AU608508B2 - 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

FORM 10 SPRUSON FERGUSON COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE: 6a 9f92 t7 Class Int. Class Complete Specification Lodged: 09 0 0 o o 0 00 0 00 0 0 0 0 o o 0 0 B 0 I 04 0 O4 00 (0 0 0 0 4 Accepted: Published: This document contains the amendments made under Section 49 and is correct for printing. Priority: Related Art: Name of Applicant: Address of Applicant: Actual Inventor(s): Address for Service: CIBA-GEIGY AG Klybeckstrasse 141, 4002 Basle, Switzerland MARTIN GEISER, ALBERT HINNEN, JAKOB BRASSEL and SILVIA SCHWEITZER-GRUTZMACHER Spruson Ferguson, Patent Attorneys, Level 33 St Martins Tower, 31 Market Street, Sydney, New South Wales, 2000, Australia Complete Specification for the invention entitled: "INSECTICIDAL PROTEINACEOUS SUBSTANCE" The following statement is a full description of this invention, including the best method of performing it known to us SBR:eah 59W I -1 1 5-15787/= Insecticidal proteinaceous substance Bacillus thuringiensis thuringiensis) is a gram-positive bacterium which is usually pathogenic to insects Changl) (Reference is made to appended bibliography which is hereby made a part hereof, the publications and other materials there correspono o dingly cited in more detail being incorporated herein by reference).

00ooo00 0 coo The varieties of B. thuringiensis differ remarkably in their se toxicity and insect host range. The insecticidal activity of 00 o c B. thuringiensis originates substantially or completely from a wO proteinaceous parasporal crystal produced at the sporulation stage of growth. The gene(s) coding for the toxic proteins (polypeptides) o of said crystal is (are) found on plasmid DNA and/or on chromosomal 00 DNA of B. thuringiensis.

o 0 SIn order to avoid any disadvantages resulting from the presence of 0 Sother compounds produced by B. thuringiensis and to gain the insecticidal polypeptide in large quantities, it is desirable to make use of the corresponding gene, the corresponding DNA 0 o 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.

i~L 2 According to a first embodiment of this invention there is provided a DNA fragment originating from Bacillus thuringiensis var. kurstaki characterized by the nucleotide sequence given in table 2 comprising a DNA sequence coding for an insecticidal toxin protein including truncated portions of the said DNA fragment, said truncated DNA portions being subject to the proviso that insecticidal activity of the corresponding toxin protein has not been lost.

According to a second embodiment of this invention there is provided a toxin protein including truncated portions thereof, said truncated DNA portions being subject to the proviso that insecticidal activity has not been lost, being encoded by at least a part of the DNA fragment of the first embodiment.

o According to a third embodiment of this invention there is provided a toxin protein containing a fragment characterized by the amino acid sequence given in table 3.

According to a fourth embodiment of this invention there is provided the protein MGE 1 as herein defined being encoded by a DNA fragment of the first embodiment.

According to a fifth embodiment of this invention there is provided the protein MGE 1 as herein defined characterized by the amino acid sequence given in table 3.

According to a sixth embodiment of this invention there is provided a DNA vector containing a DNA fragment of the first embodiment.

According to a seventh embodiment of this invention there is provided a microorganism containing a DNA fragment of the first embodiment with the proviso that, if said microorganism belongs to the group of Bacillus thuringiensis, said Bacillus has been previously transformed with the DNA on fragment of the first embodiment.

According to an eighth embodiment of this invention there is provided 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 first embodiment and the second material being represented by a whole, living or dead microorganism or by a mixture thereof with the proviso that, if said microorganism belongs to the group of Bacillus thuringiensis, said Bacillus has been previously transformed with the DNA fragment of the first embodiment.

09v

I

2A According to a ninth embodiment of this invention there is provided a method for producing a DNA fragment of the first embodiment, which method comprises the following steps: a) isolating and lysing cells of B. thuringiensis var. kurstaki HD1, separating plasmids from the material thus obtained by methods known per se and purifying and dialysing the plasmid material thus obtained; 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 as herein defined by applying an antigen/antibody test.

According to a tenth embodiment of this invention there is provided a method for combating insects which comprises applying to the insects or S their habitats an insecticidally effective amount of a toxin protein being encoded by a DNA fragment of the first embodiment, or by a truncated o portion thereof, said truncated portion being subject to the proviso that insecticidal activity of the corresponding toxin protein has not been lost.

"0.*20 According to an eleventh embodiment of this invention there is provided a method for combating insects which comprises applying to the insects or their habitats an insecticidally effective amount of the protein MGE 1 as hereinbefore defined encoded by a DNA fragment of the first embodiment.

2°oo' 5 According to a twelfth embodiment of this invention there is provided an insecticidal composition comprising an insecticidally effective amount of a toxin protein being encoded by a DNA fragment of the first embodiment, or by a truncated portion L;.ereof, said truncated portion being subject to the proviso that insecticidal activity of the corresponding toxin protein 30 has not been lost.

According to a thirteenth embodiment of this invention there is provided a method for combating insects which comprises applying to the insects or their habitats an insecticidally effective amount of a microorganism of the seventh embodiment.

According to a fourteenth embodiment of this invention there is provided a method for combating insects which comprises applying to the insects or their habitats an insecticidally effective amount of a bioencapsulation system of the eighth embodiment.

KIK:0809v 2B According to a fifteenth embodiment of this invention there is provided a hybrid vector comprising the yeast acid phosphatase promoter and a DNA fragment of the first embodiment, which is controlled by said promoter.

According to a sixteenth embodiment of this invention there is provided the yeast Saccharomyces cerevisiae GRF 18 transformed with a hybrid vector of the fifteenth embodiment.

According to a seventeenth embodiment of this invention there is provided plasmid pK36 containing a DNA fragment of the first embodiment.

The present invention describes a method for producing an insecticidal proteinaceous substance including the discovery and identification of the entire DNA sequence coding for the insecticidal o protein MGE 1 of said proteinaceous substance, which DNA sequence differs remarkably in structure from that of the B. thuringiensis genes already 1 5 known Schnepf et al.

4 M.J. Adang et al.) and Y. Shibano et S al.

30 as well as WO 86/01536).

n The instant invention provides a DNA fragment characterized by the nucleotide sequence given in table 2, said fragment coding for the protein MGE 1, as well as to the protein MGE 1 itself. The present invention also describes a DNA fragment originating from Bacillus thuringiensis var.kurstaki from Hpal to PstI (4355) coding for an insecticidal proteinaceous substance including truncated portions thereof, said o o truncated DNA portions being subject to the proviso that insecticidal activity has not been lost.

oa2 5 By the term "proteinaceous substance" the insecticidal protein MGE 1 as well as derivatives and modifications thereof 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 describes the method of constructing cloning vehicles and expression vehicles comprising the DNA fragment given in table 2 and also to said vehicles themselves. Suitable DNA vectors are, i Av9 for example, plasmids such as pBR322 and pUC8, or phages such as M13.

4/809v i I~ h~ ~i i~ I C CC- -3- Further, the invention -olatte. to living or dead microorganisms containing a DNA fragment characterized by the nucleotide sequence given in table 2, particularly to a .icroorganism belonging to the species Saccharomyces cerevisiae. The invention also e to microorganisms, particularly to a microorganism of the species Saccharomyces cerevisiae, which contain a DNA fragment originating from Bacillus thuringiensis var.kurstaki from HpaI to PstI (4355) coding for an insecticidal proteinaceous substance including truncated portions thereof, said truncated DNA portions S being subject to the proviso that insecticidal activity has not been lost. Such microorganisms are, for example, yeasts, particularly Saccharomyces cerevisiae, bacteria and phylloplane fungi with tle t proviso that, if said microorganism belongs to the group of Bacillus thuringiensis, said Bacillus has been transformed with the DNA o fragment described in table 2.

Throughout this specification, the term "transformation" shall also be understood as comprising conjugation-like mechanisms.

The invention rclatoe alsoet- a bioencapsulation system consisting of a first material completely embedded in a second material of "°oo biological origin, the first material being represented by a DNA fragment described in table 2 and the second material being represented by a whole microorganism with the proviso that, if said microorganism belongs to the group of Bacillus thuringiensis, said Bacillus has been transformed with the DNA fragment described in table 2. The DNA material may also be a DNA fragment originating from Bacillus thuringiensis var.kurstaki from Hpal to PstI (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 o0 lost. Especially suitable microorganisms are yeasts, particularly Saccharomyces cerevisiae, such as S. cerevisiae GRF 18.

i; -4- The instant invention relate further 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, 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 \O proteinaceous substance at least partially encoded by the DNA fragment coding for the protein MGE 1. The composition comprises an insecticidally effective amount of a proteinaceous substance at least partially encoded by the DNA fragment coding for the protein MGE 1.

0 0 0 00 0000) 0 00 Moreover, the invention e eseto a delivery system consisting of transformed living or dead yeast cells containing 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 0o 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 obtainable. 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 cells. Suitable yeast promoters are described in European patent specification 100,561. Particularly suitable is the PH05 promoter.

i 5 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 coli coli) PNA polymerase, said promoter being situated in front of the respective gene Wong et al. 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. coli or yeast.

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

CC) CCC 0 0 00C .0 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): L 6 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 example, S 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 a obtained with radiolabeled RNA; and i) sequencing of DNA fragments coding for the respective proteins.

Meanings of abbreviations used in the following: o bp: base pairs BSA: bovine serum albumin DEAE: diethylaminoethyl as DFP: diisopropyl fluorophosphate DTT: 1,4-dithiothreitol (1,4-dimercapto-2,3-butanediol) EDTA: ethylenediaminetetraacetic acid S* IPTG: isopropyl-0-thiogalactopyranoside (Serva) Kb: Kilo bases

PBS

1 0.01 M phosphate buffer, pH 7.4, and 0.8 NaCl 6, 49

SPBS

2 10 mM sodium phosphate, pH 7.8, and 0.14 NaCI 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 NaCI TE: solution containing 10 mM Tris*HCl (pH 7.5) and 1 mM EDTA -7 TES: 0.5 M Tris, pH 8.0, 0.005 M NaCi and 0.005 M EDIA TNE: solution containing 100 mM NaCi, 10 mM Tris'HCl (pH and 1 mM EDTA Tris*HCl: tris-(hydroxymethyl)-aminomethane, pH adjusted with HCl X-CAL: 5-bromo-4-chloro-3-indoxyl-O-D-galactoside 2xYT: 16 g Bacto Tryptone g Yeast Extract (Bacto) g NaCi Media, buffers and solutions used .)the following* GO '0 0 00 0 0 00 O 0 0 4 0 9 4 0 0 0 0 00 o ~0 00 0 00 0, 04 0 0 0 0~ 40 0

I

4- 2OxSSC 2xS SC 6xSSC Demhardt's Solution (50x) Dissolve 175.3 g of NaCl and 88.2 g of sodium citrate in 800 ml of H 2 0. Adjust pH to 7.0 with a few drops of a 10 N solution of NaOH. Adjust volume to 1 liter. Dispense into aliquots. Sterilize by autoclaving.

10 of 2OxSSC 33 of 2OxSSC Ficoll 70 (relative molecular mass approximately 700'000; Pharmacia) 5 g polyvinylpyrrolidone (Calbiochem- Behring Corp.) 5 g BSA (Sigma) 5 g

H

2 0 to 500 m' Filter through a disposable Nalgeneo filter (NalgeneO; Nalge Co.Inc., Rochester, USA). Dispense into 25 ml aliquots and store at -20 0

C.

Solutions M9 Medium a) Per liter: Na 2 HP04

KH

2

PO

4 3 NaCi N4Cl 1I Adjust pH1 to 7.4, autoclave, cool, and then add: r-il -r..rli- l1 l~ w- r -i i- l~ -LI ii -8b) 1 M MgS04 2 ml glucose 10 ml 1 M CaC12 0.1 ml vitamin BI (40 mg/lOml) 5 ml The above solutions a) and b) should be sterilized separately by filtration (glucose-vit.B1) or autoclaving.

