IE59456B1 - Insecticidal proteinaceous substance - 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

The invention relates to an insecticidal proteinaceous substance.

Bacillus thuringiensis (£. thuringiensis) ie a gram-positive bacterium vhich is usually pathogenic to insects (S. Chang’’)· (Reference is made to appended bibliography vhich is hereby made a part hereof ).

The varieties of B. thuringiensis differ remarkably in their toxicity and insect host range. The insecticidal activity of B. thuringiensis originates substantially or completely from a proteinaceous parasporal crystal produced at the sporulation stage of growth. The gene(s) coding for the toxic proteins (polypeptides) of said crystal is (are) found on plasmid DNA and/or on chromosomal DNA of B. thuringiensis.

In order to avoid any disadvantages resulting from the presence of other compounds produced by B. thuringiensis and to gain the insecticidal polypeptide in large quantities, it is desirable to make use of the corresponding gene, i.e., the corresponding DNA sequence, outside of B. thuringiensis, said gene (or DNA) coding for the desired insecticidal protein. Nevertheless, it is also possible and in special cases advantageous to transform B. thuringiensis with said DNA sequence.

In this way it is possible to obtain a protein analogeous in structure and properties to the natural one. The protein (polypeptide) obtained according to the present invention is called in the following MGE 1. υ Ine 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, which DNA sequence differs remarkably in structure from that of the B. thuringiensis genes already known (H.E. Schnepf et al. , M.J. Adang et al.53 and Y. Shibano et al.

The present invention relates also to a DNA fragment obtainable from Bacillus thuringiensis var. kurstaki HD1, ETHZ 4449, encoding an insecticidal proteinaceous substance characterized by the nucleotide sequence given in table 2, and said fragment coding for the insecticidal protein MGE1, including truncated portions thereof, said truncated DNA portions being subject to the proviso that insecticidal activity has not been lost.

By the term proteinaceous substance, the insecticidal protein MGE 1 as well as derivatives and modifications thereof exhibiting the same or comparable insecticidal activity as obtained in vitro are meant, such as, for example, protein MGE 1 combined with other protein fragments, particularly those obtained from another cloned DNA coding for other pesticidal, especially insecticidal activities, said combination of proteins being defined as fusion proteins. Besides insecticidal activity, pesticidal activity includes, for example, bactericidal, viricidal, fungicidal and herbicidal activity and particularly activity against plant pathogenic organisms. From the proteinaceous substances, the insecticidal protein MGE 1 alone is preferred.

Another aspect of the invention relates to the method of constructing cloning vehicles and expression vehicles conprising the DNA fragment given in table 2 and also to said vehicles themselves. Suitable DNA vectors are, for example, plasmids such as pBR322 and pUC8, or phages such as Ml3.

Further, the invention relates to living or dead microorganisms containing a DNA fragment characterized by the nucleotide sequence given in table 2, particularly to a microorganism belonging to the species Saccharomyces cerevisiae. The invention also relates to microorganisms, particularly to a microorganism of the species Saccharomyces cerevisiae, which contain a DNA fragment obtainable from Bacillus thuringiensis var. kurstaki HD1, ETHZ 4449, encoding an insecticidal proteinaceous substance characterized by the nucleotide sequence given in table 2 with the proviso that, if said microorganism belongs to the group of Bacillus thuringiensis var. kurstaki, said Bacillus has been transformed with the DNA fragment of the invention. Such microorgnisms are, for example, yeasts, particularly Saccharomyces cerevisiae, bacteria and phylloplane fungi.

The invention relates also to a bioencapsulation system consisting of a first material completely embedded in a second material of biological origin, the first material being represented by a DNA fragment of the invention and the second material being whole, living or dead microorganism or mixtures thereof, with the proviso that, if said microorganism belongs to the group of Bacillus thuringiensis var. kurstaki, said Bacillus has been transformed with the DNA fragment of the invention. Especially suitable microorgnisms are yeasts, particularly Saccharomyces cerevisiae, such as S. cerevisiae GRF 18.

The present invention relates further to a composition and a method for combating insects, particularly Lepidopteran insects (insects belonging to the .order Lepidoptera) and especially members of the genera Pieris, Heliothis, Spodoptera and Plutella, such as, for example, Pieris brassicae, Heliothis virescens, Heliothis zea, ρ: t;· Spodoptera littoralis, Plutella xylostella and related species, with the proteinaceous substance containing protein MGE 1 encoded by the DNA sequence given in table 2. The method comprises applying to the insects or their habitats an insecticidally effective amount of a proteinaceous substance at least partially encoded by the DNA fragment of the invention. The composition comprises an insecticidally effective amount of a proteinaceous substance at least partially encoded by the DNA fragment of the invention.

Moreover, the invention relates to a delivery system consisting of living or dead yeast cells which have been transformed with an insecticidally effective amount of a proteinaceous substance at least partially encoded by the DNA fragment coding for the protein MGE 1, said delivery system being suitable for delivering the active substance in a protected form. Thus, if the transformed yeast cells are applied in conventional manner as, for example, by spraying in the field (ground- and aerial application), a long lasting insecticidal activity is obtained. The active substance is well protected against premature decomposition originating from unfavorable conditions such as, for example, sunlight or adverse conditions on leaf surfaces.

The cloned gene can be put under the control of a yeast promoter and expressed and insecticidal activity can be demonstrated for a) extract and b) whole cell. Suitable yeast promoters are described in European patent specification 100,561. Particularly suitable is the PH05 promoter.

At least some of the genes of B. thuringiensis coding for insecticidally active proteins are known to be linked with a promoter which can be recognized by the Escherichia eoli (E. eoli) RNA polymerase, said promoter being situated in front of the respective gene (H.C. Wong jet al.2’) .

G The method of producing an insecticidal proteinaceous substance according to the invention comprises transcription and translation of the gene according to the invention with identified DNA sequence into the protein by E. eoli or yeast.

The work described herein is performed employing Bacillus thuringiensis var. kurstaki HD1, strain ETHZ 4449, for obtaining starting DNA material. Said strain is available from the Department of Microbiology at the Swiss Federal Institute of Technology in Zurich, Switzerland, and is freely accessible to anybody without any restrictions. Its origin is the H. Dulmage collection at the Cotton Insects Research Unit, U.S. Department of Agriculture, Agriculture Research Center, Brownsville, Texas, from where it is freely accessible to anybody.

According to the invention, the DNA coding for the protein MGE 1 is obtained by: a) isolating and lysing cells of B. thuringiensis var. kurstaki HD1 and separating plasmids from the material thus obtained by methods known per se. The plasmid material thus obtained is purified and dialysed; b) preparing a DNA library of B. thuringiensis var. kurstaki HD1 plasmid DNA; c) cloning the fragmented plasmid DNA obtained according to step b) in a suitable vector, preferably a plasmid; d) screening for the presence of protein MGE 1 which can be done, for example, by the following steps e) to g): e) screening of the clones for the presence of antigen responding to antibodies prepared against the crystal protein of B. thuringiensis var. kurstaki (screening for expression of the respective polypeptide); f) selecting the clones being specifically reactive with goat antiserum; and g) testing insecticidal activity of extracts of said clones obtained according to step f).

