Recombinant bHLH-PAS/JHR polypeptide and its use to screen potential insecticides
AssigneesAmerican Cyanamid Company, Colorado State University Research Foundation
InventorsThomas G. Wilson, Julia N. Heinrich
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Abstract
Promising groups of environmetally-safe insecticides consist of analogues of insect hormones, such as juvenile hormone, and antagonists of such hormones. The traditional bioassay approach for screening potential juvenile hormone analogs and antagonists is slow, expensive and inefficient. A recombinant bHLH-PAS-juvenile hormone receptor, isolated from the methoprene-tolerant locus on Drosophila, provides the basis of in vitro and in vivo binding assays that can be used to discover new juvenile hormone-type targeted insecticides. Moreover, the nucleotide sequence of the Drosophila bHLH-PAS/JHR polypeptide provides tools for isolating juvenile hormone receptor genes from other insect species.
Claims20
An isolated insect polynucleotide that encodes a bHLH-PAS polypeptide that is involved in binding juvenile hormone III, wherein said polynucleotide hybridizes under stringent conditions with a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:6, wherein the stringent conditions comprise hybridization in 1×SSC and 0.1% SDS at about 55° C. for about 60 minutes, wherein said insect is selected from the group consisting of Coleoptera, Siphonoptera, Orthoptera, Thysanoptera, Lepidoptera, Hemiptera, and Diptera, and wherein said polynucleotide has a nucleotide sequence that encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:5.
A method for screening compounds that specifically bind with a bHLH-PAS/JHR polypeptide, comprising:
- (a) incubating a test compound in a solution that comprises an isolated bHLH-PAS polypeptide, wherein said polypeptide is encoded by an isolated insect polynucleotide, wherein said polynucleotide hybridizes under stringent conditions with a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:6, wherein said insect is selected from the group consisting of Coleoptera, Siphonoptera, Orthoptera, Thysanoptera, Lepidoptera, Hemiptera, and Diptera, and wherein the stringent conditions comprise hybridization in 1×SSC and 0.1% SDS at about 55C for about 60 minutes, and
- (b) detecting the binding of said test compound with said polypeptide.
An isolated polynucleotide which comprises the sequence of nucleotide 1 through nucleotide 1291 of SEQ ID NO:1.
An isolated polynucleotide which comprises the sequence of nucleotide 1 through nucleotide 1513 of SEQ ID NO:1.
An isolated polynucleotide which comprises the sequence of nucleotide 3733 through nucleotide 6235 of SEQ ID NO:1.
An isolated polynucleotide which comprises the sequence of nucleotide 4302 through nucleotide 6235 of SEQ ID NO:1.
Dependencies are inferred from the claim text.