Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-12-15
2003-10-14
Bugaisky, Gabrielle (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600, C530S350000
Reexamination Certificate
active
06632792
ABSTRACT:
BACKGROUND OF THE INVENTION
Regular use of chemicals to control unwanted organisms can select for chemical resistant strains. This has occurred in many species of economically important insects and has also occurred in nematodes of sheep, goats, and horses. The development of chemical resistance necessitates a continuing search for new control agents having different modes of action.
In recent times, the accepted methodology for control of nematodes has centered around the drug benzimidazole and its congeners. The use of these drugs on a wide scale has led to many instances of resistance among nematode populations (Prichard, R. K. et al. [1980] “The problem of anthelmintic resistance in nematodes,” Austr. Vet. J. 56:239-251; Coles, G. C. [1986] “Anthelmintic resistance in sheep,” In
Veterinary Clinics of North America: Food Animal Practice
, Vol 2:423-432 [Herd, R. P., eds.] W. B. Saunders, New York). There are more than 100,000 described species of nematodes.
The bacterium
Bacillus thuringiensis
(B.t.) produces a &dgr;-endotoxin polypeptide that has been shown to have activity against a rapidly growing number of insect species. The earlier observations of toxicity only against lepidopteran insects have been expanded with descriptions of B.t. isolates with toxicity to dipteran and coleopteran insects. These toxins are deposited as crystalline inclusions within the organism. Many strains of B.t. produce crystalline inclusions with no demonstrated toxicity to any insect tested.
A small number of research articles have been published about the effects of delta endotoxins from
B. thuringiensis
species on the viability of nematode eggs. Bottjer, Bone and Gill (Experimental Parasitology 60:239-244, 1985) have reported that
B.t. kurstaki
and
B.t. israelensis
were toxic in vitro to eggs of the nematode
Trichostrongylus colubriformis
. In addition, 28 other B.t. strains were tested with widely variable toxicities. The most potent had LD
50
values in the nanogram range. Ignoffo and Dropkin (Ignoffo, C. M. and Dropkin, V. H. [1977] J. Kans. Entomol. Soc. 50:394-398) have reported that the thermostable toxin from
Bacillus thuringiensis
(beta exotoxin) was active against a free-living nematode,
Panagrellus redivivus
(Goodey); a plant-parasitic nematode,
Meloidogyne incognita
(Chitwood); and a fungus-feeding nematode,
Aphelenchus avena
(Bastien). Beta exotoxin is a generalized cytotoxic agent with little or no specificity. Also, H. Ciordia and W. E. Bizzell (Jour. of Parasitology 47:41 [abstract] 1961) gave a preliminary report on the effects of
B. thuringiensis
on some cattle nematodes.
At the present time there is a need to have more effective means to control the many nematodes that cause considerable damage to susceptible hosts. Advantageously, such effective means would employ biological agents.
BRIEF SUMMARY OF THE INVENTION
The subject invention concerns novel toxins active against nematodes. A further aspect of the invention concerns genes coding for nematicidal toxins. The subject invention provides the person skilled in this art with a vast array of nematicidal toxins, methods for using these toxins, and genes that code for the toxins.
One aspect of the invention is the discovery of two generalized chemical formulae common to a wide range of nematicidal toxins. These formulae can be used by those skilled in this art to obtain and identify a wide variety of toxins having the desired nematicidal activity. The subject invention concerns other teachings which enable the skilled practitioner to identify and isolate nematode active toxins and the genes which code therefor. For example, characteristic features of nematode-active toxin crystals are disclosed herein. Furthermore, characteristic levels of amino acid homology can be used to characterize the toxins of the subject invention. Yet another characterizing feature pertains to immunoreactivity with certain antibodies. Also, nucleotide probes specific for genes encoding toxins with nematicidal activity are described.
In addition to the teachings of the subject invention which define groups of B.t. toxins with advantageous nematicidal activity, a further aspect of the subject invention is the provision of specific nematicidal toxins and the nucleotide sequences which code for these toxins.
One aspect of the of the subject invention is the discovery of two groups of B.t.-derived nematode-active toxins. One group (CryV) is exemplified by the gene expression products of PS17, PS33F2 and PS63B, while the other group (CryVI) is exemplified by the gene expression products of PS52A1 and PS69D1. The organization of the toxins within each of the two groups can be accomplished by sequence-specific motifs, overall sequence similarity, immunoreactivity, and ability to hybridize with specific probes.