The DNA can be identified by methods known per se, as, for exampl by the following steps h) and i): h) mapping of the DNA of the positive clones by digestion with restriction endonucleases and hybridizing the fragments thus obtained with radiolabeled RNA; and i) sequencing of DNA fragments coding for the respective proteins Meanings of abbreviations used in the following: bp: base pairs BSA: bovine serum albumin DEAE: diethylaminoethyl DFP: diisopropyl fluorophosphate DTT: 1,4-dithiothreitol (1,4-dimercapto-2,3-butanediol) EDTA: ethylenediaminetetraacetic acid IPTG: isopropyl-B-thiogalactopyranoside (Serva) Kb: Kilo bases PBS1 0.01 M phosphate buffer, pH 7.4, and 0.8 % NaCl PBS2 10 mM sodium phosphate, pH 7.8, and 0.14 % NaCl PEG 6000 polyethylene glycol, average molecular weight 6000 PMSF: phenylmethylsulfonyl fluoride (Fluka) RT: room temperature SDS: sodium dodecyl sulphate STE: see TNE TBS: 10 mM Tris-HCl, pH 7.5, and 0.14 M NaCl 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 adjusted with HCl X-GAL: 5-bromo-4-chloro-3-indoxyl-B-D-galactoside 2xYT: 16 g Bacto Tryptone (’'Bacto is a Trade Mark) 10 g Yeast Extract (Bacto) 5 g NaCl Media, buffers and solutions used in the following: 20xSSC Dissolve 175.3 g of NaCl and 88.2 g of sodium citrate- in 800 ml of H20. Adjust pH to 7.0 vith a few drops of a 10 N solution of NaOH. Adjust volume to 1 liter. Dispense into aliquots. Sterilize by autoclaving. 2xSSC 10 X of 20 SSC 6xSSC 33 % of 20 SSC Denhardt’s Solution (50x) Ficoll 70 (relative molecular mass approximately 70*000; Pharmacia (Ficoll* is a registered Trade Mark ) . polyvinylpyrrolidone (Calbiochem- Behring Corp.) 5 g BSA (Sigma) 5 g H20 to 500 ml Filter through a disposable Nalgene filter (Nalgene ; Nalge Co.Inc., Rochester, N.Y., USA). (Nalgene is a Trade Mark). Dispense into 25 ml aliquote and store at -20°C. Solutions M9 Medium Per liter: Na2HP0i, 6 g KH2P0w 3 g NaCl 0.5 g NHkCl 1 g Adjust pH to 7.4, autoclave, cool, and then add: 1 M MgSOu 2 ml 20 % glucose 10 ml 1 M CaCl2 0.1 ml vitamin BI (40 mg/lOml) 5 ml The above solutions should be sterilized separately by filtration (glucose-vit.Bl) or autoclaving.

LB (Luria-Bertani) Medium Per liter: Bacto-tryptone 10 g Bacto-yeast extract 5 g NaCl 10 g Adjust pH to 7.5 with sodium hydroxide.

L broth see LB Medium In order to obtain sporulation of B. thuringiensis var. kurstaki, the GYS medium according to Yousten and Rogoff (A.A. Yousten and M.H. Rogoff5’) (1969) (g/l ’) is used: glucose 1 yeast extract (Difco) 2 (NHOzSOu 2 K2HPOu 0.5 MgSO<,.7H2O 0.2 CaCl2.2H2O 0.08 MnS0„.H20 0.05 (’’Difco is a Trade i4ark).

Before autoclaving, the pH is adjusted to 7.3 with potassium hydroxide.

Characterization of microorganisms used in the present invention: 1. HDl-ETHZ 4449 is a Bacillus thuringiensis subspec. kurstaki strain and is characterized first by its immunological reaction against its flagellum antigen. HDl-ETHZ 4449 belongs to the 3a, 3b serotype (A. Krieg31’)· Secondly, HDl-ETHZ 4449 is characterized by its specific pattern in a Southern blot experiment done as described below in III.5.b. Total DNA from the strain is isolated, digested to completion with the Hindlll restriction enzyme and the fragments thus obtained are separated according to size on agarose gel and transferred to a nitrocellulose sheet. The radiolabeled EcoRI fragment Pos. 423 to Pos. 1149 (Table 2) hybridizes specifically to 3 fragments with a size of 6.6 Kb, 5.3 Kb and 4.5 Kb respectively. ίθ 2. HB 101 is a hybrid between Escherichia eoli K12 x Escherichia eoli B. It is a good host for large scale DNA purification. It is used for transformation experiments and CaCl2 competent cells are commercially available from Gibco AG, Basel, Switzerland, Catalogue No. 530 8260 SA. 3. JM 103 is an Escherichia eoli K-12 and is commercially available from Pharmacia P-L Biochemicals, Catalogue No. 27-1545-xx (1984).

The E. eoli strains HB 101, and JM 103 are described in Maniatis et al. (T. Maniatis et al.**’); Strain Genotype HB101 JM103 F , hsdS2o (r^, m^), recAia, ara-14, proA2,lacYi, galK2, rpeL2o(Smr), xyl-5, mtl-1, βυρΕι,ι,, λ A(lac pro),thi, strA, supE, endA sbcB, hsdR, F’traD36, proAB, lacl^, ΖΔΜ15 In the following example, yeast refers to Saccharomyces cerevisiae.

Example I. Plasmid DNA preparation; Plasmid DNA is prepared as described below according to White and Nester (F.F. White and E.W. Nester2) 1.1. B. thuringiensis plasmids E. eoli HB 101 cells are grown in 1 1 LB medium with shaking for 12-14 hours at 37°C and prepared as described by White and Nester (F.F. White and E.W. Nester2’). After harvest, the cells are resuspended in alkaline lysis buffer and kept at 37°C for -30 minutes. The clear lysate thus obtained is neutralized by addition of 2 M Tris*HCl (pH 8). The chromosomal DNA is precipitated ii by addition of SDS and NaCl. The lysate is placed on ice and chromosomal DNA is removed by centrifugation. The plasmid DNA which is now in the supernatant is precipitated with 10 % PEG 6000. After overnight storage at 4**C the plasmid DNA is resuspended in 7-8 ml of TE. The plasmid DNA obtained from 1 1 culture is further purified on two CsCl gradients. Solid CsCl is added (8.3 g of CsCl to 8.7 ml of supernatant). After the addition of ethidium bromide (Sigma; final concentration 1 mg/ml supernatant) the solution is transferred to 13.5 ml Quick Seal polyallomer tubes (Beckman) and centrifuged in a Beckman Ti50 rotor for 40 hours at 40*000 rpm. Two fluorescent bands can be visualized vith long wave UV (366 nm). The lower band contains aupercoiled plasmid DNA which is collected by puncturing the tube from the side with a 2 ml syringe (18G needle). The ethidium bromide is removed by extracting 5 times vith equal volumes of isopropanol (saturated with CsCl) and the product is transferred to 30 ml Corex tubes. (Corex is a registered Trade Mark). 2.5 volumes of TE is added and the DNA is precipitated with ethanol. The solution is then kept for 12-15 hours at -20°C. The precipitated DNA is collected by centrifugation in a Sorvall HB-4 rotor for 30 min at 12*000 rpm at 0°C and redissolved in 200 ul of TE. (E. coli JM 103 can also be extracted and used idn the same way). 1.2. E. coli plasmids The cells from 100 ml culture (LB medium) are harvested by centrifugation (Sorvall, GSA rotor, 10 min at 6000 rpm, 4’C), resuspended in 100 ml TE (10 mM Tris«HCl, 1 mM EDTA, pH 8.0) and centrifuged again under the above conditions. The cell pellet is resuspended in 3 ml Tsuc [50 mM Tris«HCl, pH 7.5, 25 X (v/v) sucrose] and transferred to SS-34 polypropylene Sorvall tubes. All subsequent steps are carried out on ice: 0.3 ml of lysozyme solution (10 mg/ml, purchased from Vorthington, 11*000 U/mg) is add'ed, after 5' min. 1.2 ml EDTA (500 mM, pH 8.0), and after another 5 min 4.8 ml detergent [0.1 Z Triton X-lOO(Merck) (Triton is a registered Trade Mark), 50 mM EDTA, 50 mM Tris HCl, pH 8.0 ] are added. AFter 5 min the lysate is centrifuged in a precooled SS-34 rotor for 40 min at 4°C. The supernatant is careΪ2 fully removed and, after addition of solid CsCl, is purified on the CsCl gradients as described for B. thuringiensis plasmid DNA. 50-100 pg of hybrid plasmid DNA are recovered from a 100 ml culture.

II. 6-Endotoxin antigen and goat antibodies II.1. Preparation of 6-endotoxin crystal antigen: Bacillus thuringiensis (var.kurstaki HD1, strain ETHZ 4449) is cultivated in a Fernbach flask on the medium according to Yousten and Rogoff (A.A. Yousten and M.H. Rogoff^) as described above, however with increased concentration of glucose (0.3 % instead of 0.1 %). Incubation time is 4-5 days at a temperature of 30°C. The colonies are harvested immediately after sporulation (B. Trumpi ').

In order to separate parasporal bodies and spores from each other, 9) the method according to Delafield et al. (F.P. Delafield et al. ) is used. a) Separation of spores and crystals: Autolyzed cultures are suspended in 1 M NaCl/0.02 M potassium phosphate buffer (pH 7.0) containing 0.01 % Triton-X-100 (Merck). The sediment is washed repeatedly. The particles are then washed. Residual cells are removed from the suspension.

