (such as in a buffer, for the 2 Enzyme is needed).

b) Southern transfer

The sequence coding for the δ-endotoxin is determined by the hybridization reaction of radioactively labeled RNA from speculative B. thuringiensis cells, with specific restriction fragments of pK19. This is achieved by using the transfer technique described in Southern (Southern ¹⁸¹ ):

Depending on their size on an agarose gel electrophoretically separated DNA fragments are denatured, transferred to a nitrocellulose filter and immobilized. The relative position of the DNA fragments in the gel is retained in the course of their transfer to the filter. The DNA bound to the filter is then hybridized with ³² P-labeled RNA; Autoradiography then locates the position of each individual band that is complementary to the radioactive sample. The DNA transfer from the agarose gel to nitrocellulose paper is carried out according to the instructions of Maniatis et al. ⁹¹ performed.

c) Hydroslerung the Southern filter

Prehybridization and actual hybridization are performed according to the instructions of Maniatis et al. (Maniatis, T., et al., ²⁰ ) with the following modifications: the baked filters are placed in a heat sealable plastic bag

0.2 ml of a prehybridization mixture is added per cm ^{2 of} nitrocellulose filter.

Prehybridization mixture: 4x SSC

50% formamide

0.2% SDS

2OmMEDTA

25 mM potassium phosphate (pH 7.2)

5x Denhard's solution

100 μα / ml denatured calf thymus DNA

The bags are usually incubated for 3-4 hours at 37 ⁰ C.

The prehybridization mixture is removed and replaced with the following hybridization mixture (50 μl / cm ² nitroelucos <3filter).

Hybridization Mixture: Same composition as the Prehybridisierungsmixtur, but now with

³² P-labeled denatured RNA probe (10 ^e -10 ⁷ cpm / filter), prepared in section lll.5.d. below. information provided.

The bags are usually stored at 37 ^° C. overnight. After hybridization, the filters are washed for 15 minutes in 2x SSC and 0.1% SDS at RT, repeating said washing twice; then the filters are washed for 60 minutes in 0.1 χ SSC and 0.1% SDS. The filters are dried on Whatman-3 MM paper and prepared for autoradiography.

d) Isolation and radioactive labeling of B.thurlngiensls var.

B. thuringiensis var. Kurstaki cells are cultured on a rogoff medium containing 0.1% glucose (AA Yousten and MH Rogoff ⁵¹ ). 500 ml cultures are shaken in 2 liter Erlenmeier bottles at 300 rpm and 30 ° C. During cell growth, the pH decreases from pH 7 to about 4.8 and then increases again to levels of pH 7. At this time the cells start z. clumping. The time when the pH returns to baseline is considered the starting point of sporulation. The cells are cultured for another 5 to 8 hours. Add rifampicin (50pg / ml) and shake the cells for another few minutes. The ice-cold cells are harvested and resuspended in 10 ml of 4M guanidine thiocyanate, 0.5% sarcosyl, 25 mM sodium citrate pH 7 and 0.1 M 2-mercaptoethanol. The cells are frozen at -80 ° C and then broken up in a French press. After centrifugation of the cell extract at 15,000 rpm for 15 minutes (Sorvall SS34 rotor), 0.5 g / ml of CsCl is added to the supernatant, which is then incubated in a Beckman 60Ti centrifuge tube on a 5.7 M CsCl, 0.1 M EDTA Pad is piled up. After renewed centrifugation for 20 h at 38000 rpm, the RNA pellet is washed with ethanol, dried, dissolved in 7 M guanidine hydrochloride and precipitated with ethanol. The total RNA from sporulating cells is dephosphorylated and labeled with [ ³² P] ATP and T4 polynucleotide kinase according to standardized methods (Maizels N. ²¹¹ ).

RNA labeling with polynucleotide kinase

About 1 μg of RNA is subjected to mild alkaline hydrolysis by heating in 50 mM Tris-HCl (pH 9.5); Time and temperature of incubation: 20 minutes at 90 ° C.

The hydrolysis yields free 5 'hydroxyl groups which serve as substrates for the polynucleotide kinase. The hydrolysis is carried out in sealed capillary tubes with a total volume of 4μΙ. The kinase labeling is carried out in reaction units of 10μΙ, the 5OmM Tris-HCl (pH 9.5), 1OmM MgCl ₂ , 5 mM dithiothreitol, 5% glycerol and 1 μΜ [V ³² P] -ATP, labeled with a specific activity of 6000 Ci / mmol, included. Each reaction unit contains about 1 μς RNA and 2 μΙ T-4 polynucleotide kinase, the reaction is carried out at 37 ⁰ C over a period of 45 minutes. This results in an RNA with a specific activity of about 3 x 10 ⁷ Cerenkov cpm ^ g. D ^! e RNA is separated from (Y ³² P] -ATP by ethanol precipitation three times in the presence of 5 μg of a tRNA carrier.

ΙΙΙ.β. Identification of clones encoding the δ-endotoxin, in the BamHI / Clal plasmid DNA library, encoded in pBR322: The pK25 Serl

a) In-sltu hybridization of bacterial colonies

Colony hybridization (M.Grunstein and D. Hogness ¹²¹ ) is performed by transferring the bacteria from a master plate to a nitrocellulose filter, and the colonies on the filter are then lysed and the released DNA is heated by heating After hybridization with a ³² P-labeled probe, the filter is checked by autoradiography, and a colony whose DNA gives a positive result in autoradiography can then be recovered from the plate containing the basal culture the BamHI / ClaI Plasmld DNA library, cloned into pBR322, one located within the δ-endotoxin DNA fragment is used. the method is described in Maniatis et al., (T. Maniatis et al. ^23). the filter with a ³² P-labeled sample, which is prepared according to the method described in point lll.ß.b described below, hybridized.

To prepare an autoradiographic image, the filter is wrapped in Saran Wrap and exposed to X-ray film The positive clones are isolated and analyzed by the plate containing the basal culture and have the DNA flanking region next to a DNA sequence encoding the toxin This could be demonstrated by immunological methods (see point III.4 above) and by restriction maps (see point III.5 above) and in an in vivo biotest (see III.7 below) hereinafter referred to as pK25-i, where i is a number between 1-7.

b) DNA-Nick Translatlon

Radionuclide labeling of an internal DNA fragment of the δ-endotoxin gene is performed according to the following procedure:

Mixture: 3 μ! DNA (^ g)

1.5 μΙ nick translation bufferdOf concentrated: 0.5 M Tris, pH 8,

0.05M MgCl ₂ )

1.5 μΙ 2.5 mM d guanosine 5'-triphosphate (GTP) 1.5 μΙ 2.5 mM d cytidine-5'-triphosphate (CTP) 1.5 μΙ 2.5 mM d thymidine-5'- triphosphate (TTP) 2.5 μΙΗ ₂ Ο 0.75 μΙ BSAd mg / ml) 1.5 μΙ lOOmMβ-mercaptoethanol

mix and dried to 10OpCi ^[32 Pa] ATP (10mCi / mmol) to give, mix well, 0,75μΙ a 1 x 10 ~ ⁴ solution of DNAseld mg / ml) Add in nick-translation buffer, 1 Minute incubate at RT Transfer to ice, add 1 μM E. coli polymerase I (Biolabs, final volume: 15 μM). Incubate for 3 hours at 15 ° C, heated at 65 ⁰ C for 10 minutes, 35 μΙ5OmM EDTA un ^r '10μΙ tRNA stock solution add (100 pg).

Unincorporated nucleotides are separated by chromatography on a small Sephadex G 50 column.

^! c) Subl (unit does the complete δ-endotoxin gene in pUC8 vector: the PK36 clone

The pUCS V'aktor (New England Biolabs) is completely digested with the restriction enzymes Hincll and Pst I as well as by treatment with alkaline phosphatase (see point 'above). The HpaI / PstI fragment of pk25-7 (see point III.6.a) above) encoding the δ-endotoxin gene (Table 2) is linked to the vector DNA and into E. coli HB101 cells transformed. One of the correctly transformed clones is named pk36.

III.7. Biotest for the determination of S. thurlnglensls Toxlns To the purified lysate following the procedure described under point III.3. information can be obtained Add ammonium sulfate in a 30% saturation concentration. The precipitate is dissolved in 2 ml of 50 mM sodium carbonate, pH

9.5, dissolved and dialyzed against the same buffer. As a control, E. coli extracts are prepared which contain the vector DNA but without incorporated B. thuringiensis DNA. The E. coli cell extracts are first treated with ultrasound, then 4 concentrations are prepared according to the toxin content in the extracts and 0.1% (v / v) of a

Wetting agent mixed. Leaf discs of cotton plants, which under controlled conditions in a climatic chamber (25 ⁰ C, 60% relative Humidity) are immersed in the E. coli cell extract suspensions. Hellothis vlrescens Larvae of the first larval stage (30 larvae per concentration) that have been standardized in a fitness diet

then placed on the dried leaf discs and individually incubated for 3 days at 25 ⁰ C.

The mortality is measured in%, whereby only the criteria "dead" or "alive" apply. The extracts, the mortality

therefore possess bioinsecticidal activity, which is derived from the cloned β. thuringiensis DNA.