LB (Luria-Bertani) Medium Per liter: Bacto-tryptone Bacto-yeast extract NaCl 10 g 5 g 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 Yousten and M.H. Rogoff 5 (1969) (g/l 1 is used: V 0r glucose yeast extract (Difco) (NH4)2S04 KzHP04 MgS04.7H 2 0 CaCl 2 2H 2 0 MnS04.H 2 0 1 2 2 0.2 0.08 0.05 Vy 6 Before autoclaving, the pH is adjusted to 7.3 with potassium hydroxide.

Characterization of microorganisms used in the present invention: 1. HD1-ETHZ 4449 is a Bacillus thuringiensis subspec. kurstaki strain and is characterized first by its immunological reaction against its flagellum antigen. HD1-ETHZ 4449 belongs to the 3a, 3b serotype Krieg31)). Secondly, HD1-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

'I

9strain is isolated, digested to completion with the HindIII 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.

2. HB 101 is a hybrid between Escherichia coli K12 x Escherichia coli B. It is a good host for large scale DNA purification. It is used for transformation experiments and CaClz competent cells are C) commercially available from Gibco AG, Basel, Switzerland, Catalogue No. 530 8260 SA.

8 3. JM 103 is an Escherichia coli K-12 and is commercially available from Pharmacia P-L Biochemicals, Catalogue No. 27-15 4 5-xx (1984).

The E. coli strains HB 101, and JM 103 are described in Maniatis et al. Maniatis et al.6): Strain Genotype

HB

1 01 F hsdSzo (rB, mB), recA 13 ara-14, proA2,lacY1, galK 2 S rpsLzo(Sm xyl-5, mtl-l, supE44, X JM103 A(lac pro),thi, strA, supE, endA sbcB, hsdR, F'traD36, proAB, lacI, In the following example, yeast refers to Saccharomyces cerevisiae.

.L U 10 Example I. Plasmid DNA preparation: Plasmid DNA is prepared as described below according to White and Nester White and E.W. Nester I.1. B. thuringiensis plasmids E. coli 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 White and E.W. Nester After harvest, the cells are resuspended in alkaline lysis buffer and kept at 37 C for I) 20-30 minutes. The clear lysate thus obtained is neutralized by addition of 2 M Tris*HCl (pH The chromosomal DNA is precipitated by addition of SDS and NaCl. The lysate is placed on ice and chromosomal DNA is removed by centrifugation. The plasmid DNA which "e is now in the supernatant is precipitated with 10 PEG 6000. After overnight storage at 4 0 C the plasmid DNA is resuspended in 7-8 ml of J TE. The plasmid DNA obtained from 1 1 culture is further purified on Stwo CsC1 gradients. Solid CsCI is added (8.3 g of CsC1 to 8.7 ml of 0 o supernatant). After the addition of ethidium bromide (Sigma; final concentration 1 mg/ml supernatant) the solution is transferred to c0 13.5 ml Quick Seal polyallomer tubes (Beckman) and centrifuged in a 0 Beckman Ti50 rotor for 40 hours at 40'000 rpm. Two fluorescent bands o' can be visualized with long wave UV (366 nm). The lower band contains supercoiled plasmid DNA which is collected by puncturing a the tube from the side with a 2 ml syringe (18G needle). The ethidium bromide is removed by extracting 5 times with equal volumes of isopropanol (saturated with CsCI) and the product is transferred

S

e sO to 30 ml Corex tubes. 2.5 volumes of TE is added and the DNA is aGie precipitated with ethanol. The solution is then kept for 12-15 hours at -20 0 C. The precipitated DNA is collected by centrifugation in a S Sorvall HB-4 rotor for 30 min at 12'000 rpm at 0 C and redissolved in 200 pl of TE.

(E.coli JM 103 can also be extracted and used in the same way).

j litll lil 11 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, 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 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 Worthington, 11'000 U/mg) is added, after 5 min.

IO. 1.2 ml EDTA (500 mM, pH 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] are added. After 5 min the lysate is centrifuged in a precooled SS-34 rotor for 40 min at 4 0 C. The supernatant is carefully removed and, after addition of solid CsC1, is purified on the CsC1 gradients as described for B. thuringiensis plasmid DNA.

50-100 pg of hybrid plasmid DNA are recovered from a 100 ml culture.

.o II. 6-Endotoxin antigen and goat antibodies II.1. Preparation of 6-endotoxin crystal antigen: Bacillus thuringiensis (var.kurstaki HD1, strain ETHZ 4449) is 0 cultivated in a Fernbach flask on the medium according to Yousten o o and Rogoff Yousten and M.H. Rogoff 5 as described above, however with increased concentration of glucose (0.3 instead of oo 0.1 Incubation time is 4-5 days at a temperature of 30 0 C. The colonies are harvested immediately after sporulation Trumpi)).

0 In order to separate parasporal bodies and spores from each other, the method according to Delafield et al. Delafield et al.

9 is used.

ae a) Separation of spores and crystals: suspend autolyzed cultures in 1 M NaCl/0.02 M potassium phosphate -o buffer (pH 7.0) containing 0.01 Triton-X-100 (Merck) filter the suspension in order to separate particulate components, e.g. agar residues etc.

1- i I 12 centrifuge wash the sediment several times with the above solution until only traces of components which absorb at 260 nm are present in the supernatant wash the particulate components in 0.2 M NaC1/0.004 M phosphate buffer (pH 7.0)/0.01 Triton-X-100 wash again with 0.01 Triton-X-100 resuspend the particles in water remove the residual cells from the suspension centrifuge and wash the residual spores and crystals in 0.02 M phosphate buffer (pH 7.0)/0.01 Triton-X-100 three times transfer the suspension, in 182 ml of the same buffer, to a cylindrical separatory funnel containing 105 g of a 20 (w/w) aqueous solution of sodium dextran sulfate 500 (Sigma), 13.2 g of solid polyethylene glycol 6000 (Merck), 3.3 ml of phosphate buffer (pH 7.0) and 7.5 g of NaCl shake in order to dissolve the solid components adjust the volume to 600 ml by adding a well-shaken solution of the same composition, but without bacterial components 03 0 0 oon o3 0 o ao 0 I 0 3 0o So00

C

00 00 0 0 C C 0 CI O 0 oO O shake vigorously allow to stand for 30 minutes at 5 0

C

i 13 draw off the upper phase (which contains most of the spores, but very few crystals) of the separated phases centrifuge the upper phase add the supernatant to the lower phase (which contains a mixture of spores and crystals) which still remained in the separatory funnel repeat extraction After the tenth extraction, the crystals in the lower phase are virtually free of spores and can be collected by centrifugation.

The spores and crystals are both washed five times in cold distilled water. The crystals are stored at -5 0 C as suspensions in water.

b) Solubilization of the crystals: 00 0 o An aliquot of the crystal suspension is centrifuged for 10 min. at o° 0 12'000g. The sediment thus obtained is resuspended in 0.05 M carbonate buffer and 10 mM dithiothreitol (DTT, Sigma; mixture of o o carbonate buffer and dithiothreitol defined in the following as O a carbonate/DTT) in a concentration of 5 mg of sediment/ml of .o carbonate/DTT mixture.

.o After incubation for 30 min. at 37 0 C, the unsolved particles are n [0 separated by centrifugation for 10 min. at 2 5'000g. The supernatant S"is dialysed against carbonate buffer and subsequently tested for .o 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 Sconsistency. Complete solution of the protein is obtained with 0 addition of 1 mM DTT.

14 c) Inactivation of crystal-bound proteases: Serine proteases and metal proteases of the crystal suspensions (Chestukhina et al 0 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 \C 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

H

2 0 and 1 mM EDTA.

11.2. Immunisation of goats: on o Antigen is prepared from crystals of B.thuringiensis serotype H-3 by "'zo o, dissolving the crystals in carbonate/DTT, dialysing the solution S thus obtained against carbonate buffer and 1 mM DTT and purifying S' o the antigen by -4er-lfiltration using a 0.45 um millipore filter.

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

C.

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 and 80 ml on each of the other days.

L c i A- t I i 15 When the blood has coagulated, the sera are incubated at 56°C for min., thus inactivating the complement. The sera are stored at -200C.

11.3. Purification and [1 25 I]-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 Ouchterlony according to the method described 0 by Huber-Lukac Huber-Lukacl2)).

Thus, 30 ml of 3.2 M ammonium sulfate are added dropwise to 15 ml of goat anti H3 serum in 15 ml of PBS I (0.01 M phosphate buffer, pH 7.4, and 0.8 NaC1). The mixture is allowed to rest for 15 min.

After the mixture is centrifuged (10,000g, 20 min.), the sediment is o distributed in 7.5 ml of PBS', dialysed at 4°C three times against o o 1000 ml of PBS 1 then dialysed against 1000 ml of 0.01 M phosphate buffer, pH 7.8, and centrifuged (3000g, 20 min.).

u The supernatant is transferred to a 30 ml column of DEAE and eluated o at RT with 0.01 M phosphate buffer, pH 7.8 (rate of flow: n.Q 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 o a 11) o Ouchterlony against anti-goat-IgG antibodies and against .a anti-goat-serum antibodies of rabbits (Miles Laboratories).

a 0 The antibodies are further purified by absorption on a sepharose® (Pharmacia) column to which the H3-protoxin is bound. Coupling of Sthe protoxin to CNBr-sepharose® (Pharmacia) is carried out according o to the supplier's instructions as summarized below:

I-

4 Lii 16 1 g of CNBr-activated Sepharose®6MB 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 HC1. The H3 antigen protein as obtained according to part II.1.c above, is dissolved in 0.1 M NaHCO 3 and 0.5 M NaCl. 1 ml of gel contains 5-10 mg of protein. The gel suspension and the antigen are mixed at RT for 2 hours. The excess protein is eliminated by washing with 0.1 M NaHCO 3 (pH 0.5 M NaCl and 0.5 M ethanolamine. A further washing is done with 0.1 M NaHCO 3 (pH 8.3) and 0.5 M NaCl, followed S by 0.1 M CH 3 COONa (pH 4) and 0.5 M NaCl. The last washing step is done again with 0.1 M NaHCO 3 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 with 3M KSCN and dialysed against PBS 2 (10 mM sodium phosphate pH 7.8, 0.14 M NaCl). The antibodies are radiolabeled with 1251 using the chloramin-T method e iw13) ,(Amersham Buchler Review o Iodine labeling: 125 1 mCi sodium iodine- I is given to a tube containing 100 pi 0.5 M O phosphate buffer (pH Under continous stirring, 5 pg of the protein solution (0.5 mg/ml in TBS) and 50 jg chloramine T in 0.05 M phosphate buffer (pH 7.2) are added. After incubation for 1 minute at RT, 120 ig Na 2

S

2 0 5 are added. The free iodine is separated from «the labeled protein on a 0.9x12 cm column packed with o In order to avoid absorption of the labeled protein to the column, 0 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 The fractions (1 ml) are collected until the whole protein is eluted.

a I

I

17 III. Cloning of the 5-endotoxin gene: A partial Sau3A DNA library from B. thuringiensis var.kurstaki HD1, strain ETHZ 4449 plasmid DNA is essentially prepared according to Maniatis et al. Maniatis et al.1 4 and subcloned into the Bam HI site of pBR322 as vector DNA as described below: III.1. 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 (IC' the 2-10 Kb size range. This is achieved by using the method described by Maniatis et al. 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 NaC1 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.

