DD259420A5 - Process for the preparation of a DNA fragment from Bacillus thuringiensis var.kurstaki - 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

For this 16 pages tables

Field of application of the invention

The invention relates to a method for producing a DNA fragment from Bacillus thuringiensis var. Kurstaki which extends to the region between the interfaces Hpal (0) and PstI (4355) and encodes an insecticidally active proteinaceous substance including selected parts thereof, among which A prerequisite for maintaining the insecticidal activity of said selected DNA parts.

-2- 259 420 characteristic of the known prior art

A comparable prior art can not be specified at present.

Object of the invention

The aim of the invention is the enrichment of the prior art by novel substances with insecticidal activity, which are obtained by genetic engineering.

Explanation of the essence of the invention

The invention has for its object to provide a method for producing a DNA fragment from Bacillus thuringiensis var. Kurstaki 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 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. thuringiensis is substantially or completely derived 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. to use the corresponding DNA sequence coding for the desired insecticidally active protein independently of B. 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 (polypeptide) obtained in the context of the present invention is referred to below as MGE1.

The present invention relates to a method for producing an insecticidally effective proteinaceous substance including the discovery and identification of the complete DNA sequence coding for the insecticidal protein MGE1 said proteinaceous substance and (of the known B. thuringiensis genes HE Schnepfetal. ⁴¹ MJAdang et al., ³¹ and Shibano et al., ³⁰¹ as well as WO 86/01536).

The present invention further relates to a DNA fragment characterized by the nucleotide sequence shown in Table 2 which encodes the protein MGE1 and the protein MGE1 itself; Moreover, the present invention relates to a DNA fragment of the Bacillus thuringiensis var. kurstaki from the Hpal region (0) to Pstl (4355), which encodes an insecticidally active proteinaceous substance including selected portions ('trunated protions') thereof, among which A prerequisite for maintaining the insecticidal activity of said selected parts.

The term "proteinaceous substance" is understood to mean both the insecticidally active protein MGE1 itself and in vitro derivatives and modifications thereof, such as the protein MGE1 in conjunction with other protein fragments, primarily those cloned from others 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, bactericidal, viricidal, fungicidal and herbicidal activity, preferably against phytopathogenic organisms. Of the proteinaceous substances, the insecticidally active protein MGE1 alone is preferred.

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, plasmids such as pBR322 and pUC8 or phages such as MI3.

Furthermore, the present invention relates to living or dead microorganisms containing a DNA fragment characterized by the nucleotide sequence shown in Table 2, preferably a microorganism of the species Saccharomyces 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 previously been 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, wherein

the first material is 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 identified in Table 2 reproduced DNA fragment has been transformed. The DNA material may also be a DNA fragment derived from the region Hpal (0) to PstI (4355) of Bacillus thuringiensis var. Kurstaki and which codes for 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 cereyisiae GRF-IS.

Another part of the present invention relates to an agent and a method for controlling 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 xylostella 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 protein MGE1, directly to 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 protein MGE1.

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 protein MGE1, said application form for the application of the active substance in a protected form is suitable.

It is dahe'r 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 PHO5 promoter.

At least some of Bacillus thuringiensis genes encoding insecticidal proteins are known to be linked to a promoter that can be recognized by Escherichia coli (E. coli) RNA polymerase, with said promoter located in front of the respective gene (HC Wong et al. ²¹ ).

The process for producing an insecticidally active proteinaceous substance in the present invention involves the transcription and translation of an identified DNA sequence gene according to the present invention into the corresponding protein by E. coli or yeast.

The procedure for obtaining starting material described here is carried out using Bacillus thuringiensis var. Kurstaki HDI strain ETHZ 4449. This strain is obtained from the microbiology department of the Swiss Federal Institute of Technology in Zurich, Switzerland, and is freely accessible to anyone without any restriction.

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 anyone. According to the present invention, the DNA coding for the protein MGE1 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 which specifically react 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 by methods known per se, as listed, for example, under the points h) and i):

h) mapping of the DNA of positive clones by digestion with restriction endohucleases and hybridization of the resulting fragments with radioactively labeled RNA; and

i) sequencing of DNA fragments encoding the respective proteins.

Meaning of the abbreviations used below:

Bp: base pairs

BSA: bovine serum albumin (bovineserum albumin)

DEAE: diethylaminoethyl

DFP: diisopropylfluorophosphate

DTT: 1,4-dithiothreitol (1,4-dimercapto-2,3-butanedibl)

EDTA: ethylenediaminetetraacetic acid

IPTG: isopropyl-β-thiogalactopyranoside (Serva)

Kb: kilobases

PSB ¹ : 0.01 MP phosphate buffer, pH 7.4 and O, 8% NaCl

PSB ² : 1 mM sodium phosphate, pH 7.8 and 0.14% NaCl

PEG 6000: medium molecular weight polyethylene glycol 6000

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 MNaCl

TE: Solution containing 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA

TES: 0.5 M Tris, 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

2xYT: 16gBactoTrypton,

10 g yeast extract (Bacto), 5 g NaCl. Media, buffers and solutions used below:

20 χ SSC

2 χ SSC 6 χ SSC Denhardt's Solution (50x)

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

10% von20xSSC

33% of 20 χ SSC

Ficoll 70 [molecular weight approx. 700Ό00;

Pharmacia], 5 g

polyvinylpyrrolidone

(Calbiochem-Behring Corp.) 5 g

BSA (Sigma) 5 g

H ₂ O to 500 ml

Filtration through a disposable Nalgene® filter (Nalgene®, Nalge Co. Inc., Rochester, NY, USA). Separating in 25 ml Aliquotsund storing at -20 ⁰ C.

Solutions M9 Medium:

Per liter:

a) Na ₂ HPO ₄ KH ₂ PO ₄ NaCl NH ₄ Cl

6g 3g

0.5 g

ig

LB (LuHa-

Bertani)

medium

L broth

Adjust the pH to 7.4, autoclave, cool and then add: b) 1MMgSO ₄ 2 ml

20% glucose 10 ml

1MCaCl ₂ 0.1ml

Vitamin B1 (40 mg / 10 ml) 5 ml

The above-mentioned solutions a) and b) should be sterilized separately by filtration (glucose, Vit. B1) or by autoclaving.

Per liter:

Bacto tryptone 10 g

Bacto yeast extract 5 g

NaCl 10g

Adjust the pH to 7.5 with sodium hydroxide, see LB medium

For the induction of sporulation in B. thuringiensis var. Kurstaki, a GYS medium according to the data of Yousten and Rogoff (AAYousten and MHRogoff ⁵¹ ) (1969) is used (g / Γ ¹ ):

glucose 1 Yeast extract (Difco) 2 (NH ₄ ) ₂ SO ₄ 2 K ₂ HPO ₄ 0.5 MgSO ₄ -7H 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, 3 b serotype (A. Krieg ³¹¹ ) - on the other hand by its specific Southern blot pattern, which is obtained when carrying out the Southern biot 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 nitroccilulose 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 x Escherichia coli B. This strain is well suited for large scale DNA purification. It is used for transformation experiments and CaCl ₂ -competent cells are commercially available e.g. At Gibco AG, Basel, Switzerland,

Catalog no. 5308260 SA.

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

E. coli strains HB 101 and JM 103 are described in Maniatis et al. (Maniatis T. et al., ⁶¹ ): strain genotype

HB101 F ", hsdS ₂ o (rl, mi), recA ₁₃ , ara-14, proA ₂ , lacYi, galK ₂ , rpsL ₂₀ (Sm ^r ), xyl-5, ml-1, SUpE ₄₄ , λ"

JM103 VM15

In the following example, the term "yeast" is synonymous with Saccharomyces cerevisiae.

Examples I. Plasmid DNA workup:

The workup of the plasmid DNA is carried out as described below as described in White and Nester (FF White and EWNester ⁷¹ ).

1.1. B. thuringiensis plasmids

E. coli HB 101 cells are cultured in 1 liter of LB medium with shaking for 12-14 hours at 37 ⁰ C and worked up according to the standards in white and nests (FF White and EWNester ⁷¹ ). After harvest, the cells are resuspended in an alkaline lysing buffer and incubated at a temperature of 37 ° C for 20-30 minutes. A clear lysate is obtained which is neutralized by the addition of 2M Tris HCl (pH 8). The chromosomal DNA is then precipitated by the addition of SDS and NaCl. The lysate is packed on ice and the chromosomal DNA is removed by centrifugation. The plasmid DNA now in the supernatant is precipitated with 10% PEG 6000. After storage of the plasmid DNA overnight at 4 ° C resuspension in 7-8ml TE. The plasmid DNA obtained from 1 liter of culture solution is then further purified by means of 2 CsCl gradients. Solid CsCl is added to the solution (8.3 g of CsCl to 8.7 ml of supernatant). 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 40,000 rpm , Under long-wave UV light (366 nm), two fluorescent bands are visualized. The lower band contains overspiral plasmid DNA, which is collected by laterally puncturing the centrifuge tube with a 2 ml syringe (18 G needle). Ethidium bromide is removed by washing 5 times with equal volumes of isopropanol (saturated with CsCl), and the product is then transferred to 30 ml of 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 12'000rpm and a temperature of O ⁰ C and dissolved ϊη200μΙ TE. (E. coli JM 103 can also be extracted and treated in an analogous manner).