IV. DNA sequencing Both strands of the DNA fragments encoding the δ-endotoxin gene are prepared by the method of F. Sanger et al. ^MI under Using the M13 system (J. Brass ²⁶¹ ) sequenced. IV. 1. Cloning of the δ-endotoxin gene into the replicative DNA form of M13

From the restriction mapping of the cloned δ-endotoxin gene and Southern blot analysis, it can be seen that the gene is located on two DNA fragments of pK36: Hpal (position 0 on the sequence) to HindIII (position 1847) and EcoRI (Position 1732) to Pstl (position 4355). The first fragment is cloned in M13mp8 (New England Biolabs) between the unique Hincll and the unique Hind HI sites in a reaction analogous to the linking process described above.

IV.2. Production of a succession of overlapping clones

To shorten the Hpa I-Hind III DNA fragment coding for the 5 'end of the gene, the Ba 131 method (M. Poncz et al., ²⁹¹ ) is used. Said shortening is carried out as follows:

The fragment is cloned in M 13mp8 between the unique Hincll and Hind HI sites. 10 mg of the replicative DNA form is linearized with the restriction endonuclease HindIII and then treated with the endonuclease Ba 131 in 100 μM 60 mM NaCl, 12 mM CaCl ₂ , 12 mM MgCl ₂ , 20 mM Tris HCl, pH 8 and 1 mM EDTA. This mixture is preincubated at 30 ⁰ C for 5 minutes. Subsequently, units Ba 131 are added. Immediately after this addition and after 2, 4, 6, 8, 10 and 12 minutes, 13 μΙ each are taken. To prevent another reaction, 25μΙ phenol and 40μΙ TE buffer are added immediately after collection. This mixture is centrifuged, extracted with chloroform and precipitated with ethanol. The resulting DNA precipitate is resuspended in 20 μM 10 mM NaCl, 20 mM Tris HCl and 10 mM MgCl ₂ and digested with a second enzyme found on the other side of the original cloned fragment; in this case it is BamHI. After separation in an agarose gel according to size, the truncated fragments are stained with ethidium bromide and visualized under long-wave UV light at 366nm. The part of the Agerose-gel containing the shortened fragments is cut out from the gel, liquified at 65 ⁰ C, adjusted to 50OmM NaCl and incubated at 65 ⁰ C for 20 minutes. One volume of phenol (equimulated with 10 mM Tris · HCl, pH 7.5, 1 mM EDTA, 500 mM NaCl) is added.

The aqueous phase is reextracted twice with phenol and once with chloroform. The DNA is precipitated with 2.5 volumes of cold absolute ethanol and collected by centrifugation. The DNA pellet is washed with cold 80% ethanol and then dried in vacuo. The DNA will then be in 20 μ! TE resuspended.

The fragments are truncated successively by 200-300 bp and have on the one side of the fragment a single BamHI restriction site and a smooth end on the other side. These fragments are cloned in an M 13mp8 vector which has previously been linearized by digestion twice with BamHI and HincII as described above under item VI.1. In the method described above, the DNA sequence is obtained only from one strand of DNA which begins at the HindIII restriction site and proceeds towards the BamHI site.

The procedure for sequencing the complementary DNA strand of the same DNA fragment and the restriction sites of the endonucleases used for the sequencing, ie, primarily for the truncation of the second DNA fragment, further the EcoRI / Pst I fragment and I its sequencing are in Figure 1, modified according to Poncz et al. ²⁶¹ and shown in Table 1.

IV.3. Transformation of E. coli strain JM103 E. coli JM 103 cells are cultured on M 9 maximal medium. The transformation is performed as follows:

1. Inoculate a single E.coli JM103 colony in 2x YT; store at 37 ^° C. overnight with constant stirring;

2. inoculate 40ml 2x YT with 200μΙ of the culture obtained according to step 1;

3. Cultivate at 37 ^° C., with constant stirring, to an OD ₆ J ₀ of 0.5;

4. Incubate on ice for 5 minutes;

5. Centrifuge at 6000rpm for 5 minutes in a pre-cooled SS34 Sorvall rotor;

6. Suspend the cells in 20 ml of a 50 mM sterile iced CaClj solution (CaCl ₂ solution should be freshly prepared each time);

7. Incubate on ice for 40 minutes;

8. Centrifuge at 6000rpm for 5 minutes in a SS34 Sorvall rotor;

9. Suspend the cells in 3 ml of an ice-cold CaCl 2 solution;

10. Add 1-5μΙ DNA of αβΓ7-15μΙ of a ligase formulation to 200μΙ of the cells obtained according to step 9;

11. Incubate on ice for 30 minutes; /

12. incubate at a temperature of 42 ⁰ C, 3 min;

13. Add 200μΙ of the cells obtained according to step 1;

14. Bring surface agar to boil and store at 42 ^° C.

15. Fill tubes with 3 ml surface agar, 30μΙ X-GAL (20mg / ml dimethyl sulfoxide) and 30μΙ IPTG (20mg / ml H ₂ O);

16. Thoroughly mix and transfer immediately to previously heated Ix YT plates;

17. Allow plates to dry for about 1 hour;

18. Turn plates over and incubate at 37 ° C.

The plaques obtained in this way are suitable for further treatment. Controls: - 200 μΙ competent cells without exogenous DNA

+ 1 μM M 13mp8 (replicative DNA form, 10 ng) + 200 μΙ competent cells

IV.4. Preparation of the Replicative Form of a Recombinant Phage DNA

1. Carefully transfer single white plaques into 9 ml 2x YT and 1 ml of the culture obtained according to step 1 part IV.3 and incubate for 7 hours at 37 ⁰ C;

2. Centrifuge at 4000rpm for 10 minutes;

3. Store supernatant overnight at 4 ° C;

4. inoculate 10 ml of the supernatant obtained according to step 3 and 10 ml of the culture obtained according to step 1, part IV.3 in 112x YT;

5. Shake at 37 ⁰ C for 4 hours;

6. Centrifuge at 5000rpm for 15min;

7. Suspend the cells in 10 ml of a 10% sucrose solution in 50 mM Tris.HCl, pH 8 and cool;

8. Transfer to 30 ml centrifuge tube;

9. Add 2 ml freshly prepared lysozyme solution (10 μl / ml 0.25M Tris · HCl, pH 8);

10. Add 8rr.lO, 25M EDTA and mix gently;

11. Incubate for 10 min on ice;

12. Add 4ml 10% SDS (or 1.6ml 25% SDS) and mix with a glass rod;

13. Add 6 ml 5 M NaCl (final concentration: 1 M) and mix gently;

14. Incubate for 1 hour on ice;

15. Centrifuge at 20000rpm for 40min in an SS-34 Sorvall rotor;

16. Remove supernatant and add ¹ Ao volume of 5M NaCl and 15 mL of 30% PEG in TNE;

17. Incubate for 2 hours or overnight at 4 ° C;

18. Centrifuge at 8000rpm for 15 minutes;

19. Remove pellets and transfer into 18 ml of TE, pH 8;

20. Add 18 g of CsCl (1 g / ml);

21. Transfer to either Τί-50 tube and add 0.4mL ethidium bromide (10mg / ml) or transfer to Ti-60 tube adding 1.2mL ethidium bromide (10mg / ml).

22. Fill up with CsCl solution (1 g CsCl + 1 ml TE);

23. Centrifuge at 35000rpm and 2O ⁰ C 36-48 hours;

24. Remove the lower bands visible under UV light (366nm);

25. Extract 3-4 times with butanol saturated with water;

26. Dialysis at 4 ° C 3 times each against 11 sterile TE;

IV.5. Preparation of a DNA strand matrix:

1. Shake E.colkJM 103 cells in 5 ml of 2x YT medium at 37 ⁰ C overnight;

2. add 2 drops of a cell suspension obtained according to step 1 to 25 ml of 2x YT medium;

3. Fill two tubes with 2 ml each of the culture solution obtained in step 2 and 1 plaque per tube, following the instructions in section IV.3. above, admit;

4. shake for 5 '/ 2 hours at 37 ⁰ C;

5. Transfer tube contents to Eppendorf tubes and centrifuge for 5 minutes;

6. Transfer 1 ml of the supernatant to fresh Eppendorf tubes;

7. Add 200μΙ 20% PEG 6000 / 2.5M NaCl;

8. Incubate at RT for 15 minutes;

9. Centrifuge for 5 minutes;

10. Lift off the supernatant and centrifuge again briefly;

11. Carefully lift off the supernatant by aspiration with a lengthened Pasteur pipette;

12. To the remainder, add ΙΟΟμΙ TE and 50 μΙ phenol;

13. Mix for 10 seconds (with the vortex); Leave for 5 minutes; Mix for 10 seconds (with the vortex); Centrifuge for 1 min.

14. Transfer aqueous phase to fresh Eppendorf tubes;

15. Add 500 μL ethylene ether, vortex and centrifuge for 1 min;

16. Remove ether by aspiration and leave tubes uncapped for 10 minutes (if the aqueous phase is very turbid after this treatment, air should be blown through with the Pasteur pipette until the solution is clear);

17. Add 10μΙ 3M sodium acetate and 250μΙ ethanol;

At -8O ⁰ C Incubate 18 30 minutes;

19. Centrifuge for 5 minutes;

20. Wash with 80% ethanol;

21. Centrifuge for 5 minutes;

22. Lift off supernatant with extended Pasteur pipette;

23. Leave tube for 15 minutes unlocked;

24. Dissolve pellet in 25μΙ TE;

25. Apply 2μΙ of the pellet solution to a 0.6% agarose gel;

IV.6. sequencing reaction

The DNA sequence analysis of the template DNA, as described in point IV.5. is carried out according to the method described in the manual "M13 Cloning and DNA Sequencing System", published by New England Biolabs The analysis of the complete DNA sequence shows that one finds only an open reading frame that is sufficiently long is coded for a protein with a MW of 130,622 and that for 1155 amino acids.

