Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-10-24
2003-12-09
Spector, Lorraine (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C435S252300, C435S254110, C536S023100, C536S023500, C530S300000, C530S350000
Reexamination Certificate
active
06660497
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention generally relates to receptors for
Bacillus thuringiensis
(BT) toxin and thus to pesticides able to bind the receptor, and to ameliorating pesticide resistance. In particular, the invention relates to recombinant DNA and expression systems for a novel receptor and receptor elements from
Pectinophora gossypiella
, the pink bollworm.
BACKGROUND OF THE INVENTION
Without limiting the scope of the invention, its background is described in connection with uses of
Bacillus thuringiensis
toxins as cotton insect biocidal agents, as an example. Cotton insect pests reduced yields by almost 10% across the US in 1998. Insect damage reduced the overall cotton yield by more than 1.7 million bales and produced a financial loss of about $1.224 billion. One group in particular, the bollworm/budworm complex was the most damaging causing a 2.7% loss. The pink bollworm,
Pectinophora gossypiella
Saunders (“PBW”), is a lepidopteran insect that causes severe damage to cotton and is the most destructive pest of cotton worldwide.
Bacillus thuringiensis
is a gram positive, sporeforming bacterium that forms a parasporal crystal which contains insecticidal toxins (Bulla et al.,
Crit. Rev. Microbiol
. (1980) 8: 147-204; Höfte and Whiteley,
Microbiol. Rev
. (1989) 53: 242. The effect of the toxin is mediated through binding to specific receptors on the apical brush border of the midgut microvillae (BBMV) of susceptible insects.
Biological control of cotton pests using
B. thuringiensis
formulations and transgenic plants has been in use for a number of years and is growing rapidly. Recently, transgenic cotton plants carrying the toxin genes of BT have been developed and sold commercially. Such transgenic plants have a high degree of resistance to the pink bollworm (Schnepf et al.,
Microbiol. Mol. Biol. Rev
. (1998) 62: 775). However, the introduction of any new insecticide into a pest management program immediately initiates a selection process for individuals that are resistant to the pesticide. As the use of transgenic crops expressing BT toxin increases, insect resistance is expected to become more widespread. Increased tolerance for BT toxins in several species of insects has been reported by several investigators while laboratory selection experiments have shown that the use of BT toxin formulations and transgenic plants can provoke the development of resistance in the pink bollworm (Bartlett, et al.,
Beltwide Cotton Conference
(1995) 2: 766).
Concerns that BT toxin formulations or transgenic plants expressing the toxin genes may evoke emergence of either resistant or tolerant strains of insects has made the search for a better understanding of the interaction between the BT toxin proteins and their respective insect receptors a matter of considerable economic importance.
In U.S. Pat. No. 5,693,491, the present inventors disclosed the purification and cDNA cloning of a
B. thuringiensis
toxin receptor BT-R
1
from larvae of the tobacco hornworm
Manduca sexta
(
M. Sexta
). Recently, two BT toxin receptors have been identified, purified and cloned from the silkworm,
Bombyx mori
(Nagamatsu et al.,
Biosci. Biotechnol. Biochem
. (1998) 62: 727).
Heretofore in this field, there has been no structural information concerning the structure and function of BT toxin receptor of the major cotton insect pest,
P. gossypiella
. Furthermore, to the inventors' knowledge, the minimum binding fragment encoding a consensus binding domain for BT toxin on the BT receptor has not yet been identified. Isolation of the minimum binding fragment could permit cloning and structural characterization of important yet uncharacterized BT toxin receptors from other insects of worldwide economic importance such as
P. gossypiella.
SUMMARY OF THE INVENTION
The present invention provides information and materials for isolation and expression of novel BT crystal toxin receptors, herein referred to as Cry toxin receptors. Generally, the invention provides structural and functional characterization of a novel lepidopteran BT toxin receptor, herein referred to as BT-R
2
.
A cDNA that encodes an alternative glycoprotein receptor from the pink bollworm that binds specifically to a
B. thuringiensis
toxin has been cloned, sequenced and characterized. The BT-R
2
cDNA permits the analysis of receptors in pink bollworm and other insects and organisms that affect crop growth and development, as well as the design of assays for the cytotoxicity and binding affinity of potential pesticides. The clone and other methods described herein, permit the manipulation of natural and/or introduced homologous receptors and, thus, to specifically destroy organisms, tissues and/or cells of the target host, including insects resistant to toxins of
B. thuringiensis.
The invention further provides purified and cloned cDNA encoding a 200 kD receptor for the Cry1A toxins of the pink bollworm,
P. gossypiella
. An advantage of this invention is the identification of the minimum binding fragment encoding the toxin binding domain on the BT toxin receptor. Another advantage of this invention is the provision of methodologies for cloning and structural characterization of presently unknown BT receptors. Furthermore, this invention provides methods and materials for identification and design of effective toxin binding receptors for use in combating emergence of toxin resistance. Also, this invention may be used to generate transgenic organisms expressing toxin receptors.
REFERENCES:
patent: 5693491 (1997-12-01), Bulla et al.
patent: WO 98/59048 (1998-12-01), None
Lee et al., Inconsistencies in determiningBacillus thuringiensistoxin binding sites relationship by comparing competition assays with ligand blotting, Biochem. Biophys. Res. Comm., 220:575-580 (1996).*
Bartlett et al., Beltwide Cotton Conference (1995) 2:766.
Bulla et al., Crit. Rev. Microbiol. (1980) 8:147-204.
Hofte and Whiteley, Microbiol. Rev. (1989) 53:242.
Karim et al., Pesticide Biochemistry and Physiology (2000) 67(3):198-216.
Nagamatsu et al., Biosci. Biotechnol. Biochem. (1998) 62:727-734.
Schnepf et al., Microbiol. Mol. Biol. Rev. (1998) 62:775.
Bulla, Jr. Lee A.
Candas Mehmet
Kaufman Claire M.
Morrison & Foerster / LLP
Spector Lorraine
The Board of Regents The University of Texas System
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