1.2. E. coli plasmids

The cells of a 100mM culture (LB medium) are resuspended by centrifugation (Sorvall, GSA rotor, 10mM at 6,000rpm, 4 ° C) for 10μmITE (10mM Tris-HCl, 1mM EDTA, pH 8.0) and repeated under the same conditions centrifuged. The cell pellet is then resuspended in 3 ml Tsuc [50 mM Tris • HCl, pH 7.5.55% (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, 11,000 U / mg) after 5 min 1.2 ml EDTA (50OmM, pH 8.0) and after another 5 min 4.8 ml detergent [0.1% Triton X-100 (Merck), 50 mM EDTA, 50 mM Tris • HCl, pH 8.0]. After 5 minutes, the lysate is centrifuged in a precooled SS-34 rotor for 40 minutes at 4 ° C. The supernatant is carefully removed and purified on a CsCl gradient after addition of solid CsCl, according to the information given for the B. thuringiensis plasmid DNA

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

M. δ-endotoxin antigen and goat antibodies

11.1. Preparation of δ-endotoxin crystal body antigen:

Bacillus thuringiensis (var kurstaki HD 1, strain ETHZ 4449) is used in a Fernbach flask on a Yousten and Rogoff medium, (AA Yousten and MH Rogoff ⁵¹ ) as previously described, but with an increased level of glucose (0.3% instead of 0 , 1%), cultured.

The incubation period is 4-5 days at a temperature of 30 ⁰ C. The colonies are immediately after the sporulation

(B.Trürni ⁸¹ ) harvested. For the separation of spores and parasporal bodies, the method of Delafield et al. (FP Delafield et al., ⁹¹ ).

a) Separation of spores and crystal bodies:

Suspend authorized 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 the wash 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 (pH 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 of 3M phosphate. Buffer (pH 7.0) and 7.5g NaCl contains.

- Shake to dissolve the solid ingredients.

- Adjust the volume to 600 ml by adding a well-mixed solution of the same composition, but without 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 10th 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 12'00Og. The sediment thus obtained is dissolved in 0.05M carbonate buffer and 10 mM dithiothreitol (DTT, Sigma; the mixtures of carbonate buffer and dithiothreitol are hereinafter referred to as carbonate / DTT) at a concentration of 5mg sediment / ml carbonate / DTT mixture resuspended.

After 30 minutes incubation at 37 ° C, the insoluble particles are separated by centrifugation at 25'00Og for 10 minutes.

The supernatant is dialyzed against carbonate buffer and then examined for its protein content and activity in the bioassay.

For storage purposes, the protoxin solution is divided into individual portions which are deep-frozen. DTT-free protein has a gel-like consistency on thawing. Complete dissolution of the protein is achieved by addition of 1 mM DTT.

c) Inactivation of crystal-bound proteases: '

Serine proteases and metal proteases of the crystal body suspension [Chestukhina et al. ¹⁰¹ ] are inactivated by the addition of diisopropylfluorophosphate (DFP, Serva) and EDTA in the following manner:

The crystal bodies are suspended 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. The suspension is then sonicated until a monodisperse solution is obtained (this is checked by a light microscope).

In a hood, with the usual safety precautions, 1 mM DFP is added to the suspension. The tube containing 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 B. thuringiensis serotype H-3 crystal bodies by dissolving the crystal bodies in carbonate / DTT, dialyzing the resulting solution against carbonate buffer and 1 mM DTT and purifying the antigen by sterile filtration using a 0.45μιη Millipore Filters.

The antigen solution is mixed with "Freund's complete adjuvant" (Bacto) in a ratio of 1: 1 and stored at 4 ^0C.

At the experimental station of CIBA-GEIGY AG in St. Aubin (Friborg, Switzerland), 2 goats are immunized with H-3 protoxin. Each of the two goats receives 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 performed on days 0.28 and 76.

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

After coagulation of the blood, the sera are incubated for 30 minutes at 56 ° C to inactivate the complement system

Sera are stored at 2O ⁰ C. .

11.3. Purification and [ ¹²⁶ l] Labeling of Goat H3 Antibodies a) Purification of the Immunoglobulin:

The IgG (immunoglobulin G) fraction of the goat H ₃ antiserum is purified by ammonium sulfate precipitation, followed by chromatography on DEAE cellulose and analysis for Ouchterlony immunodiffusion plates (O. Ouchterlony ¹¹¹ ) according to the method described by Huber-Lukac (H. Lukac ¹²¹ ) method described.

30 ml of 3.2 M ammonium sulfate are added dropwise 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 (10'00Og, 20min), the sediment is resumed in 7.5 ml of PBS ¹ , dialyzed three times at a temperature of 4 ⁰ C against 1 000 ml of PBS ¹ , then against 1000 ml of 0.01 M phosphate Buffer pH 7.8 dialyzed and finally centrifuged (3000 g, 20 min).

The supernatant is applied to a 30 ml DEAE column and eluted at RT with 0.01 M phosphate buffer pH 7.8 (flow rate:

20-80 ml / h). The fractions of the first peak are pooled and lyophilized. The average IgG yield is 180mg / 15ml serum. The purity of the IgG fraction using the immunodiffusion (O.Ouchterlony ¹¹¹⁾ against anti-goat IgG antibody and anti-goat serum of the rabbit antibody (Miles Laboratories) checked.

Further purification of the antibodies is achieved by absorption on a Sepharose® (Pharmacia) column containing H ₃ -protoxin bound. The binding of the protoxin to CNBr-Sepharose® (Pharmacia) is carried out according to the instructions given by the manufacturer, which can be summarized as follows:

1 g CNBr-activated Sepharose® 6 ME is weighed for a gel end volume of 3 ml. The gel is washed and re-swollen on a glass frit using 200 ml of 1 mM HCl. The H ₃ -antigen protein, which is obtained as described under point 11.1. c is dissolved in 0.1 M NaHCO ₃ and 0.5 M NaCl. 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 removed 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 carried out again with 0.1 M NaHCO ₃ and 0.5 M NaCl.

The protein Sepharose® conjugate can then be packed in a Pharmacia K9 column (Pharmacia). The IgG can then be purified very effectively on this column.

Specifically bound antibodies are eluted with 3 M KSCN and dialyzed against PBS ² (10 mM sodium phosphate pH 7.8, 0, 14 M NaCl). The antibodies are then radioactively labeled with ¹²⁵ I using the chloramine-T method (Arnersham Büchler Review ¹³¹ ).

b) iodine label:

1 M Ci sodium iodide- ¹²⁵ L is placed in a tube with 100 μM 0.5M phosphate buffer (pH 7.2). With slow stirring, 5 μg of the protein solution (0.5 mg / ml in TBS) and 50 μg 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 μg of Na ₂ S ₂ O _{5 takes place} . Free iodine is separated from the labeled protein via a 0.9 x 12 cm column packed with Sephadex® G-25. To prevent absorption of the labeled protein to the column, 0.5 ml of BSA (100 mg / ml) is first passed through the packed column. Thereafter, the labeled material is quantitatively transferred to the column and eluted with phosphate buffer (pH 7.2). The fractions (1 ml) are collected until the entire protein is eluted.

III. Cloning of the δ-endotoxin genes

A partial Sau3A DNA library of B. thuringiensis var. Kurstaki HDI, strain ETHZ 4449 Plasmid DNA essentially as described in Maniatisetal. (Maniatis et al., ¹⁴¹ ) and subcloned into the BamHI restriction site of pBR322, which acts as the vector DNA, as described below:

III. 1. Partial digestion of high molecular weight B. turingiensis 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 application of Maniatisetal. (T. Maniatis et al., ¹⁴¹ ).

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 35 krpm for 3 hours in a SW40Ti Beckman rotor. The collected fractions are precipitated by addition of ethanol and analyzed on an agarose gel.

10 ug of the plasmid pBR322 are digested with 10 units of the endonuclease BamHI in 50μΙ 1OmM MgCl ₂ at 37 ⁰ C 1-2h.

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

10 μg of DNA are first dissolved in δΟμΙΤπβ · HCl, pH 8, followed by the addition of alkaline phosphatase from bovine intestine (Boehringer) in a concentration of 3 units / g of DNA. After a 30 minute incubation at 37 ° C, the DNA is treated twice with phenol and then extracted with chloroform. After ethanol precipitation, the DNA is resuspended in 20 μM 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 μg of digested DNA in 10 μl of H ₂ O.

