Patent Abstract:
Nucleotide sequences are isolated from  Drosophila melanogaster  that code for proteins essential for viability. These proteins are useful for discovering new insecticides based on the essentiality of the nucleotide sequences for  Drosophila  viability. Further provided are recombinant proteins and methods for identifying inhibitors to these proteins. Protein inhibitors active in the methods disclosed herein are useful as insecticidal, ectoparasiticidal, antiparasitic, anthementhic and acaracidal agents.

Full Description:
[0001]     This application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/422,377 filed Oct. 30, 2002, which is incorporated by reference in its entirety. 
     
    
       [0002]     The Sequence Listing associated with the instant disclosure has been submitted as a 2.62 megabyte file on CD-R (in duplicate) instead of on paper. Each CD-R is marked in indelible ink to identify the Applicants, Title, File Name (70131WOPCT.ST25.txt), Creation Date (Aug. 7, 2003), Computer System (IBM-PC/MS-DOS/MS-Windows), and Docket No. (70131 WOPCT). The Sequence Listing submitted on CD-R is hereby incorporated by reference into the instant disclosure.  
       FIELD OF INVENTION  
       [0003]     The present invention pertains to nucleic acid sequences isolated from  Drosophila melanogaster  that encode proteins essential for viability. The invention particularly relates to methods of using these proteins as insecticide targets, based on this essentiality.  
       BACKGROUND OF THE INVENTION  
       [0004]     Insects contribute or cause many human and animal diseases, and are responsible for substantial agricultural and property damage. The societal costs associated with insect pests in dollars, time and suffering are monumental. The total worldwide market size for insecticide crop protection is over $5 billion. To combat these problems, insecticidal compounds have been developed and employed.  
         [0005]     The idea to use chemicals for insect control is not new. The scientific use of pesticides started with the introduction of arsenical insecticides and organic compounds such as tar, petroleum oils, and dinitrophenol emulsions at the end of the last century. But, the systematic search for synthetic organic insecticides was only launched after the discovery of the insecticidal properties of DDT in 1939. After World War II, chemical research concentrated mainly on chlorinated hydrocarbons and cyclodienes, which all require high rates of application and have a rather broad spectrum of activity. Most of them are persistent in the environment and may pose a significant risk for accumulation in the food chain. Today the use of these chemicals is very much restricted.  
         [0006]     From this point, the major emphasis in research has been given to organophosphates and carbamates, which are readily degradable in the environment with little tendency for bioaccumulation. The toxicity of these compounds varies within a broad range from medium to highly toxic. Organophosphates and carbamates are still widely use, although the more toxic ones are banned in certain countries. The formamidines have as their major advantage a different mode of action and their selectivity, which made them suitable for use in IPM (insect pest management) programs. They are easily degradable with no accumulation potential, but for toxicological reasons some have had to be withdrawn from the market.  
         [0007]     For the past decade, insecticide research has concentrated on leadfinding for new chemical structures interfering with new target mechanisms. The chances for success are rather remote, because the hurdles for the registration of a new insecticide are set very high. Toxicological aspects, insecticide resistance, environmental behavior, and IPM fitness are some of the critical factors that have to be considered together with economical factors.  
         [0008]     Novel insecticides can now be discovered using high-throughput screens that implement recombinant DNA technology. Proteins found to be essential to insect viability can be recombinantly produced through standard molecular biological techniques and utilized as insecticide targets in screens for novel inhibitors of the enzymes&#39; activity. The novel inhibitors discovered through such screens may then be used as insecticides to control undesirable insect infestation.  
         [0009]     However, as the world population continues to grow, there will be increasing food shortages. Therefore, there exists continuing need to find new, effective and economic insecticides.  
       SUMMARY OF THE INVENTION  
       [0010]     In view of these needs, it is one object of the invention to provide essential genes in insects such as  Drosophila melanogaster . It is another object to provide the essential proteins encoded by these essential genes for assay development to identity inhibitory compounds with insecticidal activity. It is still another object of the present invention to provide an effective and beneficial method for identifying new or improved insecticides using the essential proteins of the invention.  
         [0011]     In furtherance of these and other objects, the present invention provides DNA molecules comprising nucleotide sequences isolated from  Drosophila melanogaster  that encode proteins essential for viability. The inventors are the first to demonstrate that the nucleotide sequences of the invention are essential for viability. This knowledge is exploited to provide novel insecticide modes of action. One advantage of the present invention is that the proteins encoded by the essential nucleotide sequences provide the bases for assays designed to easily and rapidly identify novel insecticides.  