The N-terminus of the protein is located 156 bp downstream of the Hpal restriction site, whereas the last amino acid of the C-terminus is encoded by a codon starting at nucleotide 3618. The DNA sequence between the Hpal and the PstI site is shown in Table 2 and the deduced amino acid sequence in Table 3.

V. Expression of the Endotoxin Gene In Yeast Cells V.1. Introduce an NcoI site before the first AUG codon of the gene

To be able to combine the protein coding sequence of the B. thuringiensis toxin gene with the PH05 yeast promoter (described in European Patent Application No. 100,561), the DNA sequence in the environment of the toxin gene is modified. This modification is achieved by oligonucleotide-mediated mutagenesis with the single-stranded phage vector M 13mp8, which has a 1.5 Kb BamHI SacI insert coding for the 5 'region of the toxin gene. 200 ng of the insert is obtained by digesting 3μg of plasmid DNA from plasmid pK36 with BamHI and SacI, and then isolating the fragment using standard methods described above. 100 ng of the replicative form (RF) of M13mp8 are digested with the same enzymes, the DNA is treated with phenol and precipitated by adding ethanol and then linked to 200 ng of the above-mentioned insert DNA. After transfection of E. coli, six white plaques are picked and analyzed by restriction digests using BamHI and SacI. A correct isolate is selected and designated M 13mp18 / Bam-Sac. An oligonucleotide of sequence (5 ') GAGGTAACCCATGGATAAC (3') is synthesized by methods known per se using an "APPLIED BIOSYSTEMS DNA SYNTHESIZER." This oligonucleotide is complementary to a sequence of M ISmpie / Bam-Sac derived from

Position 141 to position 164 of the protoxin gene are sufficient (Table 2) and have the mismatch pairs mismatched in positions 154 and 155. The general procedure for mutagenesis is given by JM Zoller and M. Smith (JM Zoller and M. Smith ²⁷¹⁾ described. Approximately 5Mgeinzelsträngiger M 13mp18 / Bam-Sac-phage DNA is mixed with 0,3pg phosphorylated oligonucleotides in a total volume of 40μΙ. This mixture is heated for 5 minutes at 65 ° C, then first cooled to 5O ⁰ C and then cooled slowly to 4 ° C. Thereafter, buffer, nucleotide triphosphates, ATP, T4 DNA ligase and large fragment of DNA polymerase are added and Nai-hi at 15 ⁰ C, as described (JM Zoller and M. Smith ²⁷¹⁾ were incubated. After agarose gel electrophoresis, circular double-stranded DNA is purified and introduced by transfection into E. coli strain JM103. the resulting plaques are analyzed for sequences out d ie hybridize with "P-labeled oligonucleotide; the phages are examined by DNA restriction endonuclease analysis. Among the resulting phages, they are referred to as clones which now correctly have C at position 154 and 155 in place of T in the pK36 DNA. These phages are termed M I3mp18 / Bom-Sac / Nco.

V.2. Linkage of the δ-endotoxin gene to the PHO5 yeast promoter

The 1.5 Kb BamHI SacI insert of the M ISmpiS / Bam-Sac / Nco is recloned into the plasmid pK36 by the wild-type BamHI-SacI fragment of pK36 through the mutated 1.5 Kb fragment using previously described standard cloning techniques is replaced. This results in the plasmid pK36 / Nco, which has a Ncol рstriction site immediately before the ATG of the protoxin gene

Nocl .... GAGGTAAC / CCATGG / ATAAC.

5 μg of this vector is digested with Ncol and the overhanging 3 'ends are filled in with Klenow polymerase, as in Maniatis et al. ²⁸¹ ). Subsequently, the plasmid is digested with Ahalll, the DNA is separated on a 0.8% low-melting agarose gel and eluted as described above. 2 μg of plasmid p31y (described in European Patent Application No. 100,561) is digested with EcoRI and the recessed ends are filled in with Klenow polymerase as previously described. The blunt-ended 3,6-Kb protoxin gene fragment is linked to the blunt-ended vector p31y by incubation of 200ng of each DNAs in 20μΙ at RT as in Maniatis et al. ²⁹ 'described. After transformation of ampicillin resistance to E. coli HB101, individual clones are subjected to restriction analysis. A correct isolate is picked out and named p31y / BL. 1 μg of this plasmid DNA is digested with BamHI and the 4 Kb fragment is isolated from a soft agarose gel. This fragment is linked to self-seeding yeast vector pJDB 207 (described in European Patent Application No. 100,561), which has also previously been digested with BamHI (0.5 μg). Positive clones are isolated by means of E. coli transformation and plasmid DNA work-up. Correct isolates can be determined by restriction analysis using BamHI. The transformation of yeast strain GRF18 / (MATa, leu 2-3, leu 2-112, his 3-11, his 3-15 can ^R ) is carried out as described in European Patent Application 100,561.

V.3. Whole yeast cells with a recombinant B.thurglnlsls toxln gene in the bioassay Whole cells with the B. thuringiensis gene represent a bioencapsulation form (an artificially engineered system that utilizes

biological material, and wherein genetic material is surrounded by protective material) for the MGE 1 product, which is now better when applied to the plants against degradation by harmful influences, such as. Light, is protected as the crystalline product formed by B. thuringiensis in the context of sporulation and by disruption of the cell into the

Medium passes. Yeast cells with and without the B. thuringiensis toxin are distilled in water to a comparable optical Density resuspended. From said suspension, 4 concentrations are provided and 0.2% (v / v) of a wetting agent

added. For assessing the insecticidal activity of these yeast cell preparations, the leaf disc test already described in section III.7 above is used.

The insecticidal activity of B. thuringiensis transformed yeast cells on first-instar heliothisis

virescens larvae is demonstrated in the following table.

Table 4

material concentration Mortality in% (N = 30) Cells with toxin 1: 5 72 1: 7.5 40 1: 11.3 37 1: 16.9 22 Cells without toxin 1: 5 3 1: 11.3 0 leaf pieces without yeast: Control 1 - 0 Control 2 - 3

Similar results are obtainable when yeast extracts prepared as described in European Patent Application 100,561 are used instead of whole yeast cells and tested in the same bioassay.

For use as insecticides, the transformed microorganisms carrying the recombinant B. thuringiensis toxin Gene, preferably transformed live or dead yeast cells, including mixtures of live and dead Yeast cells, in unchanged form or preferably together with the excipients customary in formulation technology,

used and are therefore formulated in a conventional manner, for. B. to suspension concentrates, spreadable pastes, directly sprayable or dilutable solutions, wettable powders, soluble powders, dusts. Granules, and also encapsulations in z. B. polymeric substances.

The application methods such as spraying, misting, dusting, scattering, brushing or pouring are the same as the Type of funds chosen according to the desired goals and the given circumstances. The formulations, d. h., The transformed live or dead yeast cells or mixtures thereof and optionally

solid or liquid adjuvants containing preparations οοκν preparations are prepared in a known manner, for. B. by intimate

Mixing the yeast cells with solid carriers and optionally surface-active compounds (surfactants). As solid carriers, for. For dusts and dispersible powders, ground natural minerals are generally used,

such as calcite, talc, kaolin, montmorillonite or attapulgite. To improve the physical properties, it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. As a granular, adsorptive

Granules are porous types such. As pumice, broken brick, sepiolite or bentonite, as not sorptive Support materials z. As calcite or sand in question. In addition, a variety of pre-granulated materials

inorganic or organic nature such as in particular dolomite or crushed plant residues are used.

As surface-active compounds are nonionic, cationic and / or anionic surfactants with good dispersing and Net properties into consideration. Surfactants are also surfactant mixtures. Suitable anionic surfactants can be both so-called water-soluble soaps and water-soluble synthetic

be surface-active compounds.

As soaps are the alkali, alkaline earth or unsubsti'.uierte or substituted ammonium salts of higher fatty acids (Ci ₀ -C ₂ ;),

such as As the Na or K salts of oleic or stearic acid, or of natural fatty acid mixtures, the z. B. from coconut or

Tallow oil can be obtained, called. Further mention should also be made of the fatty acid methyl taurine salts, e.g. the Sodium salt of cis -fmethyl-JJ-octadecenylamino-1-ethanesulfonic acid (content in formulations preferably about 3%). However Hö¬jiiger so-called synthetic surfactants are used, especially Fettsulfonate, Fetteoitate, sulfon'erte

denzimidazole derivatives or alkylarylsulfonates or fatty alcohols, e.g. 2,4,7,9-tetramethyl-5-decyne-4,7-diol (content in

Formulations preferably at about 2%). The fatty sulfonates or sulfates are generally as alkali metal, alkaline earth metal or optionally unsubstituted or substituted Ammonium salts and have an alkyl radical having 8 to 22 carbon atoms, wherein alkyl is also the AlkyUeil of acyl radicals

includes, for. For example, the Na or Ca salt of lignin sulfonic acid, dodecyl sulfate, or a fatty alcohol sulfate mixture made from natural fatty acids. This subheading also covers the salts of sulfuric acid esters and sulphonic acids of

Fatty alcohol ethylene oxide adducts. The sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups and

a fatty acid residue with 8-22 C atoms. Alkylarylsulfonates are z. B. the Na, Ca or triethanolamine salts of

Dodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid or a naphthalenesulfonic acid Formaldehyde condensation product. Furthermore, corresponding phosphates such. B. salts of phosphoric acid ester of a p-nonylphenol (4 to 14) -

ethylene oxide adduct in question.