The coupling reaction is achieved by addition of 50 mM HCl, pH 7.4.1 mM ATP, 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 HB101 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 Clal digestion of the plasmid DNA. This is followed by cloning in pBR322 between the CIaI and BamHI sites.

111.2. Transformation using the calcium chloride method:

Competent cells are provided by treatment of cells grown to a population density of 5χ10 ⁷ cells / ml with calcium chloride (Maniatis et al., ¹⁵ ). The transformation is achieved by adding DNA to these cells. The cells are incubated for 3 minutes at 42 ⁰ C, 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 ¹⁵¹ ).

111.3. Producing crude cell lysates ''

The colonies containing the δ-endotoxin gene express a protein with similar biological activity to the purified and dissolved toxin crystals (HE Schnepfetal 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 of LB medium in the presence of ampicillin. Ten cultures are each pooled, harvested and washed in 10 mM NaCl; Finally, the cells are suspended 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 μM 2 M Tris.HCl, pH 7.0. After centrifugation in a SS34Sorvall rotor (20 min, 10'000 rpm), the lysates are extensively dialyzed against TBS (10 mM Tris-HCl, pH 7.5, 0.14M NaCl).

111.4. Radioimmunological screening of cell extracts

The extracts are purified by the plastic cup method as described by Clarke et al. (Clarke, L., et al., ¹⁷¹ ) was radioimmunologically examined for the presence of δ-endotoxin antigen. Individual plastic cups are coated overnight with 150 ml of purified H3 goose antibodies (10 μg / ml) in 10 mM Tris HCl, pH 9.3, and stored overnight at 4 ° 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 ⁰ C. After washing the cups with 150μΙ are ^[125 I] labeled rabbit anti-goat antibodies HS (60ng, 10 ⁵ cpm) in TBS containing 25% horse serum, enable and incubated overnight at room temperature. After washing again with TBS / Tween, 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 pK19

a) Restriction mapping:

The restriction map of the DNA clone pK19, which is obtained according to the above-described immunological screening of the Sau3A library of B. thuringiensis HD1, ETHZ 4449, can be derived from the products of single, double and triple 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 (I pg / 50pl) 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 ° C on 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 conditions (such as in a buffer needed for the second enzyme).

b) Southern transfer:

The sequence coding for the δ-endotoxin is determined by means of the hybridization reaction of radiolabelled RNA from sporulating 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 maintained during 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

DNA transfer from the agarose gel to nitrocellulose paper is carried out according to the instructions of Maniatis et al. ¹⁹¹ performed.

c) Hybridization of Southern Filters:

Prehybridization and actual hybridization are performed according to the instructions of Maniatis et al. (Maniatis, T., et al., ²⁰¹ ) 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: 4 x SSC

50% formamide

0.2% SDS

2OmMEDTA

25mM potassium phosphate (pH7,2)

5 x Denhard's solution

100 μg / ml denatured calf thymus DNA

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

The prehybridization mixture is removed and replaced by the following hybridization mixture (50μΙ / οιτι ² nitrocellulose filter).

Hybridization Mixture: Same composition as the Prehybridization Mixture, but now with ³² P-labeled

denatured RNA sample (10 ^e -10 ⁷ cpm / filter), prepared according to the procedures below under point lll.5.d. information provided.

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

d) Isolation and radioactive labeling of B. thuringiensis var. kurstaki RNA.

B. thuringiensis var. Kurstaki cells are cultured on a rogoff medium containing 0.1% glucose (AA Yousten and MHROgoff ⁵¹ ). 500ml cultures are shaken in 2 liter conical Meier bottles bei300rpm and 30 ⁰ C. During cell growth, the pH decreases from pH 7 to about 4.8 and then increases again to pH 7. At this point the cells begin to clump together. The time when the pH returns to baseline is considered the starting point of sporulation. The cells are cultured for another 5 to 6 hours. Add rifampicin (50 μg / ml) and shake the cells for a further 10 minutes. The ice-cooled 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 -8O ⁰ C and then broken in a "Frensh Press". After a 15minütigenZentrifugation the cell extract at 15000rpm

(Sorvall SS34 rotor), 0.5 g / ml CsCl is added to the supernatant, which is then layered in a Beckman 60Ti centrifuge tube on a pad consisting of 5.7M CsCl, 0.1M EDTA. After renewed centrifugation for 20 h at 38,000 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 standard procedures (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 10 μΙ, the 5OmM Tris-HCl (pH9.5), 1OmM MgCl ₂ , 5 mM dithiothreitol, 5% glycerol and 1 μΜ [γ ³² Ρ] -ΑΤΡ, labeled with a specific activity of 6000 Ci / mmol , contain. Each reaction unit contains about 1 μg of RNA and 2 μΙ Τ4 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 / Vg. The RNA is separated from the Iy ³² P] -ATP by three ethanol precipitations in the presence of 5 μg of a tRNA carrier.

111.6. Identification of clones encoding the δ-endotoxin in the BamHI / CIal plasmid DNA library cloned in pBR322: The pK 25 series

a) In situ hybridization of bacterial colonies:

Colony hybridization (Grunstein and D. Hogness ²²¹ ) 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 is positive in autoradiography can then be recovered from the plate containing the basal culture The BamHI / CIal plasmid DNA library, cloned into pBR322, utilizes a DNA fragment located within the δ-endotoxin, as described in Maniatis et al., (T. Maniatis et al., ²³¹ ).

The filters are hybridized with a ³² P-labeled probe prepared according to the method described in Section III.6.b below.

To produce an autoradiographic image, the filter is wrapped in "Saran Wrap" and exposed to X-ray film.

The positive clones are isolated from the plate containing the base culture and analyzed. They have, in addition to a DNA sequence coding for the toxin, the DNA flanking region, as determined by immunological methods (see Item 111.4., Top) and by restriction maps (see Section III.5., Above) and in an in vivo biotest (see Point III.7. Below) could be detected. These clones are referred to below as pK25-i, where i stands for a number between 1-7.

b) DNA-nick translation:

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

Mixture: 3μΙ DNA (^ g)

1.5 μΙ Nick translation buffer (10x concentrated: *

0.5M Tris, pH 8, 0.05M MgCl ₂ ) 1.5μΙ, 2.5mM d guanosine 5'-triphosphate (GTP) 1.5μΙ, 2.5mM d cytidine 5'-triphosphate (CTP ) 1.5μΙ, 2.5mMdthymidine-5'-triphosphate (TTP) 2.5μΙ H ₂ O

0.75 μΙ BSAd mg / ml) 1.5 μΙ, 10 μM β-mercaptoethanol

mix thoroughly and add to 100μΦ dried [ ³² P-Ci] -ATP [10mCi / mmol] mix well,

Add 0.75μΙ of 1 χ 10 ~ "solution of DNAsel (1mg / ml) into Nick Translations buffer, incubate for 1 minute at RT, bring to ice

1 μM E. coli polymerase I (Biolabs, final volume: 15 μΙ).

Incubate for 3 hours at 15 ° C,

Heat at 65 ⁰ C for 10 minutes, add 35μΙ 5OmM EDTA and 10μΙ tRNA stock solution (100μg).

Unincorporated nucleotides were separated by chromatography on a small Sephadex G50 column.

c) Subcloning of the complete δ-endotoxin gene in the pUC8 vector: of the pK36 clone

The pUC8 vector (New England Biolabs) is completely digested with the restriction enzymes HincII and PstI as well as by treatment with alkaline phosphatase (see point III.1 above). The Hpal / PstI fragment from pK25-7 (see point III.6.a] above) which codes for the δ-endotoxin gene (Table 2) is linked to the vector DNA and transformed into E. coli HB101 cells. One of the correctly transformed clones is named pk36.

111.7. Biotest for the determination of B. thuringiensis toxin:

To the purified lysate following the procedure described under point III.3. information can be obtained, ammonium sulfate is added in a 30% saturation concentration. The precipitate is dissolved in 2 ml of 50 mM sodium carbonate, pH 9.5, and dialyzed against the same buffer. As a control, E. coli extracts are produced, which contain the vector DNA but without incorporated B. thuringiensis DNA. The E. coli cell extracts are first sonicated, then 4 concentrations are prepared according to the toxin content in the extracts and mixed with 0.1% (v / v) of a wetting agent.

Leaf discs of cotton plants, which in a climate chamber under controlled conditions (25 ⁰ C, 60% relative humidity) have been used, are immersed in the E. COELI cell extract suspensions. Heliothis virescens larval larvae (30 larvae per concentration) previously standardized in a fitness test are then placed on dried leaf discs and incubated individually for 25 days at 25 ° C.

The mortality is measured in%, whereby only the criteria "dead" or "alive" apply. The extracts that cause mortality therefore have bioinsecticidal activity derived from the cloned B. 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. ²⁴¹ using the M13 system (J. Messing ²⁶¹ ).

