WO1988009812A1 - Sequences de nucleotidiques codant pour des polypeptides dotes d'une activite larvicide vis-a-vis de lepidopteres - Google Patents

Sequences de nucleotidiques codant pour des polypeptides dotes d'une activite larvicide vis-a-vis de lepidopteres Download PDF

Info

Publication number
WO1988009812A1
WO1988009812A1 PCT/FR1988/000292 FR8800292W WO8809812A1 WO 1988009812 A1 WO1988009812 A1 WO 1988009812A1 FR 8800292 W FR8800292 W FR 8800292W WO 8809812 A1 WO8809812 A1 WO 8809812A1
Authority
WO
WIPO (PCT)
Prior art keywords
littoralis
leu
asn
aat
polypeptide
Prior art date
Application number
PCT/FR1988/000292
Other languages
English (en)
French (fr)
Inventor
Vincent Sanchis
Didier Lereclus
Ghislaine Menou
Marguerite-Marie Lecadet
Daniel Martouret
Raymond Dedonder
Original Assignee
Institut Pasteur
Institut National De La Recherche Agronomique (Inr
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from FR8708090A external-priority patent/FR2616444B1/fr
Application filed by Institut Pasteur, Institut National De La Recherche Agronomique (Inr filed Critical Institut Pasteur
Publication of WO1988009812A1 publication Critical patent/WO1988009812A1/fr
Priority to US08/461,551 priority Critical patent/US5792928A/en
Priority to US08/461,750 priority patent/US6110734A/en
Priority to US10/632,973 priority patent/US20050091714A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins

