Screening Assay for Insecticides

Abstract

The present invention relates to polypeptides, preferably from Drosophila melanogaster (DmShal) as target for insecticides.

1 - 19 . (canceled) 
     
     
         20 . A method for identifying an insecticidal active compound that reduces the activity of a G-protein coupled receptor (GPCR) with the activity of an octopamine receptor, which method comprises:
 a) assembling in a membrane a polypeptide with the activity of an octopamine receptor which is originally not present in said membrane;   b) applying at one side of the membrane a compound suspected of having the ability to inhibit the activity of said polypeptide which is originally not present said membrane;   c) determining the activity of said polypeptide; and   d) identifying the compound applied in (b) that reduces the activity of said polypeptide as an insect-controlling compound,   
       wherein the polypeptide has the activity of an octopamine receptor selected from the group consisting of oa2, Oamb, Oct-beta-2R and Oct-beta-3R, and such receptor is from a species selected from the group consisting of  Drosophila melanogaster, Myzus persicae , and  Tribolium castaneum.   
     
     
         21 . The method of  claim 20 , wherein a gene coding for the polypeptide is expressed in a host cell and the polypeptide is assembled in the membrane of the host cell. 
     
     
         22 . The method of  claim 20 , wherein the polypeptide is encoded by a nucleic acid molecule selected from the group consisting of:
 a) a nucleic acid molecule encoding a polypeptide shown in SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 or 174;   b) a nucleic acid molecule shown in SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 or 173;   c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 or 174;   d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule of b);   e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c);   f) a nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;   g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of an octopamine G-protein coupled receptor (GPCR) and the receptor is selected from the group consisting of oa2, Oamb, Oct-beta-2R and Oct-beta-3R, and such receptor is from a species selected from the group consisting of  Drosophila melanogaster, Myzus persicae , and  Tribolium castaneum;      h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 177, 178 or 179, or one or more motifs selected from the group consisting of SEQ ID NO: 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and 226;   i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers selected from the group consisting of SEQ ID NO: 131, 132; 135, 136; 139, 140; 143, 144; 147, 148; 151, 152; 155, 156; 159, 160; 163, 164; 167, 168; 171, 172, 175, and 176; and   j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 200 nucleotides of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e).   
     
     
         23 . The method of  claim 20 , wherein the activity of the polypeptide is determined by fluorescence measurement. 
     
     
         24 . The method of  claim 21  wherein the activity of the polypeptide is determined with calcium concentration sensitive dyes. 
     
     
         25 . The method of  claim 21  wherein the host cell is a mammalian cell. 
     
     
         26 . The method of  claim 25  wherein the host cell is selected from the group consisting of CHO-cells and HEK293 cells. 
     
     
         27 . The method of  claim 20 , further comprising:
 e) treating insects, populations of insect pest or the location wherein insect pests are to be controlled by applying to an insect pest, to a population of insect pests or to the location wherein said insect pest is to be controlled, an insect-controlling amount of the insect-controlling compound identified in step d);   f) comparing the growth or the viability of the treated insect pest or population of insect pests or of insects pest or population of insect pests on said location with the growth and viability of untreated insects, population of insects or location of insects; and   g) selecting the insect-controlling compounds that reduce the growth or the viability of the treated insect pest or population of insect pests or of insects pest or population of insect pests on said location following application of the insect-controlling compound of step e).   
     
     
         28 . An assay system comprising a host organism, tissue, cells or a cell digest thereof or a membrane, which has embedded, assembled, intercalated or incorporated a nucleic acid molecule selected from the group consisting of the nucleic acid molecules depicted in  claim 22  and, based on the expression of the nucleic acid molecule, a polypeptide having the biological activity of a G-protein coupled receptor (GPCR) with the activity of an octopamine receptor selected from the group consisting of oa2, Oamb, Oct-beta-2R and Oct-beta-3R, and such receptor is from a species selected from the group consisting of  Drosophila melanogaster, Myzus persicae , and  Tribolium castaneum , for identifying insecticidally active compound that reduces the activity of a G-protein coupled receptor (GPCR) with the activity of an octopamine receptor selected from the group consisting of oa2, Oamb, Oct-beta-2R and Oct-beta-3R, and such receptor is from a species selected from the group consisting of  Drosophila melanogaster, Myzus persicae , and  Tribolium castaneum.   
     
     
         29 . The assay system of  claim 28  whereby the host organism is a stably transfected mammalian cell which expresses a nucleic acid molecule selected from the group consisting of the nucleic acid molecules as depicted in  claim 22 . 
     
     
         30 . The assay system of  claim 29  wherein the mammalian cell is selected from the group consisting of CHO-cells, HEK293, COS, HeLa, NIH3T3, BAK21, Jurkat, CV-1, HepC-2-,  Xenopus  oocyte, Sf9, S2, Sf21, Hi5, Pc12, and U2OS. 
     
     
         31 . A method for killing or inhibiting the growth or viability of an insect pest comprising applying to the insect pest the compound identified in  claim 20 . 
     
     
         32 . A nucleic acid molecule selected from the group consisting of:
 a) a nucleic acid molecule encoding the polypeptide shown in 2, 6, 10, 14, 18, 22, 26, 30, 34,   b) a nucleic acid molecule shown in SEQ ID NO: 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169 or 173;   c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a polypeptide sequence according to SEQ ID NO: 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170 or 174;   d) a nucleic acid molecule having at least 50% identity with the nucleic acid molecule of b);   e) a nucleic acid molecule encoding a polypeptide having at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c);   f) a nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under stringent hybridization conditions;   g) a nucleic acid molecule encoding a polypeptide which can be isolated with the aid of monoclonal or polyclonal antibodies made against a polypeptide encoded by one of the nucleic acid molecules of (a) to (e) and having the activity of an octopamine G-protein coupled receptor (GPCR) and the receptor is selected from the group consisting of oa2, Oamb, Oct-beta-2R and Oct-beta-3R, and such receptor is from a species selected from the group consisting of  Drosophila melanogaster, Myzus persicae , and  Tribolium castaneum;      h) a nucleic acid molecule encoding a polypeptide comprising the consensus sequence as shown in SEQ ID NO: 177, 178 or 179, or one or more motifs selected from the group consisting of SEQ ID NO: 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and 226;   i) nucleic acid molecule which comprises a polynucleotide, which is obtained by amplifying a cDNA library or a genomic library using the primers selected from the group consisting of SEQ ID NO: 131, 132; 135, 136; 139, 140; 143, 144; 147, 148; 151, 152; 155, 156; 159, 160; 163, 164; 167, 168; 171, 172, 175, and 176; and   j) a nucleic acid molecule which is obtainable by screening a suitable nucleic acid library under stringent hybridization conditions with a probe comprising a complementary sequence of a nucleic acid molecule of (a) or (b) or with a fragment thereof, having at least 200 nucleotides of a nucleic acid molecule complementary to a nucleic acid molecule sequence characterized in (a) to (e).   
     
     
         33 . A transgenic cell comprising a vector comprising a nucleic acid molecule according to  claim 32 . 
     
     
         34 . A transgenic cell comprising a vector according to  claim 33 . 
     
     
         35 . A transgenic cell comprising a nucleic acid molecule according to  claim 32 . 
     
     
         36 . A polypeptide encoded by a nucleic acid molecule according to  claim 32 .