The remaining crystals and spores are centrifuged and washed three times in 0.02 M phosphate buffer (pH 7.0)/0.01 % Triton-X-100. The suspension is added to a cylindrical separatory funnel containing 105 g of a 20 % (w/w) aqueous solution of sodium dextran sulfate 5000 (Sigma), 13.2 g of solid polyethylene glycol 6000 (Merck), 3.3 ml of 3 M phosphate buffer (pH 7.0), and 7.5 g of NaCl. After shaking to dissolve the solids, the volume is adjusted to 600 ml by adding a well-shaken solution of the same composition, but without bacterial particles. The complete mixture is vigorously shaken and placed at 5°C for 30 min.

The lower phase contains a mixture of crystals and spores. The upper phase contains most of the spores initially present, but very few crystals. The upper phase is drawn off. The supernatant solution is poured back into the separatory funnel and the extraction is repeated. After the tenth extraction, the crystals in the lower phase are virtually free of spores and are collected by centrifugation. The spores and crystals are both washed five times in cold distilled water. The crystals are stored at -5°C as suspensions in water. b) Solubilization of the crystals: An aliquot of the crystal suspension is centrifuged for 10 min. at 12’000g. The sediment thus obtained is resuspended in 0.05 M carbonate buffer and 10 mM dithiothreitol (DTT, Sigma; mixture of carbonate buffer and DTT defined in the following as carbonate/DTT) in a concentration of 5 mg of sediment/ml of buffer/DTT mixture.

After incubation for 30 min. at 37°C, the unsolved particles are separated by centrifugation for 10 min. at 25'000g. The supernatant is dialysed against carbonate buffer and subsequently tested for protein content and for activity in a biotest.

For storage, the protoxin solution is partitioned to portions which are deep frozen. At thawing, protein free from DTT has a gel-like consistency. Complete solution of the protein is obtained with addition of 1 mM DTT. c) Inactivation of crystal-bound proteases: Serine proteases and metal proteases of the crystal suspensions (Chestukhina et βΐ’θ’) are inactivated by addition of diisopropyl fluorophosphate (DFP, Serva) and EDTA in the following procedure: The crystals are suspended in 0.01 M phosphate buffer, pH 8.0, and 1 mM EDTA in a concentration of 5-10 mg of crystals/ml of buffer/EDTA mixture. The suspension is sonicated until monodisperse suspension is obtained (tested with light-optical microscope) .

In a fume cupboard, with common precautionary measures, 1 mM DFP is added to the suspension. The tube containing the suspension is airtightened and vigorously shaken. After incubation at RT overnight, the inactivated suspension is dialysed up to balance against H2O and 1 mM EDTA.

II.2. Immunisation of goats: Antigen is prepared from crystals of B.thuringiensis serotype H-3 by dissolving the crystals in carbonate/DTT, dialysing the solution thus obtained against carbonate buffer and 1 mM DTT and purifying the antigen by steril filtration using a 0.45 pm millipore filter.

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

At the experimental station of CIBA-GEIGY AG in St. Aubin (Fribourg, Switzerland), two goats are immunised with H-3 protoxin. Each of the goats is treated by intracutaneous injection of 0.5 mg of antigen and by subcutaneous injection of 1.5 ml of Pertussis (Behring), the latter being made in order to increase immunological reaction. The whole treatment is made on days 0, 28 and 76. Blood samples are taken on days 35, 40, 84 and 89, taking amounts of 5 ml on day 35 and 80 ml on each of the other days.

When the blood has coagulated, the sera are incubated at 56°C for 30 min., thus inactivating the complement. The sera are stored at -20°C. 11.3. Purification and P** l)-labeling of the goat H3 antibodies: a) Purification of immunoglobulin: The IgG (immunoglobulin G) fraction of goat anti H3 serum is purified by ammonium sulfate precipitation followed by chromatography on DEAE cellulose and analysis on Ouchterlony immunodiffusion plates (0. Ouchterlony’") according to the method 12) described by Huber-Lukac (H. Huber-Lukac Thus, 30 ml of 3.2 M ammonium sulfate are added dropwise to 15 ml of goat anti H3 serum in 15 ml of PBS1 (0.01 M phosphate buffer, pH 7.4, and 0.8 % NaCl). The mixture is allowed to rest for 15 min. After the mixture is centrifuged (10,000g, 20 min.), the sediment is distributed in 7.5 ml of PBS1, dialysed at 4°C three times against 1000 ml of PBS1, then dialysed against 1000 ml of 0.01 M phosphate buffer, pH 7.8, and centrifuged (3000g, min.).

The supernatant is transferred to a 30 ml column of DEAE and eluated at RT with 0.01 M phosphate buffer, pH 7.8 (rate of flow: 20-80 ml/h). The fractions of the first peak are pooled and lyophilized. Average yield of IgG is 180 mg/15 ml of serum. The IgG fraction is checked for degree of purity by immunodiffusion (0. Ouchterlony"’) against anti-goat-IgG antibodies and against anti-goat-serum antibodies of rabbits (Miles Laboratories).

The antibodies are further purified by absorption on a sepharose (Pharmacia) column to which the H3-protoxin is bound. Coupling of the protoxin to CNBr-aepharose (Pharmacia) is carried out according to the supplier’s instructions as summarized below: g of CNBr-activated Sepharose 6MB (’’Sepharose" is a Trade Mark) is weighed out for about 3 ml of final gel volume. The gel is washed and allowed to reswell on a sintered glass filter by using 200 ml of 1 mM HCl. The H3 antigen protein is obtained according to part II.l.c above, is dissolved in 0.1 M NaHC03 and 0.5 M NaCl. 1 ml of gel contains -10 mg of protein. The gel suspension and the antigen are mixed at RT for 2 hours. The excess protein is eliminated by washing vith 0.1 M NaHC03 (pH 8.3), 0.5 M NaCl and 0.5 M ethanolamine. A further washing is done with 0.1 M NaHCOj (pH 8.3) and 0.5 M NaCl, followed by 0.1 M CHjCOONa (pH 4) and 0.5 M NaCl. The last washing step is done again with 0.1 M NaHCOj and 0.5 M NaCl. The Protein-Sepharose conjugate is now ready for packaging into a Pharmacia column K9 (Pharmacia). The IgG can now be effectively purified on that column.

Specifically bound antibodies are eluted vith 3M KSCN and dialysed against PBS2 (10 mM sodium phosphate pH 7.8, 0.14 M NaCl). The antibodies are radiolabeled vith X25I using the 13) chloramin-T method (Amersham Buehler Review ). b) Iodine labeling: 125 mCi sodium iodine- I is given to a tube containing 100 μΐ 0.5 M phosphate buffer (pH 7.2). Under continous stirring, 5 pg of the protein solution (0.5 mg/ml in TBS) 50 pg chloramine T in 0.05 M phosphate buffer (pH 7.2) are added. After incubation for 1 minute at RT, 120 pg NajSjOj are added. The free iodine is separated from the labeled protein on a 0.9x12 cm column packed with Sephadex G-25 (Sephadex is a Trade Mark ) .

In order to avoid absorption of the labeled protein to the column, 0.5 ml BSA (100 mg/ml) is first run through the packed column. After this run, the labeled material is quantitatively transferred on top of the column and eluted with the phosphate buffer (pH 7.2). The fractions (1 ml) are collected until the whole protein is eluted.