IV. 1. Cloning of the δ-endotoxin gene into replicative DNA _: M13 form

From the restriction mapping of the cloned δ-endotoxin gene and the 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 HincII and the unique HindIII site in a reaction analogous to the linking process described above.

IV.2. Production of a succession of overlapping clones

To shorten the Hpal-HindIII DNA fragment coding for the 5 'end of the gene, the Bal31 method (M. Poncz et al., ²⁶¹ ) is used. Said shortening is carried out as follows:

The fragment is cloned in M13mp8 between the unique Hincll and HindIII sites. 10 mg of the replicative form DNA is linearized with restriction endonuclease HindIII and then treated with the endonuclease Bal31 in 100 μΙ 600 mM NaCl, 12 mM CaCl _2, 12 mM MgCl _2, 2OmM Tris HCl, pH 8 and ImMEDTA. This mixture is pre-incubated for 5 minutes at 30 ⁰ C for. Then 5 units of Bal31 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 40μΙΤΕ 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 portion of the agarose gel containing the truncated fragments is excised from the gel, liquefied at 65 ° C, adjusted to 50 mM NaCl, and incubated at 65 ° C for 20 minutes. One volume of phenol (equilibrated with 10 mM Tris · HCl, pH 7.5, 1 mM EDTA, 50 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 is then resuspended in 20μΙ TE.

The fragments are truncated successively by 200-300 bp and have on one side of the fragment a single BamHI restriction site and a blunt end on the other side. These fragments are cloned in a M13mp8 vector which has previously been linearized by digestion twice with BamHI and HincII as described under point IV.1 above. In the method described above, the DNA sequence is obtained only from one strand of DNA starting at the HindIII restriction site and proceeding towards the BamHI site.

The procedure for sequencing the complementary DNA strand of the same DN fragment as well as 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 its sequencing 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 M9 maximal medium. The transformation is performed as follows:

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

2. inoculate 40 ml 2 χ ΥΤηηΓΐ200μΙ of the culture obtained according to step 1;

3. Cultivate at 37 ° C with constant stirring to an OD ₅₅₀ of 0.5;

4. Incubate on ice for 5 minutes;

5. Centrifuge rotor at 6000 rpm for 5 minutes in a pre-cooled SS34Sorvall;

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

7. Incubate on ice for 40 minutes;

8. Centrifuge rotor at 6000 rpm for 5 minutes in a SS34Sorvall;

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

10. Add 1-5μΙ DNA or 7-15μΙ of ligase formulation to 200μΙ of cells obtained according to step 9;

11. Incubate on ice for 30 minutes;

• 12th Incubate at a temperature of 42 ⁰ C for 3 min;

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

14. Boil surface agar and store at 42 ° C;

15. Fill tubes with 3 ml surface agar, 30μΙ X-GAL (20mg / ml dimethylsulfoxide) υηα30μΙ IPTG (20mg / ml H ₂ O);

16. Thoroughly mix and transfer immediately to previously heated 1 x YT plates;

17. Allow plates to dry for about 1 hour; -

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

The plaques obtained in this way are suitable for further treatment.

Controls: -

200 μΐ competent cells without exogenous DNA

1 μM M13mp8 (replicative DNA form, 10 ng) + 200 μΐ competent cells IV.4. Production of the Replicative Form of a Recombinant Phage DNA

'. 1. Carefully transfer single white plaques into 9 ml 2 x YT and 1 ml of the culture obtained after step 1, part IV.3. Incubate for 7 hours at 37 ° C

2. Centrifuge at 4000rpm for 10 minutes,

3. Store the supernatant at 4 ^° C. overnight;

4. inoculate 10 ml of the supernatant obtained according to step 3 and of 10 ml of the culture obtained according to step 1, part IV.3, in 11 2 x YT; ->

5. shaking at 37 ° C 4 ^1/2 hours;

6. Centrifuge at 5000rpm for 15min;

7. Suspend the cells in 10 ml of a 10% sucrose solution in 50 mM Tris.HCl, pH8 and cool;

8. Transfer to 30 ml centrifuge tube;

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

10. Add 8ml 0.25M 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 6ml 5M NaCl (final concentration: 1M) and mix gently;

14. Incubate for 1 hour on ice;

15. Centrifuge 40min at 20000rpm in a SS34Sorvall rotor;

16. Remove supernatant and add volume to 5M NaCl and 15 ml of 30% PEG.

17. Incubate 2 hours or overnight at 4 ⁰ 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 Ti-50 tube and add 0.4 mL of ethidium bromide (10 mg / mL) or transfer to Ti-60 tube with 1.2 mL of ethidium bromide (10 mg / mL).

22. Fill with CsCI solution (1g CsCI + ImITE);

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 water, saturated butanol;

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

IV.5. Making a single-stranded DNA template:

1. Shake E. coli JM103 cells in 5 ml of 2 × ^V T medium at 37 ° C. overnight; ,

2. add 2 drops of a cell suspension obtained according to step 1 to 25 ml of 2 × 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 5V2 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 NaCI;

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 100μΙ TE and 50μΙ phenol;

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

14. Transfer aqueous phase to fresh Eppendorf tubes;

15. Add 500μΙ 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;

18. Incubate at -80 ° C for 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 2% pellet solution to a 0.6% agarose gel;

IV.6. sequencing reaction

The DNA sequence analysis of the template DNA, according to the information in Section 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 Endotoxin Gene in Yeast Cells V.1. Introduce an NcoI site before the first AUG codon of the gene

In order 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 M13mp8, which has a 1.5 Kb BamHI SacI insert encoding the 5 'region of the toxin gene. 200 ng of the insert is obtained by digesting 3 μg 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 as M13mp18 / Bam-Sac. An oligonucleotide having the sequence (5 ') GAGGTAACCCATGGATAAC (3') is synthesized by methods known per se using an APPLIED BIOSYSTEM DNA SYNTHESIZER. This oligonucleotide is complementary to a sequence of the M13mp18 / Bam-Sac that extends from position 141 to position 164 of the protoxin gene (Table 2) and has the mismatch pairs mismatched at positions 154 and 155. The general procedure for mutagenesis is J. Μ ·. Zoller and M. Smith (JMZoller and M. Smith ²⁷ '). Approximately 5μg of single stranded MISmpiS / Bam-Sac phage DNA is mixed with 0.3pg phosphorylated oligonucleotides in a total volume of νοη40μΙ. This mixture is heated to 65 ⁰ C for 5 minutes, then initially cooled to 50 ⁰ C and then gradually cooled down to 4 ° C. Thereafter, buffer, nucleotide triphosphates, ATP, T4 DNA ligase and the large fragment of DNA polymerase are added and incubated overnight at 15 ° C as described (JM Zoller and M.Sniith ²⁷ '). After agarose gel electrophoresis, circular double-stranded DNA is purified and transfected into E. coli strain JM103. The resulting plaques are probed for sequences that hybridize with ³² 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 p! <36 DNA. These phages are referred to as MISrnpie / Bam-Sac / Nco.

V.2. Linkage of the δ-endotoxin gene to the PH05 yeast promoter

The 1.5 Kb BamHI-Sacl insert of M13mp18 / Bam-Sac / Nco is recloned into plasmid pK36 by replacing the wild-type BamHI-SacI fragment of pK36 with the mutated 1.5Kb fragment using standard cloning techniques previously described becomes. This results in the plasmid pK36 / Nco, which has an Ncol restriction site just before the ATG of the protoxin gene

Noc I · GAGGTAAC / CCATGG / ATAAC.

5 μg of this vector is digested with NcoI and the overhanging 3 'ends are filled in with Klenow polymerase, as in Maniatis et al. ²⁸ ). Subsequently, the plasmid is digested with Ahalll, the DNA is separated on a 0.8% low-melting agarose gel and eluted as described above. 2pg 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 p31y vector by incubation of 200 ng of both DNAs in 20 μΙ 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 / Bt. 1 μg of this plasmid DNA is digested with BamHI and the 4Kb fragment isolated from a soft agarose gel. This fragment is linked to the self-replicating yeast vector pJDB207 (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. thuringiensis toxin gene in the bioassay

Whole cells with the B. thuringiensis gene represent a bioencapsulation form (an engineered system consisting of biological material, and surrounding genetic material of protective material) for the MGE1 product, which now better when applied to the plants against degradation by harmful influences, such. As light, is protected as the crystalline product, which is formed by B. thuringiensis in the context of sporulation and enters the medium by breaking the cell.

Yeast cells with and without the B. thuringiensis toxin are resuspended in distilled water to a comparable optical density. From said suspension, 4 concentrations are provided and 0.2% (v / v) of a wetting agent is admixed. To assess the insecticidal activity of these yeast cell preparations, the leaf disk test already described in Section III.7 above is used.