Nonionic surfactants are primarily polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols,

saturated or unsaturated fatty acids and alkylphenols which may contain 3 to 30 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon radical and 6 to 18 carbon atoms in the alkyl radical of the alkylphenols.

Other suitable nonionic surfactants are the water-soluble, 20 to 250 ethylene glycol ether groups and 10 to

100 polyethylene glycol adducts containing propylene glycol ether groups on polypropylene glycol,

Ethylenediaminopolypropylen glycol and alkylpolypropylene glycol having 1 to 10 carbon atoms in the alkyl chain. The

The compounds mentioned usually contain 1 to 5 ethylene glycol units per propylene glycol unit.

Examples of nonionic surfactants are nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, polypropylene / Polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxypolyethoxyethanol

mentioned. Also suitable are fatty acid esters of polyoxyethylene sorbitan, such as the polyoxyethylene sorbitan trioleate.

The cationic surfactants are, above all, quaternary ammonium salts which, as N-substituents, are at least

contain an alkyl radical having 8 to 22 carbon atoms and have as further substituents unsubstituted or halogenated lower alkyl, - benzyl or lower hydroxyalkyl radicals. The salts are preferably as halides, methyl sulfates or

Ethyl sulfates before, z. For example, stearyltrimethylammonium chloride or benzyldi (2-chloroethyl) ethylammonium bromide.

The surfactants commonly used in formulation technology are i.a. in the following publications: Mc Cutcheon's Detergents and Emulsifiers Annual

MC Publishing Corp., Ridgewood New Jersey, 1980; Helmut Stäche "Tensid-Taschenbuch" Carl-Hanser-Verlag Munich / Vienna 1981.

The agrochemical compositions usually contain 0.1 to 99%, in particular 0.1 to 95%, of the transformed, living or

dead yeast cells or mixtures thereof, 99.9 to 1%, in particular 99.8 to 5%, of a solid or liquid additive and 0 to 25%, in particular 0.1 to 25%, of a surfactant.

While merchandise tends to be more concentrated, the end user typically uses diluted ones Formulations. Such agents may contain other additives such as stabilizers, defoamers, viscosity regulators, binders, adhesives

as well as fertilizers or other active ingredients for achieving special effects.

The transformed live or dead yeast cells or mixtures thereof containing the recombinant B. thuringiensis toxin Genes are excellent for insect pest control. Preferably, they are

Plant-destroying insects of the order Lepldoptera, in particular those of the genera Pieris, Heliothis, Spodoptera and Plutella, such as, for example, Pieris brassicae, Heliothis virescens, Heliothis zea, Spodoptera littoralis and Plutella xylostella.

The application rates in which the yeast cells are used depend on the respective conditions, such as, for example, the weather conditions, the nature of the soil, the growth of the plants and the time of application.

Based on preliminary tests carried out in the greenhouse, it can be assumed that application rates of 1 to 10 kg, in particular 3 to 9 kg, of the yeast cells per hectare are advantageous.

Formulation Examples for B.thurliensiens toxin-containing material

In the following formulation examples, the term "yeast cells" is to be understood as meaning those which contain the recombinant j.thuringiensis gene. {The data are by weight% throughout.)

F1. Granules a) b)

Yeast cells 5% 10%

Kaolin 94% fumed silica 1%

Attapulgite - 90%

The yeast cells are first suspended in methylene chloride, then the suspension is sprayed onto the support material and then the suspending agent is evaporated in vacuo.

F2. Dusts a) b)

Yeast cells 2% 5%

Highly disperse silica 1% 5%

Talc 97%

Kaolin - 90%

Ready-to-use dusts are obtained by intimately mixing the excipients with the yeast cells.

F 3. Spray powder a) b) c)

Yeast cells 25% 50% 75%

Sodium lignosulfonate 5% 5%

Sodium lauryl sulfate 3% - 5%

Sodium diisopropyl-

naphthalenesulfonate - 6% 10%

Octylphenolpolyethylen-

glycol ether (7-8 mol

Ethylene oxide) - 2%

Highly disperse silica 5% 10% 10%

Kaolin 62% 27%

The yeast cells are mixed thoroughly with the additives and then the mixture is ground well in a suitable mill.

This gives wettable powders which can be diluted with water to give suspensions of any desired concentration.

F4. Extruder granules 10% Yeast cells 2% Sodium lignosulfonate 1% carboxymethylcellulose 87% kaolin

The yeast cells are mixed with the excipients, carefully ground and moistened with water. This mixture is extruded and then dried in a stream of air.

F5. Coated granules

Yeast cells 3%

Polyethylene glycol 200 3%

Kaolin 94%

The homogenously mixed yeast cells are uniformly applied in a mixer to the kaolin moistened with polyethylene glycol. In this way, dust-free coating granules are obtained.

F6. Suspension concentrate 40% Yeast cells 10% ethylene glycol Nonylphenolpolyethylenglykol 6% (15 moles of ethylene oxide) Alkylbenzolsulfonsäure- 3% triethanolamine * 1% carboxymethylcellulose Silicone oil in the form of a 0.1% 75% aqueous emulsion 39% water

* Alkyl is preferably linear with 10 to 14, especially 12-11, carbon atoms, such as n-dodecylbenzenesulfur of urethane thiethanolamine salt.

The homogeneously mixed yeast cells are intimately mixed with the additives. This gives a suspension concentrate from which suspensions of any desired concentration can be prepared by dilution with water. Of each of the microorganisms listed below that are used in the present invention, a culture was recognized by the German Collection of Microorganisms in Göttingen, West Germany, as recognized by the International Depository, in accordance with the requirements of the Budapest Treaty for the International Recognition of Deposit of microorganisms for the purpose of patenting, deposited. A statement on the viability of the deposited samples was made by the said International Depository.

microorganisms Deposit date Deposit number * Date of viability certificate HDI-ETHZ 4449 (Bacillus thuringiensisvar. Kurstaki HDI strain ETHZ4449) March 4, 1986 DSM 3667 March 7, 1986 HB101 (pK36) (Escherichia coli HBIOI transformed with pK36 plasmid DNA) March 4, 1986 DSM 3668 March 7, 1986 GRF18 (Saccharomyces cerevisiae) March 4, 1986 DSM 3665 March 7, 1986

* Entry number issued by the International Depository Office referred to above.

Limitations on the accessibility of said microorganisms have not been required by the depositor.

Literature:

1) S.Chang, Trends in Biotechnology 1 (4) 100 (1983)

2) H.C. Wong, H.E. Schnepf and M.R. Whiteley, The Journal of Biological Chemistry 258 (3), 1960 (1983)

3) MY. Adang, MJ. Staver, T.A. Rocheteau, J. Leighton, R.F. Barker and D.V. Thompson, Gene 36,29 (1985)

4) H.E. Schnepf, H.C. Wong and H.R. Whiteley, The Journal of Biological Chemistry 260 (10), 6264 (1985)

5) A.A. Yousten and M.H. Rogoff, Journal of Bacteriology 100, 122 (1969)

6) T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, USA, Appendix C: Commonly Used Bacterial Strains, pp. 504,506 (1982).

7) F.F. White and E.W. Nester, Journal of Bacteriology 141 (3), 1134 (March 1980)

8) B.TrOmpi, Zentralblatt f. Bacteriology Dept. II, 305 (1976)

9) F.P. Oelafield, HJ. Sommerville and S.C. Rittenberg, Journal of Bacteriology 96,713 (1968)

10) G.G. Chestukhina, I.A. Zalunin, L.I. Kostina, T.S. Kotova, S.P. Katrukha, L.A. Lyublinskaya and V.M. Stepanov, Brokliniya43, 857 (1978)

11) O.Ouchterlony, Handbook of Immune Diffusion and Immunoelectrophoresis, Ann Arbor Science Publishers, Ann Arbor, Mich., USA (1968)

12) H. Huber-Lukac, Doctoral Thesis, Swiss Federal Institute of Technology, Zurich, Switzerland, No.7050 (1982)

13) Amereham Buchler Review no. 18, Amereham Ruchler GmbH & Co. KG, Braunschweig, Federal Republic of Germany (1979)

14) T. Maniatis, E.F. Fritsch and J. Sambrook, Mole jular Cloning: A Laboratory Manual, Coid Spring Harbor Laboratory, Cold Spring Harbor, USA, p. 282 (1982)

15) T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, USA, p. 252 (1982)

IS) H.E. Schnepf and U.R. Whiteley, Proc.Natl.Acad. Be. USA 78, 2893 (1981) 17) L. Clarke, R. Hitzeman and J. Carbon, Methods in Enzymology 68, 446 (1979) IS) E.M. Southern, J. Molec. Biol. 98, 503 (1975)

19) T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, USA, p.383 (1982).

20) T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, USA, p. 387 (1982)

21) N. Maizels, Cell 9,431 (1976)

22) M.Grunsteln and D.Hogness, PNAS 72,396 (1975)

23) T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, USA, p.318,0982).