III. Cloning of the ^-endotoxin gene: A partial Sau3A DNA library from B. thuringiensis (var.kurstaki) HD1, strain ETHZ 4449 plasmid DNA is essentially prepared according 14) to Maniatis et al. .(T. Maniatis et al. ) and subcloned into the Bam HI site of pBR322 as vector DNA as described below: III.l. Partial digestion of high-molecular-weight B. thuringiensis DNA Digestions with Sau3A are done in such a way that ethidium bromide coloration of the cleaved DNA on agarose gel is mostly present in the 2-10 Kb size range. This is achieved by using the method 14) described by Maniatis et al. (T. Maniatis et al ). The fractionation of the partially cleaved DNA is done on preparative agarose gel as described below in part IV.2. or preferably on a NaCl salt gradient. The linear salt gradient is made between 5 and 20 % NaCl in TE buffer and is run at 35 Krpm for 3 hours in a SW 40 Ti Beckman rotor. The collected fractions are precipitated by addition of ethanol and analysed on an agarose gel. pg of pBR322 plasmid are digested with 10 units of Bam HI endonuclease in 50 pi of 10 mM Tris-HCl, pH 7.4, 100 mM NaCl and 10 mM MgCli at 37°C for 1-2 h. The phosphatase treatment of the cleaved DNA is done as follows: 10’ pg of the DNA are dissolved in 50 pi of Tris-HCl, pH 8, and intestine alkaline phosphatase (Boehringer) is added in an amount of 3 units/pg of DNA. After incubation for 30 min at 37°C, the DNA is phenolized two times and finally extracted with chloroform. After ethanol precipitation, the DNA is resuspended in 20 pi of H2O and is used in the ligation reaction with partial Sau 3A derived fragment. The reaction is set up as follows: to 0.4 pg of Sau 3A digested DNA in 10 pi of H20, 0.100 pg of the phosphatase-treated vector are added. The ligation reaction is brought about with addition of 50 mM Tris-HCl, pH 7.4, mM ATP, 10 mM MgCl2 and 15 mM DTT followed by addition of 20 units of T4 DNA ligase (Biolabs). After incubation at 15°C overnight, the DNA is used to transform E.coli HB101 competent cells.

Alternatively to the method of preparing a partial Sau 3A library, a DNA library from B.thuringiensis (var. kurstaki HD1, strain ETHZ 4449) is prepared by digesting the plasmid DNA to completion with Bam HI and partially with Clal. Subsequently it is cloned into pBR322 between the Clal and Bam HI sites. 111.2. Transformation by the calcium chloride procedure: The preparation of competent cells is done by treating cells growing to a density of 5 χ 107 cells/ml with calcium chloride (Maniatis et al’5’). The transformation is achieved by addition of the DNA to these cells after which said cells are maintained at 42eC for 3 minutes, diluted vith 1 ml LB medium, incubated at 37°C for 60 minutes and spread on selective media by using common procedure (T. Maniatis et al’5’). 111.3. Preparation of cell crude lysates The colonies containing the δ-endotoxin gene will express a protein vith a biological activity similar to the purified and dissolved toxin crystals (H.E. Schnepf et al.’"). They are therefore immunologically screened with goat antibodies (the H3 antibodies) prepared against the B.thuringiensis var.kurstaki crystal protein. The bacterial colonies are grown individually in 5 ml LB-medlum in the presence of ampicillin. Ten cultures are pooled, harvested, washed in 10 mM NaCl and finally the cells are lysed in 2 ml of 400 mM NaCl, 0.1 M NaOH, 1 mM PMSF. After 20 min incubation at room temperature the lysates are neutralized by the addition of 20 μΐ of 2 M Tris*HCl, pH 7.0. After centrifugation in a SS34 Sorvall rotor (20 min, 10'000 rpm) the lysates are extensively dialysed against TBS (10 mM Tris«HCl, pH 7.5, 0.14 M NaCl). 111.4. Radioactive immunological screening of the cell extracts The extracts are tested for the presence of the δ-endotoxin antigen radioimmunologically using the plastic well method described by Clarke et al. (L. Clarke et al.’2’). Single plastic wells are coated overnight with 150 μΐ of purified H3 goat antibodies (10 pg/l ml) in 10 mM Tris'HCl, pH 9.3, and kept overnight at 4’C. The wells are washed three times with TBS/Tween, (TBS + 0.5 Z Tween 20) (Tween is a registered Trade Mark ) filled with 150 μΐ of the bacterial extract and incubated for 6 hrs at 37°C. After washing, the wells are filled with 150 jil of [’251] labelled rabbit anti-goat H3 antibodies (60 ng, 105 cpm) in TBS containing 25 Z horse serum and incubated overnight at room temperature. After washing with TBS/Tween, the wells are counted in a scintillation counter. 111.5. Restriction map and localization of the 6-endotoxin gene on pK 19 recombinant plasmid a) Restriction mapping: The restriction map of pK 19 DNA clone obtained from the immunological screening procedure described above for the Sau3A library of B. thuringiensis HD1, ETHZ 4449 is deduced from single, double and triple digests of the plasmid DNA with various restriction enzymes. The digestions are all done according to the instructions of the enzyme suppliers. Thus, in short, the DNA (1 pg/50 μΐ) is dissolved in the buffer suitable for the pertinent restriction endonuclease and after 1-2 hours incubation at 37°C, the digested DNA is loaded on agarose gel and electrophoresed. If treatment with a second enzyme is necessary under conditions which are incompatible with the first enzyme (wrong buffer, for instance), the DNA is first extracted with a 1:1 mixture of phenol and chloroform, precipitated in ethanol and then exposed to conditions incompatible before (such as, for instance, the buffer required for the second enzyme). b) Southern transfer: The coding sequence for the 6-endotoxin is localized by hybridizing radiolabeled RNA isolated from sporulating B.thuringiensis cells to specific restriction fragments of pK 19. This is accomplished by the transfer technique described by Southern ( Southern3): DNA fragments which have been separated according to size by electrophoresis through an agarose gel are denatured, transferred to a nitrocellulose filter, and immobilized. The relative positions of the DNA fragments in the gel are preserved during their transfer to the filter. The DNA attached to the filter is then hybridized to 3ZP-labeled RNA, and autoradiography is used to locate the position of any bands complementary to the radioactive probe.

The transfer of DNA from agarose gels to nitrocellulose paper is done as described in Maniatis et al. 19) c) Hybridization of Southern filters: The prehybridization and hybridization are done according to 20) Maniatis et al. (T. Maniatis et al. ) with the following modifications: The baked filters are put into a heat-sealable plastic bag. 0.2 ml of polyhybridization mixture is added per square cm of nitrocellulose filter. prehybridization mixture: 4 x SSC % formamide 0.2 % SDS 20 mM EDTA mM Potassiumphosphate (pH 7.2) x Denhardt’s solution 100 pg/ml calf thymus denaturated 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 (50 pl/cm2 of nitrocellulose filter). hybridization mixture: as the prehybridization mixture but now containing the 3ZP labeled denaturated RNA probe (106-107 cpm/filter) as prepared according to part III.5.d below.

The bags are usually kept at 37°C overnight. After the hybridization, the filters are washed for 15 minutes in 2 x SSC and 0.1 % SDS at RT, said washing procedure being repeated two times and then the filters are washed in O.lxSSC and 0.1 % SDS for 60 minutes. The filters are dried on Whatman 3MM paper and prepared for autoradiography. (Whatman is a Trade Mark) . d) Isolation and radioactive labeling of RNA from B. thuringiensis var. kurstaki B. thuringiensis var.kurstaki cells are grown on Rogoff medium containing 0.1 % glucose (A.A. Yousten and M.H. Rogoff z). 500 ml cultures are shaken in a 2 1 Erlenmeyer flask at 300 rpm and 30°C. During growth the pH drops from 7 to about 4.8, then rises again to 7. At this point the cells start to clump. The time point at which the pH reaches again its original value is taken as the starting point of sporulation. The cells are further grown for 5-6 hours. Rifampicin (50 gg/ml) is added and the cells are shaken for 10 min. The ice-chilled cells are harvested and resuspended in 10 ml 4 M guanidium thiocyanate, 0.5 % sarcosyl, mM sodium citrate pH 7 and 0.1 M 2-mercaptoethanol. The cells are frozen at -80eC and then disrupted in a French Press. After centrifugation of the extract for 15 min at 15*000 rpm (Sorvall SS34 rotor) 0.5 g/ml CsCl is added to the supernatant, vhich is then overlayed on a 5.7 M CsCl, 0.1 M EDTA cushion in a Beckmann 60Ti centrifugation tube. After centrifugation at 38*000 rpm for 20 hours, the RNA pellet is washed with ethanol, dried, dissolved in 7 M guanidium hydrochloride and precipitated with ethanol. Total RNA from eporulating cells is dephosphorylated and labeled with [33p] ATP and T4 polynucleotide - 21) kinase according to a standard procedure (N. Maizels ).

RNA Labeling with Polynucleotide Kinase: About 1 pg RNA is subjected to mild alkaline hydrolysis by heating in 50 mM Tris-HCl (pH 9.5); time and temperature of incubation: 20 min at 90°C.

