Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Patent
1997-05-13
2000-06-06
Carlson, Karen Cochrane
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
530350, 536 2371, 536 231, 4352523, 4352542, 4353201, A61K 3800, C07K 100, C07H 2104, C12N 1500
Patent
active
060718773
DESCRIPTION:
BRIEF SUMMARY
The biological control of insects of the Diptera family, which comprises for example the mosquitoes and the simuliids, vectors of tropical diseases, is principally conducted with the aid of the bacteria Bacillus thuringiensis ser. israelensis (Bti) of the H14 serotype or with the aid of Bacillus sphaericus. During sporulation these two bacteria synthesize proteins assembled in the form of crystals, which are toxic for the insect larvae on ingestion. The crystals of B. thuringiensis ser. israelensis are composed of 4 major polypeptides CryIVA (125 kDa), CryIV B (135 kDa), CryIV D (68 kDa) and Cyt A (28 kDa) (Hofte H et al., 1989 Microbiol. Rev. 53:242-255). The crystals of B. sphaericus are constituted of 2 polypeptides of 51 and 42 kDa. These proteins have different specificities and each of them contributes to the total toxicity, acting if necessary in synergy. With the objective of neutralizing the possible appearance of insects resistant to the Bti toxins, the search for novel strains exhibiting an activity against mosquitoes was undertaken.
The B. thuringiensis strains active against mosquitoes may be classed in four groups depending on their larvicidal activity, the protein composition of their crystals and the presence of genes related to those of the B. thuringiensis ser. israelensis strain: respectively as morrisoni PG14 (H8a8b), canadensis 11S2-1 (H5a5c), thompsoni B175 (H12), malaysiensis IMR81.1 (H36), K6 (autoagglutinating), and B51 (autoagglutinating) which have a similar larvicidal activity and polypeptides of the crystal similar to those of B. thuringiensis ser. israelensis, (H30) and jegathesan 367 (H28a28c) which are almost as toxic as the B. thuringiensis ser. israelensis strain but which produce different polypeptides, synthesizes polypeptides different from those found in the crystals of Bacillus thuringiensis ser. israelensis but which is active on only one species of mosquito, and Kyushuensis 74 F6-18 (H11a11c) which are only weakly toxic.
Given the low toxicity of the strains of groups 3 and 4, these strains have not been studied in detail.
Of all the strains isolated the Bacillus thuringiensis ser jegathesan 367 (Btjeg) strain, of the H28a 28c serotype, seems interesting both from the point of view of its activity and from the polypeptide composition of its crystals. Like Bti this bacterium produces crystals during sporulation which are toxic to mosquito larvae when ingested. This strain was isolated in Malaysia by L. LEE and identified as belonging to a new subtype.
The crystals of B. thuringiensis ser. jegathesan contain 7 major polypeptides having a molecular weight of 80, 72-70, 65, 37, 26 and 16 kDa, respectively. The 37 kDa protein is immunologically related to a constituent of the crystal of B. thuringiensis ser. israelensis whereas the other proteins only give weak or variable cross-reactions. No gene related to those of B. thuringiensis ser. israelensis was detected in this strain, indicating that the proteins of the crystal might be encoded in a new class of toxin genes.
The inventors have identified within the total DNA of a Btjeg 367 strain, sequences coding for polypeptides capable of inducing and/or contributing to the toxic activity of the strain against insects of the Diptera family in particular.
Hence the object of the invention is nucleotide sequences coding for polypeptides with toxic activity against insects. Target insects are for example insects of the Diptera family, in particular mosquitoes or simuliids and especially the larvae of these insects. However, it is not excluded that the polypeptides obtained from the sequences of the invention may exhibit an activity against insects of other families.
The application also concerns polypeptides having a larvicidal activity against insects, or polypeptides capable of contributing to such a toxic activity, if necessary by acting in synergy with polypeptides determining this activity.
Thus the polypeptides of the invention are capable either of inducing the toxic activity or of enhancing the level of toxicity agains
REFERENCES:
Creighton. Proteins: Structures and Molecular Properties, 2nd edition. WH Freeman and Company, New York, pp. 23-28, 1993.
Kawalek et al. Isolation and identification of novel toxins from a new mosquitocidal isolate from Malaysia, Bacillus thuringiensis subsp. jegathesan. Appl. Envrion. Microbiol. 61(8):2965-2969, Aug. 1, 1995.
Arantes et al. Construction of cloning vectors for Bacillus thuringiensis. Gene 108:115-119, 1991.
Donovan et al. Molecular characterization of a gene encoding a 72-kilodalton mosquito-toxic crystal protein from Bacillus thuringiensis. J. Bacteriol. 170:4732-4738, 1988.
Yong-man YU, et al., Appl. Environ. Microbiol., vol. 57, No. 4, pp. 1075-1081, "Characterization of Mosquitocidal Activity of Bacillus thuringiensis Subsp. Fukuokaensis Crystal Proteins", Apr. 1991.
HL Lee, et al., Southeast Asian J Trop. Med. Public Health, vol. 21, No. 2, pp. 281-287, "Isolation of Indigenous Larvicidal Microbial Control Agents of Mosquitos: The Malaysian Experience", Jun. 1990.
Michael D. Kawalek, et al., Appl. Environ. Microbio., vol. 61, No. 8, pp. 2965-2969, "Isolation and Identification of Novel Toxins From a New Mosquitocidal Isolate From Malaysis, Bacillus shuringiensis Subsp. jegathesan", Aug. 1995.
Armelle Delecluse, et al., Appl. Environ. Microbiol., vol. 61, No. 12, pp. 4230-4235, "Cloning and Expression of a Novel Toxin Gene From Bacillus thuringiensis Subsp. jegathesan Encoding a Highly Mosquitocidal Protein", Dec. 1995.
Delecluse Armelle
Thiery Isabelle
Carlson Karen Cochrane
Institut Pasteur
Srivastava Devesh
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