The genes or gene fragments of the invention encode
Bacillus thuringiensis
&dgr;-endotoxins which have nematicidal activity. The genes or gene fragments can be transferred to suitable hosts via a recombinant DNA vector.
BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO. 1 discloses the DNA of 17a.
SEQ ID NO. 2 discloses the amino acid sequence of the toxin encoded by 17a.
SEQ ID NO. 3 discloses the DNA of 17b.
SEQ ID NO. 4 discloses the amino acid sequence of the toxin encoded by 17b.
SEQ ID NO. 5 is the nucleotide sequence of a gene from 33F2.
SEQ ID NO. 6 is the amino acid sequence of the protein expressed by the gene from 33F2.
SEQ ID NO. 7 is the nucleotide sequence of a gene from 52A1.
SEQ ID NO. 8 is the amino acid sequence of the protein expressed by the gene from 52A1.
SEQ ID NO. 9 is the nucleotide sequence of a gene from 69D1.
SEQ ID NO. 10 is the amino acid sequence of the protein expressed by the gene from 69D1.
SEQ ID NO. 11 is the nucleotide sequence of a gene from 63B.
SEQ ID NO. 12 is the amino acid sequence of the protein expressed by the gene from 63B.
SEQ ID NO. 13 is the amino acid sequence of a probe which can be used according to the subject invention.
SEQ ID NO. 14 is the DNA coding for the amino acid sequence of SEQ ID NO. 13.
SEQ ID NO. 15 is the amino acid sequence of a probe which can be used according to the subject invention.
SEQ ID NO. 16 is the DNA coding for the amino acid sequence of SEQ ID NO. 15.
SEQ ID NO. 17 is the N-terminal amino acid sequence of 17a.
SEQ ID NO. 18 is the N-terminal amino acid sequence of 17b.
SEQ ID NO. 19 is the N-terminal amino acid sequence of 52A1.
SEQ ID NO. 20 is the N-terminal amino acid sequence of 63B.
SEQ ID NO. 21 is the N-terminal amino acid sequence of 69D1.
SEQ ID NO. 22 is the N-terminal amino acid sequence of 33F2.
SEQ ID NO. 23 is an internal amino acid sequence for 63B.
SEQ ID NO. 24 is a synthetic oligonucleotide derived from 17.
SEQ ID NO. 25 is an oligonucleotide probe designed from the N-terminal amino acid sequence of 52A1.
SEQ ID NO. 26 is the synthetic oligonucleotide probe designated as 69D1-D.
SEQ ID NO. 27 is the forward oligonucleotide primer from 63B.
SEQ ID NO. 28 is the reverse oligonucleotide primer from 63B.
SEQ ID NO. 29 is the nematode (NEMI) variant of region 5 of Höfte and Whiteley.
SEQ ID NO. 30 is the reverse complement primer to SEQ ID NO. 29, used according to the subject invention.
SEQ ID NO. 31 is a peptide used according to the subject invention.
SEQ ID NO. 32 is an oligonucleotide coding for the peptide of SEQ ID NO. 31.
SEQ ID NO. 33 is oligonucleotide probe 33F2A.
SEQ ID NO. 34 is oligonucleotide probe 33F2B.
SEQ ID NO. 35 is a reverse primer used according to the subject invention.
SEQ ID NO. 36 is a forward primer according to the subject invention.
SEQ ID NO. 37 is a probe according to the subject invention.
SEQ ID NO. 38 is a probe according to the subject invention.
SEQ ID NO. 39 is a probe according to the subject invention.
SEQ ID NO. 40 is a forward primer according to the subject invention.
DETAILED DISCLOSURE OF THE INVENTION
The subject invention concerns a vast array of B.t. &dgr;-endotoxins having nematicidal activity. In addition to having nematicidal activity, the t
Foncerrada Luis
Narva Kenneth E.
Payne Jewel
Schnepf H. Ernest
Schwab George E.
Bugaisky Gabrielle
Mycogen Corporation
Saliwanchik Lloyd & Saliwanchik
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