This hydrolysis generates free 5’hydroxyl groups which are available as substrates for polynucleotide kinase. Hydrolysis is in a sealed capillary in a total volume of 4 pi. Kinase labeling is in 10 pi reactions containing 50 mM Tris-HCl (pH 9.5), 10 mM MgCl2, 5 mM dithiothreitol, 5 % glycerol, and lpM [y32P]-ATP labeled at a specific activity of 6000 Ci/mmole. Each reaction contains about 1 pg of RNA and 2 pi T4 polynucleotide kinase and continues for 45 min at 37°C. This generates RNA with a specific activity of about 3 χ 107 Cerenkov cpm/pg. The RNA is separated from the [y32P]-ATP by three ethanol precipitations in the presence of 5 pg tRNA carrier. 111.6. Identification of clones coding for the 6-endotoxin in the Bam ΗΙ/Cla I plasmid DNA library cloned in pBR322: The pK 25 serie a) In situ hybridization of bacterial colonies: 22) Colony hybridization (M. Grunstein and D. Hogness ) is accomplished by transferring bacteria from a master plate to a nitrocellulose filter. The colonies on the filter are then lysed and the liberated DNA is fixed to the filter by baking. After hybridization to a 32P-labeled probe, the filter is monitored by autoradiography. A colony whose DNA gives a positive autoradiographic result may then be recovered from the master plate.

A 6-endotoxin gene internal DNA fragment is used to screen the Bam ΗΙ/Cla I plasmid DNA library cloned in pBR322. The procedure 23) is described in Maniatis et al. (T. Maniatis et al. ).

The filters are hybridized to a 3zP-labeled probe prepared as described below in part III.6.b.

Wrap the filter in Saran Wrap and apply to X-ray film to obtain an autoradiographic image.(Saran" is a registered Trade Mark) .

The positive clones are isolated from the master plate and are analysed. They contain the DNA sequence coding for the toxin as well as the DNA flanking region as found immunologically (see part III.4. above) by restriction mapping (see part III.5. above), and in an in vivo biotest (see part III.7 below). These clones are now called pK 25-i with i being 1 to 7.

DNA Nick translation: An internal DNA fragment of the δ-endotoxin gene is radiolabeled by following the procedure described below: mixture: 3 pi DNA (1 pg) 1.5 pi of nick translation buffer (lOx concentrated: 0.5 M Tris, pH 8, 0.05 M MgClj) 1.5 pi 2.5 mM d guanosine triphosphate (GTP) 1.5 pi 2.5 mM d cytidine 5’-triphosphate (CTP) 1.5 pi 2.5 mM d thymidine triphosphate (TTP) 2.5 pi Hz0 0.75 pi BSA (1 mg/ml) 1.5 pi 100 mM P-mercaptoethanol mix and add to 100 pCi dried [32P-a]-ATP [10 mCi/mmol] mix well add 0.75 pi of a 1x10 solution of DNase 1(1 mg/ml) in Nick translation buffer incubate at RT for 1 minute, transfer to ice add 1 μΐ E.coli polymerase I (Biolabs; final volume: 15 μΐ) incubate at 15°C for 3 hours heat at 65°C for 10 min; add 35 μΐ 50 mM EDTA and 10 μΐ tRNA stock (100 pg) separate the non-incorporated nucleotides by chromatography on a small Sephadex G50 column. c) Subcloning of the complete δ-endotoxin gene in the pUC8 vector: the pK 36 clone The pUC8 vector (New England Biolabs) is digested to completion with the restriction enzymes Hindi and Pstl and by alkaline phosphatase treatment (see part III.l above). The Hpal/Pstl fragment from pK 25-7 (see part III.6.a above) coding for the δ-endotoxin gene (Table 2) is ligated to the vector DNA and transformed into E.coli HB101 cells. One of the correct transformed clones is called pK 36.

Ill. 7 . Biotest to evaluate B.thuringiensis toxins: To the cleared lysate as obtained according to part III.3. above, ammonium sulfate is added to a concentration of 30 % saturation. The precipitate is dissolved in 2 ml 50 mM sodium carbonate, pH 9.5, and dialysed against the same buffer. As control E.coli extracts harboring the vector DNA without B.thuringiensis DNA is prepared.

The E.coli cell extracts are sonified, 4 concentrations according to the toxin content In the extracts prepared and 0.1 % (v/v) wetting agent admixed. Leaf discs from cotton plants which are grown under controlled conditions (25°C, 60 % relative humidity) in a growth chamber are dipped in the E.coli cell extract suspensions. First instar larvae of Heliothis virescens (30 larvae per concentration) which were standardized in a fitness test are then put onto the dried leaf discs and individually incubated at 25°C for 3 days. Mortality is measured in % using the criteria dead and alive. The extracts causing mortality therefore possess bioinsecticidal activity originating from the cloned B. thuringiensis DNA.

IV. DNA sequencing; The DNA fragments encoding the δ-endotoxin gene were sequenced on 24) both strands according to F. Sanger et al. using the M13 system ,, m · 25h (J. Messing ).

IV.1. Cloning of the 6-endotoxin gene into Ml3 replicative form DNA From the restriction mapping of the cloned 6-endotoxin gene and the Southern blotting analysis thereof it can be seen that the gene is located in two DNA fragments of pK. 36: Hpa I (position 0 on the sequence) to Hind III (position 1847) and EcoRI (position 1732) to Pstl (position 4355). The first fragment is cloned in M13 mp8 (New England Biolabs) between the single Hindi site and the single Hindlll site in a reaction similar to the ligation process described above.

IV. 2. Generation of a sequential series of overlapping clones: The Bal 31 method (M. Poncz et al; ') is used to shorten the Hpal-Hindlll DNA fragment coding for the 5' end of the gene. Said shortening is made as follows: The fragment is cloned in M13rap8 between single Hindi and Hindlll. 10 mg of the replicative form of the DNA is linearized with restriction endonuclease Hindlll and subjected to the action of the endonuclease Bal 31 in 100 μΐ of 600 mM NaCl, 12 mM CaCl2, 12 mM MgCl2, 20 mM Tris-HCl, pH 8, and 1 mM EDTA. The mixture is preincubated at 30°C for 5 min. Subsequently, 5 units of Bal 31 are added. Immediately after said addition and after 2, 4, 6, 8, 10 and 12 min., 13 μΐ are removed each time. Immediately after taking-off, 25 μΐ of phenol and 40 μΐ of TE buffer are added in order to stop further reaction. The mixture is centrifuged, extracted with chloroform and then precipitated with ethanol. The DNA precipitate thus obtained is resuspended in 20 μΐ of 100 mM NaCl, 20 mM Tris-HCl and 10 mM MgCl2 and digested with a second enzyme found on the other site of the originally cloned fragment, which second enzyme in the present case is Bam HI. After size separation in agarose gel, the shortened fragments are stained with ethidium bromide and visualized under long wave UV light at 366 nm. The piece of agarose containing the shortened fragments is cut out from the gel, liquified at 65°C, adjusted to 500 mM NaCl and incubated at 65ώϋ for 20 min. One volume of phenol (equilibrated with 10 mM Tris«HCl pH 7.5, 1 mM EDTA, 500 mM NaCl) is added. The aqueous phase is reextracted twice with phenol and once with chloroform. The DNA is precipitated with 2.5 volumes of cold absolute ethanol and collected by centrifugation. The DNA pellet is washed with cold 80 % ethanol and then dried in vacuum. The DNA is resuspended in 20 μΐ of TE.

The fragments are successively shortened by 200-300 bp at each standpoint and have a single Bam HI site on one side of the fragment and a blunt end on the other. These fragments are cloned in a M13mp8 vector linearized after double digestion with Bam HI and Hindi as described above in part IV.1.

By the process described above, the DNA sequence is obtained as one DNA strand starting at the Hindlll site and going toward the BamHI site. Strategy followed to sequence the complementary strand of the same DNA fragment and the endonuclease restriction sites used for the sequencing, that is first for the shortening of the second DNA fragment, the Eco Rl/Pstl fragment and its sequencing is given in 26) figure 1 as modified from M. Poncz et al. and on table 1. 26) Fig. 1 as modified from M. Poncz et al. . Construction of the deletion mutant library. Steps: 1, the insert (thick lines) is cloned into the cohesive end A site in >113; 2, after linearization at site B, the phage DNA is digested with BAL-31 for various times [broken lines, extent of BAL-31 digestion into insert (thick lines) and >113 (thin lines)]; 3, the digest is cleaved at site A, and the BAL-31-induced continuum of inserts is isolated, resulting in a family of differently sized fragments each of which has a BAL-31-induced blunt end and a cohesive end A; 4, the fragments are subcloned into >113 so that blunt end is proximal to the primer site P used for DNA sequence analysis.