         [0012]     Disruption of the nucleotide sequences or messenger RNA of the invention demonstrates that the activity of each corresponding encoded protein is essential for  Drosophila  viability. Genetic results show that when each nucleotide sequence of the invention is mutated in  Drosophila  or disrupted at the transcription level, the resulting phenotype is lethal. This demonstrates a critical role for the protein encoded by the mutated nucleotide sequence. This further implies that chemicals that inhibit the expression of the protein when in contact with insects are likely to have detrimental effects on insects and are potentially good insecticide candidates. The present invention therefore provides methods of using the disclosed nucleotide sequences or proteins encoded thereby to identify inhibitors thereof. The inhibitors can then be used as insecticides to kill undesirable insect populations where crops are grown, particularly agronomically important crops such as maize, and other cereal crops such as wheat, oats, rye, sorgum, rice, barley, millet, turf and forage grasses and the like, as well as cotton, sugar cane, sugar beet, oilseed rape, soybeans, vegetable crops and fruits.  
         [0013]     The present invention accordingly provides cDNA sequences derived from  Drosophila melanogaster . In one embodiment, the present invention provides an isolated DNA molecule comprising a nucleotide sequence selected from the group consisting of the even numbered SEQ ID NOs:14-380. In another embodiment, the present invention provides an isolated DNA molecule comprising a nucleotide sequence that encodes a protein selected from the group consisting of the odd numbered SEQ ID NOs:15-381.  
         [0014]     The present invention also provides a chimeric construct comprising a promoter operatively linked to a DNA molecule according to the present invention, wherein the promoter is preferably functional in a eukaryote, wherein the promoter is preferably heterologous to the DNA molecule. The present invention further provides a recombinant vector comprising a chimeric construct according to the present invention, wherein said vector is capable of being stably transformed into a host cell. The present invention still further provides a host cell comprising a DNA molecule according to the present invention, wherein said DNA molecule is preferably expressible in the cell. The host cell is preferably selected from the group consisting of an insect cell, a yeast cell, and a prokaryotic cell.  
         [0015]     The present invention also provides proteins essential for  Drosophila melanogaster  viability. In one embodiment, the present invention provides an isolated protein comprising an amino acid sequence selected from the group consisting of the odd numbered SEQ ID NOs:15-361. In accordance with another embodiment, the present invention also relates to the recombinant production of proteins of the invention and methods of using the proteins of the invention in assays for identifying compounds that interact with the protein.  
         [0016]     In another preferred embodiment, the present invention describes a method for identifying chemicals having the ability to inhibit the activity of the disclosed proteins. In a preferred embodiment, the present invention provides a method for selecting compounds that interact with a protein of the invention, comprising: (a) expressing a DNA molecule according to the present invention to generate the corresponding protein of the invention, (b) testing a compound suspected of having the ability to interact with the protein expressed in step (a), and (c) selecting compounds that interact with the protein in step (b).  
         [0017]     Other objects and advantages of the present invention will become apparent to those skilled in the art and from a study of the following description of the invention and non-limiting examples. The entire contents of all publications mentioned herein are hereby incorporated by reference.  
       BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING  
       [0018]     SEQ ID NOs:1-13 are PCR primers.  
         [0019]     Even numbered SEQ ID NOs:14-380 are nucleotide sequences described in the table below.  
         [0020]     Odd numbered SEQ ID NOs:15-381 are protein sequences encoded by the immediately preceding nucleotide sequence, e.g., SEQ ID NO:15 is the protein encoded by the nucleotide sequence of SEQ ID NO:14, SEQ ID NO:17 is the protein encoded by the nucleotide sequence of SEQ ID NO:16, etc.  