Definitions

  • the subject of the invention is nucleotide sequences coding for polypeptides endowed with larvicidal activity vis-à-vis lepidoptera.
  • S. littoralis vis-à-vis Spodoptera littoralis
  • M. brassicae Mamestra brassicae
  • This activity results from the ability of B. thuringiensis strains to synthesize, at the time of sporulation, crystal inclusions of protein nature, or ⁇ -endotoxins, under the control of one or more types of genes.
  • the work carried out has shown the high specificity of ⁇ -endotoxins vis-à-vis larvae of a given species.
  • ⁇ -endotoxin genes identified to date do not code for a polypeptide which is active preferentially with respect to S. littoralis.
  • the object of the invention is therefore to provide nucleotide sequences capable of coding for at least the NH 2 -terminal part of a ⁇ -endotoxin toxic against Noctuelles, preferably against S. littoralis or
  • the invention further relates to a process for obtaining such a sequence and to a polypeptide having the desired activity as well as the intermediate means such as vectors and bacterial strains which can be used for obtaining the polypeptide.
  • the invention further relates to the applications of these sequences and polypeptides for the preparation of larvicidal compositions with regard to Noctuelles, in particular of S. littoralis and for the transformation of plants liable to be infected by these larvae.
  • the invention relates to a nucleotide sequence coding for at least a part of the N-terminal region of a polypeptide which is specifically toxic to Lepidopteran larvae of the family Noctuelles, preferably vis-à-vis from S. littoralis. characterized by its ability to hybridize with a gene capable of expressing a polypeptide toxic to larvae of S. littoralis.
  • the nucleotide sequence is characterized in that it is carried by a nucleotide sequence of approximately 3 kb as obtained by genetic in vitro recombination of nucleotide sequences of B. thuringiensis capable of s hybridize with pHTA2 probes 1, 2 and 3 reported in FIG. 2.
  • the 3 kb fragment corresponds more specifically to the HindIII-PstI restriction fragment.
  • the nucleotide sequences of the invention are further characterized in that they have sites in the following order: HindlII - HincII - BglII Kpnl - HindIII - PstI.
  • these nucleotide sequences are obtained by genetic recombination in vitro of DNA sequences originating from at least one strain of B. thuringiensis.
  • two different strains of B. thuringiensis are used.
  • Strains of B. thuringiensis which are particularly suitable for obtaining these nucleotide sequences are the strains corresponding to aizawai 7-29 and entomocidus 6-01, deposited on April 21, 1987 under Nos. 1-661 and Nos. 1-660 to the National Collection of Culture of Microorganisms (CNCM) in Paris.
  • nucleotide sequences of the invention code for a polypeptide capable of forming an immunological complex with antibodies directed against polypeptides exhibiting larvicidal activity against S. littoralis.
  • a nucleotide sequence according to the invention is characterized in that it has the capacity to hybridize with a probe formed from the sequence (I) having the following sequence:
  • CAA ATT GAA CAA TTA ATT AAT GAA AGA ATA GCT GAA TTT GCT
  • Nucleotide sequences coding for at least part of the N-terminal region of a polypeptide which is specifically toxic to Leptidopteran larvae of the family Noctuelles, preferably vis-à-vis S. littoralis are characterized in that they include the sequence (I) defined above.
  • nucleotide sequence characterized by the sequence defined above codes for a part of a polypeptide having a larvicidal activity vis-à-vis S. littoralis higher than that of the polypeptides coded by isolates currently known for their effects on S. littoralis.
  • This sequence is also characterized by an attachment site for the GGAGG ribosomes at positions 230 to 234.
  • the nucleotide sequence of the invention is characterized in that it includes, upstream of the ATG codon, a sequence going from the nucleotide at position 137 to the nucleotide at position 177, highly homologous to the region found by Wong and al.
  • the invention also relates to a nucleotide sequence coding for the following amino acid sequence (II):
  • CNCM has shown that the nucleotide located at position 273 is guanine (G), the amino acid resulting from the GTA codon then being valine.
  • nucleotide sequence of the invention is characterized by its capacity for hybridization with a probe formed from the sequence (III) having the following sequence:
  • nucleotide sequences of the invention coding for a polypeptide which is toxic specifically with regard to the larvae of Lepidoptera of the family Noctuelles, preferably with regard to S. littoralis include or are made up by the sequence (III) defined above.
  • sequence (III) included in the nucleotide sequence of the invention, contains 2711 nucleotides. This fragment notably comprises the potential promoter of the ⁇ -endotoxin gene active on S. littoralis.
  • nucleotide sequences modified with respect to the sequences (I) or (III) described above insofar as these modifications do not generate appreciable variations in the toxicity of the polypeptide encoded by the modified sequence, with respect to S. littoralis.