The insecticidal activity of B. thuringiensis transformed yeast cells on first instar Heliothis 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 containing the recombinant B. thuringiensis toxin gene are preferably transformed live or dead yeast cells, including mixtures of living and dead yeast cells, in unaltered form or preferably together with the excipients customary in the art of formulation , 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 Sto.ffen.

The application methods such as spraying, misting, dusting, scattering, brushing or pouring are chosen in the same way as the type of means according to the desired goals and the given conditions.

The formulations, i. the means or preparations containing the transformed live or dead yeast cells or mixtures thereof and optionally solid or liquid adjuvants are prepared in a known manner, e.g. B. by intimately mixing the yeast cells with solid carriers and optionally surface-active compounds (surfactants). As solid carriers, for. As for dusts and dispersible powders, ground natural minerals are commonly used, such as calcite, talc, koaline, montmorillonite or attapulgite. To improve the physical properties, it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. As granular, adsorptive granules are porous types such. As pumice, broken brick, sepiolite or bentonite, as non-sorptive carrier materials such. As calcite or sand in question. In addition, a variety of vorgranulierten materials of inorganic or organic nature, in particular dolomite or crushed plant residues can be used. Suitable surface-active compounds are nonionic, cationic and / or anionic surfactants having good dispersing and wetting properties. Surfactants are also surfactant mixtures. Suitable anionic surfactants may be both so-called water-soluble soaps and water-soluble synthetic surface-active compounds.

As soaps are the alkali, alkaline earth or unsubstituted or substituted ammonium salts of higher fatty acids (C ₁₀ -C ₂₂ ), such as. As the Na or K salts of oleic or stearic acid, or of natural fatty acid mixtures, the. B. can be obtained from coconut or tallow oil, called. Furthermore, the fatty acid methyl-taurine salts are to be mentioned, such as. As the sodium salt of cis ^ -methyl-g-octadecenylaminoj-ethanesulfonic acid (content in formulations preferably about 3%). More often, however, so-called synthetic surfactants are used, especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates or fatty alcohols, such as. B. 2,4,7,9-tetramethyl-5-decyne-4,7-diol (content in formulations preferably at about 2%).

The fatty sulfonates or sulfates are generally present 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 also includes the alkyl moiety of acyl radicals, for. As the Na or Ca salt of lignin, the dodecyl sulfate or a fatty alcohol sulfate mixture prepared from natural fatty acids. This subheading also covers the salts of sulfuric acid esters and sulfonic acids of fatty alcohol / ethylene oxide adducts. The sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups and a fatty acid residue having 8-22 C atoms. Alkylarylsulfonates are z. As the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid or a naphthalenesulfonic acid-formaldehyde condensation product.

Furthermore, corresponding phosphates such. B. salts of the phosphoric acid ester of a p-nonylphenol (4 to 14) -ethylene oxide adduct in question.

Suitable nonionic surfactants are primarily polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, saturated or unsaturated fatty acids and alkylphenols containing 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 can.

Further suitable nonionic surfactants are the water-soluble polyethylene oxide adducts containing from 20 to 250 ethylene glycol ether groups and from 10 to 100 propylene glycol ether groups, with polypropylene glycol, ethylenediaminopolypropylene glycol and alkylpolypropylene glycol having from 1 to 10 carbon atoms in the alkyl chain. The compounds mentioned usually contain 1 to 5 ethylene glycol units per propylene glycol unit.

Nonylphenol polyethoxyethanols, castor oil polyglycol ethers, polypropylene / polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxypolyethoxyethanol may be mentioned as examples of nonionic surfactants. 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 contain as N-substituents at least one alkyl radical having 8 to 22 carbon atoms and which have unsubstituted or halogenated lower alkyl, benzyl or lower hydroxyalkyl radicals as further substituents. The salts are preferably in the form of halides, methylsulfates or ethylsulfates, e.g. stearyltrimethylammonium chloride or benzyldi (2-chloroethyl) ethylammonium bromide.

The surfactants commonly used in formulation technology are i.a. described in the following publications:

"McDeckcheon's Detergents and Emulsifiers Annual" MC Publishing Corp., Ridgewood New Jersey, 1980; Helmut Stäche "Tensid-Taschenbucfi" Carl Hanser-Verlag Munich / Vienna 1981.

The agrochemical compositions generally contain from 0.1 to 99%, in particular from 0.1 to 95%, of the transformed, living or dead yeast cells or mixtures thereof, from 99.9 to 1%, in particular from 99.8 to 5%, of a solid Boderflüssigen 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 dilute 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.thurigiensis toxin genes are eminently suitable for the control of noxious insects. Preferably plant-destroying insects of the genus Lepidoptera are mentioned, in particular those of the genera Pieris, Heliothis, Spodoptera and Plutella, such as 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 of B. thuringiensis toxin-containing material

In the following formulation examples, the term "yeast cells" is to be understood as meaning those which are the recombinant B.

Thufingiensis gene included. (The information is throughout by wt .-%.)

FI. granules a) b) Yeast cells 5% 107 ' kaolin 94% - Highly dispersed silicic acid 1% - attapulgite - 90 7 <

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

F2. dusts a) b) a) b) I 67o derTrägi Yeast cells 27o 57o 25 7o 50 % c) Highly dispersed silicic acid 17o 57o 57o 57o - 75 % talc 977c - 37o - 2% - kaolin - 90 7o 107o 5 7o Ready-to-use dusts are obtained by intimate mixing - 27 % F3. wettable powder 107c Yeast cells Sodium lignosulfonate - Sodium Lauryl sulfate 57o - Sodium diisopropyl- 627 » 107c naphthalenesulfonate Octylphenolpolyethylen- glycol ether (7-8 mol ethylene oxide) Highly dispersed silicic acid kaolin

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

Yeast cells 10%

Sodium lignosulfonate 2%

Carboxymethylcellulose 1%

Kaolin 87%

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 glycol200 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) Alkyl benzenesulfonic acid 3% triethanolamine * 1% carboxymethylcellulose Silicone oil in the form of a 75% 0.1 ° / aqueous emulsion 39% water

* Alkyl is preferably linear with 10 to 14, especially 12-14 carbon atoms such as n-Dodecylbenzolsulfonsäuretriethanolaminsalz.

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, which are used in the present invention, a culture was recognized by the "German Collection of Microorganisms" in Göttingen, West Germany, according to the requirements of the Budapest Treaty for the International Recognition of the Deposit of microorganisms for the purpose of patenting A statement on the viability of the deposited samples was made by said International Depository.

Microorganisms deposit background date of

date number * Viable

keitsbescheinigung

HD1-ETHZ 4449 March 4, 1986 DSM 3667 March 7, 1986 (Bacillus thuringiensis vr. kurstaki HD1 Strain ETHZ4449) HB101 (pK36) March 4, 1986 DSM 3668 March 7, 1986 (Escherichia coli HB101 transformed with pK36 plasmid DNA)

GRF18 4 March 1986 DSM 3665 7 March 1986

(Saccharomyces

cerevisiae)

* Entrance 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 H.R. Whiteley, The Journal of Biological Chemistry 258 (3), 1960 (1983)

3) M.J. Adang, M.J. Staver, T.A. Rocheteau, J. Leighton, R.F. Barker and D.V. Thompson, Gene 36, 289 (1985)

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

5) A. A. Youstenand 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, p. 504,506 (1982)

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

8) B. Trümpi, Zentralblatt f. Bacteriology Dept. 11,305 (1976)

9) F.P.Delafield, H.J. 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.Lyublinskaja and V. M.Stepanov, Brokliniya 43, 857 (1978)

11) O. Ouchterlony, Handbook of Immunodiffusion 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) Amersham Buchler Review No. 18, Amersham Buchler GmbH & Co. KG, Braunschweig, Federal Republic of Germany (1979)

14) T. Maniatisi E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold 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)

16) H.E. Schnepf and H.R. Whiteley, Proc. Natl. Acad. Be. USA 78, 2893 (1981)

17) L. Clarke, R. Heitzeman and J. Carbon, Methods in Enzymology 68,436 (1979)

18) 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.GrunsteinandD.Hogness, PNAS72,396 (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 (1982).

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

25) J. Messing, Methods of Enzymology 101, 20 (1983)

26) M. Poncz, D. Solowieczyk, M. Ballantine, E. Schwartz and S. Surrey, Proc. Natl. Acad. Be. USA, 79,4298 (1982)

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

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

29) T. Maniatis, 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. lizuka, H. Sugisaki and M. Takanami, Gene 34, 243 (1985)

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

1) digestion with. Endonuclease B

2) Bal-31

Digestion with endonuclease A

Linkage, transformation and production ·

single-stranded DNA (matrix)

Fig. 1 modified according to M. Poncz et al. ²⁶ '. Construction of the deletion mutant library. Procedure: 1, the insert (thick line) is spliced into the Α-site of the MT3, which has cohesive ends; 2, after linearization at the B site, the phage DNA is digested with Bal-31 for varying lengths of time (the dashed line indicates the extent of Bal-31 digestion relative to the insert [thick line] and to M13 [thin line] ); 3, the digestion product is cleaved at the Α site and the Bal-31-induced insert continuum is isolated. This results in a whole family of fragments of different sizes, each having 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 is proximal to the primer site P used for DNA sequence analysis. X and C = smooth ends.