10 ig of pBR322 plasmid are digested with 10 units of Bam HI endonuclease in 50 pl of 10 mM Tris-HCl, pH 7.4, 10C mM NaCl and &O 10 mM MgC12 at 37 0 C for 1-2 h. The phosphatase treatment of the 1 on cleaved DNA is done as follows: 10 jg of the DNA are dissolved in Il of Tris.HCl, pH 8, and bovine intestine alkaline phosphatase (Boehringer) is added in an amount of 3 units/pg of DNA. After So t incubation for 30 min at 37 0 C, the DNA is phenolized two times and 0 finally extracted with chloroform. After ethanol precipitation, the DNA is resuspended in 20 pl of H20 and is used in the ligation reaction of fragments derived from partial Sau 3A digestion. The reaction is set up as follows: to 0.4 ig of Sau 3A digested DNA in 0# a0 10 pl of H 2 0, 0.1 pg of the phosphatase-treated vector are added.

The ligation reaction is brought about with addition of 50 mM Tris.HCl, pH 7.4, 1 mM ATP, 10 mM MgC12 and 15 mM DTT followed by addition of 20 units of T4 DNA ligase (Biolabs). After incubation at 0 C overnight, the DNA is used to transform E.coli HB101 competent cells.

I, i l i i ~II 18 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 ClaI. Subsequently it is cloned into pBR322 between the Clal and Bam HI sites.

III.2. Transformation by the calcium chloride procedure: The preparation of competent cells is done by treating cells growing to a density of 5 x 107 cells/ml with calcium chloride 15) (Maniatis et al The transformation is achieved by addition of C the DNA to these cells after which said cells are maintained at 42°C for 3 minutes, diluted with 1 ml LB medium, incubated at 37 0 C for minutes and spread on selective media by using common procedure Maniatis et al 15 111.3. Preparation of cell crude lysates The colonies containing the 6-endotoxin gene will express a protein with a biological activity similar to the purified and dissolved 16) toxin crystals 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-medium in the presence of ampicillin. Ten cultures are pooled, harvested, washed Uo in 10 mM NaCl and finally the cells are lysed in 2 ml of 400 mM NaCI, 0.1 M NaOH, 1 mM PMSF. After 20 min incubation at room temperature the lysates are neutralized by the addition of 20 1l of co eo 2 M Tris-HCl, pH 7.0. After centrifugation in a SS34 Sorvall rotor 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 6-endotoxin antigen radioimmunologically using the plastic well method described by Clarke et al. Clarke et al. 17). Single plastic wells are coated overnight with 150 pl of purified H3 goat antibodies (10 pg/ml) in ^i ^1^c i 19 mM Tris.HC1, pH.9.3, and kept overnight at 4 0 C. The wells are washed three times with TBS/Tween, (TBS 0.5 Tween 20) filled with 150 pl of the bacterial extract and incubated for 6 hrs at 37 0 C. After washing, the wells are filled with 150 pl of [125I] labeled rabbit anti-goat H3 antibodies (60 ng, 10 5 cpm) in TBS containing 25 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 l 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 pl) is dissolved a in the buffer suitable for the pertinent restriction endonuclease o; o" and after 1-2 hours incubation at 37 0 C, the digested DNA is loaded o 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 extrac- S'o ted with a 1:1 mixture of phenol and chloroform, precipitated in o oa o o ethanol and then exposed to conditions incompatible before (such as, for instance, tha buffer required for the second enzyme).

00 O o 0 0 b) Southern transfer: The coding sequence for the 6-endotoxin is localized by hybridizing S0,° 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 (Southern 20 DNA fragments which have been separated according to size by electrophoresis 'hrough 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 32 P-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) 1iQ c) Hybridization of Southern filters: The prehybridization and hybridization are done according to 20) Maniatis et al. Maniatis et al.

2 with the following modifications: The baked filters are put into a heat-sealable plastic bag.

0.2 ml of prehybridization mixture is added per square cm of nitrocellulose filter.

o Cd 0

O

C C1 C *3 IC C Cr c prehybridization mixture: 4 x SSC formamide 0.2 SDS 20 mM EDTA mM Potassiumphosphate (pH 7.2) 5 x Denhardt's solution 100 ig/ml calf thymus denaturated DNA The bags are usually incubated for 3-4 hours at 37 0

C.

The prehybridization mixture is removed and replaced by the following hybridization mixture (50 pl/cm 2 of nitrocellulose filter).

t t I I t CCe C hybridization mixture: as the prehybridization mixture but now containing the 32 P labeled denaturated RNA probe (106-107 cpm/filter) as prepared according to part III.5.d below.

21 The bags are usually kept at 37 0 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.1xSSC and 0.1 SDS for 60 minutes. The filters are dried on Whatman 3MM paper and prepared for autoradiography.

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 Yousten and M.H. Rogoff5)). 500 ml cultures are shaken in 2 1 Erlenmeyer flasks at 300 rpm and 30 0

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 Spoint .f sporulation. The cells are further grown for 5-6 hours.

°o Rifam, Lcin (50 ig/ml) is added and the cells are shaken for 10 min.

Sa The ice-chilled cells are harvested and resuspended in 10 ml 4 M Sguanidinium thiocyanate, 0.5 sarcosyl, 25 mM sodium citrate pH 7 and 0.1 M 2-mercaotoethanol. The cells are frozen at -80 0 C and then o 0o0 disrupted in a French Press. After centrifugation of the extract for min at 15'000 rpm (Sorvall SS34 rotor) 0.5 g/ml CsCI is added to the supernatant, which is then overlayed on a 5.7 M CsC1, 0.1 M EDTA cushion in a Beckmann 60Ti centrifugation tube. After centrifugation 0o at 38'000 rpm for 20 hours, the RNA pellet is washed with ethanol, dried, dissolved in 7 M guanidine hydrochloride and precipitated with ethanol. Total RNA from sporulating cells is dephosphorylated and labeled with 32 P] ATP and T4 polynucleotide kinase according to the following standard procedure described by N. Maizels 2 1) RNA Labeling with Polynucleotide Kinase: 0. About 1 pg RNA is subjected to mild alkaline hydrolysis by heating in 50 mM Tris-HCl (pH time and temperature of incubation: min at 90 0

C.

22 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 il. Kinase labeling is in 10 il reaction samples containing 50 mM Tris-HCl (pH 10 mM MgC12, mM dithiothreitol, 5 glycerol, and 1pM [y 32 P]-ATP labeled at a specific activity of 6000 Ci/mmole. Each reaction sample contains about 1 ig of RNA and 2 il of T4 polynucleotide kinase and reaction takes place for 45 min at 37 0 C. This generates RNA with a specific activity of about 3 x 107 Cerenkov cpm/pg. The RNA is separated from the [y 32 P]-ATP by three ethanol precipitations in the presence of jg tRNA carrier.

111.6. Identification of clones coding for the 6-endotoxin in the Bam HI/Cla I plasmid DNA library cloned in pBR322: The pK 25 serie a) In situ hybridization of bacterial colonies: a 22) S Colony hybridization Grunstein and D. Hogness 22) is accomplished by transferring bacteria from a master plate to a nitrocellulose filter. The colonies on the filter are then lysed and the .o liberated DNA is fixed to the filter by baking. After hybridization to a 32 P-labeled probe, the filter is monitored by autoradiography.

O oa A colony whose DNA gives a positive autoradiographic result may then be recovered from the master plate.

o A 6-endotoxin gene internal DNA fragment is used to screen the Bam HI/Cla I plasmid DNA library cloned in pBR322. The procedure is 0^o, 0 described in Maniatis et al. Maniatis et al.2 3 The filters are hybridized to a 32 P-labeled probe prepared as described below in part III.6.b.

ao0 o In order to obtain an autoradiographic image, the filter is wrapped in Saran Wrap and applied to X-ray film.

23 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) and 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.

b) DNA Nick translation: An internal DNA fragment of the 6-endotoxin gene is radiolabeled by following the procedure described below: mixture: no I B O O tr jn n~Yn (7 O i) on o

J

ooo o on D wouo 3 pl DNA (1 Ig) p1 of nick translation buffer concentrated: 0.5 M Tris, pH 8, 0.05 M MgCI 2 1p 2.5 mM d

(GTP)

1.5 pl 2.5 mM d

(CTP)

1.5 il 2.5 mM d

(TTP)

2.5 11 H 2 0 0.75 p1 BSA (1 mg/ml) 1l 100 mM B-mercaptoethanol Bo 0 00 o ao Oa 00 0 0r t0o100 mix and add to 100 pCi dried 32 P-a]-ATP [10 mCi/mmol] mix well -4 add 0.75 1i of a 1x10 4 solution of DNase I (1 mg/ml) in Nick translation buffer incubate at RT for 1 minute, transfer to ice add 1 p1 E.coli polymerase I (Biolabs; final volume: 15 1l) 2 incubate at 15°C for 3 hours heat at 65°C for 10 min; add 35 p1 50 mM EDTA and 10 pi tRNA stock (100 pg) The non-incorporated nucleotides are separated by chromatography on a small Sephadex G50 column.

i cu. i" i -1~113-. i ii li; 'IL- Ir^a~~ 24 c) Subcloning of the complete 6-endotoxin gene in the pUC8 vector: the pK 36 clone The pUC8 vector (New England Biolabs) is digested to completion with the restriction enzymes HinclI and PstI and by alkaline phosphatase treatment (see part III.1 above). The HpaI/PstI fragment from pK 25-7 (see part III.6.a above) coding for the 6-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.

111.7. Biotest to evaluate B.thuringiensis toxins: To the cleared lysate as obtained according to part 111.3. 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 0 a the toxin content in the extracts prepared and 0.1 wetting 0 agent admixed. Leaf discs from cotton plants which are grown under o controlled conditions (25°C, 60 relative humidity) in a growth o° 00 chamber are dipped in the E.coli cell extract suspensions. First .0C3 instar larvae of Heliothis virescens (30 larvae per concentration) o 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 Sextracts causing mortality therefore possess bioinsecticidal oa activity originating from the cloned B. thuringiensis DNA.

IV. DNA sequencing: The DNA fragments encoding the 6-endotoxin gene were sequenced on both strands according to F. Sanger et al.

24 using the M13 system 0 0 0 IV.1. Cloning of the 6-endotoxin gene into M13 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 c i

I_

25 sequence) to Hind III (position 1847) and EcoRI (position 1732) to PstI (position 4355). The first fragment is cloned in M13mp8 (New England Biolabs) between the single HincII site and the single HindIII 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 Poncz et al.

26 is used to shorten the HpaI-HindIII DNA fragment coding for the 5' end of the gene. Said shortening is made as follows: [C The fragment is cloned in M13mp8 between single HincII and HindIII.

mg of the replicative form of the DNA is linearized with restriction endonuclease HindIII and subjected to the action of the endonuclease Bal 31 in 100 pl of 600 mM NaC1, 12 mM CaCl 2 12 mM MgCl 2 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 pl are removed each time. Immediately after taking-off, pl of phenol and 40 pl of TE buffer are added in order to stop further reaction. The mixture is centrifuged, extracted with aooO chloroform and then precipitated with ethanol. The DNA precipitate thus obtained is resuspended in 20 lI of 100 mM NaC1, 20 mM Tris*HCl and 10 mM MgC1 2 and digested with a second enzyme found on the other site of the originally cloned fragment, which second enzyme in the present case is BamHI. After size reparation in agarose gel, the "o 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, liqiifled at 65 0

C,

adjusted to 500 mM NaCl and incubated at 65°C for 20 min. One volume of phenol (equilibrated with 10 mM Tris*HCl pH 7.5, 1 mM EDTA, 3o 500 mM NaCl) is added. The aqueous phase is reextracted twice with phenol and once with chloroform. The DNA is precipitated with 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 il of TE.

c i i- 26 The fragments are successively shortened by 200-300 bp at each standpoint and have a single BamHI 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 BamHI and HinclI as described above in part IV.I.

By the process described above, the DNA sequence is obtained as one DNA strand starting at the HindIII 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 EcoRI/PstI fragment and its sequencing is given in figure 1 as modified from M. Poncz et al.

26 and on table 1.