  • nucleotide sequences (I) and (III) comprising in their position 611 a variable nucleotide corresponding to adenine (A) in the sequence (I) and to cytosine (C) in the sequence (III).
  • These nucleotides enter into the composition of the respective codons GAA and GCA which code respectively for the amino acids glutamic acid (GLU) and alanine (ALA) in the respective sequences II and IV.
  • any nucleotide sequence which can be hybridized with that of the sequences (I) or (III), as obtained by reverse enzymatic transformation of the corresponding RNA or also by chemical synthesis also comes within the scope of the definitions of the invention.
  • nucleotide sequence of formula (III) begins with an ATG initiation codon located at position 241 and which represents the start of an open phase of reading of 2470 nucleotides.
  • the invention further relates to a nucleotide sequence characterized in that it codes for a polypeptide comprising the following amino acid sequence (IV):
  • the invention also relates to the recombinant expression and cloning vectors comprising more particularly at least one nucleotide sequence as defined above, in particular at least part of the sequence of approximately 3 kb.
  • a particular recombinant vector is for example a plasmid comprising the HindIII-PstI fragment of the nucleotide sequence of the invention, inserted into a vector pUC9.
  • a first preferred vector is the plasmid pHT71, the construction of which is reported in the sets below, which comprises a DNA fragment HindIII-PstI according to the invention consisting solely of DNA originating from the strain aizawai 7-29.
  • Another recombinant vector consists of the plasmid pHT 671, the construction of which is given in FIG. 4.
  • This plasmid comprises a chimeric HindIII-Pstl fragment, obtained by fusing a 1.1 kb HindIII-HindIII DNA fragment originating from the entomocidus 6-01 strain with a 1.9 kb HincII-PstI fragment from the aizawai strain 7-29.
  • modified bacterial strains which comprise one of the nucleotide sequences defined above or else a recombinant expression and cloning vector defined above, preferably the plasmid pHT 671 or the plasmid pHT71.
  • the invention further relates to a polypeptide which is toxic with regard to lepidopteran larvae and preferably with respect to S. littoralis, attacking the cotton leaves or other crops as listed above. , characterized in that it is capable of forming an immunological complex with antibodies directed against polypeptides with larvicidal activity vis-à-vis S. littoralis.
  • the invention relates more particularly to the NH 2 -terminal part of this polypeptide which contains the larvicidal activity.
  • the end of the active NH 2 -terminal part responds to the amino acid sequence (II) given above.
  • a preferred polypeptide of the invention is that which responds to this sequence (II) and responds to the amino acid sequence (IV) given in the preceding pages.
  • This polypeptide corresponding to the sequence (IV) comprises 823 amino acids. Its calculated molecular mass is 92906 Da.
  • This ⁇ -endotoxin sequence has been compared to the amino acid sequences of ⁇ -endotoxins from other strains of B. thuringiensis active on lepidoptera and whose genes have been isolated and previously sequenced: these are ⁇ -endotoxins strains kurstaki HD1 (19), kurstaki HD73 (20), berliner 1715 (21) and (22) Sotto (23) and aizawai IPL7 (24).
  • nucleotide sequence falling within the scope of the invention, coding for at least the active part of a polypeptide having a specific toxicity with respect to Lepidopteran larvae of the family Noctuelles, preferably with - screw of S. littoralis.
  • the following steps are used, in accordance with the invention, namely: - carrying out molecular hybridization between on the one hand a nucleotide sequence of a strain of B. thuringiensis active against S. littoralis, and d on the other hand at least two nucleotide sequences, used as probes, originating from the 5 'part of a restriction fragment of a consequent-endotoxin gene from B. thuringiensis, this part coding for the NH 2 -terminal part of the polypeptide active on the larvae of lepidoptera and of the 3 ′ part of this fragment coding for the COOH part of the polypeptide,
  • the hybridization probes used are obtained from a ⁇ -endotoxin gene originating from the aizawai 7-29 strain coding for a protein of 130 kDa, active against P. brassicae and inactive vis-à-vis of S. littoralis, this gene having been cloned in the recombinant plasmid pHTA2.
  • the fragment recombined with the vector in the step of cloning is carried out using a HindIII-PstI restriction fragment originating from a single strain of B. thuringiensis. preferably aizawai 7-29.
  • this fragment is preferably carried by the recombinant plasmid pHTA6 as isolated using a probe constituted by a PvuII fragment of 2kb from the plasmid pBT15-88 corresponding to the internal part of a chromosomal crystal gene of the strain berliner 1715, from transforming clones containing nucleotide sequences derived from B. thuringiensis strains active against larvae of inter-alia lepidoptera of S. littoralis.
