Recombinant bHLH-PAS/JHR polypeptide and its use to screen potential insecticides

Abstract

Promising groups of environmentally-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 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.

1 - 14 . (canceled)  
     
     
         15 . An isolated polypeptide selected from the group consisting of: 
 (a) a conservative amino acid variant of SEQ ID NO:3,    (b) a functional fragment of a polypeptide having the amino acid sequence of SEQ ID NO:3,    (c) a polypeptide having an amino acid sequence of SEQ ID NO:3,    (d) a conservative amino acid variant of SEQ ID NO:4,    (e) a functional fragment of a polypeptide having the amino acid sequence of SEQ ID NO:4,    (f) a pol ypeptide having an amino acid sequence of SEQ ID NO:4, and    (g) a Met-JHR alternatively-spliced isoform.    
     
     
         16 . The isolated polypeptide of  claim 15 , wherein said conservative amino acid variant is a polypeptide having an amino acid sequence that differs from the amino acid sequence of SEQ ID NO:3 by containing at least one amino acid substitution selected from the group consisting of (1) the substitution of an alkyl amino acid for an alkyl amino acid in SEQ IN NO:3, (2) the substitution of an aromatic amino acid for an aromatic amino acid in SEQ ID NO:3, (3) the substitution of a sulfur-containing amino acid for a sulfur-containing amino acid in SEQ ID NO:3; (4) the substitution of a hydroxy-containing amino acid for a hydroxy-containing amino acid in SEQ ID NO:3; (5) the substitution of an acidic amino acid for an acidic amino acid in SEQ ID NO:3; (6) the substitution of a basic amino acid for a basic amino acid in SEQ ID NO:3; (7) the substitution of a dibasic monocarboxylic amino acid for a dibasic monocarboxylic amino acid in SEQ ID NO:3.  
     
     
         17 . The isolated polypeptide of  claim 15 , wherein said conservative amino acid variant is a polypeptide having an amino acid sequence that differs from the amino acid sequence of SEQ ID NO:4 by containing at least one amino acid substitution selected from the group consisting of (1) the substitution of an alkyl amino acid for an alkyl amino acid in SEQ IN NO:4, (2) the substitution of an aromatic amino acid for an aromatic amino acid in SEQ ID NO:4, (3) the substitution of a sulfur-containing amino acid for a sulfur-containing amino acid in SEQ ID NO:4; (4) the substitution of a hydroxy-containing amino acid for a hydroxy-containing amino acid in SEQ ID NO:4; (5) the substitution of an acidic amino acid for an acidic amino acid in SEQ ID NO:4; (6) the substitution of a basic amino acid for a basic amino acid in SEQ ID NO:4; (7) the substitution of a dibasic monocarboxylic amino acid for a dibasic monocarboxylic amino acid in SEQ ID NO:4.  
     
     
         18 - 29 . (canceled)  
     
     
         30 . A method for detecting JH-resistant individuals in an insect population, said method comprising: 
 (a) obtaining a representative biological sample of said population; and    (b) detecting a nucleic acid sequence in said sample that corresponds to a predetermined sequence within a polynucleotide encoding a bHLH-PAS polypeptide that is altered in JH analog-resistant individuals, wherein said polypeptide is involved in binding juvenile hormone III.    
     
     
         31 . A method according to  claim 30 , wherein said detecting step comprises: 
 (i) amplifying a nucleic acid sequence from said sample, wherein said sequence corresponds to a predetermined sequence within a polynucleotide encoding a bHLH-PAS/JHR polypeptide and wherein said sequence comprises at least one RFLP characteristic of JH analog resistance;    (ii) incubating said amplified nucleic acid with at least one predetermined restriction endonuclease, to form fragments;    (iii) size-separating said fragments to form a detectable pattern; and    (iv) comparing said pattern with a predetermined pattern obtained from JH analog-resistant individuals to detect the appearance of one or more RFLP characteristic of JH analog resistance.    
     
     
         32 . An in vivo method for screening compounds that specifically bind with a bHLH-PAS polypeptide that is involved in binding juvenile hormone III, 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, SEQ ID NO:6, and SEQ ID NO:7, wherein said insect is selected from the group consisting of  Coleoptera, Siphonaptera, Orthoptera, Thysanoptera, Lepidoptera, Hemiptera,  and  Diptera,  and wherein the stringent conditions comprise hybridization in 1×SSC and 0.1% SDS at about 55° C. for about 60 minutes, comprising: 
 (a) providing a host cell comprising (1) DNA encoding a fusion polypeptide comprising said bHLH-PAS polypeptide and a second polypeptide comprising a DNA binding domain, and (2) a reporter gene under the control of a minimal promoter driven by the response element for said second polypeptide;    (b) incubating a test compound with said host cell; and    (c) detecting the binding of the test compound to said bHLH-PAS polypeptide by monitoring expression of the reporter gene.    
     