Fig. 1 as modified from M. Poncz et al. 2. Construction of the deletion mutant library. Steps: 1, the insert (thick lines) is cloned into the cohesive end A site in M13; 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 M13 (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 S BAL-31-induced blunt end and a cohesive end A; 4, the fragments are o° subcloned into M13 so that blunt end is proximal to the primer site P used o for DNA sequence analysis.

SX and C blunt ends 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: 8 1. inoculate into 2xYT a single colony of E.coli JM 103; keep over night at 370C with stirring 2. inoculate 40 ml of 2xYT with 200 pV of the culture obtained according S° to step 1 3. keep at 370C with stirring until ODss0 is 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 CaC12 (CaC12 should be fresh prepared) 7. keep 40 min. on ice SBR:eah 136 i. Illl~-~~inh?*-*r-iYD 27 8. centrifuge at 6000 rpm for 5 min in a SS34 Sorvall rotor 9. suspend the cells in 3 ml of ice-cold CaCl 2 to 200 il of cells obtained according to step 9 add 1-5 il of DNA or 7-15 pl of ligase formulation 11. keep 30 min. on ice 12. keep at 42°C for 3 min.

13. add 200 pl of cells obtained according to step 1 14. bring top agar to the boil and keep at 42°C tubes are filled with 3 ml of top agar, 30 il of X-GAL (20 mg/ml 10 dimethylsulfoxid) and 30 il of IPTG (20 mg/ml H 2 0) 16. mix thoroughly and transfer immediately onto IxYT plates warmed-up before using o 17. let dry disks for ca. one hour 18. Turn upside down and incubate at 37 0

C.

oa Oa BThe plaques thus obtained are suitable for further treatment.

°o Controls: 200 il competent cells without exogenous DNA o 1 il M13mp8 (replicative form of DNA, 10 ng) 200 ll of competent cells.

IV.4. Preparation of the replicative form of recombinant phage DNA: O 0 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 IV.3 above and keep at 37 0 C for seven hours 2. centrifuge for 10 min. at 4000 rpm 28 3. keep supernatant over night at 4 0

C

4. inoculate 10 ml of supernatant obtained according to step 3 and ml of the culture obtained according to step 1 of part IV.3 above into 1 1 of 2xYT shake at 37 0 C for 41 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 o 11. keep on ice for 10 min.

12. add 4 ml of 10 SDS (or 1.6 ml of 25 SDS), mix with glass I. rod 13. add 6 ml of 5 M NaC1 (final concentration: 1 mix cautiously 14. keep on ice for one hour 15. centrifuge at 20 000 rpm for 40 min. in a SS34 Sorvall rotor t 4 t I 16. take supernatant and add an amount corresponding to 1/10 of the X0. volume of 5 M NaCl and 15 ml of 30 PEG in TNE 17. keep at 4 C for two hours or overnight _1 I 29 18. centrifuge at 8000 rpm for 15 min.

19. take pellets and transfer to 18 ml of TE, pH 8 add 18 g of CsC1 (Ig/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 CsC1 solution (1 g CsCl 1 ml TE) 23. centrifuge at 35 000 rpm at 20 0 C for 36-48 hours 24. take the lower bands below UV (366 nm) 25. extract 3-4 times with butanol saturated with water 26. dialyse 3 times at 4 0 C against 1 1 of sterile TE each time u a Preparation of the single stranded template DNA: 1. shake E.coli JM 103 cells in 5 ml of 2xYT medium at 37 0 C overo night 2. add two drops of cell suspension obtained according to step 1 to So" 25 ml of 2xYT medium 3. fill tubes with 2 ml of culture solution obtained according to t o "step 2 each and add one plaque per tube as obtained according to part IV.3. above <cd 4. shake for 5 hours at 37°C I_ I~ 30 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 pl 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 SJp 12. to the remaining, add 100 pl of TE and 50 1l of phenol 13. mix for 10 sec. (by vortex); allow to stand for 5 min.; mix for 10 sec. (by vortex); centrifuge for 1 min.

S 0 o 14. transfer aqueous phase into fresh Eppendorf tube add 500 p1 of ethylene ether, mix (vortex) and centrifuge for 0 1 min.

16. withdraw ether by suction, keep tubes unclosed for 10 min. (if, 0\o after this treatment, aqueous phase is very turbid, air should 0 0 be blown through with the pasteur pipette, until solution is clear) o 0 17. add 10 p of 3 M sodium acetate and 250 pl of ethanol 18. keep at -80 0 C for 30 min.

19. centrifuge for 5 min.

31 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 pl of TE transfer 2 pl 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 mannual "M13 cloning and o 0 DNA sequencing system", published by New England Biolabs.

o" 4 An analysis of the complete DNA sequence shows that only one open n° reading frame long enough for a protein with a MW of 130 622 and coding for 1155 amino acids is found. The N-terminus of the protein o. 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 HpaI site and the PstI site is given in tab. 2. The deduced amino acid sequence is given in tab. 3.

Sa'o V. Expression of the 6-endotoxin gene in yeast cells V.1 Introduction of a Ncol site before the first AUG of the gene °o "o In order to combine the protein coding sequence of the i 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 I_ 32 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 SacI and isolating of the fragment using standard techniques described above. 100 ng of replicative form (RF) of M13mpl8 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. coli, 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.

O An oligonucleotide with the sequence GAGGTAACCCATGGATAAC 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) o, o J.M. Zoller and M. Smith Zoller and M. Smith Approximately five pg of single-stranded phage M13mpl8/Bam-Sac DNA is mixed with 0.3 pg of phosphorylated oligonucleotide in a volume of 40 p1.

The mixture is heated to 65°C for 5 min., cooled to 50 0 C, and slowly So'i o) cooled to 4 0 C. Next, buffer, nucleotide triphosphates, ATP, T4 DNA ligase and large fragment of DNA polymerase are added and incubated overnight at 15 0 C as described (J.M Zoller and M. Smith 2 7 After agarose gel electrophoresis, circular double-stranded DNA is purified and transfected into E.coli strain JM103. The resulting plaques are screened for sequences which hybridize with 3 2 P-labeled oligonucleotide, and phage are analyzed by DNA restriction endoo nuclease analysis. Among the resulting phages, clones will be ones which have correctly now a C at position 154 and 155 instead of the T in the pK 36 DNA. This phage is called M13mpl8/Bam-Sac/Nco.

L io I 33 V.2. Ligation of the 6-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/Nco having a Ncol site in front of the ATG of the protoxin NcoI gene GAGGTAAC/CCATGG/ATAAC. 5 pg of this vector is digested with NcoI and the 3' recessed ends are filled in with Klenow polymerase as described by Maniatis et al.

28 Then, the plasmid is digested with AhaIII, 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 with EcoRI and the 3' recessed ends are filled in with 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 1l 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 this plasmid DNA is digested with BamHI and the a 4 Kb fragment is isolated from a soft agarose gel. This fragment is ligated to the self-replicating yeast vector pJDB207 (described in European patent specification No. 100,561), which previously has also been digested with BamHI (0.5 ig). 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 (MATa, leu 2-3, leu 2-112, his 3-11, his 3-15, can.

R

is performed as described in European patent specification 100,561.

_L ~L i 34 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 bioencapsulation form (an artificially constructed system consisting of biological material and comprising genetic material surrounded by protecting material) 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 by B. thuringiensis at sporulation and reaching the culture medium by breaking up the cell.

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 the suspensions thus obtained, some concentrations are prepared and 0.2 wetting agent admixed. The same leaf disc test as S described above in part III.7 is used to evaluate the insecticidal activity of these yeast cell preparations. The insecticidal activity S of B. thuringiensis-transformed yeast cells on Heliothis virescens first instar larvae is shown in the following table.

I O Table 4 0 0- BE ii BP a a r rl ri 11~ d t material concentration mortality in cells with toxin 1 5 72 1 :7.5 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 xl 35 Similar results are obtainable if yeast extracts which are prepared as described in European patent specification 100,561 are used instead of whole yeast cells 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 LO 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.

0 0 0 o The formulations, i.e. the compositions or preparations containing O" the transformed, living or dead yeast cells or mixtures thereof and, where appropriate, a solid or liquid adjuvant, are prepared in known cO, manner, e.g. by homogeneously mixing the ye,,st cells with solid S*o carriers and, where appropriate, surface-active compounds (surfac- 0o0 tants).

S 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, Ssepiolite 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.

36 Suitable surface-active compounds are nonionic, cationic and/or anionic surfactants 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 (Cio-C 22 e.g. the sodium or potassium salts of oleic or 0& 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)-eth-nesulfonic acid (amount in formulations preferably about 3 S, o More frequently, however, so-called synthetic surfactants are used, especially fatty sulfonates, fatty sulfates, sulfonated benzimidso azole derivatives, alkylarylsulfonates or fatty alcohols, such as, Sj for example, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (amount in ao' o formulations preferably about 2 4 o 0 C 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-C 2 2 alkyl 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 S 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 naphthalene-

I-

37 sulfonic acid and formaldehyde. Furthermore, corresponding phosphates, for example salts of the phosphoric acid ester of a p-nonylphenol-(4 to 14)-ethylene oxide adduct, are also suitable.

Non-ionic surfactant? are preferably polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, or saturated or unsaturated fatty acids and alkylphenols, said derivatives containing 3 to 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.

iD 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 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.

o° Representative examples of non-ionic surfactants are nonylphenol- .0 polyethoxyethanols, castor oil polyglycol ethers, polypropylene/ polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, o 0 '0O polyethylene glycol and octylphenoxypolyethoxyethanol. Fatty acid esters of polyoxyethylene sorbitan such as polyoxyethylene sorbitan trioleate are also suitable non-ionic surfactants.

44 Cationic surfactants are preferably quaternary ammonium salts which contain, as N-substituent, at least one Cs-C 2 2 alkyl radical and, as ,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.

li I 38 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 \l 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 Uop 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.

S* 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 a a time of application. Based on experiments conducted in the greenhouse, it is estimated that rates of application of 1 to 10 kg, in particular of 3 to 9 kg, of the yeast cells per hectare should prove Qo. advantageous.

i; c 1 39 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) 5% 94 1 b) 10 90 The yeast cells are suspended in methylene chloride, the is sprayed onto the carrier, and the suspending agent is ly evaporated off in vacuo.

suspension subsequent- F2. Dusts yeast cells highly dispersed silicic acid talcum kaolin a) 2% 1% 97 0 0 0 o o 4 0 0 0 e a o o so 0 0 0 laQ o 90 mixing the carriers Ready-for-use dusts are obtained by intimately with the yeast cells.

4 00 0 0 04 4 9 0 F3. Wettable powders yeast cells sodium lignosulfonate sodium lauryl sulfate sodium diisopropylnaphthalenesulfonate ontylphenol polyethylene glycol ether (7-8 moles of ethylene oxide) highly dispersed silicic acid kaolin c) 75 3% 6 2 5 10 62 27 10 n 40 The yeast cells are thoroughly mixed with the adjuvants and the mixture 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 carboxymethylcellulose 1 kaolin 87 \O 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.

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

o F6. Suspension concentrate yeast cells 40 ethylene glycol 10 nonylphenol polyethylene glycol (15 moles of ethylene oxide) 6 44 0 alkyl benzene sulfonic acid triethanol- 3 amine salt* carboxymethylcellulose 1% silicone oil in the form of a 75 aqueous emulsion 0.1 water 39 i 41 alkyl preferably being linear, containing 10-14, preferably 12-14, carbon atoms, such as, for example, n-dodecyl benzene sulfonic acid 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), Gittingen, 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 number of date of viabio deposition deposit* lity statement o 44 4 I I, 3C HD1-ETHZ 4449 March 4, 1986 DSM 3667 March 7, 1986 (Bacillus thuringiensis var. kurstaki HD1 strain ETHZ 4449) HB 101 (pK 36) March 4, 1986 DSM 3668 March 7, 1986 (Escherichia coli HB 101 transformed with pK 36 plasmid DNA) GRF 18 March 4, 1986 DSM 3665 March 7, 1986 (Saccharomyces cerevisiae) accession number given by the above-identified Intern:tional Depositary Authority.