  • the fragment recombined with the vector in the cloning step is produced from several nucleotide sequences originating from recombinant vectors containing nucleotide sequences from at least two different strains of B. thuringiensis. having the same restriction maps and themselves containing all or part of the nucleotide sequences capable of coding for an active polypeptide, preferably, with respect to S. littoralis.
  • the recombinant fragment used in the cloning step is a fragment of approximately 3kb, advantageously produced from a HindlII-HincII restriction fragment of approximately 1.1kb originating from the strain entomocidus 6-01 and d 'a HincII-PstI fragment of approximately 1.9 kb of the aizawai strain 7.29. It corresponds to a truncated ⁇ -endotoxin gene.
  • HindIII-HincII and HincII-PstI restriction fragments are more particularly carried by the respective recombinant plasmids pHTE6 and pHTA6 as isolated using the probe constituted by the PvuII fragment mentioned above.
  • the invention therefore relates to the application, for the preparation of toxic larvicidal compositions preferably vis-à-vis S. littoralis. of these modified strains, of recombinant vectors containing the nucleotide sequences defined above, in particular of the plasmid pHT671 and of the plasmid pHT71, and of these sequences themselves.
  • compositions of the invention are then characterized in that they contain an effective amount of polypeptides as defined above or expressed by the nucleotide sequences mentioned above.
  • the truncated ⁇ -endotoxin genes are advantageously used. corresponding to the nucleotide sequences of the invention.
  • genes can be used to produce excess toxic polypeptide in microorganisms allowing expression of the above recombinant vectors.
  • Suitable strains of microorganisms include E. coli or B. subtilis.
  • truncated genes can be reintroduced into strains of B. thuringiensis or in related species such as B. cereus, according to conventional techniques, for example, by transformation, conjugation or transduction. These techniques make it possible to produce the toxic polypeptide in large quantities without having to first modify the natural region of the promoter of the th-endotoxin genes of B. thuringiensis.
  • This transformation can be carried out using methods derived from the transformation of the protoplasts of B. subtilis according to (11) or of the vegetative cells of B. thuringiensis as described in (12).
  • the introduction of recombinant plasmids by a conjugation type system can be carried out using B. thuringiensis as host strain and B. subtilis or Streptococcus faecalis as donor type strains, operating according to (13) and (14).
  • the nucleotide sequences are introduced into microorganisms living in the environment or in association with plants and capable of expressing recombinant vectors containing these sequences.
  • the introduction can be carried out in microorganisms such as Pseudomonas by operating according to the method described in (17), by means of plasmid vectors containing the Tn5 transposon.
  • the truncated genes are alone in the Bacilli strains, or in a variant are associated with different ⁇ -endotoxin genes which makes it possible to obtain crystals synthesized by these strains specifically toxic, with respect to given species of Noctuelles, or toxic to both Noctuelles and insects sensitive to other ⁇ -endotoxins.
  • These recombinations carried out in vitro or in vivo with the nucleotide sequences of the invention and other ⁇ -endotoxin genes having different toxic specificities, lead to the construction of new genes coding for new toxic hybrid proteins with a broad spectrum activity vis-à-vis insects. These new genes and these new proteins also come within the scope of the invention.
  • nucleotide sequences of the invention can be transferred and expressed in plants sensitive to S. littoralis in order to reduce the ravages caused by these insects.
  • the transfer of the truncated gene into cotton plants can be carried out by transformation involving strains such as Agrobacterium as described in (15).
  • the invention further relates to plant cells, plants and seeds containing the nucleotide sequences defined above.
  • the plant cells according to the invention are cells whose genome, after transformation by a process which is not essentially biological, has a stable nucleotide sequence capable of expressing a polypeptide toxic towards S. littoralis. such as defined above.
  • the invention also relates to plant cells resulting from their division.
  • the plants according to the invention are plants transformed by a non-essentially biological process, having in particular the predator S. littoralis, the genome of which has a stable nucleotide sequence as defined above, capable of expressing a polypeptide toxic towards S. littoralis. They are also plants resulting from their reproduction, multiplication or hybrid crosses.
  • the invention relates to plants having in particular the predator S. littoralis, having in addition to their initial genotypic and phenotypic characters the property of expressing a toxic polypeptide preferentially with respect to S. littoralis . this property resulting from the insertion into their genome by genetic manipulation of a nucleotide sequence capable of expressing said polypeptide.