                                                                   TABLE 1                             Drosophila  Sequences            seq   Inventor&#39;s                       ID   reference   function   Domains   Best blast hit   score                    14-15   CT28483   CG10260   PI3Ka, PI3_4_KINASE_1,   (D83538) 230 kDa   1600               EG: BACR7C10.2 protein   PI3_4_KINASE_2,   phosphatidylinositol 4-kinase               kinase, 1-   PI3_4_KINASE_3,   [ Rattus norvegicus ]               phosphatidylinositol 4-   PI3_PI4_kinase               kinase       16-17   CT28925   CG10365 unknown       hypothetical protein MGC4504   185                       [ Homo sapiens ]       18-19   CT29122   CG10370 Tbp-1 Tat-   AAA, ATP_GTP_A,   Q63569|PRSA_RAT 26S   720               binding protein-1,   MITOCH_CARRIER   PROTEASE REGULATORY               Proteasome 26S       SUBUNIT 6A (TAT-BINDING               regulatory subunit 6A,       PROTEIN 1) (TBP-1)               multicatalytic               endopeptidase,       20-21   CT29492   CG10545 Gb13F G   GPROTEINB,   GBB1_CAEEL GUANINE   619               protein b-subunit 13F, G-   GPROTEINBRPT, WD40,   NUCLEOTIDE-BINDING               protein coupled receptor,   WD40_REGION,   PROTEIN BETA SUBUNIT 1               protein signaling pathway   WD_REPEATS       22-23   CT30008   CG10701 Moe Dmoesin,   BAND41, BAND_41_1,     Homo sapiens  ‘moesin’               motor involved in   BAND_41_2, BAND_41_3,   gi: 4505257               cytoskeleton organization   Band_41, ERM, ERMFAMILY               and biogenesis       24-25   CT30208   CG10776 wit   PROTEIN_KINASE_ATP,   NP_031587.1| (NM_007561)   362               Serine/threonine kinase-   PROTEIN_KINASE_DOM,   bone morphogenic protein               D; wishful thinking, a type   TGFB_RECEPTOR, pkinase   receptor, type II               II transforming growth               factor beta receptor               involved in protein               phosphorylation       26-27   CT30807   CG10997 chloride       NP_001280.2| (NM_001289)   119               channel?       chloride intracellular channel 2                       [ Homo sapiens ]       28-29   CT30887   CG11033 unknown       NP_036440.1| (NM_012308) F-   431                       box and leucine-rich repeat                       protein 11       30-31   CT31117   CG11130 Rtc1 RNA 3′       Q9Y2P8|RCL1_HUMAN RNA   326               terminal phosphate       3′-TERMINAL PHOSPHATE               cyclase, Rtc1       CYCLASE-LIKE PROTEIN                       (HSPC338)       32-33   CT1249   CG1114 Weak similarity       NP_071334.1| (NM_022051)   249               with apoptosis protein RP-       egl nine homolog 1 ( C. elegans )               8,       34-35   CT1483   CG1119 Gnf1 Germ line   ATP_GTP_A, BRCT,   A49651 replication factor C   661               transcription factor 1,   BRCT_DOMAIN, NLS_BP,   large subunit - human               DNA binding/DNA   RFC               replication factor       36-37   CT7860   CG11190 unknown       BAB60854.1| (AB057724)   387                       phosphatidyl inositol glycan                       class T [ Homo sapiens ]       38-39   CT1834   CG1135 unknown   FHA, FHA_DOMAIN   NP_006328.1| (NM_006337)   383                       microspherule protein 1; cell                       cycle-regulated factor       40-41   CT31875   CG11418 EG: 8D8.8       NP_060579.1| (NM_018109)   252               involved in cell cycle       hypothetical protein FLJ10486                       [ Homo sapiens ]       42-43   CT36241   CG11452 unknown       none       44-45   CT1993   CG1149 MstProx   LRRNT     Homo sapiens  ‘toll-like               MstProx, transmembrane       receptor1’ gi: 4507527               receptor involved in               defense response       46-47   CT34608   CG11511 similarity to   ZINC_FINGER_C2H2,   AAC78286.1| (AF032674)   128               broad-complex Z2-   ZINC_FINGER_C2H2_2, zf-   broad-complex Z2-isoform               isoform   C2H2   [ Manduca sexta ]       48-49   CT5404   CG11595 unknown       none       50-51   CT17728   CG11779 receptor -       XP_049282.1| (XM_049282)   436               mitochondrial       translocase of inner               transporter???       