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

L I- 42 Table 1 A B C M13mpi (BamilI) l-paI-H-indIII a) BamlI HindIIl luncII 8 HindIII BamnIl SinaI 9 EcoRI-PstI EcoRI PstI SinaI 8 >Psti EcoRI SinaI 9 a) according to given example o 3 3. 3. 3 33 3 33 ~*3 3 4 33 43 .4 43 44 43 43 I 43 43 0 OOC 00 C

C

0~0.0 0 OOe 0 000 0 00 0 0 C C C 00 000 0 0 0 00 0 0 0 C 0' 0- 00' nO 0 00 0 0 0 0 0 0 0 0 0 0 ~0 0 c 0 0 C C C 0 0 0 Table 2: Nucleotide sequence of DNA coding for protein MGE 1

GTTAAPCACCC

TCATAAi8ATG 130 AATTGGTtATC 190

AATGCATTCC

250

TAGAAACTGG

310 AfATTTGTTCC 370

GTCCCTCTCA

430

AAGAATTCGC

490

TTTACGCAGA~

TGTCAAAA

AGTQIAT(TGT

1110

TTAAT~AAA

200

TTATAATTGT

260

TTACACCCCA~

CGGTCT8iGA 300

ATGGGACGCA

440 500 ATCTTTTAG3A ATT8IATPTTT 90

TTTAAITTGT

150 iA8ATGGAGT 210

TTAAGTAACC

270 ATCGPATiTTT 330

TTTGTGTTAG

390 TTTCTT3T(AC 115 0

GCCATTTCTA'

5 10 GAG3TRGGAA

ATAAAATTA

100 160 AAtCTTATGGA 2120 CTGAATAt8

CCTTGTCGCT

GCTAGTT6#A 400 AAATT6CAACA 460 GATTAiGAAGG 520

CAGATCCTACQ

GTTGCACTTT

110 170

TAA"CAATCCG

230

PAGTATTAGGT

2190 AACfCAATTT 350

TIATAATATGG

410 GTTAA1TTPSAC 470

ACTAACAAPT

53 0

TAATCCAGCA

GTGCATTTTT

1,20

TIATCATPAATG

ACATCAAiTG 240

GGGAAA

300 CTTTTIAGT3

GGAATTTTTG

400

CTTTATCAAA~

540 TTAAGA3GA 550 560 57 0 0 09( 600 AGA~TGCGTAT TCAA2TTCAT GCIATAAA GTGCCCTTAC A4ACCGCTATT CCTCTTTTTG age 0 0~ 00 4 0 0 0 0 0 0 4 0 5 C 0 0 0 o 0 0 0 0 0 0 0 0 0 0 0 00 000 Table 2 (continuation) 610

CAGTTCAAAA

670 TATCAt3TTTT 73-,0

TCAATAGTCG

790

GCTGGTACA

ATAATCAATT

910 1CTATGATAG 970

CAAACCCAGT

1030

GAATATTAG

1090 CTCATA0GM3G 620

TTIATCAAG~TT

680 6PA6PAGPTGTT 740

TTATAATGAT

800 TACGGGiTTA 860 TA6IVAAGA 920 TAGAACGTiiT 980

ATTGAAAT

1040

GAGTCCACT

1100 AGA~ATATTAtT 63:0

CCTCTTTTA~T

690 TCAGiTGTTTG TTAACTA3GC 810 GAGCGTGTtAT 870

TTAATTCGAA

990

TTTGATGGTA

10 50

TTGATGGATA

1110

TGGTCAGGGC

610(-

CIAGTAT(ATGT

700 GAiCAAAGG3TG 760

TTATTGGCAA

820 G6S6PACCGGA 000 CTGT(ATTfM3 910 CtAGTTTCCCA 1000

GTTTTCGA(GG

1060

TIACTTWACAGV

1120:

ATCAATAAT

650 TCAAG3CTGC 710

GGGATTTGAT

770

CTPJITAAAT

TTCT~AAAT

890

TATCGTTTCT

950o 1010 CTCGGCTCM3 1070

TATAACCPATC

1130

GGICTTCTCCT

66C0

ATTTCATT

720)

GCCCGAPCTA-

CATGCTGTAC

900 CTAcTTTCCGA 960 1020 GGCATiAM3AB TI\TAC6GGTG 1140 GTA~GiGTTTT 1150J 1160 1170 1100 1190) 1200 CGGGCCAGAP ATTCACTTTT CCGCTPATATG GAATATGG AAATGCAGCT CCAQCAAC Table 2 (continuation) 1210 12220 123 0 1240 1250 1 26 0 GTATTGTTGC TCAACTAGGT CAGGGCGTGT ATAGAACATT ATCGTCCACT TTATATAGAA 1270 1,280 1290 1300 1310 1320 GACCTTTTAA IATAGGGATA ATATCAA~C AACTATCTGT TCTTGACGGG ACAGAATTTG 1330 13040 13 50 13-T6 0 13S70 1380o CTTAT6BGAAC CTCCTCAAAT TTGCCA~TCCG CTGTATtACAG AAAAACGA ACGGTA~GATT 1390 1400 1410 1420 1430 1440 CGCTGGATIA £ATACCGCCA CATAACA ACGTGCI7ACC TAGGCAAGGA TTTAG.xTATC

U-I

XO1450 1460 1470 1480 1490 15 00 GATTAAGCCA TGTTTCAATG TTTCGTTCAG GCTTTAGTAA TAGTAGTGTA AGAAAA 1510 1520 15 30 1540 15 50 1560 GAGCTCCTAT GTTCTCTTGG ATACATCGTA GTGCTGAATT TTATATA ATTCCTTCAT 1570 1580 1590 1600 1610 1620 CACAAATTAC ACAAT.CCT TTAACAAAAT CTPACTATCT TGGCTCTGG ACTTCTGTCG 1630 1640 1650 1660 1670 1600 TTAAAGACC CAGGATTTACA 68 GAAT TTCTTCGAAG AACTTCACCT GGCCAGATTT 1690 1700 1710 1720' 1730 1740 CAACCTTAAG (ATAAATATT fACTGCA'~CCAT TATCACAAAG A'TTCGGGTA fMGfATTCGCT 1750 1760 1770 1700 1790 1800 ACGCTTCTAC CACATTTTA CAATCCATA CATCAATTGA CS fA8PhiCT ATTATCA~G I 1; 101 Table 2 (continuation) 18310 1820 1830 18410 12350 1860 GGATTTTTC AGCAACTATG AGTASTGGGA GTAATTTACA GTCCGGAAGC

TTTAGGACTG

18E70 1880 1890 1900 1910 192:0 TAGGTTTTAC TACTCCGTTT AACTTTTCAA ATGGATCAAG TGTATTTACG TTAG8TGCTC 1930 1940 1950 1960 197o 1980 ATGTCTTCAA TTCAGGCAAT GAAGTTTATA TAGATCGAAT TGAATTTGTT

C~CGGCGA

1990 21000 2010-)I 2020 -1030 21040 TAACCTTTGA GGCAGAATAT GATTTA~GAA GACCAA GCGGTGAT

GAGCTGTTTA

2050 2060 2070 2080 2090 2100 CTTCTTCA TCAAATCGGG TTAACAG ATGTGACGGA TTATCATATT

GATCAAGTAT

2110 2120 210 214 2115 0 2160 CCAATTAG TGAGTGTTTA TCTGATGAAT TTTGTCT8GG TGAAA

GAATTGTCCG

2170 21002920 A 6AATCAA ACATGCGAAG CGACTTAJST6

(ATAC~GG

22 ;O 2240 2250o 22 TTAGAGGGAT CAATAGACAA CTAGACCGTG

GCTGGAGAGG

2290 2-00 231 232"0 AAGGAGGCGA TGACGTATTC AAGAGAATT

ACTTACGCT

2 350C 2 36 0 2370 2 GCTATCCAAC GTATTTATAT CAAAATA ATfMMTCA 2210 2220 TTTACTTCAA

GATCCAAACT

,2270 22 AATCGA

ATTACCA~TCC

ATTGGGTACC

TTTLGATGAGT

23 90 2400 ATAAAC TATCCC6TT Table 2 (continuation) 2410 24 20 2431C 2440 '24 50 246 0 ACCAATTAG AGGGTATATC GTAGTC AAGACTTAGA ATCTATTTA

ATTCGCTACA

2470 2480 2490 2 5 00 2510 2520 ATGCCAAACA CGAAACAGTA ATGTGCCAG GTAC6GGTTC CTTATGGCCG CTTTCAGCcC 2530 21540 2550J 2560 25 70 2580O CAAGTCCAAT CG6AATGT GCCCATCATT CCATCATTT CTCCTT8GC

ATTGATGTTG

2590 2600 216 10 2 62-l0 26 30 2640 GATGTACAGA CTTAAATGAG GA~CTTAt38TG TA"TGGGTGAT A'TTCAGAMgTT

AAGCGCAG~

I02650 2660 2670 2600 2690 2700 ATGGCCATGC AAGACTAGGA A4ATCTGAAT TTCTCAAGA GAACCATTA GT88G8AG 2710 2720 2730 2740 2-750 27 CACTAGCTCG TGTGAAAAGA GCGAGAAA ATGGA8GG CAAACGTGA

AAATTGGAT

2770 2780 2790 2800 2810 2820 668 AACAAA TATTGTTTAT AAAGAGG3CAA( AAfnA8(TGiT O'G(IT6CTTTA

TTTGTAACT

2830 28410 2850 2060 28070 24880 CTCAATATGA TAGATTACAA GCGTACCA ACATCGCr3AT tiATTCA~TGCG 6GCAGTAAAC 2890 2900 2910 29210 299 240 GCGTTCATAG CATTCGAGAA GCTTATCTGC CTBGA8CTGTC TGTGATTCCG 2950 2960 2 9 70 2980 2990 3,C00 CGGCTATTTT TGAAGATTA -GAGCT TTTTCA~CTGC (ATTCTCCCTA TATGATGCGA Table 2 (continuation) 3010 3-10 20 3030. 3040 3 050 3 6 0 GAAATGTCAT T AATGGT GATTTTAATA ATGGCTTATC CTGCTGnC GTGA~8BGSC 3-070 30830 3-1090 3100 3110 3 120- C ATGTAGATGT AGAGACA ACAACCACC GTTCGGTCCT TGTTGTTCCO OrinTOGGAAG 3130 31410 150 3160 3170 ::18 CAGAAGTGTC ACAGAATT CGTGTCTGTC CGTCGTGG CTA-ThTCCTT CGTGTQCCAG 3 190 3200 3210 3220 3 2 30 32-40 CRGTACAAGGA GGGATtATGGA GAAG~GTTGCG TACCATTCn\ TGAGAPTC~GG ACATCAG 4 1032-0 3260 =270 3200 =290 ACGAACTGAA GTTTAGCAAC TGTGTAGAAG A6GG 6TATA TCCACAAC ACGGTAACGT 33 10 3320 3330 330 0 36 P3TAATGATTA TACTGCGACT CAGAAGAT ATGAGGGTAC GT..CACTTCT CGTAATCGAG 3370 3330 339 340 40 342 0 GATAT6PACGG AGCTfATG(- AUCAI)T1C:TT CTGTPACCCOGC TtMATTAiTGCA' TCAGCCTPATG 343 340-15 160 3470 3480 AGAdnAAGC ATATACAMGAT GGACG 6PGLG ACARTCCTTG TGATCTAAC AGAGGM3ATATG 3490 3500j 3510 3 52 0 -530 3540 GGGATTACAC ACCA"CTACCA GCTGf3CTATG TGACAAAAGA ATTAGAGMTA~C TTCCCAGAA 3550 3560 3570 3580 3590 36 00C CCGAT5VA6GT fATGGATTGAG ATCGGAfAAgA CGAAGGAAC ATTCATCGTG GACAGCt3T8G o OO~' 00 0 p 0 t- 000 0 00C 0 00 00 00 0 0 0 0 U C 0 0 U C 0 0 0 ~o p 0 0~ U- PC 000 Table 2 (continuation) 3610 3620 363-10 36 40 365C0 3660 AATTACTTCT TATGBGGAA TAATATATGC TTTAAATGT AAGGTGTGCA AATAAAAT 3670 37600 3690 37 00 3 710 37720 GATTACT6TAC TTGTATTGAC AGTAAA GG6ATTTTT ATATAATA AAACGGGCA 3730 3740 3730 3760 3770 .3780 TCACTCTTA AAGATGATG TCCGTTTTTT GTATGATTTA ACGAGTGATA TTTAAATGTT -3790 .'8900 33)O10 3-720 323 03840 TTTTTTGCGA -AGGCTTTACT T ACGGTA CCGCC(ACATG CCCATCAACT TAJAGAATTTG 3 85 0 Ze60 3870 3880 3090 3900 CACTACCCCC AAGTGTCAAA AAGTTATT CTTTTAA AGCTAGCTAG A82GTGAC 3910 ,920 3930 3 9 40 395o -396C0 ATTTTTTATG AATCTTTCA TTCAAGATGA (ATTACTA TTTTCTGAAG AGCTGTATCG 3970 3980 3 990 '1000 4010 10--20 TCATTTACC CCTTCTCTTT TGGAAGA(WTC CGCTAAAGAAM '*rTAGG3TTTIf3 TAAAAA 4030