  • the invention further relates to seeds capable of giving a plant as defined above or originating from such a plant, characterized in that they have integrated into their genome, by genetic manipulation a nucleotide sequence described above.
  • FIG. 1 represents the restriction map of plasmids pHTA6 and pHTE6,
  • Figure 2 the restriction map of a gene for a crystal protein of the strain aizawai 7.29 cloned in the plasmid pHTA2 and defining the DNA fragments which are used as probe
  • - Figure 3 presents the fragment of 6.6kb cloned in pHTA6 and the result of a hybridization carried out between this fragment and the probes described in FIG. 2
  • FIG. 4 the restriction map of the plasmid PHT671, and - FIG. 5, the photographs of immunodiffusion tests.
  • the hybridization experiments carried out for the implementation of the invention were carried out at 42 ° C for 24 h. in a solution containing 5 x SSC, 30% formamide and 1 Denhardt (7) in the presence of the DNA probe labeled with 32 p.
  • the filters are washed at 42oC, 20 un, successively using the following solutions:
  • This construction includes: 1 / the preparation of B. thuringiensis gene banks 2 / the selection and characterization of the transforming clones containing the genes of a crystal protein and of the nucleotide sequences responsible for the larvicidal activity,
  • the total DNA of the aizawai 7-29 and entomocidus 6-01 strains of Bacillus thuringiensis is purified using the method reported in (1) and 50 ⁇ g of each purified DNA are completely digested with the restriction enzyme PstI.
  • the DNA digested with PstI is analyzed by horizontal electrophoresis on 0.8% agarose gel and DNA fragments of a size of 5 to 8 kb are recovered from the agarose gels, by electroelution, from as described in (2).
  • the purified DNA fragments of 5-8 kb from the aizawai 7-29 strain are ligated to the DNA of the cloning vector pUC18 digested with PstI according to (3).
  • the purified 5-8 kb DNA fragments of the entomocidus 6-01 chain are ligated to the DNA of the cloning vector pUC9 digested with PstI.
  • E.coli JM83 cells are transformed with the ligation mixture as described in (4).
  • the transforming clones of E. coli are selected on LB medium containing 100 ⁇ g / ml of ampicillin.
  • Transforming clones containing recombinant plasmids carrying the crystal gene are detected by colony hybridization, according to the method described in (5), using as probe a 2 kb PvuII fragment of the plasmid pBT 15-88 corresponding to an internal part of the gene for the crystal protein, located on the chromosome of the strain berliner 1715.
  • the restriction map of the two plasmids is given in FIG. 1.
  • the comparison of the restriction sites shows that the two cloned DNA fragments appear to be identical.
  • the plasmids pHTA6 and pHTE6 are hydrolyzed by various restriction endonucleases, analyzed by horizontal electrophoresis on 0.8% agarose gel and hybridized with the various probes.
  • the transfer of DNA to nitrocellulose filters is carried out according to the SOUTHERN method described in (6).
  • the hybridization is carried out at 42oC for 24 hours in a solution containing: 5X SSC, 30% formamide and a 1X Denhardt mixture described in (7) in the presence of a DNA probe labeled with 32P.
  • the filters are then washed at 42oC for 20 minutes, successively using the following solutions: 5 SSC in 50% formamide, 5 SSC, 2 SSC, 1 SSC and 0.5 SSC before being dried at room temperature.
  • the 1.1 kb HindiII-HinçII DNA fragment from the plasmid pHTE6 and the 1.9 kb HincII-PstI DNA fragment from the plasmid pHTA6 are purified on agarose gels. Equal quantities of the two purified DNA fragments and pUC9 DNA digested with HindIII and PstI are mixed and ligated. The ligation mixture is used to transform competent cells of E.coli JM83, then the transformed cells of E.coli are selected on LB medium containing ampicillin.
  • One of the recombinant clones of interest examined contains a plasmid designated by pHT671, the restriction map of which has been determined and is shown in FIG. 4.
  • This plasmid contains a 3 kb DNA fragment inserted into the vector pUC9 .
  • This DNA sequence has the same restriction map as the 3 kb HindIII-PstI fragments contained in the plasmids pHTA6 and pHTE6, but corresponds to a reconstituted DNA molecule constructed by recombination in vitro from DNA sequences originating from strains aizawai 7-29 on the one hand and entomocidus 6-01 on the other.
  • the HindIII-HincII fragment of pHT671 is sequenced according to the method described in (8) using an M13 system. To obtain partially overlapping cloned DNA fragments which will be used in DNA sequencing, use is made of the deletion subcloning method in M13, developed by DALE et al (9).
  • the 940 nucleotide sequence of the HindIII-HincII fragment which is approximately 1 kilobase in length corresponds to sequence I above.
  • a potential GGAGG ribosome binding site is six base pairs upstream of this codon ATG (position 230 to 235).