24) F. Sänger, S. Nicklen and A.R. Coulson, Proc. Nail. Acad. ScI. USA, 74, 5463 (1977)

25) J. Messing, Mothodsof Enzymology 101, 20 (1983)

26) M. Ponce, D. Solowieczyk, M. Balantine, E. Schwartz and S. Surrey, Proc. Natl. Acad. Be. USA79,4298 (1982)

27) MY. Zoller and M. Smith, Nucl. Acids Res. 10,6487 (1982)

28) T. Manlatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, USA, p. 394 (1982)

29) T. Manlatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, USA, p. 392 (1982)

30) Y.Shibano, A. Yamagata, N.Nakamura, T.llzuka, H.Sugisakl and M.Takanaml, Gene 34,243 (1985)

3I) A. War In: The Procaryotes, a Handbook on Habitats, Isolation and Identification of Bacteria (Eds. Starr, PM, Stolp, H., Trueper, GH, Baulows, Α., And Schlegel, HG), Springer Verlag New York, Heidelberg, Berlin, p. 1743 (1981)

Digestion with Kndunuklaaso Λ

I) Verdmiuiti '. With

KiulonukUMse B I) Β.ιΙ-, Ή

Linkage, tt; inaforma tion and finalization iiiylgur DMA (Ma tritzo)

σσ

Fig. 1 modified according to M. Poncz et al. ² "Construction of Deletlons Mutant Library Procedure: 1, the insert (thick line) is spliced into the Α site of the M13, which has cohesive ends, 2, after linearization at the B site, the phage DNA digested with Bal-31 for varying amounts of time [the dashed line indicates the extent of Bal-31 digestion relative to the insert (thick line) and M13 (thin line)]; 3, the digestion product becomes at the A site and isolating the insert's Bal-31-induced insert, resulting in a whole family of fragments of different sizes, each with a Bal-31-induced smooth end and a cohesive end at the Α site; 4 the fragments are subcloned into M13 such that the smooth end comes to lie proximal to primer site P, which is used for DNA sequence analysis

 X and C = smooth ends.

Table 1

A B C M13mpi (BamHI) Hpal-Hindlll <* ' BamHI HindIII EcoRI Pstl Hindll BamHI Pstl EcoRI Hincll Smal Smal Smal 8 9 8 9 EcoRI-PstI

a) according to the example given.

20 TGGGTCAAAA 30 ATTGATATTT 40 AGTAAAATTA 50 GTTGCACTTT -19-7673 80 AGTCATATGT 90 TTTAAATTGT 100 AGTAATGAAA 110 AACAGTATTA 140 TTAATAAAAG 150 AGATGGAGGT 160 AACTTATGGA 170 TAACAATCCG 60 GTGCATTTTT 200 TTATAATTGT 210 TTAAGTAACC 220 CTGAAGTAGA 230 AGTATTAGGT 120 TATCATAATG Rebell 2 nucleotide sequence of the DNA coding for the protein MGEI 260 TTACACCCCA 270 ATCGATATTT 280 CCTTGTCGCT 290 AACGCAATTT 180 AACATCAATG 10 GTTAACACCC 320 CGGTGCTGGA 330 TTTGTGTTAG 340 GACTAGTTGA 350 TATAATATGG 240 GGAGAAAGAA 70 TCATAAGATG 380 ATGGGACGCA 390 TTTCTTGTAC 400 AAATTGAACA 410 GTTAATTAAC 300 CTTTTGAGTG 130 AAHGGTATC 440 TAGGAACCAA 450 GCCATTTCTA 460 GATTAGAAGG 470 ACTAAGATE 360 GGAAT Il ITG 190 AATGCATTCC 500 ATCTTTTAGA 510 GAGTGGGAAG 520 CAGATCCTAC 530 TAATCCAGCA 420 CAAAGAATAG 250 TAGAAACTGG 560 TCAATTCAAT 570 GACATGAACA 580 GTGCCCTTAC 590 AACCGCTATT 480 CTTTATCAAA 310 AATTTGTTCC 620 TTATCAAGTT 630 CCTCTTTAT 640 CAGTATATGT 650 TC.AAGCTGCA 540 TAGAGAGAAG 370 GTCCCTCTCA 680 GAGAGATGTT 690 TCAGTGTTTG 700 GACAAAGGTG 710 GGGATTTGAT 600 CCTCTTTTG 430 AAGAATTCGC 740 TT, TAATGAT 750 TTAACTAGGC 760 TTATTGGCAA 770 CTATACAGAT 660 AATTTACATT 490 TTTACGCAGA 800 TACGGGATTA 810 GAGCGTGTAT 820 GGGGACCGGA 830 TTCTAGAGAT 720 GCCGCGACTA 550 AGATGCGTAT 860 TAGAAGAGA 870 TTAACACTAA 880 CTGTATTAGA 890 TATCGTTTCT 780 CATGCTGTAC 610 CAGTTCAAAA 920 TAGAACGTAT 930 CCAATTCGAA 940 CAGTTTCCCA 950 ATTAACAAGA 840 TGGATAAGATE 670 TATCAGTTTT 080 ATTAGAAATE 990 TTTGATGGTA 1000 GTTTTCGAGG 1010 CTCGGCTCAG 900 CTATTTCCGA 730 TCAATAGTCG 1040 GAGTCCACAT 1050 TTGATGGATA 1060 TACTTAACAG 1070 TATAACCATC 960 GAAATTTATA 790 GC7GGTACAA 1100 AGAATATE 1110 TGGTCAGGGC 1120 ATCA DATA 1130 GGCTTCTCCT 1020 GGCATAGAAG 850 ATAATCAATT 1160 ATTCACTTTT 1170 CCGCTATATG 1180 GAACTATGGG 1190 AAATGCAGCT 1080 TATACGGATG 910 ACTATGATAG 1220 TCAACTAGGT 1230 CAGGGCGTGT 1240 ATAGAACATT 1250 ATCGTCCACT 1140 GTAGGGTTTT 970 CAAACCCAGT 1280 TATAGGGATA 1290 AATAATCAAC 1300 AACTATCTGT 1310 TCTTGACGGG 1200 CCACAACAAC 1030 GAAGTATTAG 1260 TTATATAGAA 1090 CTCATAGAGG 1320 ACAGATE TAG 1150 CGGGGCCAGA 1210 GTATTGTTGC 1270 GACCTTTTAA

1340 CTCCTCAAAT 1350 TTGCCATCCG 1360 CTGTATACAG 1370 AAAAAGCGGA -20- 297 763 1400 AATACCGCCA 1410 CAGAATAACA 1420 ACGTGCCACC 1430 TAGGCAAGGA Table 2 (continued) 1460 TGTTTCAATG 1470 TTTCGTTCAG 1480 GCTTTAGTAA 1490 DAY DAY 1380 ACGOTAGATT 1330 CTTATGGAAC 1520 GTTCTCTTGG 1530 ATACATCGTA 1540 GTGCTGAATT 1550 TAATAATATA 1440 TTTAGTCATC 1390 CGCTGGATGA 1580 ACAAATACCT 1590 TTAACAAATE 1600 CTACTAATCT 1610 TGGCTCTGGA 1500 AGTATAATAA 1450 GATTAAGCCA 1640 AGGATTTACA 1650 GGAGGAGATA 1660 TTCTTCGAAG 1670 AACTTCACCT 1560 ATTCCTTCAT 1510 GAGCTCCTAT 1700 AGA 1710 ACTGCACCAT 1720 TATCACAAAG 1730 ATATCGGGTA 1620 ACTTCTGTCG 1570 CACAAATTAC 1760 CACAAATTTA 1770 CAATTCCATA 1780 CATCAATTGA 1790 CGGAAGACCT 1680 GGCCAGATTT 1630 TTAAAGGACC 1820 AGCAACTATG 1830 AGTAGTGGGA 1840 GTAATTTACA 1850 GTCCGGAAGC 1740 AGAATTCGCT 1690 CAACCTTAAG 1880 TACTCCGTTT 1890 AACTTTTCAA 1900 ATGGATCAAG 1910 TGTATTTACG 1800 ATTAATCAGG 1750 ACGCTTCTAC 1940 TTCAGGCAAT 1950 GAAGTTTATA 1960 TAGATCGAAT 1970 TGAATTTGTT 1860 TTTAGGACTG 1810 GöAATTTTC 2000 GGCAGATE 2010 GATTTAGAAA 2020 GAGCACAAAA 2030 GGCGGTGAAT 1920 TTAAGTGCTC 1870 TAGGTTTTAC 2060 TCAAATCGGG 2070 TTAAAAACAG 2080 ATGTGACGGA 2090 TTATCATATT 1980 CCGGCAGAAG 1930 ATGTCTTCAA 2120 TGAGTGTTTA 2130 TCTGATGAAT 2140 TTTGTCTGGA 2150 TGAAAAAAAA 2040 GAGCTGTTTA 1990 TAACCTTTGA 2180 ACATGCGAAG 2190 CGACTTAGTG 2200 ATGAGCGGAA 2210 TTTACTTCAA 2100 GATCAAGTAT 2050 CTTCTTCCAA 2240 CAATAGACAA 2250 CTAGACCGTG 2260 GCTGGAGAGG 2270 AAGTACGGAT 2160 GAATTGTCCG 2110 CCAATTTAGT 2300 TGACGTATTC 2310 aaagagaAtt 2320 ACGTTACGCT 2330 ATTGGGTACC 2220 GATCCAAACT 2170 AGAAAGTCAA 2360 GTATTTATATE 2370 CAAAAAATAG 2380 ATGAGTCGAA 2390 ATTAAAAGCC 2280 ATTACCATCC 2230 TTAGAGGGAT 2420 AGGGTATATC 2430 GAAGATAGTC 2440 AAGACTTAGA 2450 AATCTATTTA 2340 TTTGATGAGT 2290 AAGGAGGCGA 2480 CGAAACAGTA 2490 AATGTGCCAÜ 2500 GTACGGGTC 2510 CTTATGGCCG 2400 TATACCCGTT 2350 GCTATCCAAC 2540 CGGAAAATGT 2550 GCCCATCATT 2560 CCCATCATTT 2570 CTCCTTGGAC 2460 ATTCGCTACA 2410 ACCAATTAAG 2600 CTTAAATGAG 2610 GACTTAGGTG 2620 TATGGGTGAT 2630 ATTCAAGATT 2520 CTTTCAGCCC 2470 ATGCCAAACA 2580 ATTGATGTTG 2530 CAAGTCCAAT 2640 AAGACGCAAG 2590 GATGTACAGA