ACGAAAGTTT

4040

TCAGGAAATG

4050 4060 4070 4080 AA~TTf8CTAC CATATGTATC TGGGGCAGTC AACGTACAGC 4090 4100 41110 4120 4130> 4140 GAGTGATTCT CTCGTTCGAC TATGCAGTCA TACACGCC GCCACAGCAC TCTTATGAGT 4150 4160 4170 4180 4190 4200 CCAGAGGAC TCAATAAACG CTTTGATAAA AAAGCGGTTG APATTTTTGA ATIATATTTTT a 00 0 a 00 a t a

S

00 3CO Table 2 (continuation)* 4210 4220 42 -0 424C:) 4250 4260 TCT6CATTAT GGAGST4A AMTTTTgAA ACATC1AGCCA TTTCAAGTGC AGCACTCACiG 427 0 42130 4290 4 300 43 ,10 4320 TATTTTCAAC GAATCCGTAT TTTIAGITGCG ACI3ATTTTCC AATCCGAAP ACATTTAGCA '1330 4340 4350 4360 CATGTATATC CTBGGTCAd3G TGGTTGTGCA CAAACTG3CAG( c' 0 400 0 000 0 0 0 0 a c 0 0 408 Table 3: Amino acid sequence of protein MGE 1 LIMITS: 1.56 3623 195 g 1 TG GAT AAC~ AT CCG AAC ATC AVAT GAAV T(3C ATT CCT TAT AAT TGT TTA nGT M~C CCT Met A~sp Asn Asn Pro Asn Ile Asn Gilu Cys :Ile Pro Tyr Asn Cys LELLU 9cr Asn Pro GTlA GAA GTvA TTSA GGT GGrA GPA/' AGA~ iAT Val Chitj Val Leu Gly Gly Gilu (rg Ile TCG CTA ACG CAA TTT CTT TTG AGT GAA 9cr LeU~ Th r Gin Phe LeU LeU~ S9er GILU GTT GA\T AT e 1 T( TOO GGrA AT TTT GGT Val A~sp Ilie Ile Trp Gly 1ie Phe Gly Glu Gin Letu Ile A-sn Gin A~rg Ile GIl GAA GGA CTA (AGC AAT CTT TAT CAA ATT Glu Giy Leu Ser Asn Leu Tyr Gin Ilie CCT ACT AAT CCA GCA TTA AG GAA GAG Pro Th r Asn iro Alia LCLL Arg G1.Lt GILI 245 305

TTT

Ph e 365

CCC

Pro 425 4985 TgAC Tyr Met ACT GOT TLAC A~CC CCA ATC GAT ATT TCC Thr Giv Tyr Thr Pro Ile Asp Ile Ser GTT CCC GGT GCT GGA TTT GTG TT/A GOA Val Pro Gly Ala Gly Ph P Val LeLI Gly TCT CAA( TGG GAC BC(A TTT CTT GTA CAA 9cr Gin Trp Asp Alia Phe LeLI Val Gin TTC GCT AGG AAC CAA GCC (ATT TCT AG FPic Alia (Arg Asn (11 ni Al a Ile Ser (-'rg OCA G(YA TCT TTT AGAI GAG TGG GAAy~ GCA Ala Gilu Ser Phe (Arg SiLU Trp SiLL Alia COT iATT CAA TTC AAT GAC ATG AAC AGT A'rg Ile Gin Phe (Asn A~sp Met Asn 9cr 215 275

TTG

Le Lt

CTA

3.95

ATT

Ile 455

TTIA

Leu 515

GA~T

A'sp 575

GCC

Alia C9 0 a a -z Table 3 (continuation) CTT ACAl ActCC GOT ATT CCT CTT TTT GCA Leu Thr Thr Ala lie Pro LeuFhe Ala TAT GTT CAA GCT GCA AAT TTA CAT TTA Tyr Val Giin Ala Ala Asn Leu His Le AGG TGS GSA TTT GAT GCC GCG ACT ATC Arg Trp (31y Fhe Asp Ala AlF Thr Ile GGC AAC TAT ACA GAT CAT GCT GTA CGC Sly Asn Tyr Thr Asp His Ala Val Arg CCG GAT TCT AGR GAtT TGG ATA AGA TAT Pro Asp Ser Arg Asp Trp Ile Arg Tyr TTA GAT ATC GTT TCT CTA TTT CCG AAC Leu Asp Ile Val Ser Le Fle Pro (Asn TCC CAA TTA ACA AGA GAA ATT TA'T ACA Ser 31 n Leu Thr Arg Gl. U Ile Tyr liii- CGA GGC TC GC CAG GOC ATA GAA GGR Arg Sly Ser AIa Bin Sly Ile G1.u Sly 605

GTT

Val 665

TCA

Ger 725

AAT

ASn 705

TGG

045

AAT

Asn 905 Tyr 965

AAC

Osn 025

AGT

Ser CAA A(Y TAT CAAYP GTT CCT CTT TTO TCA Bin Asn Tvr Gin Val Pro LeU Leu Ser STT TTG AGA GAT OTT TCA GTG TTT GGA Val Leu Arg Asp Val Ser Val Phe Sly AST COT TAT AAT GAT TTA ACT AGG CTT Ser Arg Tyr Asn Asp Leu Thr Arg Leu TAC AAT ACG GSA TTA GAG CST GTA TGG Tyr Asn Thr Sly Leu Glu Arg Val Trp CAA TTT AGA AGA GAA TTA ACA CTA ACT Bin The -rg Arg GLlU LeLt Thr Le Thr GT AGT AGA A-CG I-ArT CCA ATT CGA ACA Asp 8cr Arg Thr Tyr Pro Ile Arg Thr CC( GTA TTA GA AAT TTT GAT GGT AST Pro Val LRLt (31ii U sn PIFe A~sp Sly Ser 635

GT(R

Va1 695

CAA

Bin 755 PtTT I. e 815

GG(-A

Sly 975

S-TA

Va1 935

STT

Val 995

TTT

Phe 055

CTT

LeU In

I"

I

lie ATT AGG AGT CCA CA-T TTG ATG GAT Ile Arg Ser Pro His LeLl Met Asp 7 Table 3 (continuation) 0 0 0 4' U AAC AGT AT ACC ATC IAsn Ser Ilie Thr Ilie AT(A ('JO OCT TCT CCT I:le Met Alia Ger Pro ('JO GGA A(T GC(A OCT Met Gly Ys~n Al~a Ala AtCA TTPA TCG TCC ACT Thr Leu Ser Ser Thr TCT GTT CTT GAC 000 Ser Val Leu Asp Oly TAC AGAY AAA AGC OOA Tyr Arg Lys Ser Oly C:CA CCT (AOO CAA GGA Pro Pr o Arg Gln Gly 1085 TAT ACG GAT OCT CAT AGA~ Tyr Thr Asp Ala His Arg 1145 GT(A 000 TTT -rCG 000 CCA Val Oly Phe Ser Oly Pro 1205 CCtA CAA~ CAA COT ATT OTT Pro Oin Oh-i (rg Ilie Val 12 65 TTA TA'T AGA~ AGAO CCT TTT LeLL Tyr Ar (Arg Pro Fhe 1325 A~CA GAA TTT OCT TA~T OO(A Thr 01Lu Phe Al a Tyr 01 y 1305 (ACG OT(A GAT TCG CTO GA~T Thy- Val A-SP S cr Le FU SP 1441 TTT (AOT CAT CGA TTA (AOC Phe Ser His (hrg LeLIG(_ er 1115 GGA GAA TAT TAT TOO TCA 000 CAT CAA Oly Olu Tyr Tyr Trp Ser Oly His Oln 1175 SAA TTm A'CT TTT CCO CTA TAT OO(A ACT GlLI Phe Thr Fhe Pro LeLI Tyr Oly Thr 1235 OCT CAA CT(A OOT CAG GO--C OTO TAT AGA( Al1a Gin L(LI Oly Gln Oly Val Tyr (Arg 1295 AAT PATA 000 OTO AAT AAT CAA CAA CTPA Asn Ile Oiy Ile Asn (Asn Gln Oh-i Let 1355 ACC TCC TC(A AT TTG CCA I-CC OCT GTA Thr Ser Ocr Asn L e U Pr o Ser Al]a Val 1415 GAA AT( CCO CCA CAG AAT (VC AAC OTO GiU Ile Pro Pro G1 i (Osn A! (Asn Val 1475 CrAT OTT TCA ATq TTT COT TCA GGC TTT Hi s Val Ser Met Phe (Arg Ser Gly Phe 0c0 Table 3 (continuation) AG3T P~AT AGT AGT GTA AGT A'TPA AT Ser Asri Ser Ser Val Ser Ile Ile GAA TTT PAT PAT AT ATT CCT TCA GI u Ph e, Asn (Asn 1Tie Ile Pro SOr PAT CTT GGC TCT Gn A iCT TCT GTC Pisn LCLL Gly Ser Gly Thr Ser Val CG(A AGA~ ACT TCA CCT (36C CfA6 ATT Arg Ar-q Thr Ser Pro Gly Gin Ile CAA AG TAT CGG GTA AG ATT CGC Gin (Arg Tyr Ar-o Val rg Ile Arg ATT GAC GGA AGA3 CCT ATT PA~T CAG Ile A~sp Gly Arg Pro Ile Asn Gin TTA CAIG TCC GGA AGC TTT AGG ACT LeLL Gin Ser 3 iy Scr Plie Arg Thr 1505 AGAr~ GCT CCT ATG TTC TCT TGG AT(A Arg Ala Pro Met Phe 8cr Trp Ie 1565 TCA CPA ATT AC CAA AT CCT TTA Ser Gin ]Ie Thr Gin Ile Pro Leu 1625 GTT AA GGA CCA~ GG(- TTT AC GGAP Val Lys (My Pro Gly Flie Thr Gly 16805 TCA A~CC TTA AGA( GTA PA~T (ATT A~CT Ser *rhr LCLI A-rg Val iAsn le Thri 1745 TAC GCT TCT ACC AC PA~T TTA CAA Tyr Al a 8cr Th r Thr 0-sn* LeLI Gin I 105 GG6( APT TTT TCPL (3C( A'CT A'TG AGT Glv Amis Plie SerI Ala ihr Met Ser 1005 (3TA' GGT TTT A~CT A'CT CCG TTT PAJ'C Val (Ily Phe Thr Thr Pro Phe A"sn 1535 CA~T COT A3T (3CT His A'rg 8cr Ala 1595 Thr Lys 9cr Thr 1655 GG(A GAT ATT CTT Giy Asp Ile LCLL 1715 GCA~ CCA TTIA TCA Ala Fro Leu 8cr 1775 TTC CAT A~CA TCiA Phe His Thr Ser 1835 8cr Gly 8cr rAsn 1895 TTT TCAPA T GGA' Fhe 8cr Asn (Riy 1955 GTT TAT AT GAiT Vai Tyr Ile A'sp 1925 TCA (M3T GTA TTT TTA A:MT (3CT CAT (3TC TTC PA('T TC(\ G;(C PAiT G3AP Ser Ser Val Phe Thr LCLI 8cr Alia Hi~s Val The Ser Gly (Asn (MU 1 I r I ~g :L F ~I~x.