  • the region located between nucleotides 137 and 177 is highly homologous to the region present upstream of the crystal gene of the kurstaki HD1 Dipel (BTK) strain sequenced by WONG et al ( 1983) and described in (16) and the authors of which have shown that it contains three promoters Btl, BtlI. and Ec functional in B. thuringiensis and E.coli respectively.
  • EXAMPLE III Construction of a DNA sequence of approximately 2.7 kb containing a gene for a larvicidal toxin.
  • the HindIII-PstI fragment of about 2.7 kb obtained from the plasmid pHTA6 was then subcloned into the vector pUC9, previously hydrolyzed by the restriction enzymes HindIII-PstI, to give the plasmid PHT71.
  • EXAMPLE IV Study of the nucleotide sequence constituting the plasmid pHT71 coding for a polypeptide toxic to Lepidopteran larvae of the family Noctuelles.
  • nucleotide sequence of this 2.7kb fragment which corresponds to the sequence (III) given above, was determined on the 2 strands of DNA, except for the last 212 nucleotides (position 2500 to 2711) which have not been streaked on only 1 strand.
  • the nucleotide sequence of this Hind-III-PstI fragment is 2711 nucleotides in length. This fragment contains the potential promoter as well as most of the litt-endotoxin gene active on S. littoralis.
  • the toxicity of the recombinant clones of E.coli JM83 containing pHT671 and of E.coli JM83 containing pHT71 was determined by biological tests on caterpillars of the species P. brassicae and S. littoralis as described by LECADET and MARTOURET in (10 ). The results were compared with the specific toxicity of the native crystal proteins and purified from the berliner 1715 and aizawai 7-29 entomocidus 6.01 B cereus 569 strains (containing the plasmid pBT45, pAM ⁇ l) against the two insect species. The specific toxicity of the recombinant clone and of the strains of B. thuringiensis is expressed in terms of the "specificity index" defined above.
  • concentration 1 corresponds to a 14 hour bacterial culture concentrated 20 times, disintegrated by ultrasound; for B. thuringiensis strains the concentration is expressed in ⁇ g of crystal protein per ⁇ l of preparation.
  • the toxic activity of the preparations was tested by forced ingestion on caterpillars at the fifth stage of development with 5 ⁇ l of preparation, or by a free ingestion technique using larvae at the second stage of development.
  • JM83 pHT671
  • JM83 pHT71
  • the LC50 value is 0.02 , using second stage larvae.
  • the crystal inclusions produced by a strain of Bacillus cereus which received the plasmid pBT45, one of the plasmids of the strain aizawai 7-29 which also carries a gene for ⁇ -endotoxin (the gene of plasmid origin of the strain aizawai 7-29), are also specifically active on P. brassicae.
  • the LD50 value has been estimated at 2.4 ⁇ g / gram of larva for the native crystals of the strain aizawai 7-29.
  • the extracts of the recombinant clone E.coli JM83 (pHTA2) are weakly active with regard to S. frugiperda and S. littoralis and not at all toxic with regard to M. brassicae.
  • the extracts of the recombinant clone JM83 (pHTA4) are not toxic towards M. brassicae and S.littoralis and are slightly toxic towards S. frugiperda.
  • Soluble extracts of proteins from E. coli clones containing the plasmids pHT671, ⁇ HTA4, pHTA2 or pHT71, pUC18 were placed respectively in wells 2, 3, 4, 5, 6.
  • a sample of a purified crystal dissolved in aizawai 7-29 was placed in well # 1 to serve as a positive control.
  • the antiserum used contained polyclonal rabbit antibodies against the proteins of the berliner 1715 crystal.
  • crystal genes isolated in pHTA4 and pHTA2 express polypeptides having antigenic determinants common with the proteins of crystal berliner 1715, a strain which is not specifically active with respect to S. littoralis.
  • the crude extracts of E. coli containing the plasmids pHT671 and pHT71 contain polypeptides having antigenic determinants common with the crystal proteins of the strain aizawai 7.29, which are not linked immunogenically with the proteins of the crystal of crystal the 1715 berliner strain.
  • Antigen-antibody precipitation tests made it possible to determine the level of expression of the ⁇ -endotoxin genes in different recombinant clones.
  • the results obtained showed that the crystal protein represents between 7 and 10% of total cellular proteins of E. coli JM83 (pHTA2), between 2 and 3% in E. coli JM83 (pHT671) and between 0.5 and 1% in E. coli JM83 (pHTA4) and E. coli. JM83 (pHT71).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)
PCT/FR1988/000292 1987-06-10 1988-06-09 Sequences de nucleotidiques codant pour des polypeptides dotes d'une activite larvicide vis-a-vis de lepidopteres WO1988009812A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US08/461,551 US5792928A (en) 1987-06-10 1995-06-05 Nucleotide sequences coding for polypeptides endowed with a larvicidal activity towards lepidoptera
US08/461,750 US6110734A (en) 1987-06-10 1995-06-05 Nucleotide sequences coding for polypeptides endowed with a larvicidal activity towards lepidoptera
US10/632,973 US20050091714A1 (en) 1987-06-10 2003-08-04 Methods for obtaining nucleotide sequences coding for polypeptides specifically active for larvae of S. littoralis