X and C = blunt end-s IV.3. Transformation of E. coli strain JM 103: E. coli JM 103 cells are kept on M9 maximal medium.

The transformation is made as follows: 1. inoculate into 2xYT a single colony of E. coli JM 103; keep over night at 37° with stirring 2. inoculate 40 ml of 2xYT with 200 pi of the culture obtained according to step I 3. keep at 37° with stirring until 0D^ $ θ is 0.5 4. keep 5 min on ice . centrifuge at 6000 rpm for 5 min in a SS34 Sorvall rotor (cool down before using) 6. suspend the cells in 20 ml of 50 mM sterile, ice-cold CaCl^ (CaC^ should be freshly prepared) 7. keep 40 min. on ice 8. centrifuge at 6000 rpm for 5 min in a SS34 Sorvall rotor 9. suspend the cells in 3 ml of ice-cold CaC^ . to 200 pi of cells obtained according to step 9 add 1-5 pi of DNA or 7-15 pi of ligase formulation 11. keep 30 min. on ice 12. keep at 42°C for 3 min. 13. add 200 pi of cells obtained according to step 1 14. bring top agar to the boil and keep at 42°G . tubes are filled with 3 ml of top agar, 30 ul of X-GAL (20 mg/mldimethylsulfoxide) and 30 yl of IPTG (20 mg/ml 1^0) 16. mix thoroughly and transfer immediately onto lxYT plates warmed-up before using 17. let dry disks for ca. one hour 18. Turn upside down and incubate at 37°C.

The plaques thus obtained are suitable for further treatment. Controls: - 200 μΐ competent cells without exogenous DNA + 1 μΐ M13 mp8 (replicative form of DNA, 10 ng) + 200 μΐ of competent cells.

IV. 4. Preparation of the replicative form of recombinant phage DNA: 1. transfer carefully separated single white plaque into 9 ml of 2xYT and 1 ml of the culture obtained according to step 1 of part a) above and keep at 37°C for seven hours 2. centrifuge 10 min. at 4000 rpm 3. keep supernatant over night at 4°C 4. inoculate 10 ml of supernatant obtained according to step 3 and 10 ml of the culture obtained according to step 1 of part a) above into 1 1 of 2xYT . shake at 37°C for 4{ hours 6. centrifuge at 5000 rpm for 15 min. 7. suspend the cells in 10 ml of 10 % sucrose in 50 mM Tris-Cl, pH 8 and cool down 8. transfer into 30 ml-tubes for centrifugation 9. add 2 ml of freshly prepared lysozyme (10 mg/ml 0.25 M Tris-HCl, pH 8) . add 8 ml of 0.25 M EDTA, mix cautiously 11. keep on ice for 10 min. 12. add 4 ml of 10 % SDS (or 1.6 ml of 25 % SDS), mix with glass rod 13. add 6 ml of 5 M NaCl (final concentration: 1 Μ), mix cautiously 14. keep on ice for one hour . centrifuge at 20 000 rpm for 40 min. in a SS34 Sorvall rotor 16. take supernatant and add an amount corresponding to */io of the volume of 5 M NaCl and 15 ml of 30 % PEG in TNE 17. keep at 4°C for two hours or overnight 18. centrifuge at 8000 rpm for 15 min. 19. take pellets and transfer to 18 ml of TE, pH 8 . add 18 g of CsCl (lg/ml) 21. transfer either to Ti-50-tubes and add 0.4 ml of ethidium bromide (10 mg/ml) or to Ti-60-tubes and add 1.2 ml of ethidium bromide (10 mg/ml) 22. fill up with CsCl solution (1 g CsCl + 1 ml TE) 23. centrifuge at 35 000 rpm at 20°C for 36-48 hours 24. take the lower bands below UV (366 nm) . extract 3-4 times with butanol saturated with water 26. dialyse 3 times at 4°C against 1 1 of sterile TE each time IV.5. Preparation of the single stranded template DNA: night 1. shake E.coli JM 103 cells in 5 ml of 2xYT medium at 37°C overC ο ti J 2. add two drops of solution obtained according to step 1 to 25 ml of 2xYT medium 3. fill tubes with 2 ml of solution each and add one plaque per tube as obtained according to part IV.3. above 4. shake for 5| hours at 37°C . transfer contents of tubes into Eppendorf tubes and centrifuge for 5 min. 6. transfer 1 ml of supernatant into fresh Eppendorf tubes 7. add 200 μΐ of 20 % PEG 6000/2.5 M NaCl 8. keep at RT for 15 min. 9. centrifuge for 5 min. . discard supernatant and centrifuge again briefly 11. withdraw supernatant cautiously by suction with extended pasteur pipette 12. to the remaining, add 100 μΐ of TE and 50 μΐ of phenol mix for 10 sec. (by vortex); allow to stand for 5 min.; mix for 10 sec. (by vortex); centrifuge for 1 min. 14. transfer aqueous phase into fresh Eppendorf tube . add 500 μΐ of ethylene ether, mix (vortex) and centrifuge for 1 min. 4 ¢-)1 16. withdraw ether by suction, keep tubes unclosed for 10 min. (if, after this treatment, aqueous phase is very turbid, air should be blown through with the pasteur pipette, until solution is clear) 17. add 10 gl of 3 M sodium acetate and 250 μΐ of ethanol 18. keep at -80°C for 30 min. 19. centrifuge for 5 min. . wash with 80 % ethanol 21. centrifuge for 5 min. 22. withdraw supernatant with extended pasteur pipette 23. keep tubes unclosed for 15 min. 24. dissolve pellet in 25 μΐ of TE . transfer 2 μΐ of pellet solution to 0.6 % agarose gel.

IV.6. Sequencing reaction The DNA sequence analysis on the template DNA obtained according to part IV.5. above is done according to the inanual M13 cloning and DNA sequencing system, published by New England Biolabs.

An analysis of the complete DNA sequence shows that only one open reading frame long enough for a protein with a MW of 130 622 and coding for 1156 amino acids is found. The N-terminus of the protein is localized at 156 bp downstream from the Hpal site and the last amino acid of the C-terminus is coded by the codon started at nucleotide 3618. The DNA sequence between the Hpal site and the Pstl site is given in tab. 2. The deduced amino acid sequence is given in tab. 3.

V. Expression of the δ-endotoxin gene in yeast cells V.l Introduction of a Ncol site before the first AUG of the gene In order to combine the protein coding sequence of the B. thuringiensis toxin gene with the yeast PH05 promoter (described in European patent specification No. 100,561), the DNA sequence around the toxin gene is modified. The modification is achieved by oligonucleotide-directed mutagenesis with the single-stranded phage vector M13mpl8 containing a 1.5 Kb BamHI-SacI insert coding for the 5' region of the toxin gene. 200 ng of insert are obtained’from plasmid pK 36 by digesting 3 pg of plasmid DNA with BamHI and Sacl and isolating of the fragment using standard techniques described above. 100 ng of replicative form (RF) of Ml3mpl8 is digested with the same enzymes, the DNA is phenolized and precipitated by addition of ethanol and then ligated with 200 ng of insert DNA from above. After transfection of E. eoli, six white plaques are picked and analyzed by restriction digestion using BamHI and Sacl. One correct isolate is picked and is called M13mpl8/Bam-Sac.

An oligonucleotide with the sequence (5') GAGGTAACCCATGGATAAC (3') is synthesized by common procedures using an APPLIED BIOSYSTEM DNA SYNTHESIZER. This oligonucleotide is complementary to the sequence in M13mpl8/Bam-Sac from position 141 to position 164 of the protoxin gene (Table 2) and has a mismatch at positions 154, 155. The general procedure for the mutagenesis is that described in 27) J.M. Zoller and M. Smith (J.M. Zoller and M. Smith ). Approximately five pg of single-stranded phage mpl8/Bam-Sac DNA is mixed with 0.3 pg of phosphorylated oligonucleotide in a volume of 40 pi. The mixture is heated to 65°C for 5 min., cooled to 50°C, and slowly cooled to 4°C. Next, buffer, nucleotide triphosphates, ATP, T4 DNA ligase and large fragment of DNA polymerase are added and incubated 27) overnight at 15°C as described (J.M Zoller and M. Smith After agarose gel electr.ophoresis, circular double-stranded DNA is purified and transfected into E.coli strain JM103. The resulting plaques are screened for sequences which hybridize with 32P-labeled oligonucleotide, and phage are analyzed by DNA restriction endonuclease analysis. Among the resulting phages, clones will be ones which have correctly now a C at position 154 and 155 instead of the I In the pK 36 DNA. This phage is called M13mpl8/Bam-Sac/Nco.