et c, Table 3 (continuation) CGA ATT GAA TTT GTT CCG GCrA GA Arg Ile Glu he Val Pro Ala Glu CAA AAG GCG GTG AAT GAG CTG TTT Gin Lys Ala Val Asn Giu Leu Phe AUG GAT TAT CAT ATT GAT CAA GTA Thr Asp Tyr I- s lie Asp Gin Val CTG GAT GAA AAA AAA GAA TTG TCC Leu Asp G].u Lys Lys Glu Leu 6er CGG AAT TTA CTT CAA GAT CCA AAC Arg Asn Leu Leu Gin Asp Pro Asn AGA GBG GT ACG GAT ATT ACC PATC Arg 31y Ser Thr Asp Ile Thr 1le 1985 GTA ACC Val Thr 2045 ACT TCT Thr Ser 2105 TCC AAT Ser Asn 2165 GAG AAA GIu Lys TTT AGA Ple Arg 2205 Gin G Iy 234 5 TG TAT Cys Tyr TTT GAG GCA GAA TAT GAT TTA GAA Phe Glu Ala Glu Tyr Asp Le Gits TCC AAT CAA ATC GGG TTA AA CA Ser Asn Gin Ile G1.y Let Lys Thr TTA GTT GAG TGT TTA TCT GAT GAA LeL Val GILU Cys Le Ser- Asp SIt.

(TC AAA CAT GCG AAG CGA CTT AGT Val Lys His Ala Lys Arg Leu Cer GGG ATC AAT AGA CAA CTA GAC CGT Gly Ile Asn Arg GIn LeU ASP Arg GGC GAT GAC GrA TTC AAAS GAG AAT Gly Asp Asp Y~l Fhe Lys GIL A5sn CCA ACG TAT TTA TAT CAA AAA ATA Fro Thr Tyr Le Tyr Gin Lys Ile 2015 AGA GCA Arg Ala 2075 GAT GTG Asp Val 2135 TTT TGT Phe Cys 2195 GAT GAG Asp Glt GGC TGG Gly Trp 2315 TAC GTT Tyr Val 2375 GAT GAG Asp Glu A) ACG CTA TTG GGT ACC TTT GAT GAG Thr Leu Leu Gly Thr Ple Asp Glu Table 3 (continu'ition) 2405 2405 TCG AAAIP TTPI AAAI (CC TAIT ACC CGT TPIC CAAP TTPI AGAP 006 TAIT PTC GP GAT PIT CAAP GPC Ser

TTPI

L eu

GT

Gay

CAT

Hi s

GTG

Val1

OGAA

61 u

AGAI

TCT

Ser Lys

GPAA

TCC

Ser

TTC

Phe

PITA

Ilie

GAG

Asp

GTA

Val1

LELI

PITC

Ilie

TTA

Le LI

TCC

S er

TTC

Ph e

AAAIP

Ly s

AAAIP

Lys

GAT

Asp Lys

TAT

TG

Tr p TT6 L eLI

PIPG

Lys

CCA

Pro

CGT

PArg

OCT

Al a Ala

TTPI

LeLI

CCG

Pro

OPIC

Asp

PITT

Ile

TTA

LELI

GAA

61 LI

TTPI

LELI

Tyr

PITT

Ile

CTT

L eLI

PITT

I Ie

APIG

Lys

GTA

Val

PAAA

Lys

TTT

Ph e Th r

CGC

Arg

TCA

Ser

GAT

s p PIC6 Thr 66A 61 y

TTG

Le LI

GTA

Val

TAC

Tyr 6CC Al a

GTT

Val1

CAPI

Gln

GAP

AC

Tyr Gin 2465 PAPT GCC PAsn Al a CCPI AGT Pro Ser 2505 GGAP T(3T 3 i y Cys 245 OAT GGC Asp (31 y 2705 GCA (ZTA h~l a LeII 2 7625 TOO GAPI Trp GI Lt 2B325 TCT CAP Ser G].n LeLI Arg Gly Tyr Ilie GiLI ASP Ser Gin Asp Lys

CCPI

A CA Th r

CAT

His B CT Al a

AICA

Thr

TAT

Tyr

CAC

Hi s

ATC

Ile

GAC

A SI]

GCA

CGT

A sn

OAT

Asp

GPA

O i LL

GOA

Gi y

TTA

L ELI

AGAP

Arg (3T Val

PITT

lIeR

AGAI

Ar q

PICA

Thr

AAAP

Lys

PAPT

PIsn

CTPI

LeuL

PAAA

L-ys

OTT

Val

TTPI

L eLI

OTA

Val

TOT

Cys

GAGG

O 1LI

GGA

Giy

AGAI

A rg

TAT

Tyr

CAP

Gin

PAPT

Asn (3CC A~la

SAC

Asp

APIT

PAsn

GCG

Alia

AAAP

Lys

GCG

Al a GT(3 Val

CAT

His

TTPI

L eLI

CTPI

LELI

GAG

GilI

IG

61 LI

GAT

Asp

CCPI

Pr o

COT

Hi s

GGT

01 y

GAPI

(31LI

AAAP

Lys

GCPI

A I a

ACC

Th r 2495 GOT ACG GI y Thr TCC CAIT Ser Hi s 2615 (3TP T(GG Val Trp 2675 TTT CTC Phe LeLI 2735 PAAA TG Lys Trp 2795 PIPIP GAPI Lys Gi Lt 2855 AC PITC PAsn Ilie

C

C C C C C C

C

2.

Table 3 (continuation) GCE (ATG fTT CAT GCE GCA SAT AA A~la Met Ile His Ala Alia Asp Lys CTO TCT GTE ATT CCG GGT ETC AAT Leu ESer Val Ile Pro Sly Val Asn ACT GCA TTC TCC CTA TA~T GAT BCG Thr A1a Phe 9cr LeU Tyr (Asp Ala TTA TCC TGC TGO AAC GTE AAA( GG Leu 9cr Cys Trp Asn Val Lys Sly ETC CTT OTT GTT CCG GAA TEE GAA Val LeLI Val Val Pro GilU Trp GilU CST EEC TAT PATC CTT CST GTC ACA'- (Arg Sly Tyr Ile Leu Arg Val Thr (ATT CA~T GAS ATC GAG AAC An(T OC(A Ile Hi~s GiLu Ile Elu A~sn (Asn Thr 2885 CEC GTT Arg Val 2945 BCE OCT Alia Ala 0005s CAT GTA His Val 31.25 GCA GOA AI a i1 1105 ECG TIAC Ala Tyr 3245 GA(C GOA (Asp Eiu C(AT (AGC (ATT CGA (A(A OCT T(AT CTG His cr Ile (Arg Gi.t UAla Tyr Let- (ATT TTT O(AA G(AA TTA G(AA GEE COT Ile Flie Glu L iLt ElU ly (Arg ETC (ATT (AAYA (AAT GET G(AT TTT (AAT Val Ile Lys (Asn Sly (Asp The (Asn G(AT ETA E(AA (A(A C(AA (AAC (AAC C(AC (Asp Val Gu EluI,Gln (Asn (Asn His GTE TCA C(AA (A(A OTT CST ETC TOT Val 9cr Gin GI U Val (Arg Val Cys (A(A G(AG GGA T(AT GGA E(AA EET TEC Lys Giu Ely Tyr Sly G1u Efly Cys CTE (A(A TTT (AGC (AAC TOT GTA (A(A Leit Lys Phe Ser (Asn Cys Val GOh 2915 CCT G(AG Pro ElU 2975 (ATT TTC Ile Phe- 3035 (AAT EEC (Asn El y 3095 CET TCE eArg Ser 3155 CCE GET Pro Ely 3215 ETA (ACC Val Thr 32? 1 EGAE OGAA GilU EIA!' Table 3 (continuation) GTA TAT CCA AAC OVOC (CG GTA A~CO Val Tyr Pro /Asn Asn Thr Val1 Thr GGT OCG TAC ACT TCT C(3T AAT CGA" Gly Thr Tyr Thr S er Arg O~sn Argj CCPA GCT SAT TAT GCA TCA 6CC TAT Pro Al Asp Tyr- Ala Ser Ala Tyr CCT TGT GAA TCT AAC AGA~ GL3 TA~T Pro Cys G1U Ser PAsn Arg Gly Tyr AAA GAA TTIA GAG TAiC TTC CC(A GAA Lys GilU LeU G1 L Tyr Flie Pro GIuL GGIA AC TTC ATC GTG G(AC AGC GTG Gly Thr Fhe I Ie Val Asp Ser Val 3C)o TGT AAT Cys gAsn GtA I-AT GIly Tyr 3425 EGtA GA(-A Glu GiL( GEE GA~T 6i y iAsi ACC GAT Thr Asp 5.60 GOO TTPA GI L Leuf SAT TAT A~CT GCG ACT CAA~ GAA GA Asp Tyr Thr Ala Thr Gin GiUt GIU GAC GGA (3CC TAT GAAV AGC AAT TCT A-sp Ely A~la Tyr GI U Ser fAsn Ser AAA~ GCA TAT AC GAT GGAt CGA AGA~ Lys Ala Tyr Thr Asp 012 Arg Arg TAC AC CCA CTA CCA GCT GGC TAT Tyr Thr Pro Leut Pro Ala Gly Tyr AA GTA TGG ATT GAG ATC GGA GAA Lys Val Trp Ile Gil Ilie Gly GIL( CTT CTT ATG GAG GAA TAA Leut Leul Met GILI GlU End TAT GAG Tyr G1iL TCT GTA Ser Val GAC AAT GTG A~CA Val. Thr 357 PCG GAA Thr GlU I I: 59 Bibliography 1) S. Chang, Trends in Biotechnology 1 100 (1983) 2) H.C. Wong, H.E. Schnepf and H.R. Whiteley, The Journal of Biological Chemistry 258 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) SH.E. Schnepf, H.C. Wong and H.R. Whiteley, The Journal of Biological Chemistry 260 6264 (1985) A.A. Yousten and M.H. Rogoff, Journal of Bacteriology 100, 1229 (1969) oo o 6) 6 )o T. Maniatis, E.F. Fritsch and J. Sambrook, Molekular Cloning: S°u A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, USA, Appendix C: Commonly used bacterial strains, pp. 504, 506 (1982) .5 7) 7 F.F. White and E.W. Nester, Journal of Bacteriology 141 1134 (March 1980) 8) B. TrUmpi, Zentralblatt f. Bakteriologie Abt. II, 305 (1976) 0 00 9) SF.P. Delafield, H.J. Sommerville and S.C. Rittenberg, Journal S of Bacteriology 96, 713 (1968) 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) 0. Ouchterlony, Handbook of Immunodiffusion and Immunoelectrophoresis, Ann Arbor Science Publishers, Ann Arbor, Mich., USA (1968) H. Huber-Lukac, PhD thesis Swiss Federal Institute of Technology Zurich, Switzerland, No. 7050 (1982) Amersham Buchler Review No. 18, Amersham Buchler GmbH Co.