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
FR8708090A FR2616444B1 (fr) 1987-06-10 1987-06-10 Sequences nucleotidiques codant pour des polypeptides dotes d'une activite larvicide vis-a-vis de lepidopteres
FR87/08090 1987-06-10
EP88401121A EP0295156B1 (fr) 1987-06-10 1988-05-06 Séquences de nucléotides codant pour des polypeptides dotés d'une activité larvicide vis-à-vis de lépidoptères
FR88401121.4(EP) 1988-05-06

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US9438293A Continuation 1987-06-10 1993-07-21

Publications (1)

Publication Number Publication Date
WO1988009812A1 true WO1988009812A1 (fr) 1988-12-15

Family

ID=26117677

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/FR1988/000292 WO1988009812A1 (fr) 1987-06-10 1988-06-09 Sequences de nucleotidiques codant pour des polypeptides dotes d'une activite larvicide vis-a-vis de lepidopteres

Country Status (3)

Country Link
JP (2) JP3177237B2 (ja)
OA (1) OA09149A (ja)
WO (1) WO1988009812A1 (ja)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5914318A (en) * 1996-11-27 1999-06-22 Ecogen, Inc. Transgenic plants expressing lepidopteran-active δ-endotoxins

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988009812A1 (fr) * 1987-06-10 1988-12-15 Institut Pasteur Sequences de nucleotidiques codant pour des polypeptides dotes d'une activite larvicide vis-a-vis de lepidopteres

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0178151A2 (en) * 1984-10-09 1986-04-16 Agricultural Genetics Company Limited Preparation of strains of bacillus thuringiensis having an improved activity against certain lepidopterous pests and novel strain produced thereby
EP0192319A2 (en) * 1985-01-22 1986-08-27 Mycogen Corporation Cellular encapsulation of biological pesticides
EP0224331A1 (en) * 1985-10-28 1987-06-03 Sumitomo Chemical Company, Limited Production of insecticidal protein of bacillus thuringiensis subsp. aizawai IPL by the expression of insecticidal protein gene in host cells
EP0228838A2 (en) * 1985-12-12 1987-07-15 Mycogen Corporation Bacillus thuringiensis toxins

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988009812A1 (fr) * 1987-06-10 1988-12-15 Institut Pasteur Sequences de nucleotidiques codant pour des polypeptides dotes d'une activite larvicide vis-a-vis de lepidopteres

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0178151A2 (en) * 1984-10-09 1986-04-16 Agricultural Genetics Company Limited Preparation of strains of bacillus thuringiensis having an improved activity against certain lepidopterous pests and novel strain produced thereby
EP0192319A2 (en) * 1985-01-22 1986-08-27 Mycogen Corporation Cellular encapsulation of biological pesticides
EP0224331A1 (en) * 1985-10-28 1987-06-03 Sumitomo Chemical Company, Limited Production of insecticidal protein of bacillus thuringiensis subsp. aizawai IPL by the expression of insecticidal protein gene in host cells
EP0228838A2 (en) * 1985-12-12 1987-07-15 Mycogen Corporation Bacillus thuringiensis toxins