     
         33 . An in vivo method for screening compounds that specifically bind with a bHLH-PAS polypeptide that is involved in binding juvenile hormone III, 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, SEQ ID NO:6, and SEQ ID NO:7, 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 55° C. for about 60 minutes, comprising the steps of: 
 (a) providing a host cell comprising (1) DNA encoding a fusion polypeptide comprising said bHLH-PAS polypeptide and a second polypeptide comprising a DNA binding domain;    (2) a reporter gene under the control of a minimal promoter driven by the response element for said second polypeptide; and (3) DNA encoding a polypeptide that is a heterodimeric partner of said bHLH-PAS polypeptide;    (b) incubating a test compound with said host cell; and    (c) detecting the binding of the test compound to said bHLH-PAS polypeptide by monitoring expression of the reporter gene.    
     
     
         34 . An in vivo method for screening compounds that specifically bind to a multimeric complex comprising a bHLH-PAS polypeptide that is involved in binding juvenile hormone III, wherein said polyneptide 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, SEQ ID NO:6, and SEQ ID NO:7, 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 55° C. for about 60 minutes, and the heteromultimeric partner of said polypeptide, comprising the steps of: 
 (a) providing a host cell comprising (1) DNA encoding a fusion polypeptide comprising said bHLH-PAS polypeptide and a second polypeptide comprising a DNA binding domain; (2) DNA encoding a heteromultimeric partner of said bHLH-PAS polypeptide and the activation domain of said second polypeptide, and (3) a reporter gene under the control of a minimal promoter driven by the response element for said second polypeptide;    (b) incubating a test compound with said host cell; and    (c) detecting the binding of the test compound to said complex by monitoring expression of the reporter gene.    
     
     
         35 . An in vivo method for screening compounds that specifically bind to a multimeric complex comprising a bHLH-PAS polypeptide that is involved in binding juvenile hormone III, 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, SEQ ID NO:6, and SEQ ID NO:7, 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 55° C. for about 60 minutes, and the heteromultimeric partner of said polypeptide, comprising the steps of: 
 (a) providing a host cell comprising (1) DNA encoding a fusion polypeptide comprising bHLH-PAS polypeptide and the activation domain of a second polypeptide; (2) DNA encoding a heteromultimeric partner of said bHLH-PAS polypeptide and the DNA binding domain of said second polypeptide, and (3) a reporter gene under the control of a minimal promoter driven by the response element for said second polypeptide;    (b) incubating a test compound with said host cell; and    (c) detecting the binding of the test compound to said complex by monitoring expression of the reporter gene.    
     
     
         36 . An in vivo method for screening compounds that specifically bind with a bHLH-PAS polypeptide that is involved in binding juvenile hormone III, 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, SEQ ID NO:6, and SEQ ID NO:7, 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 55° C. for about 60 minutes, comprising the steps of: 
 (a) providing a host cell comprising (1) DNA encoding a fusion polypeptide comprising bHLH-PAS polypeptide and the DNA binding domain of a second polypeptide; (2) DNA encoding a bHLH-PAS polypeptide and the activation domain of said second polypeptide, and (3) a reporter gene under the control of a minimal promoter driven by the response element for said second polypeptide;    (b) incubating a test compound with said host cell; and    (c) detecting the binding of the test compound with said bHLH-PAS polypeptide by monitoring expression of the reporter gene.    
     
     
         37 . A method according to  claim 32 , wherein said host cell is selected from the group of an insect cell, a yeast cell, and a mammalian cell.  
     
     
         38 . A method according to  claim 33 , wherein said host cell is selected from the group of an insect cell, a yeast cell, and a mammalian cell.  
     
     
         39 . A method according to  claim 34 , wherein said host cell is selected from the group of an insect cell, a yeast cell, and a mammalian cell.  
     
     
         40 . A method according to  claim 35 , wherein said host cell is selected from the group of an insect cell, a yeast cell, and a mammalian cell.  
     
     
         41 . A method according to  claim 36 , wherein said host cell is selected from the group of an insect cell, a yeast cell, and a mammalian cell.  
     
     
         42 - 51 . (canceled)