Table 1

ν. A HindIII BasHI PstI EcoRI C M13mpi (BamHI) Hpal-Hindlll s . , , Λ BarnHI Hindlll EcoRI Pstl HincII Smal Smal Smal 8 9 8 9 v a; EcoRI-PstI

according to the example given

Table 2: Nucleotide sequence of the DNA coding for the protein MGIiI

10 20 30 4 O 50 6ύ

GTTAACACCC TGGGTCAAAA ATTGATATTT AGTAAAATTA GTTGCACTTT GTGCATTTTT

7Ο OO 9Ο 1OO IIO 12Ο

TCATAAGATG AGTCATATGT TTTAAATTGT AGTAATGAAA AACAGTATTA TATCATAATG

130 140 150 160 17Ο 100 _ ^ AATTGGTATC TTAATAAAAG AGATGGAGGT AACTTATGGA TAACAATCCG AACATCAATG

ex * 190 200 210 220 230 240

^ AATGCATTCC TTATAATTGT TTAAGTAACC CTGAAGTAGA AGTATTAGGT GGAGAAAGAA

250 260 270 200 290 300 ^J: ~ TAGAAACTGG TTACACCCCA ATCGATATTT CCTTGTCGCT AACGCAATTT CTTTTGAGTG

^cn 310 320 330 340 350 360

- ^ AATTTGTTCC CGGTGCTGGrt TTTGTGTTAG GACTAGTTGA TATAATATGG GGAATTTTTG

^ ⁰ 370 300 39Ο 400 4 10 42Ο

GTCCCTCTCA ATGGGACGCA TTTCTTGTAC AAATTGAACA GTTAATTAAC CAAAGAATAG

430 440 450 460 470 400 AAGAATTCGC TAGGAACCAA GCCATTTCTA GATTAGAAGG ACTAAGCAAT CTTTATCAAA

490 500 .510 520 530 54Ο TTTACGCAGA ATCTTTTAGA GAGTGGGAAG CAGATCCTAC TAATCCAGCA TTAAGAGAAG

550 560 570 500 590 6OO AGATGCGTAT TCAATTCAAT GACATGAACA GTGCCCTTAC AACCGCTATT CCTCTTTTG

Table 2: (continued)

610 620 630 610 650 660 CAGTTCAAAA TTATCAAGTT CCTCTTTTAT CAGTATATGT TCAAGCTGCA AATTTACATT

670 600 69O 700 7IO 720 TATCAGTTTT GAGAGATGTT TCAGTGTTTG GACAAAGGTG GGGATTTGAT GCCGCGACTA

730 740 750 760 770 - 700 TCAATA6TCG TTATAATGAT TTAACTAGGC TTATTGGCAA CTATACAGAT CATGCTGTAC

790 BOO ΟΙΟ Π20 Q30 GCTGGTACAA TACGGGATTA GAGCGTGTAT GGGGACCGGA TTCTAGAGAT TGGATAAGATE

850 060 070 000 890 900 ATAATCAATT TAGAAGAGAA TTAACACTAA CTGTATTAGa TATCGTTTCT CTATTTCCGA

910 920 930 940 950 960 ACTATGATAG TAGAACGTAT CCAATTCGAA CAGTTTCCCA ATTAACAAGA 6ΛΑΛΤΤΤΑΤΑ

970 9QO 990 1000 1010 1020 CAAACCCAGT ATTAGAAAT TTTGATGGTA GTTTTCGAGG CTCGGCTCAG GGCATAGAAG

1030 1040 1050 1060 1070 1000 GAAGTATTAG GAGTCCACAT TTGATGGATA TACTTAACAG TATAACCATC TATACGGATG

1090 1100 1110 1120 1130 1140 CTCATAGAGG AGAATATE TGGTCAGGGC ATCAAATAAT GGCTTCTCCT 6TAGGGTTTT

1150 CGGGGCCA6A

1160 ATTCACTTTT

1170 CCGCTATATG

1180 GAACTATGGG

1 190 AAATGCAGCT

1200 CCACAACAAC

Table 2: (continued)

1210 1220 1230 124O 125O 1260 GTATTGTTGC TCAACTAGGT CAGGGCGTGT ATAÜAACATT ATCGTCCACT TTATATAGAA 127O 12ΘΟ 1290 1300 1310 132o GACCTTTTAA 1 ATAGGGATA AATAATCAAC AACTATCTGT TCTTGACGGG ACAGAATTTG 1330 1340 1350 1360 1370 1300 CTTATGGAAC CTCCTCAAAT TTGCCATCCG CTGTATACAG AAAAAGCGGA ACGGTAGATT 1390 1400 14 10 1420 1430 14 4O CGCTGGATGA AATACCGCCA CAGAATAACA ACGTGCCACC TAGGCAAGGA TTTAGTCATC 1450 1460 1470 140o 1490 1500 GATTAAGCCA TGTTTCAATG TTTCGTTCAG GCTTTAGTAA TAGTAGTGTA AGTATAATAA

1520 1530 1540 1550 i 56Ο GA6CTCCTAT GTTCTCTTGG ATACATCGTA GTGCTGAATT ΤΛΑΤΛΑΤΑΤΛ ATTCCTTCAT

1570 CACAAATTAC

1630 TTAAAGGACC

1690 CAACCTTAAG

15Q0 ACAAATACCT

1640 AGGATTTACA

1700 AGA

1750 1760 ACGCTTCTAC.CACAAATTTA

159Ο TTAACAAATE

1650 GGAGGAGATA

1710 ACTGCACCAT

1770 CAATTCCATA

1600 CTACTAATCT

1660 TTCTTCGAAG

1720 TATCACAAAG

1700 CATCAATTGA

IA10 TGGCTCTGGA

1670 AACTTCACCT

1 730 ATATCGGGTA

1790 CGGAAGACCT

162Ο ACTTCTGTCG

16B0 GGCCAGATTT

1740 AGAATTCGCT

1800 ATTAATCAGG

Table 2: (continued)

lOlO GGAATTTTTC

1070 TAGGTTTTAC

1930 ATGTCTTCAA

1020 AGCAACTATG

! 0OO TACTCCÜTTT

1940 TTCAGGCAAT

1030 AGTAGTGGGA

1O9O AACTTTTCAA

1950 GAAGTTTATA

1010 GTAATTTACA

I 9OO ATGGATCAAG

1960 TAGATCGAAT

in 1060

GTCCGGAAGC TTTAGGACT

191Ο TGTATTTACG

1970 TGAATTTGTT

1 92Ο TTAAGTGCTC

1900 CCGGCAGAAG

1990 2000 2010 2020 2030 204 0

TAACCTTTGA GGCAGATE GATTTAGAAA GAGCACAAAA GGCGGTGAAT GAGCTGTTTA

2050 · 2060 2070 2000 2090 2100

CTTCTTCCAA TCAAATCGGG TTAAAAACAG ATGTGACGGA TTATCATATT GATCAAGTAT

2110 2120 2130 2140 2150 21 60 CCAATTTAGT TGAGTGTTTA TCTGATGAAT TTTGTCTGGA TGAAAAAAAA GAATTGTCCG 2170 2100 219O 22oo 2210 222O AGAAA6TCAA ACATGCGAAG CGACTTAGTG ATGAGCGUAA TTTACTTCAA GATCCAAACT 2230 2240 2250 2260 2270 2200

TTAGAGGGAT CAATAGACAA CTAGACCGTG GCTGGAGAGG AAGTACGGAT ATTACCATCC

2290 230O 2310 2320 2330 234Ο AAGGAGGCGA TGACGTATTC AAAGAGAATT ACGTTACGCT ATTGGGTACC TTTGATGAGT

2350

2360

2370

2300

2390

GCTATCCAAC GTATTTATAT CAAAAAATAG ATGAGTCGAA ATTAAAAGCC TATACCCGTT

Table 2 : (continued)