KG, Braunschweig, Federal Republic of Germany (1979) 14) T. Maniatis, A Laboratory Harbor, USA, E.F. Fritsch and J. Sambrook, Molecular Cloning: Manual, Cold Spring Harbor Laboratory, Cold Spring p. 282 (1982) E.F. Fritsch and J. Sambrook, Molecular Cloning: Manual, Cold Spring Harbor Laboratory, Cold Spring p. 252 (1982) T. Maniatis, A Laboratory Harbor, USA, oon 0o 0 *0 0 o 6o 0o o 0 0 00 0 0 o 50 o1 e uj H.E. Schnepf and H.R. Whiteley, Proc.Natl.Acad.Sci. USA 78, 2893 (1981) 1 L. Clarke, R. Hitzeman and J. Carbon, Methods in Enzymology 68, 436 (1979) 18) E.M. Southern, J. Molec.Biol. 98, 503 (1975) T. Maniatis, A Laboratory Harbor, USA, T. Maniatis, A Laboratory Harbor, USA, E.F. Fritsch and J. Sambrook, Molecular Cloning: Manual, Cold Spring Harbor Laboratory, Cold Spring p. 383 (1982) E.F. Fritsch and J. Sambrook, Molecular Cloning: Manual, Cold Spring Harbor Laboratory, Cold Spring p. 387 (1982) 21) N. Maizels, Cell 9, 431 (1976) 22) M. Grunstein and D. Hogness, PNAS 72, 396 (1975) c ,r i ll 61 ZL) 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. Sanger, S. Nicklen and A.R. Coulson, Proc.Natl.Acad.Sci. USA, 74 5463 (1977) J. Messing, Methods of Enzymology 101, 20 (1983) 26) M. Poncz, D. Solowieczyk, M. Ballantine, E. Schwartz and S. Surrey, Proc.Natl.Acad.Sci. USA, 79, 4298 (1982) 27) M.J. Zoller and M. Smith, Nucl. Acids Res. 10, 6487 (1982) on 0 0 0a o 0 o 40 oa 0 O O D 0n 0 o 0 0* 00 6 0 a T. Maniatis, A Laboratory Harbor, USA, T. Maniatis, A Laboratory Harbor, USA, E.F. Fritsch and J. Sambrook, Molecular Cloning: Manual, Cold Spring Harbor Laboratory, Cold Spring p. 394 (1982) E.F. Fritsch and J. Sambrook, Molecular Cloning: Manual, Cold Spring Harbor Laboratory, Cold Spring p. 392 (1982) Y. Shibano, A. Yamagata, N. Nakamura, T. lizuka, H. Sugisaki and M. Takanami, Gene 34, 243 (1985) A. Krieg in: The Procaryotes, a handbook on habitats, isolation and identification of Bacteria (Eds. Starr, P.M., Stolp, Trueper, Balows, A. and Schlegel, Springer Verlag New York, Heidelberg, Berlin, p. 1743 (1981) I -L i W I-

Claims (39)

1. A DNA fragment originating from Bacillus thuringiensis var. kurstaki characterized by the nucleotide sequence given in table 2 comprising a DNA sequence coding for an insecticidal toxin protein including truncated portions of the said DNA fragment, said truncated DNA portions being subject to the proviso that insecticidal activity of the corresponding toxin protein has not been lost.

2. A truncated DNA fragment according to claim 1 ranging from HpaI to PstI (4355).

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

4. A toxin protein including truncated portions thereof, said truncated DNA portions being subject to the proviso that insecticidal activity has not been lost, being encoded by at least a part of the DNA fragment according to claim 1. A toxin protein including truncated portions thereof, said truncated DNA portions being subject to the proviso that insecticidal activity has not been lost being encoded by at least a part of the DNA fragment according to claim 2.

6. A toxin protein containing a fragment characterized by the amino acid sequence given in table 3. o.o1 7. The protein MGE 1 as hereinbefore defined being encoded by a DNA fragment according to claim 1.

8. The protein MGE 1 as hereinbefore defined being encoded by a DNA fragment according to claim 2. KW iiiii.- i i. ii -Ilil i _1__II 63

9. The protein MGE 1 as hereinbefore defined characterized by the amino acid sequence given in table 3. A DNA vector containing a DNA fragment according to claim 1.

11. A vector according to claim 10 which is a plasmid.

12. A vector according to claim 10 which is a phage.

13. A DNA vector containing a DNA fragment according to claim 2.

14. A vector according to claim 13 which is a plasmid. A vector according to claim 13 which is a phage. S 16. A microorganism containing a DNA fragment according to claim 1 with the proviso that, if said microorganism belongs to the group of Bacillus thuringiensis, said Bacillus has been previously transfocmed with the DNA fragment according to claim 1.

17. A microorganism according to claim 16, said microorganism belonging to the species Saccharomyces cerevisiae. S18. A microorganism containing a DNA fragment according to claim 2 with the proviso that, if said microorganism belongs to the group of Bacillus Sthuringiensis, said Bacillus has been previously transformed with the DNA fragment according to claim 2. o o 19. A microorganism according to claim 18, said microorganism belonging to the species Saccharomyces cerevisiae. A bioencapsulation system consisting of a first material completely e'nbedded 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 represented by a whole, living or dead microorganism or by 64 a mixture thereof with the proviso that, if said microorganism belongs to the group of Bacillus thuringiensis, said Bacillus has been previously I transformed with the DNA fragment according to claim 1.

21. A bioencapsulation system according to claim 20, wherein the micro- organism is Saccharomyces cerevisiae.

22. 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 2 and the second material being represented by a whole, living or dead microorganism or by a mixture thereof with the proviso that, if said microorganism belongs to the group of Bacillus thuringiensis, said Bacillus has been previously nao transformed with the DNA fragment according to claim 2.

23. A bioencapsulation system according to claim 22, wherein the micro- 0 organism is Saccharomyces cerevisiae.

24. 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 HD1, separating plasmids from the material thus obtained by methods known per se and purifying and dialysing the plasmid material thus obtained; 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 as hereinbefore defined by I applying an antigen/antibody test. 4 A Imethod according to claim 24 wherein step d) comprises the following particular steps: e) screening of the clones for the presence of antigen responding to antibodies prepared against the crystal protein of B. thuringiensis var. kurstaki; 65 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).

26. A method according to claim 24 wherein, in step the vector is a plasmid.

27. A method for producing a DNA fragment according to claim 2, which method comprises the following steps: a) isolating and lysing cells of B. thuringiensis var. kurstaki HD1 and separating plasmids from the material thus obtained by methods known per se and purifying and dialysing the plasmid material thus obtained; 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 as hereinbefore defined by applying an antigen/antibody test. 8 8 I 0 0 O o a u8 o 88? 8 a 98 ^o a 0 0 0 00 0 co o

28. A method according to claim 27 wherein step d) comprises the following particular steps: 88 e) screening of the clones for the presence of antigen responding to antibodies prepared against the crystal protein of B. thuringiensis var. kurstaki; f) selecting the clones being specifically reactive with goat antiserum; and g) testing insecticidal activity of extracts of said clones obtained 00 4 0 0 according to step f). 4O

29. A method according to claim 27 wherein in step c) the vector is a plasmid. A method for combating insects which comprises applying to the insects or their habitats an insecticidally effective amount of a toxin protein being encoded by a DNA fragment according to claim 1, or by a i I i -66 truncated portion thereof, said truncated portion being subject to the proviso that insecticidal activity of the corresponding toxin protein has not been lost.

31. A method according to claim 30 for combating insects of the order Lepidoptera.

32. A method for combating insects which comprises applying to the insects or their habitats an insecticidally effective amount of a toxin protein being encoded by a DNA fragment according to claim 2, or by a truncated portion thereof, said truncated portion being subject to the proviso that insecticidal activity of the corresponding toxin protein has not been lost.

33. A method according to claim 32 for combating insects of the order o o Lepidoptera. o 0 0 o 2o 34. A method for combating insects which comprises applying to the o 0 insects or their habitats an insecticidally effective amount of the protein MGE 1 as hereinbefore defined encoded by a DNA fragment according to claim 1.

35. A method according to claim 34 for combating insects of the order Lepidoptera. i

36. A method for combating insects which comprises applying to the insects or their habitats an insecticidally effective amount of the protein MGE 1 as hereinbefore defined encoded by a DNA fragment according Sto claim 2. II t 1 i! I i

37. A method according to claim 36 for combating insects of the order Lepidoptera. -67

38. An insecticidal composition comprising an insecticidally effective amount of a toxin protein being encoded by a DNA fragment according to claim 1, or by a truncated portion thereof, said truncated portion being subject to the proviso that insecticidal activity of the corresponding toxin protein has not been lost.

39. An insecticidal composition comprising an insecticidally effective amount of a toxin protein being encoded by a DNA fragment according to claim 2, or by a truncated portion thereof, said truncated portion being subject to the proviso that insecticidal activity of the corresponding toxin protein has not been lost. A method for combating insects which comprises applying to the S:a o insects or their habitats an insecticidally effective amount of a microorganism according to any one of claims 16 to 19. S0 0 co o 41. A method for combating insects which comprises applying to the insects or their habitats an insecticidally effective amount of a 0 o" bioencapsulation system according to any one of claims 20 to 23.

42. A hybrid vector comprising the yeast acid phosphatase promoter and a DNA fragment according to claim 1, which is controlled by said promoter.

43. A hybrid vector according to claim 42 comprising the yeast acid phosphatase promoter PH05 and a DNA fragment according to claim 2 which is controlled by said promoter. O, 44. The yeast Saccharomyces cerevisiae GRF 18 transformed with a hybrid vector according to claim 42. The yeast Saccharomyces cerevisiae GRF 18 according to claim 44 transformed with a hybrid vector according to claim 43. .i -I i- i 1 -i WI- i i I- 68

46. Plasmid pK36 containing a DNA fragment according to claim 1.

47. A DNA fragment originating from Bacillus thuringiensis var. kurstaki characterized by the nucleotide sequence given in table 2 comprising a DNA sequence coding for an insecticidal toxin protein, substantially as hereinbefore described with reference to the Example or the Formulation Examples.

48. A toxin protein including truncated portions thereof, said truncated DNA portions being subject to the proviso that insecticidal activity has not been lost, substantially as hereinbefore described with reference to the Example.

49. The protein MGE 1, as hereinbefore defined, substantially as hereinbefore described with reference to the Example.

50. A DNA vector containing a DNA fragment according to claim 47, Oo substantially as hereinbefore described with reference to the Example or the Formulation Examples.

51. An insecticidal composition comprising an insecticidally 0 effective amount of a toxin protein being encoded by a DNA fragment t according to claim 47, or by a truncated portion thereof, said truncated portion being subject to the proviso that insecticidal activity of the corresponding toxin protein has not been lost, together with an insecticidally acceptable carrier, diluent and/or adjuvant.

52. A method for producing a DNA fragment, substantially as o hereinbefore described with reference to the Example and Fig. 1.

53. A method for combating insects which comprises applying to the insects or their habitats an insecticidally effective amount of a toxin protein being encoded by a DNA fragment according to claim 47, or by a truncated portion thereof, said truncated portion being subject to the proviso that insecticidal activity of the corresponding toxin protein has not been lost or a composition according to claim 51.

54. A method for combating insects which comprises applying to the insects or their habitats an insecticidally effective amount of the protein MGE 1 as hereinbefore defined encoded by a DNA fragment according to claim 47. DATED this FOURTEENTH day of AUGUST 1990 Ciba-Geigy AG Patent Attorneys for the Applicant IA" SPRUSON FERGUSON KWK/ R809v I L -C ~I 1_1 i ~I--1