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5914318A (en) * 1996-11-27 1999-06-22 Ecogen, Inc. Transgenic plants expressing lepidopteran-active δ-endotoxins
US6033874A (en) * 1996-11-27 2000-03-07 Ecogen, Inc. CRY1C polypeptides having improved toxicity to lepidopteran insects
US6153814A (en) * 1996-11-27 2000-11-28 Monsanto Company Polypeptide compositions toxic to lepidopteran insects and methods for making same
US6177615B1 (en) 1996-11-27 2001-01-23 Monsanto Company Lepidopteran-toxic polypeptide and polynucleotide compositions and methods for making and using same
US6313378B1 (en) 1996-11-27 2001-11-06 Monsanto Technology Llc Lepidopteran-resistent transgenic plants
US6423828B1 (en) 1996-11-27 2002-07-23 Monsanto Technology Llc Nuclei acid and polypeptide compositions encoding lepidopteran-toxic polypeptides
US6809078B2 (en) 1996-11-27 2004-10-26 Monsanto Technology Llc Compositions encoding lepidopteran-toxic polypeptides and methods of use
US6825006B2 (en) 1996-11-27 2004-11-30 Monsanto Technology Llc Nucleic acid and polypeptide compositions encoding lepidopteran-toxic polypeptides
US7256017B2 (en) 1996-11-27 2007-08-14 Monsanto Technology Llc Methods for generating lepidopteran-toxic polypeptides

Also Published As

Publication number Publication date
JPH08275784A (ja) 1996-10-22
JP3183622B2 (ja) 2001-07-09
JPH02503745A (ja) 1990-11-08
JP3177237B2 (ja) 2001-06-18
OA09149A (fr) 1991-10-31

Similar Documents

Publication Publication Date Title
US6686149B1 (en) Methods for obtaining nucleotide sequences coding for polypeptides specifically active for larvae of S. littoralis
US5424410A (en) Bacillus thuringiensis isolates for controlling acarides
JPH04505250A (ja) 細菌遺伝子
JP4338057B2 (ja) 殺虫剤
EP0759469B1 (fr) Bactériocine
EP0295156B1 (fr) Séquences de nucléotides codant pour des polypeptides dotés d'une activité larvicide vis-à-vis de lépidoptères
CA2117270A1 (en) Use of bacillus thuringiensis isolates for controlling pests in the family aphididae
CA2409940C (fr) Bacteriocine anti-listeria
AU668685B2 (en) Novel bacillus thuringiensis isolates for controlling acarides
JP2531913B2 (ja) バシルス チュリンギエンシス（Ｂａｓｉｌｌｕｓ ｔｈｕｒｉｎｇｉｅｎｓｉｓ） ｃｒｙＩＩＩＣ（ｂ）毒素遺伝子及び甲虫類昆虫に毒性のタンパク質
WO1988009812A1 (fr) Sequences de nucleotidiques codant pour des polypeptides dotes d'une activite larvicide vis-a-vis de lepidopteres
US6110734A (en) Nucleotide sequences coding for polypeptides endowed with a larvicidal activity towards lepidoptera
BE1001877A4 (fr) Composes nouveaux.
EP0777734B1 (fr) Nouveaux polypeptides a activite toxique vis-a-vis d'insectes de la famille des dipteres
CA2103248C (en) Novel bacillus thuringiensis isolates active against hymenopteran pests and genes encoding hymenopteran-active toxins
WO1990010071A1 (fr) Expression de sequences de nucleotides codant pour des vesicules a gaz
EP0585396A1 (en) NOVEL $i(BACILLUS THURINGIENSIS) ISOLATES ACTIVE AGAINST HYMENOPTERAN PESTS AND GENES ENCODING HYMENOPTERAN-ACTIVE TOXINS
JPH10506522A (ja) 鱗翅目及び鞘翅目害虫に対して活性な新規バシラス・チューリンジエンシス株
AU668687C (en) Novel (bacillus thuringiensis) isolates active against hymenopteran pests and genes encoding hymenopteran-active toxins
JPH08511686A (ja) 殺虫活性に関連するタンパク質をコードする遺伝子を支持するＣｌｏｓｔｒｉｄｉｕｍｂｉｆｅｒｍｅｎｔａｎｓのＤＮＡフラグメント

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BJ CF CG CM GA ML MR SN TD TG