2410 2420 243O 2440 2450 2460

ACCAATTAAG AGGGTATATC GAAGATAGTC AAGACTTAGA AATCTATTTA ATTCGCTACA

2470 24G0 2490 2500 2510 252Ο

ATGCCAAACA CGAAACAGTA AATGTGCCAG GTACGGGTTC CTTATGGCCG CTTTCAGCCC

2530 2540 2550 2560 2570 25B0

CAAGTACATE CGGAAAATGT GCCCATCATT CCCATCATTT CTCCTTGGAC ATTGATGTTG

2590 2600 2610 2620 2630 2640

GATGTACAGA CTTAAATGAG GACTTAGGTG TATGGGTGAT ATTCAAGATT AAGACGCAAG

2650 2660 2670 2600 2690 2700

ATGGCCATGC AAGACTAGGA AATCTAGATE TTCTCGAAGA GAAACCATTA GTAGGAGAAG

^ 2710 2720 2730 2740 2750 2760

^ CACTAGCTCG TGTGAAAAGA GCGGAGAAAA AATGGAGAGA CAAACGTGAA AAATTGGAAT

2770 2780 2790 2000 2B10 2G20

.4-- GGGAAACAAA TATTGTTTAT AAAGAGGCAA AAGAATCTGT AGATGCTTTA TTTGTAAACT

tn 2830 2B4O 2050 2Ο6Ο 2Π7Ο 2000

- * CTCAATATGA TAGATTACAA GCGGATACCA ACATCGCGAT GATTCATGCG 6CA6ATAAAC

^M. 2890 2900 2910 2920 2930 2940

GCGTTCATAG CATTCGAGAA GCTTATCTGC CTGAGCTGTC TGTGATTCCG GGTGTCAATG

2950 2960 2970 2900 2990 3000

CGGCTATTTT TGAAGAATTA GAAGGGCGTA TTTTCACTGC ATTCTCCCTA TAT6ATGCGA

Table 2: (continued)

Cn ._v

3010 3020 3030 3040 3050 3O6O GAAATGTCAT TAAAATGGT GATTTTAATA ATGGCTTATC CTGCTGGAAC GTGAAAGGGC

3070 30Q0 3090 3100 3110 3120 AT6TAGAT6T AGAAGAACAA AACAACCACC GTTCGGTCCT TGTTGTTCCG GAATGGGAAG

3130 3MO 3150 3160 3170 3100 CAGAAGTGTC ACAAGAAGTT C6TGTCTGTC CGGGTCGTGG CTATATCCTT CGTGTCACAG

3190 3200 3210 3220 3230 324 0 CGTACAAGGA GGGATATGGA GAAGGTTGCG TAACCATTCA TGAGATCGAG AACAATACAG

3250 3260 3270 3200 3290 3300 ACGAACTGAA GTTTAGCAAC TGTGTAGAAG AGGAAGTATA TCCAAACAAC ACGGTAACGT

3310 3320 3330 334O 3350 3360 GTAATGATTA TACTGCGACT CAAGAAGATE ATGAGGGTAC GTACACTTCT CGTAATCGAG

3370 3300 3390 3400 3410 3420 GATATGACGG AGCCTATGAA AGCAATTCTT CTGTACCAGC TGATTATGCA TCAGCCTATG

3430 3440 345O 3460 347O 34G0 AAGAAAAAGC ATATACAGAT GGACGAAGAG ACAATCCTTG TGAATCTAAC AGAGGATATG

3490 3500 35IO 3520 3530 354O GGGATTACAC ACCACTACCA GCTGGCTATG TGACAAAAGA ATTAGAGTAC TTCCCAGAAA

3550 3560 3570 "3500 3590 3600 CCGATAAGGT ATGGATTGAG ATCGGAGAAA CGGAAGGAAC ATTCATCGTG GACAGCGTGG

Table 2: (continued)

3610 3620 363 3610 3650 3660 AATTACTTCT TATGGAGGAA TAATATATGC TTTAAAATGT AAGGTGTGCA AATAAAGAAT

3670 360O 3690 3700 3710 3720 GATTACTGAC TTGTATTGAC AGATAAATAA GGAAATTTT ATATGAATAA AAAACGGGCA

3730 3740 ^v 3750 3760 377O 3700 TCACTCTTAA AAGAATGATG TCCGTTTTT GTATGATTTA ACGAGTGATA TTTAAATGTT

3790 3000 3BlO 3020 3B30 3B4O TTTTTTGCGA AGGCTTTACT TAACGGGGTA CCGCCACATG CCCATCAACT TAAGAATTTG

3B50 3060 3070 3000 3090 3900 CACTACCCCC AAGTGTCAAA AAACGTTATT CTTTCTAAAA AGCTAGCTAG AAAGGATGAC

3910 3920 393 O 394 O 395O 3960 ATTTTTATG AATCTTTCAA TTCAAGATGA ATTACAACTA TTTTCTGAAG AGCTGTATCG

3970 3900 3990 4 000 4010 4O2O TCATTTAACC CCTTCTCTTT TGGAAGAACT CGCTAAAGAA TTAGGTTTTG TAAAAAGAAA

4030 4040 4050 4060 4070 4000 ACGAAAGTTT TCAGGAAATG AATTAGCTAC CATATGTATC TGGGGCAGTC AACGTACAGC

4090 4100 4110 4 120 4 130 4140 GAGTGATTCT CTCGTTCGAC TATGCAGTCA ATTACACGCC GCCACAGCAC TCTTATGAGT

4150 4160 4170 4100 4190 4200 CCAGAAGGAC TCAATAAACG CTTTGATAAA AAAGCGGTTG AATTTTTGAA ATATATTTTT

Table 2 : (continued)

4210 4220 4230 4240 4250 4260

TCTGCATTAT GGAAAAGTAA ACTTTGTAAA ACATCAGCCA TTTCAAGTGC AGCACTCACG

4270 4280 4290 4300 4310 4 320

TATTTTCAAC GAATCCGTAT TTTAGATGCG ACGATTTTCC AAGTACCGAA ACAT-TTAGCA

4330 4340 4350 43ώΟ

CATGTATATC CTGGGTCAGG TGGTTGTGCA CAAACTGCAG

Table 3: Amino acid sequence of the protein MGE1

LIMITS: 156 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 Ser 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 GIy GIu Arg lie GIu Thr GIy Tyr Thr Pro He Asp He 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

Z ^,. 365 395

ω GTT GAT ATA ATA TGG GGA ATT TTT GGT CCC TCT CAA TGG GAC GCA TTT CTT GTA CAA ATT

oa VaI Asp He He Trp GIy He Phe GIy Pro Ser GIn Trp Asp Ala Phe Leu VaI Gin He

425 455

- ¹ - GAA CAG TTA ATT AAC CAA AGA ATA GAA GAA TTC GCT AGG AAC CAA GCC ATT TCT AGA TTA

"GlLi GJn Leu He? Asn GIn Arg lie GIu GIu Phe Ala Arg Asn Gin Ala He Ser Arq Leu Cn"

7t "485 515

_M 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 lie 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 He GIn Phe Asn Asp Met Asn Ser AIa

Table 3 : (continued)

605 635

CTT 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 Flsri 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 AGT CTT ATT

Arg Trp GIy Phe Asp Ala Ala Thr He Asn Ser ArQ Tyr Asn Asp Leu Thr Arg Leu He

^. 785 θ 15

f ^ 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 VaJ. Arg Trp Tyr Asn Thr GIy Leu GIu Arg VaI Trp GIy

045 875

^ CCG GAT TCT AGA GAT TGG ATA AGA ACT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA

_Cr) Pro Asp Eggs Arg Asp Trp He Arg Tyr Asn GIn Phe Arg Arg Glu Leu Thr Leu Thr VaI

_co 905 935

M TTA GAT ATC GTT TCT CTA TTT CCG AAC TAT GAT AGT AGA ACG ACT CCA ATT CGA ACA GTT

Leu Asp He VaI Ser Leu Phe Pro Asn. Tyr Asp Ser Arg Thr Tyr Pro Arg Arg 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 Glu Tyr Thr Asn Pro VaI Leu GIu Asn Phe Asp GIy Ser Phe

1025 1055

CGA GGC TCG GCT CAG GGC 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

Table 3 : (continued)

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 ATS GCT TCT CCT GTA GGG TTT TCG GGG CCA GAA TTC ACT TTT CCG CTA ACT GGA ACT

Ue Met Ala Ser Pro VaI GIy Phe .Ser GIy Pro GIu Phe Thr Phe Pro Leu Tyr GIy Thr

- ¹ - '1205 1235

^ ATG GGA AAT GCA GCT CCA CAA CAA CGT ATT GTT GCT CAA CTA GG CAG GGC GTG TAT AGA

I ^ Met GIy Asn Ala Ala Pro Gin Gin Arg lie Val Ala Gin Leu Giy Gin Giy VaI Tyr Arg

12ώ5 1295

ACA TTA TCG TCC ACT TTA TAT AGA AGA CCT TTT AAT ATA GGG ATA AAT AAT CAA CAA CTA

-a. Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn He Giy Lie Asn Asn Gin Gin Leu

-V 13. ^ 5 1355

CO TCT GTT CTT GAC GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AAT TTG CCA TCC GCT GTA

* ° 'Ser VaI Leu Asp GIy Thr GIu Phe Ala 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 Glu Pro Pro Gin Asn Asn Asn VaI

1445 1475

CCA CCT AGG CAA GGA TTT AGT CAT CGA TTA AGC 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 Ser Gly Phe

