Primary sequence and cDNA of insecticidally effective toxins...

Details Primary sequence and cDNA of insecticidally effective toxins... Primary sequence and cDNA of insecticidally effective toxins...

1998-04-01

2001-08-07

Wortman, Donna C. (Department: 1648)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Biocides; animal or insect repellents or attractants

C530S324000, C530S350000

Reexamination Certificate

active

06270785

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the general field of insecticidally effective peptides that can be used as bioinsecticides for pest control, in particular to insecticidally effective toxins isolated from the venom of Mexican scorpions of the genus Centruroides.
BACKGROUND OF THE INVENTION
Insect pests have detrimental effects on both agriculture and environment. Therefore, there has always been a need for new effective insecticides. Conventional synthetic insecticides widely used for several decades are toxic to a relatively broad spectrum of insects and may have negative consequences for the environment like groundwater contamination, toxicity to non-target organisms and potential human health-hazards caused by acute or chronic exposure. Consenquently, researchers continuously search for new more specific and environmentally non-hazardous insecticidal agents.
A natural microbial source of the insecticidal peptide delta endotoxin is the soil-dwelling bacterium
Bacillus thuringiensis
(B.t.) that is used to control pests of leaf-eating caterpillars, beetles and mosquitos (Priest, 1992; Aronson, 1993). There exist various studies dealing with the improvement of the insecticidal properties of B.t., e.g. the development of hybride bacterial cells containing the genes of the delta-endotoxins of B.t.
kurstaki
and B.t.
tenebrionis
(U.S. Pat. No. 4,797,279 Karamata et al., 1989) and the expression of a hybrid delta-endotoxin gene comprising parts of two variants of B.t.
kurstaki
(European Patent 0325400, 1989). Baculoviruses are major insect pathogens that exhibit a potential for insect control (Huber, 1986). Furthermore, they have been used in several studies as a vector to express an exogenous gene whose product is specifically toxic to insects, e.g. juvenile hormone esterase of
Heliothis virescens
(Hammock et al., 1990) as well as insecticidally effective toxins from the spider
Tegenaria agrestis
(Krapcho et al., 1995), from female mites of the species
Pyemotes tritici
(Tomalski and Miller, 1991) and from the scorpion
Androctonus australis
(Medea et al., 1991; Stewart et al., 1991).
The insect-specific toxins from arthropods like spiders, mites, scorpions or centipedes or predator insects like wasps as well as other insecticidally effective agents can also be applied introducing them directly to the target to be protected. The first examples of the expression of natural biological insecticides in transgenic plants were the expression of delta endotoxin from
Bacillus thuringiensis
(Barton et al., 1987) and of a cowpea trypsin inhibitor (Hilder et al., 1987) both in tobacco. Later, Barton et al. (U.S. Pat. No. 5,177,308, 1993) patented the development of transgenic plants which express an insect-specific toxin from the scorpions
Antroctonus australis
and
Buthus eupeus.
The expression of spider insecticides in transgenic plants and insect virus was patented in World-patent 9315108 (1993) and U.S. Pat. No. 5,441,934 (1995).
As mentioned above scorpions are natural sources of insecticidally effective peptides. Their venom contains a cocktail of various toxins (Possani, 1984) that serve to immobilize its insect prey as well as a defense against possible scorpion predators. The individual toxins display a certain specificity towards mammals, insects or crustaceans. Several toxins have been isolated that show activity in several species (De Lima et al., 1986; Eitan et al., 1990; Loret et al., 1991). The use of these toxins although effective as insecticides is not advisable because of possible side effects on domestic or other animals and humans. Scorpion toxins are required that are highly specific for insects and do not display toxic effects in mammals. Several of these have been described in the literature (Babin et al., 1976; Darbon et al., 1982; Lester et al., 1982; Kopeyan et al., 1990; Loret et al., 1990; Zlotkin et al., 1991). The Canadian patent 2,005,658 (1990) discloses an insecticidally effective peptide of the scorpion
Leirus quinquestriatus hebraeus.
The European patent 374753 (1990) reports insect-selective toxins of the scorpion
Androctonus australis, Leiurus quinquestriatus quinquestriatus, Buthotus judaicus, Leirus quinquestriatus hebraeus
and
Scorpiomaurus palmatus
as well as their expression in transgenic plants and microorganisms. Nevertheless, because of the need of specific and potent insecticides there is still a necessity to search and characterize new natural insecticidally effective toxins.
SUMMARY OF THE INVENTION
There is provided by this invention the amino acid sequences of novel toxins from scorpions of the genus Centruroides which are effective in insects but non-toxic to mammals. These basic polypeptides comprise about 60 to 70 amino acids, they contain eight cysteine residues at equivalent positions and show a similarity of 59%. Further provided by this invention is a novel cDNA sequence coding for one of the toxins of this invention.


REFERENCES:
patent: 4797279 (1989-01-01), Karamata et al.
patent: 5177308 (1993-01-01), Barton et al.
patent: 5441934 (1995-08-01), Krapcho et al.
Possani et al., The Amino Terminal Sequence of Several Toxins from the Venom of the Mexican Scorpion Centruroides Noxius Hoffman. Carlsberg Res. Commun. 46:207-214, 1981.*
Alagon et al., Isolation and Characterization of Two Toxins from the Mexican Scorpion Centruroides Limpidus Limpidus Karsch. Comp. Biochem. Physiol. 89B:153-161, 1988.*
Stewart; Lorna M. et al., “Construction Of An Improved Baculovirus Insecticide Containing An Insect-Specific Toxin Gene”,Nature, vol. 352, pp. 85-88 (Jul. 1991).
Hammock; Bruce D. et al., “Expression And Effects OF The Juvenile Hormone Esterase In A Baculovirus Vector”,Nature, vol. 344, pp. 458-461 (Mar. 1990).
Hilder; Vaughan A. et al., “A Novel Mechanism Of Insect Resistance Engineered Into Tobacco”,Nature, vol. 330, pp. 160-163 (Nov. 1987).
Becerril; Baltazar et al., “Cloning and Characterization of cDNAs That Code For Na+-Channel-Blocking Toxins Of The ScorpionCentruroides NoxiusHoffman”,Gene, vol. 128. pp. 165-171 (1993).

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