V.2. Ligation of the δ-endotoxin gene to the yeast PH05 promoter The 1.5 Kb BamHI-SacI insert of M13mpl8/Bam-Sac/Nco is cloned back to plasmid pK 36 by replacing the wild type BamHI-SacI fragment of pK 36 with the mutated 1.5 Kb fragment using standard cloning techniques described above. This gives rise to plasmid pK 36/Neo having a Ncol site in front of the ATG of the protoxin Ncol gene ...........GAGGTAAC/CCATGG/ATAAC. 5 pg of this vector is digested with Ncol and the 3' recessed ends are filled in with 28) Klenov polymerase as described by Maniatis et al. . Then, the plasmid is digested with Ahalll, the DNA is separated on a 0.8 % low melting agarose gel and eluted as described above. 2 pg of plasmid p31y (described in European patent specification No. 100,561) is digested vith EcoRI and the 3' recessed ends are filled in vith Klenow polymerase as described above. The ligation of the blunt ended 3.6 Kb protoxin gene fragment with the blunt ended vector p31y is performed by incubating 200 ng of each DNA in 20 pi at RT as 29) described by Maniatis et al. . After transformation of E. coli HB101 to ampicillin resistance individual clones are analyzed by restriction analysis. One correct isolate is picked and called p31y/B.t. 1 pg of thia plasmid DNA is digested with BamHi and the 4 Kb fragment is isolated from a soft agarose gel. This fragment is ligated to 0.5 pg BamHi and pJDB207 DNA and positive clones are isolated via E. coli transformation and plasmid preparation. Correct isolates are verified by restriction analysis using BamHi.

Transformation of yeast strain GRF18 (MATo, leu 2-3, leu 2-112, his 3-11, his 3-15, can. ) is performed as described in European patent specification 100,561.

V.3. Yeast whole cells containing the recombinant B. thuringiensis toxin gene in the biotest The whole cells containing the B. thuringiensis gene are a bioencapsidation form of the MGE 1 product, which now as applied on the plants will better be protected from degradation by damaging influences such as, for example, light, than the crystal produced directly by B. thuringiensis at sporulation.

Yeast cells containing the B. thuringiensis toxin and also yeast cells without the B. thuringiensis toxin are resuspended in distilled water to a corresponding optical density. From said suspension, 4 concentrations are prepared and 0.2 % (v/v) wetting agent admixed. The same leaf disc test as described above in part III.7 is used to evaluate the insecticidal activity of these yeast cell preparations. The insecticidal activity of B. thuringiensis-transformed yeast cells on Heliothis virescens first instar larvae is shown 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 discs without yeast: control 1 - 0 control 2 - 3 Similar results are obtainable with yeast extracts which are prepared as described in European patent specification 100,561 and tested in the same biotest.

For application as insecticides, the transformed microorganisms containing the recombinant B. thuringiensis toxin gene, preferably transformed living or dead yeast cells, including mixtures of living and dead yeast cells, are used in unmodified form or, preferably, together with the adjuvants conventionally employed in the art of formulation, and are therefore formulated in known manner e.g. to suspension concentrates, coatable pastes, directly sprayable or dilutable suspensions, wettable powders, soluble powders, dusts, granulates, and also encapsulations in e.g. polymeric substances. As with the nature of the compositions, the methods of application, such as spraying, atomising, dusting, scattering, coating or pouring, are chosen in accordance with the intended objectives and the prevailing circumstances.

The formulations, i.e. the compositions or preparations containing the transformed, living or dead yeast cells or mixtures thereof and, where appropriate, a solid or liquid adjuvant, are prepared in known manner, e.g. by homogeneously mixing the yeast cells with solid carriers and, where appropriate, surface-active compounds (surfactants) .

The solid carriers used e.g. for dusts and dispersible powders, are normally natural mineral fillers such as calcite, talcum, kaolin, montmorillonite or attapulgite. In order to improve the physical properties it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. Suitable granulated adsorptive carriers are porous types, for example pumice, broken brick, sepiolite or bentonite; and suitable nonsorbent carriers are materials such as calcite or sand. In addition, a great number of pregranulated materials of inorganic or organic nature can be used, e.g. especially dolomite or pulverised plant residues. 3S Suitable surface-active compounds are nonionic, cationic and/or anionic surfactante having good dispersing and wetting properties. The term surfactants will also be understood as comprising mixtures of surfactants.

Suitable anionic surfactants can be both water-soluble soaps and water-soluble synthetic surface-active compounds.

Suitable soaps are the alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts of higher fatty acids (C10-C22), e.g. the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures which can be obtained e.g. from coconut oil or tallow oil. Mention may also be made of fatty acid methyltaurin salts, such as, for example, the sodium salt of cis-2-(methyl-9-octadecenylamino)-ethanesulfonic acid (amount in formulations preferably about 3 %).

More frequently, however, so-called synthetic surfactants are used, especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives, alkylarylsulfonates or fatty alcohols, such as, for example, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (amount in formulations preferably about 2 %).

The fatty sulfonates or sulfates are usually in the form of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts and contain a Cs-C22alkyl radical which also includes the alkyl moiety of acyl radicals, e.g. the sodium or calcium salt of lignosulfonic acid, of dodecylsulfate or of a mixture of fatty alcohol sulfates obtained from natural fatty acids. These compounds also comprise 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 one fatty acid radical containing 8 to 22 carbon atoms. Examples of alkylarylsulfonates are the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid, or of a condensation product of naphthalene37 sulfonic acid and formaldehyde. Also suitable are corresponding phosphates, e.g. salts of the phosphoric acid ester of an adduct of p-nonylphenol with 4 to 14 moles of ethylene oxide.

Non-ionic surfactants are preferably polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, or saturated or unsaturated fatty acids and alkylphenols, said derivatives containing 3 to 30 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols.

Further suitable non-ionic surfactants are the water-soluble adducts of polyethylene oxide with polypropylene glycol, ethylenediamine propylene glycol and alkylpolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethylene glycol ether groups and 10 to 100 propylene glycol ether groups. These compounds usually contain 1 to 5 ethylene glycol units per propylene glycol unit.

Representative examples of non-ionic surfactants are nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, polypropylene/ polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxyethoxyethanol. Fatty acid esters of polyoxyethylene sorbitan and polyoxyethylene sorbitan trioleate are also suitable non-ionic surfactants.

Cationic surfactants are preferably quaternary ammonium salts which contain, as N-substituent, at least one Ce-Cejalkyl radical and, aa further substituents, unsubstituted or halogenated lower alkyl, benzyl or hydroxy-lower alkyl radicals. The salts are preferably in the form of halides, methylsulfates or ethylsulfates, e.g. stearyltrimethylammonium chloride or benzyldi(2-chloroethyl)ethylammonium bromide.

The surfactants customarily employed in the art of formulation are described e.g. in McCutcheon's Detergents and Emulsifiers Annual, MC Publishing Corp. Ridgewood, New Jersey, 1980; Helmut Stache, Tensid-Taschenbuch (Handbook of Surfactants), Carl Hanser Verlag, Munich/Vienna, 1981.

The agrochemical compositions usually contain 0.1 to 99 %, preferably 0.1 to 95 %, of the transformed, living or dead yeast cells or mixtures thereof, 99.9 to 1 %, preferably 99.8 to 5 %, of a solid or liquid adjuvant, and 0 to 25 %, preferably 0.1 to 25 %, of a surfactant.

Whereas commercial products are preferably formulated as concentrates, the end user will normally employ dilute formulations.

The compositions may also contain further ingredients such as stabilisers, antifoams, viscosity regulators, binders, tackifiers as well as fertilisers or other active ingredients for obtaining special effects.