Table 3 : (continued)

1505 1535

AGT AAT AGT AGT GTA AGT ATA ATA AGA GCT CCT ATG TTC TCT TGG ATA CAT CGT AGT GCT

Ser Asn Eggs Ser VaI Eggs 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

Gius Phe Asn Ann He loves? Pro Ser Ser Gin He Thr Gin Pro Leu Thr Lys Ser Thr

1625 1655

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

  '

Γ 1685 1715

CGA AGA ACT TCA CCT GGC CAG ATT TCA ACC TTA AGA GTA AAT ATT ACT GCA CCA TTA TCA

Arg Arg Thr Ser Pro GIy Gin lie Ser Thr Leu Arg VaI Asn He Thr AIa Pro Leu Ser

- 1745 1775

CAA AGA TAT CGG GTA AGA ATT CGC TAG GCT TCT ACC ACA AAT TTA CAA TTC CAT ACA TCA

Gin Arg Tyr Arg VaI Arg He Arg Tyr AIa Ser Thr Thr Asn Leu GIn Phe His Thr Ser

, 1005 · 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

1065 1B95

TTA CAG TCC GGA AGC TTT AGG ACT GTA GGT TTT ACT ACT CCG TTT AAC TTT TCA A.AT GGA

Leu Gin Ser 61y Ser Phe Arg Thr VaI GIy Phe Thr Thr Per Phe Asn 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 lie Asp

Table 3 :, (continued)

1985 2015

CGA ATT RAA TTT RTT CCG GCA GAA GTA ACC TTT RAG GCA GAA TAT GAT TTA RAA AGA GCA

Arg He Glu Phe VaI Pro Al a Glu VaI Thr Phe 61 u Ala 61 Ti Tyr Asp Leu GIu Arg Ala

2045 2075

Γ TCT TCC AAT CAA ATC GGG TTA AAA ACA GAT GTG Gin Lys Ala VaI Asn Glu Leu Phe Thr Ser Ser Asn Gin lie Gly Leu Lys Thr Asp VaI

CAA AAG GCG GTG AAT GAG CTG TTT ACT TCT TCC AAT CAA ATC GGG TTA AAA ACA GAT GTG Up

f ^, 2105 2135

f ^ ACG GAT TAT CAT ATT GAT CAA GTA TCC AAT TTA GTT GAG TGT TTA TCT RAT GAA TTT TGT

ou 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 AAR CGA CTT AGT GAT GAG

- ^ 'Leu Asp GIu Lys Lys Glu Leu Eggs GIu Lys K> al Lys His Ala Lys Arg Leu Eggs Asp GIu cn

^ CGG AAT TTA CTT CAA GAT CCA AAC TTT AGA GGG ATC AAT AGA CAA CTA GAC CGT GGC TGG

Species] Asn Leu Lei Gin Asp Pro Asn Phe Arg GIy lie 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 He Gin Gly GIy Asp Asp VaI Phe Lys GIu Asn Tyr VaI

2345 2375

ACG CTA TTG GGT ACC TTT COUNCIL GAG TGC ACT CCA ACG TAT TTA ACT CAA AAA ATA RAT RAG

Thr Leu Lei GIy Thr Phe Asp GIu Cyss Tyr Pro Thr Tyr Leu Tyr Bin Lys lie Asp GIu

Table 3 : (continued)

2405 2435

TCG AAA TTA AAA GCC TAT ACC CGT TAC CAA TTA AGA GGG TAT ATC GAA GAT AGT CAA GAC

Ser Lys Leu Lys AIa Tyr Thr Arg Tyr GIn Leu Arg GIy Tyr lie 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 Glu Tyr Leu He Arg Tyr Asn Ala 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

^ 2505 2615

tZ 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 He Asp VaI Giy Cys Thr Asp Leu Asn Giu Asp Leu Giy VaI Trp

* 2nd ² ^ S 2675

_m GTG ATA TTC AAG ATT AAG ACG CAA GAT GGC CAT GCA AGA CTA GGA AAT CTA GAA TTT CTC

_i. Vai Phe Lys was Lys Thr Gin Asp Giy His Ala Arg Leu Giy Asn Leu Giu Phe Leu er.

ro '2705 · 2735

_N GAA GAG AAA CCA TTA GTA GGA GAA GCA CTA GCT CGT GTG AAA AGA GCG GAG AAA AAA TGG

GIu GIu Lys Pro Leu VaI GIy GIu AIa Leu AIa 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 GIu Lys Leu GIu Trp GIu Thr Asn He VaI Tyr Lys GIu Ala Lys GIu

2825 2S55

TCT GTA GAT GCT TTA TTT GTA AAC TCT CAA TAT GAT AGA TTA CAA GCG GAT ACC AAC ATC

Ser VaI Asp Ala Leu Phe VaI Asn Ser GIn Tyr Asp Arg Leu Gin Ala Asp Thr Asn lie

Table 3 : (continued).

2885 2915

GCG ATG ATT CAT GCG GCA GAT AAA CGC GTT CAT AGC ATT CGA GAA GCT TAT CTG CCT GAG

Ala Met; He His Ala Ala Asp Lys Arg VaI His Ser He Arg GIu AIa Tyr Leu Pro GIu

2945 ·. 2975

CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA TTA GAA GGG CGT ATT TTC

Leu Ser Vali Pro GIy VaI Asn Ala Ala lie Phe Giu Giu Leu GIu Giy Arg He Phe t

^. 3005 3035

^ ACT GCA-TTC TCC CTA TAT GAT GCG AGA AAT GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC

- ^ ¹ Thr AIa Phe Ser Leu Tyr Asp Ala Arg Asn VaI He. Lys Asn GIy Asp Phe Asn Asn GIy

_ * _- 3065. 3095

~ i- TTA TCC TGC TGG AAC GTG AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAC CGT TCG

CD Leu Ser Cys Trp Asn VaI Lys GIy His VaI Asp VaI GIu GIu Gin Asn Asn His Arg Ser

C-O 3125 3155

t ° C) TC CTT 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

3105 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 He GIu Asn Asn Thr Asp Giu Leu Lys Phe Ser Asn Cys VaI GIu GIu GIu

Table 3 : (continued)

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

- * Gly Tyr Thr Thr Ser Arg Asn Arg Gly Tyr Gly Asp Ala Tyr Glu Asn Ser Ser Ser Val

£ 3425 3455

-J 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

Ii. 34B5 3515

Cn CCT TGT GAA TCT AAC AGA GGA TAT GGG OAT TAC ACA CCA CTA CCA GCT GGC TAT GTG ACA

- ^ Pro Cyo GIu Ser Asn Arg GIy Tyr GIy Asp Tyr Thr Pro Leu Pro Ala GIy Tyr VaI Thr

(Γ ·

f ° 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 lie 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 lie VaI Asp Ser VaI Giu Leu Leu Leu Met GIu GIu End

Claims (9)

A method of producing a DNA fragment from Bacillus thuringiensis var. Kurstaki which extends to the region between the interfaces H ρ a1 (0) and Pst I (4355) and which encodes an insecticidally active proteinaceous substance including selected portions thereof the condition that the insecticidal activity of said selected DNA parts is maintained, and which is characterized by the following process steps: a) isolation and lysis of Bacillus thuringiensis var. 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 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. 2. The method according to claim 1, characterized in that said DNA fragment has the sequence of Table 2. A method according to claim 1, characterized in that said DNA fragment comprises the MGEI insecticidal protein coding region starting 156 bp downstream of the Hpal restriction site and terminating at nucleotide 3620, including selected portions thereof, provided that the insecticidal activity of said selected DNA parts is preserved. 4. The method according to claim 3, characterized in that said DNA fragment has the sequence of Table 3. 5. The method according to any one of claims 1 to 4, characterized in that method step d) is carried out according to the following individual steps: e) screening the clones for the presence of antigen that reacts with antibodies prepared against the crystal body protein of B. thuringiensis var. kurstaki; · f) reading the clones that react specifically with goat antiserum; and g) Examination of the insecticidal activity of extracts of the clones obtained according to step f). 6. The method according to claim 5, characterized in that in step c) a plasmid serves as a vector. 7. The method according to claim 5, characterized in that in step c) a phage serves as a vector. 8. The method according to claim 1, characterized in that in the Verfahrenssch ritte) described cloning of Bacillus thuringiensis var. Kurstaki HDI plasmid DNA library and subsequent splicing of the thus fragmented plasmid DNA into a suitable restriction site of pBR322 takes place. 9. The method according to claim 1, characterized in that the method described in step c) cloning and expression of B. thuringiensis DNA is carried out by a) modifies the DNA sequence in the environment of the toxin gene by oligonucleotide-mediated mutagenesis, b) linked to the PH05 promoter from yeast and c) yeast cells are subsequently transformed by means of known methods with said DNA sequence.