2660 AAGACTAGGA 2670 AATCTAGATE 2680 TTCTCGAAGA 2690 GAAACCATTA -21- 297 763 2720 TGTGAAAAGA 2730 GCGGAGAAAA 2740 AATGGAGAGA 2750 CAAACGTGAA Table 2 (continued) 2780 TATTGTTTAT 2730 AAAGAGGCAA 2800 AAGAATCTGT 2810 AGATGCTTTA 2700 GTAGGAGAAG 2650 ATGGCCATGC 2840 TAGATTACAA 2850 GCGGATACCA 2860 ACATCGCGAT 2870 GATTCATGCG 2760 AAATTGGAAT 2710 CACTAGCTCG 2900 CATTCGAGAA 2910 GCTTATCTGC 2920 CTGAGCTGTC 2930 TGTGATTCCG 2820 TTTGTAAACT 2770 GGGAAACAAA 2960 TGAAGAATTA 2970 GAAGGGCGTA 2980 TTTTCACTGC 2990 ATTCTCCCTA 2880 GCAGATAAAC 2830 CTCAATATGA 3020 TAAAAATGGT 3030 GATTTTAATA 3040 ATGGCTTATC 3050 CTGCTGGAAC 2940 GGTGTCAATG 2890 GCGTTCATAG 3080 AGAAGAACAA 3090 AACAACCACC 3100 GTTCGGTCCT 3110 TGTTGTTCCG 3000 TATGATGCGA 2950 CGGCTATTTT 3140 ACAAGAAGT 3150 CGTGTCTGTC 3160 CGGGTCGTGG 3170 CTATATCCTT 3060 GTGAAAGGGC 3010 GAAATGTCAT 3200 GGGATATGGA 3210 GAAGGTTGCG 3220 TMCCATTCA 3230 TGAGATCGAG 3120 GAATGGGAAG 3070 ATGTAGATGT 3260 GTTTAGCAAC 3270 TGTGTAGAAG 3280 AGGAAGTATA 3290 TCCAAACAAC 3180 CGTGTCACAG 3130 CAGAAGTGTC 3320 TACTGCGACT 3330 CAAGAAGATE 3340 ATGAGGGTAC 3350 GTACACTTCT 3240 MCAATACAG 3190 CGTACAAGGA 3380 AGCCTATGAA 3390 AGCAATTCTT 3400 CTGTACCAGC 3410 TGATTATGCA 3300 ACGGTMCGT 3250 ACGAACTGAA 3440 ATATACAGATE 3450 GGACGAAGAG 3460 ACAATCCTTG 3470 TGAATCTAAC 3360 CGTMTCGAG 3310 GTAATGATTA 3500 ACCACTACCA 3510 GCTGGCTATG 3520 TGACAAAAGA · 3530 ATTAGAGTAC 3420 TCAGCCTATG 3370 GATATGACGG 3560 ATGGATTGAG 3570 ATCGGAGAAA 3580 CGGAAGGAAC 3590 ATTCATCGTG 3480 AGAGGATATG 3430 AAGAAAAAGC 36? 0 TATGGAGGAA 3630 TAATATATGC 3640 TTTAAAATGT 3650 AAGGTGTGCA 3540 TTCCCAGAM 3490 GGGATTACAC 3680 TTGTATTGAC 3690 AGATAAATAA 3700 GGAAAII 111 3710 ATATGAATAA 3600 GACAGCGTGG 3550 CCGATAAGGT 3740 AAGAATGATG 3750 TCCGTTTTT 3760 GTATGATTTA 3770 ACGAGTGATA 3660 AATAAAGATE 3610 AATTACTTCT 3800 AGGCTTTACT 3810 TAACGGGGTA 3820 CCGCCACATG 3830 CCCATCAACT 3720 AAMCGGGCA 3670 GATTACTGAC 3860 AAGTGTCAAA 3870 AAACGTTATT 3880 CTTTCTAAAA 3890 AGCTAGCTAG 3780 TTTAAATGTT 3730 TCACTCTTAA 3920 AATCTTTCAA 3930 TTCAAGATGA 3940 ATTACAACTA 3950 TTTTCTGAAG 3840 TMGAATTTG 3790 TTTTTTGCGA 3900 AMGGATGAC 3850 CACTACCCCC 3960 AGCTGTATCG 3910 ATTTTTATG

3980 CCTTCTCTTT 3990 TGGAAGAACT 4000 CGCTAAAGAA 4010 TTAGGTTTG -22- 297 763 4040 TCAGGAAATG 4050 AATTAGCTAC 4060 CATATGTATC 4070 TGGGGCAGTC abello2 (continued) 4100 CTCGTTCGAC 4110 TATGCAGTCA 4120 ATTACACGCC 4130 GCCACAGCAC 4020 TAAAAAGAAA 3970 TCATTTAACC 4160 TCAATAAACG 4170 CTTTGATAAA 4180 AAAGCGGTTG 4190 AATTTTTGAA 4080 AACGTACAGC 4030 ACGAAAGTT 4? 20 GGAAAAGTAA 4230 ACTTTGTAAA 4240 ACATCAGCCA 4250 TTTCAAGTGC 4140 TCTTATGAGT 4090 GAGTGATTCT 4280 GAATCCGTAT 4290 TTTAGATGCG 4300 ACGATTTTCC 4310 AAGTACCGAA 4200 ATATATTTTT 4150 CCAGAAGGAC 4340 CTGGGTCAGG 4350 TGGTTGTGCA 4360 CAAACTGCAG 4260 AGCACTCACG 4210 TCTGCATTAT 4320 ACATTTAGCA 4270 TATTTTCAAC 4330 CATGTATATC

Table 3 Amino acid sequence of the protein MGEI

LIMITS: 106 3623

185 215

ATG GAT AAC AAT CCG AAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA AGT AAC CCT GAA Met Asp Asn Asn Pro Asn He Asn Giu Cys He Pro Tyr Asn Cys Leu 3 Asn Pro GIu

245 275

GTA GAA GTA TTA GGT GGA GAA AGA ATA GAA ACT GGT TAC ACC CCA ATC GAT ATT TCC TTG VaI GIu VaI Leu GIy GIu GIu Arg He GIu Thr GIy Tyr Thr Pro Ho Asp Ue Ser Leu

305,335

TCG CTA ACG CAA TTT CTT TTG AGT GAA TTT GTT CCC GGT GCT GGA TTT GTG TTA GGA CTA Ser Lei Thr GIn Phe Leu Leu Ser GIu Phe VaI Pro GIy Ala GIy Phe VaI Leu GIy Leu

365 395

GTT GAT ATA ATA TGG GGA ATT TTT GGT CCC TCT CAA TGG GAC GCA TTT CTT GTA CAA ATT VaI Asp He He Trp GIy He Phe GIy Pro Ser GIn Trp Asp AIa Phe Leu VaI Gin He

425 455

GAA CAG TTA ATT AAC CAA AGA ATA GAA GAA TTC GCT AGG AAC CAA GCC ATT TCT AGA TTA GIu GIu GIu Phe AIa Arg Asn Gin Ala He Ser Arg Leu

485 515

GAA GGA CTA AGC AAT CTT TAT CAA ATT TAC GCA GAA TCT TTT AGA GAG TGG GAA GCA GAT GIu GIy Leu Ser Asn Leu Tyr Gin He Tyr Ala GIu Ser Phe Arg GIu Trp GIu Ala Asp

545 575

CCT ACT AAT CCA GCA TTA AGA GAA GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC Pro Thr Asn Pro AIa Leu Arg GIu GIu Met Arg lie GIn Phe Asn Asp Met Asn Ser AIa

605 635

CCT ACA ACC GCT ATT CCT CTT TTT GCA GTT CAA AAT ACT CAA GTT CCT CTT TTA TCA GTA Leu Thr Thr Ala He Pro Leu Phe Ala Vai GIn Asn Tyr Gin VaI Pro Leu Leu Ser VaI

665 695

TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTT TCA GTG TTT GGA CAA Tyr VaI Gin Ala Ala Asn Leu His Leu Ser VaI Leu Arg Asp VaI Ser VaI Phe GIy GIn

725 755

AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT TAT AAT GAT TTA ACT AGG CTT ATT Arg Trp GIy Phe Asp Ala Ala Thr He Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu He

-23- 297 763 Table 3 (continued)

785,815

GGC AAC TAT ACA GAT CAT GCT GTA CGC TGG TAC AAT ACG GGA TTA GAG CGT GTA TGG GGA GIy Asn Tyr Thr Asp His Ala VaI Arg Trp Tyr Asn Thr GIy Leu GIu Arg VaI Trp GIy