The transformed, living or dead yeast cells or mixtures thereof containing the recombinant B. thuringiensis toxin gene are preeminently suitable for combating noxious insects. Particularly to be mentioned are plant destructing insects of the order Lepidoptera, especially of the genera Pieris, Heliothis, Spodoptera and Plutella, such as, for example, Pieris brassicae, Heliothis virescens, Heliothis zea, Spodoptera littoralis and Plutella xylostella.

The rates of application in which the yeast cells are employed depend on the respective conditions, for example in particular the weather conditions, the nature of the soil, the plant growth and the time of application. In general, rates of application of 1 to 10 kg, in particular of 3 to 9 kg, of the yeast cells per hectare have proved advantageous.

Q ϋ Formulation Examples for B. thuringiensis-toxin containing material In the following formulation examples yeast cells means yeast cells containing the recombinant B. thuringiensis toxin gene, (throughout, percentages are by weight) Fl. Granulates yeast cells kaolin highly dispersed silicic acid attapulgite a) b) % 10 % % % % The yeast cells are suspended in is sprayed onto the carrier, and ly evaporated off in vacuo.

F2. Dusts yeast cells highly dispersed silicic acid talcum kaolin methylene chloride, the suspension the suspending agent is subsequental b) % 5 % % 5 % % % Ready-for-use dusts are obtained by intimately mixing the carriers with the yeast cells.

F3. Wettable powders yeast cells sodium lignosulfonate sodium lauryl sulfate sodium diisopropylnaphthalenesulfonate octylphenol polyethylene glycol ether (7-8 moles of ethylene oxide) highly dispersed silicic acid kaolin a) b) c) % 50 % 75 % % 5 % 3 % - 5 % % 10 % % 5 % 10 % 10 % % 27 % 40 The yeast cells are thoroughly mixed with the adjuvants and the mixtures is thoroughly ground in a suitable mill, affording wettable powders which can be diluted with water to give suspensions of the desired concentration.

F4. Extruder granulate yeast cells 10 % sodium lignosulfonate 2 % carboxymethyleellulose 1 % kaolin 87 % The yeast cells are mixed and thoroughly ground with the adjuvants, and the mixture is subsequently moistened with water. The mixture is extruded and then dried in a stream of air.

F5. Coated granulate yeast cells 3 % polyethylene glycol 200 3 % kaolin 94 % The homogenously mixed yeast cells are uniformly applied, in a mixer, to the kaolin moistened with polyethlene glycol. Non-dusty coated granulates are obtained in this manner.

F6. Suspension concentrate yeast cells 40 % ethylene glycol 10 % nonylphenol polyethylene glycol (15 moles of ethylene oxide) 6 % alkyl benzene sulfonate triethanolamine salt* 3 % carboxymethyleellulose 1 % silicone oil in the form of a 75 % aqueous emulsion 0.1 % water 39 % * preferably linear, containing 10-14, preferably 12-14, carbon atoms, such as, for example, n-dodecyl benzene sulfonate triethanolamine salt .

The homogenously mixed yeast cells are intimately mixed with the adjuvants, giving a suspension concentrate from which suspensions of any desired concentration can be obtained by dilution with water.

A culture of each of the following microorganisms used in the present invention has been deposited at the International Depositary Authority Deutsche Sammlung von Mikroorganismen (DSM; German Collection of microorganisms), Gottingen, Germany, according to the requirements of Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure, and a viability statement has been issued by said International Depository Authority: Microorganism date of deposition number of deposit* date of viability statement HD1-ETHZ 4449 (Bacillus thuringiensis var. kurstaki HD1 strain ETHZ 4449) March 4, 1986 DSM 3667 March 7, 1986 HB 101 (pK 36) (Escherichia coli HB 101 transformed with pK 36 plasmid DNA) March 4, 1986 DSM 3668 March 7, 1986 GRF 18 (Saccharomyces cerevisiae) March 4, 1986 DSM 3665 March 7, 1986 * accession number given by the above-identified International Depositary Authority.

A restriction with respect to accessibility of said microorganisms has not been requested by the depositor.

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Claims (31)

Claims

1. A DNA fragment obtainable from Bacillus thuringiensis var.kurstaki HD1, ETHZ 4443, encoding an insecticidal proteinaceous substance characterized by the nucleotide sequence given in table 2.

2. A DNA fragment according to claim 1 coding for the insecticidal protein MGE 1 including truncated portions thereof, said truncated DNA portions being subject to the proviso that insecticidal activity of the corresponding protein has not been lost.

3. A proteinaceous substance at least partially being encoded by a DNA fragment according to claim 1.

4. A proteinaceous substance containing a fragment characterized by the amino acid sequence given in table 3.

5. A proteinaceous substance according to claim 3, which is the protein MGE 1 being encoded by a DNA fragment according to claim 1.

6. A proteinaceous substance according to claim 4, which is the protein MGE 1 characterized by the amino acid sequence given in table 3.

7. A DNA vector containing a DNA fragment according to claim 1.

8. A vector according to claim 7 vhich is a plasmid.

9. A vector according to claim 7 vhich is a phage.

10. A microorganism containing a DNA fragment according to claim 1 with the proviso that, if said microorganism belongs to the group of Bacillus thuringiensis var.kurstaki, said Bacillus has been transformed with the DNA fragment according to claim 1.

11. A microorganism according to claim 10, said microorganism belonging to the species Saccharomyces cerevisiae.

12. A bioencapsulation system consisting of a first material completely embedded in a second material of biological origin, the first material being represented by a DNA fragment according to claim 1 and the second material being whole, living or dead microorganism or mixtures thereof with the proviso that, if said microorganism belongs to the group of Bacillus thuringiensis var.kurstaki, said Bacillus has been transformed with the DNA fragment according to claim 1.

13. A bioencapsulation system according to claim 12, wherein the microorganism is Saccharomyces cerevisiae.

14. A method for producing a DNA fragment according to claim 1, which method comprises the following steps: a) isolating and lysing cells of B. thuringiensis var. kurstaki HD 1, ETHZ 4449, separating plasmids from the material thus obtained and purifying and dialysing the plasmid material thus obtained by methods known per se: b) preparing a DNA library of B. thuringiensis var. kurstaki HD1 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 which can be done, by methods known per se; e) sequencing the thus obtained DNA fragment.

15. Method according to claim 14, wherein the screening for the presence of protein MGE 1 comprises a) screening of the clones for the presence of protein MGE 1 by an immunological assay using antibodies prepared against the crystal protein of B. thuringiensis var. kurstaki; b) selecting the clones being specifically reactive with goat antiserum; and c) testing insecticidal activity of extracts of said clones obtained according to step, b) .

16. A method for combating insects which.comprises applying to the insects or their habitats an insecticidally effective amount of a proteinaceous substance at least partially encoded by a DNA fragment according to claim 1.

17. A method according to claim 16 for combating insects of the order Lepidoptera.

18. A method for combating insects which comprises applying to the insects or their habitats an insecticidally effective amount of the protein MGE 1 encoded by a DNA fragment according to claim 1.

19. A method according to claim 18 for combating insects of the order Lepidoptera.

20. An insecticidal composition comprising an insecticidally effective amount of a proteinaceous substance at least partially encoded by a DNA fragment according to claim 1.

21. A method for combating insects which comprises applying to the insects or their habitats an insecticidally effective amount of a microorganism according to claim 10 or 11.

22. A method for combating insects which comprises applying to the insects or their habitats an insecticidally effective amount of a bioencapsulation system according to claim 12 or 13.

23. A DNA fragment according to claim 1, substantially as hereinbefore described and exemplified.

24. . A proteinaceous substance according to claim 3 or 4, substantially as hereinbefore described and exemplified.

25. A DNA vector according to claim 7, substantially as hereinbefore described and exemplified.

26. A microorganism according to claim 10, substantially as hereinbefore described and exemplified.

27. A bioencapsulation system according to claim 12, substantially as hereinbefore described and exemplified. 5

28. A method according to claim 14 for producing a DNA fragment, substantially as hereinbefore described and exemplified.

29. A DNA fragment whenever produced by a method claimed in any one of claims 14, 15 or 28. 10

30. . A method according to any one of claims 16, 18, 21 or 22, substantially as hereinbefore described and exemplified.

31. An insecticidal composition according to claim 20, substantially as hereinbefore described with particular 15 reference to the accompanying formulation Examples.