845 875

CCG GAT TCT AGA GAT TGG ATA AGA ACT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA Pro Asp Ser Arg Asp Trp He Arg Tyr Asn GIn Phe Arg Arg Glu Leu Thr Leu Thr VaI

905 935

TTA GAT ATC GTT TCT CTA TTT CCG AAC TAT GAT AGT AGA ACG TAT CCA ATT CGA ACA GTT. Leu Asp He VaI Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro He Arg Thr VaI

965,995

TCC CAA TTA ACA AGA GAA ATT ACT ACA AAC CCA GTA TTA GAA AAT TTT GAT GGT AGT TTT Ser GIn Leu Thr Arg GIu He Tyr Thr Asn Pro VaI Leu GIu Asn Phe Asp GIy Ser Phe

1025 1055

CGA GGC TCG GCf CAG C ATA GAA GGA AGT ATT AGG AGT CCA CAT TTG ATG GAT ATA CTT Arg GIy Ser Ala Gin GIy He GIu GIy Ser He Arg Ser Pro His Leu Met Asp He Leu

1085 1115

AAC AGT ATA ACC ATC TAT ACG GAT GCT CAT AGA GGA GAA ACT TGG TCA GGG CAT CAA Asn Ser He Thr He Tyr Thr Asp Ala His Arg GIy GIu Tyr Tyr Trp Ser GIy His Gin

1145 1175

ATA ATG GCT TCT CCT GTA GGG TTT TCG GGG CCA GAA TTC ACT TTT CCG CTA ACT GGA ACT He Met Ala Sar Pro Vai GIy Phe Ser GIy Pro Giu Phe Thr Phe Pro Leu Tyr GIy Thr

1205 1235

ATG GGA AAT GCA GCT CCA CAA CAA CGT ATT GTT GCT CAA CTA GG CAG GGC GTG TAT AGA Met GIy Asn Ala Ala Pro Gin Gin Arg He VaI Ala Gin Leu Giy Gin Giy VaI Tyr Arg

1265 1295

ACA TTA TCG TCC ACT TTA TAT AGA AGA CCT TTT AAT ATA GGG ATA AAT AAT CAA CAA CTA Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn lie Giy He Asn Asn Gin GIn Leu

1325 · 1355

TCT GTT CTT GAC GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AAT TTG CCA TCC GCT GTA Ser VaI Leu Asp Gly GIu Phe AIa Tyr GIy Thr Ser Ser Asn Leu Pro Ser Ala VaI

1385 1415

TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAA ATA CCG CCA CAG AAT AAC AAC GTG Tyr Arg Lys Ser Gly Thr VaI Asp Ser Leu Asp GIu He Pro Pro GIn Asn Asn Asn VaI

1445 1475

CCA CCT AGG CAA GGA TTT AGT CAT CGA TTA AC C CAT GTT TCA ATG TTT CGT TCA GGC TTT Pro Pro Arg Gin Gly Phe Ser His Arg Leu Ser His VaI Ser Met Phe Arg Sei GIy Phe

, 1505 1535

AGT AAT AGT AGT GTA AGT ATA ATA AGA GCT CCT ATG TTC TCT TGG ATA CAT CGT AGT GCT Ser Asn Ser Ser VaI Ser He lie Arg AIa Pro Met Phe Ser Trp He His Arg Ser AIa

1565 '1595

GAA TTT AAT AAT ATA ATT CCT TCA TCA CAA ATT ACA CAA ATA CCT TTA ACA AAA TCT ACT GIu Phe Asn Asn He He Pro Ser Ser Gin He Thr Gin He Pro Leu Thr Lys Ser Thr

1625 165S

AAT CTT GGC TCT GGA ACT TCT GTC GTT AAA GGA CCA GGA TTT ACA GGA GGA GAT ATT CTT Asn Leu GIy Ser GIy Thr Ser VaI VaI Lys GIy Pro GIy Phe Thr GIy GIy Asp He Leu

1685 1715

CGA AGA ACT TCA CCT GGC CAG ATT TCA ACC TTA AGA GTA AAT ATT ACT GCA CAA TTA TCA Arg Arg Thr Ser Pro GIy Gin He Ser Thr Leu Arg VaI Asn He Thr AIa Pro Leu Ser

1745 1775

CAA AGA TAT CGG GTA AGA ATT CGC TAC GCT TCT ACC ACA AAT TTA CAA TTC CAT ACA TCA Gin Arg Tyr Arg Va ¹ Arg He Arg Tyr Al Ser Thr Thr Asn Leu G Pho His Thr Ser

-24- 297 763 Table 3 (Application)

1805 1835

ATT GAC GGA AGA CCT ATT AAT CAG GGG AAT TTT TCA GCA ACT ATG AGT AGT GGG AGT AAT He Asp GIy Arg Pro He Asn Gin GIy Asn Phe Ser AIa Thr Met Ser Ser GIy Ser Asn

1865 1895

TTA CAG TCC GGA AGC TTT AGG ACT GTA GGT TTT ACT ACT CCG TTT AAC TTT TCA AAT GGA Leu Gin Ser Giy Ser Phe Arg Thr VaI Giy Phe Thr Thr Per Phe Asr. Phe Ser Asn GIy

1925 1955

TCA AGT GTA TTT ACG TTA AGT GCT CAT GTC TTC AAT TCA GGC AAT GAA GTT TAT ATA GAT Ser Ser VaI Phe Thr Leu Ser Ala His VaI Phe Asn Ser GIy Asn GIu VaI Tyr He Asp

1985 2015

CGA ATT GAA TTT GTT CCG GCA GAA GTA ACC TTT GAG GCA GAA TAT GAT TTA GAA AGA GCA Arg He GIu Phe VaI Pro Ala GIu VaI Thr Phe GIu Ala GIu Tyr Asp Leu GIu Arg Ala

2045 2075

CAA AAG GCG GTG AAT GAG CTG TTT ACT TCT TCC AAT CAA ATC GGG TTA AAA ACA GAT GTG Gin Lys Ala VaI Asn Giu Leu Phe Thr Ser Ser Asn Gin lie Gily Leu Lys Thr Asp VaI

2105 2135

ACG GAT TAT CAT ATT GAT CAA GTA TCC AAT TTA GTT GAG TGT TTA TCT GAT GAA TTT TGT Thr Asp Tyr His He Asp Gin VaI Ser Asn Leu VaI Giu Cys Leu Ser Asp GIu Phe Cys

2165 2195

CTG GAT GAA AAA AAA GAA TTG TCC GAG AAA GTC AAA CAT GCG AAG CGA CTT AGT GAT GAG Leu Asp Glu Lys Lys Glu Leu Ser Glu Lys Vali Lys His Ala Lys Arg Leu Ser Asp Glu

2225 2255

CGG AAT TTA CTT CAA GAT CCA AAC TTT AGA GGG ATC AAT AGA CAA CTA GAC CGT GGC TGG Arg Asn Leu Lei Gin Asp Pro Asn Phe Arg GIy He Asn Arg Gin Leu Asp Arg GIy Trp

2285 2315

AGA GGA AGT ACG GAT ATT ACC ATC CAA GGA GGC GAT GAC GTA TTC AAA GAG AAT TAC GTT Arg Gly Ser Thr Asp He Thr Gin Gly GIy Asp Asp VaI Phe Lys GIu Asn Tyr VaI

2345 2375

ACG CTA TTG GGT ACC TTT GAT GAG TGC ACT CCA ACG TAT TTA ACT CAA AAA ATA GAT GAG Thr Lei Lei GIy Thr Phe Asp Giu Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys He Asp GIu

2405 2435

TCG AAA TTA AAA GCC TAT ACC CGT TAC CAA TTA AGA GGG TAT ATC GAG GAT AGT CAA GAC Ser Lys Leu Lys AIa Tyr Thr Arg Tyr GIn Leu Arg GIy Tyr He GIu Asp Ser Gin Asp

2465 2495

TTA GAA ATC TAT TTA ATT CGC TAC AAT GCC AAA CAC GAA ACA GTA AAT GTG CCA GGT ACG Leu GIu He Tyr Leu He Arg Tyr Asn AIa Lys His GIu Thr VaI Asn VaI Pro GIy Thr

2525 2555

GGT TCC TTA TGG CCG CTT TCA GCC CCA AGT CCA ATC GGA AAA TGT GCC CAT CAT TCC CAT GIy Ser Leu Trp Pro Leu Ser AIa Pro Ser Pro He Gly Lys Cys Ala His His Ser His

2585 2615

CAT TTC TCC TTG GAC ATT GAT GTT GGA TGT ACA GAC TTA AAT GAG GAC TTA GGT GTA TGG His Phe Ser Leu Asp was Asp VaI GIy Cys Thr Asp Leu Asn GIu Asp Leu GIy VaI Trp

2645 2675

GTG ATA TTC AAG ATT AAG ACG CAA GAT GGC CAT GCA AGA CTA GGA AAT CTA GAA TTT CTC VaI He Phe Lys He Lys Thr Gin Asp GIy His AIa Arg Leu GIy Asn Leu GIu Phe Leu

2705 2735

GAA GAG AAA CCA TTA GTA GGA GAA GCA CTA GCT CGT GTG AAA AGA GCG GAG AAA AAA TGG G ¹ U GIu Lys Pro Leu VaI GIy GIu AIa Leu AIn Arg VaI Lys Arg Ala GIu Lys Lys Trp

2765 2795

AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA ACA AAT ATT GTT ACT AAA GAG GCA AAA GAA Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn lie Vai Tyr Lys Glu Ala Lys Glu

-25- 297 763 Table 3 (continued)

2825 2855

TCT GTA GAT GCT TTA TTT GTA AAC TCT CAA TAT GAT AGA TTA CAA GCG GAT ACC AAC ATC Ser VaI Asp AIa Leu Phe VaI Asn Ser GIn Tyr Asp Arg Leu Gin Ala Asp Thr Asn He

2885 2915

GCG ATG ATT CAT GCG GCA GAT AAA CGC GTT CAT AGC ATT CGA GAA GCT TAT CTG CCT GAG Ala Met His His Ala Ala Asp Lys Arg VaI His Ser || _e Arg GIu AIa Tyr Leu Pro GIu

2945 2975

CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT ΊΤΤ GAA GAA η «GAA GGG CGT ATT TTC Leu Ser VaI He Pro GIy VaI Asn Ala Ala He Phe GIu Giu Leu GIu GIy Arg De Phe

3005 3035

ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC Thr AIa Phe Ser Leu Tyr Asp AIa Arg Asn VaI Me Lys Asn GIy Asp Phe Asn Asn GIy

3065 3095

TTA TCC TGC TGG AAC GTG AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAC CGT TCG Leu Ser Cys Trp Asn VaI Lys GIy His VaI Asp VaI GIu GIu Gin Asn Asn His Arg Ser

3125 3155

GTC GTT GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT CCG GGT VaI Leu VaI VaI Pro GIu Trp GIu Ala GIu VaI Ser Gin GIu VaI Arg VaI Cys Pro GIy

3185 3215

CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAG GGA TAT GGA GAA GGT TGC GTA ACC Arg GIy Tyr He Leu Arg VaI Thr AIa Tyr Lys GIu GIy Tyr GIy GIu GIy Cys VaI Thr

3245 3275

ATT CAT GAG ATC GAG AAC AAT ACA GAC GAA CTG AAG TTT AGC AAC TGT GTA GAA GAG GAA He His GIu lie GIu Asn Asn Thr Asp Giu Leu Lys Phe Ser Asn Cys VaI GIu GIu GIu

3305 3335

GTA ACT CCA AAC AAC ACG GTA ACG TGT AAT GAT ACT ACT GCG ACT CAA GAA GAA ACT GAG VaI Tyr Pro Asn Asn Thr VaI Thr Cys Asn Asp Tyr Thr AIa Thr Gin GIu GIu Tyr GIu

3365 3395

GGT ACG TAC ACT TCT CGT AAT CGA GGA TAT GAC GGA GCC TAT GAA AGC AAT TCT TCT GTA GIy Thr Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly AIa Tyr Glu Ser Asn Ser Ser VaI

3425 3455

CCA GCT GAT ACT GCA TCA GCC TAT GAA GAA AAA GCA TAT ACA GAT GGA CGA AGA GAC AAT Pro Ala Asp Tyr AIa Ser AIa Tyr GIu GIu Lys AIa Tyr Thr Asp GIy Arg Arg Asp Asn

3485 3515

CCT TGT GAA TCT AAC AGA GGA TAT GGG GAT TAC ACA CCA CTA CCA GCT GGC TAT GTG ACA Pro Cys GIu Ser Asn Arg GIy Tyr GIy Asp Tyr Thr Pro Leu Pro Ala GIy Tyr VaI Thr

3545 3575

AAA GAA TTA GAG TAC TTC CCA GAA ACC GATE AAG GTA TGG ATT GAG ATC GGA GAA ACG GAA Lys GIu Leu GIu Tyr Phe Pro GIu Thr Asp Lys VaI Trp Hc GIu He GIy GIu Thr GIu

3605

GGA ACA TTC ATC GTG GAC AGC GTG GAA TTA CTT CTT ATG GAG GAA TAA GIy Thr Phe He VaI Asp Ser VaI Giu Leu Leu Leu Met GIu GIu End

Claims (23)

1. Insecticidal agent, characterized in that it contains, in addition to the usual excipients and carriers, an insecticidally effective amount of a proteinaceous substance, at least partially by a DNA fragment from Bacillus thuringiensisvar. kurstaki, which extends to the area between the interfaces Hpa l (0) and Pstl (4355), which can be obtained by the following procedures: a) Isolation and lysis of Bacillus thuringiensisvar. kurstaki HDI cells, separating the plasmids' from the material thus obtained by means of known measures and purifying and dialyzing the plasmid material thus obtained; b) preparation of a DNA library of Bacillus thuringiensis var. kurstaki MDI plasmid DNA; c) cloning the fragmented plasmid DNA obtained according to step b) in a suitable vector; ' d) Screening for the presence of protein MGE 1 by means of antigen / antibody test. 2. Insecticidal composition according to claim 1, characterized in that it contains in addition to the usual excipients and carriers an insecticidally effective amount of a proteinaceous substance which is at least partially encoded by a DNA fragment having the following nucleotide sequence: (Insert sequence from table 2) An insecticidal composition according to claim 1, characterized in that said insecticidally active proteinaceous substance is the protein MGEI encoded by a Bacillus thuringiensis var. Kurstaki DNA fragment starting 156 bp downstream of the Hpal restriction site and ends at nucleotide 3623. 4. Insecticidal composition according to claim 3, characterized in that said insecticidally active protein MGEI has the following amino acid sequence: (Insert amino acid sequence from Table 3) An insecticidal composition according to any one of claims 1 or 3, characterized in that said insecticidally active proteinaceous substance is a fusion protein consisting of the protein MGEI and another protein portion other than the protein MGEI. An insecticidal composition according to claim 5, characterized in that said protein portion other than the protein MGEI has a herbicidal activity. An insecticidal composition according to claim 5, characterized in that said protein portion other than the protein MGEI has an insecticidal activity. 8. Insecticidal composition according to one of claims 1 or 3, characterized in that said insecticidally active proteinaceous substance is completely surrounded by a protective biological material, so that said proteinaceous substance is protected when applied to the plant against degradation by harmful environmental influences. 9. An insecticidal composition according to claim 8, characterized in that said biological material is a microorganism which is transformed with a DNA fragment according to claim 1 and contains said proteinaceous substance due to the expression of the introduced DNA fragment. An insecticidal composition according to claim 9, characterized in that said microorganism is a yeast which has been previously transformed with a hybrid vector containing a DNA fragment according to any one of claims 1 or 3 under the control of the yeast acid phosphatase. Contains promoter. 11. An insecticidal composition according to claim 10, characterized in that said yeast is Saccharomyces cerevisiae GRF18, a sample of which has been deposited with the German Collection of Microorganisms, FRG under the accession number DSM 3665. 12. A method for controlling insects, characterized in that treating insects or their habitat with an insecticidally effective amount of a proteinaceous substance which is at least partially encoded by a DNA fragment from Bacillus thuringiensis var. Kurstaki, which is in the range between extends the interfaces Hpa l (0) and Pst 1 (4355) and which can be obtained by the following procedures: a) isolation and lysis of Bacillus thuringiensis var. kurstaki HDI cells, separating the plasmids from the material thus obtained by known means and purifying and dialyzing the plasmid material thus obtained; b) preparation of a DNA library of Bacillus thuiingiensis var. kurstaki HDI plasmid DNA; c) cloning the fragmented plasmid DNA obtained according to step b) in a suitable vector; d) Screening for the presence of protein MGE 1 by means of antigen / antibody test. 13. The method according to claim 12, characterized in that treating insects or their habitat with an insecticidally effective amount of a proteinaceous substance which is at least partially encoded by a DNA fragment having the following nucleotide sequence: (Insert sequence from table 2) 14. The method according to claim 12, characterized in that said insecticidally active proteinaceous substance is the ptotein MGEI encoded by a Bacillus thuringiensis var. Kurstaki DNA fragment starting 156 bp downstream of the Hpal restriction site and at nucleotide 3623 ends. 15. The method according to claim 14, characterized in that said insecticidally active protein MGEI has the following amino acid sequence: (Insert amino acid sequence from Table 3) A method according to any one of claims 12 or 14, characterized in that said insecticidally active proteinaceous substance is a fusion protein consisting of the protein MGEI and another protein portion other than the protein MGEI. 17. The method according to claim 16, characterized in that said protein portion, which is different from the protein MGEI, has a herbicidal activity. A method according to claim 16, characterized in that said protein portion other than the protein MGEI has an insecticidal activity. id. A method according to any one of claims 12 or 14, characterized in that said insecticidally active proteinaceous substance is completely surrounded by a protective biological material, so that said proteinaceous substance is protected against degradation by harmful environmental influences when applied to the plant. 20. The method according to claim 19, characterized in that said biological material is a microorganism which is transformed with a DNA fragment according to claim 17 and contains due to the expression of the introduced DNA fragment said proteinaceous substance. A method according to claim 20, characterized in that said microorganism is a yeast which has been previously transformed with a hybrid vector containing a DNA fragment according to any one of claims 12 or 14 under the control of the yeast acid phosphatase promoter , 22. The method according to claim 21, characterized in that said yeast is Saccharomyces cerevisiae GRF18, of which a sample has been deposited with the German Collection of Microorganisms, FRG under the accession number DSM 3665. 23. The method according to any one of claims 12 to 22, characterized in that they are insects of the order Lepidoptera.