mitochondrial membrane 44                       homolog       52-53   CT1465   CG12007       NP_004572.1| (NM_004581)   278               geranylgeranyltransferase,       Rab geranylgeranyltransferase,               alpha subunit       alpha subunit [ Homo sapiens ]       54-55   CT5438   CG12079 NADH   complex1_30 Kd   AAD40386.1| (AF100743)   323               dehydrogenase       NADH-Ubiquinone reductase               (ubiquinone)       [ Homo sapiens ]       56-57   CT43008   CG12085 pUbsf DPUF68   RBD, RNP_1, rrm   NP_525123.1| (NM_080384)   1037               Puf60 polyU binding       poly-U-binding splicing factor               splicing factor, poly(U)               binding involved in               mRNA splicing       58-59   CT5902   CG12093 unknown   CRYSTALLIN_BETAGAMMA   NP_499515.1| (NM_067114)   137                       Y41C4A.8.p [ Caenorhabditis                           elegans ]       60-61   CT6734   CG12113 unknown   ATP_GTP_A   AAH08013 (BC008013) Similar   498                       to CG12113 gene product                       [ Homo sapiens ]       62-63   CT7760   CG12135 c12.1 unknown       AF110775_1 (AF110775)   252                       adrenal gland protein AD-002                       [ Homo sapiens ]       64-65   CT9355   CG12181 Sgs4 sgs-4         Mus musculus  Sap62               salivary gland secretion       MGI: 104912               protein 4, pupal glue               protein       66-67   CT12665   CG12225 Spt6 spt6,   S1     Caenorhabditis elegans                 promoter-associated       T04A8.14 WP: CE13120               pausing and               transcriptional elongation       68-69   CT13424   CG12238 &#39;probable       NP_060758.1| (NM_018288)   222               transcription factor       hypothetical protein FLJ10975                       [ Homo sapiens ]       70-71   CT14932   CG12251 AQP AQP       XP_059490.1| (XM_059490)   62.4               aquaporin, water channel       hypothetical protein XP_059490                       [ Homo sapiens ]       72-73   CT23511   CG12348 Sh open               rectifying potassium               channel, shaker       74-75   CT32757   CG12482 unknown       NP_076113.1| (NM_023624)   40.8                       lecithin-retinol acyltransferase                       [ Mus musculus ]       76-77   CT33237   CG12497   LDLRA_1, LDLRA_2,   CAC86027.1| (AJ313389) tsetse   90.9               EG: BACR25B3.2 low-   LDLRECEPTOR, NLS_BP,   EP protein [ Glossina morsitans                 density lipoprotein   PRO_RICH, ldl_recept_a     morsitans ]               receptor-like       78-79   CT33996   CG12537 unknown       AAK31375.1|AC084329_1   116                       (AC084329) ppg3 [ Leishmania                           major ]       80-81   CT34671   CG12600 unknown   WW_rsp5_WWP   AF213258_1 (AF213258)   56.2                       membrane-associated guanylate                       kinase-related MAGI-3 [ Mus                            musculus ]       82-83   CT2591   CG1265 unknown       XP_059471.1| (XM_059471)   67.8                       similar to MANNOSE-P-                       DOLICHOL UTILIZATION                       DEFECT 1       84-85   CT35764   CG12701 unknown   NLS_BP, PRO_RICH,   NM_078717) kismet   117                   ZINC_FINGER_C2H2,   [ Drosophila melanogaster ]                   ZINC_FINGER_C2H2_2, zf-                   C2H2       86-87   CT28931   CG12750 nucampholin,   RNA binding   (AB046824) KIAA1604 protein   833               transcription factor?       [ Homo sapiens ]       88-89   CT32253   CG13034 unknown       (AC084329) ppg3 [ Leishmania     94.4                         major ]       90-91   CT32701   CG13372 EG: 171D11.6       none               unknown       92-93   CT40992   CG13372 EG: 171D11.6       none               unknown       94-95   CT32721   CG13380 unknown       NP_499428.1| (NM_067027)   43.5                       W09D6.5.p [ Caenorhabditis                           elegans ]       96-97   CT33014   CG13620 unknown   CYTOCHROME_C, NLS_BP,     Caenorhabditis elegans  ‘similar                   ZINC_FINGER_C2H2,   to Zinc finger, C2H2 type                   ZINC_FINGER_C2H2_2, zf-                   C2H2       98-99   CT33019   CG13625 histone   NLS_BP   NP_498982.1| (NM_066581)   265               protein?       R08D7.1.p [ Caenorhabditis                           elegans ]       100-101   CT33241   CG13760   Cysteine proteinases   (AK054681) unnamed protein   144               EG: BACR25B3.6       product [ Homo sapiens ]               unknown       102-103   CT33317   CG13818 unknown   ATP_GTP_A   T26047 hypothetical protein   39.3                       W01C8.5 -  Caenorhabditis                           elegans         104-105   CT3228   CG1405 cg1405 ATP   HELICASE, helicase_C   XP_008088.1| (XM_008088)   825               dependent helicase       pre-mRNA splicing factor Prp16                       [ Homo sapiens ]       106-107   CT33819   CG14206 structural       AF400207_1 (AF400207)   225               protein of ribosome       ribosomal protein S10                       [ Spodoptera frugiperda ]       108-109   CT3352   CG1422 p115 vesicular       P41541|VDP_BOVIN General   725               transporter, membrane       vesicular transport factor p115               docking       110-111   CT33841   CG14226 CT33841   fn3   NP_075214.1| (NM_022925)   93.6               protein tyrosine       protein tyrosine phosphatase,               phosphatase       receptor type, Q [ Rattus         112-113   CT34063   CG14411 protein   CRYSTALLIN_BETAGAMMA   AAK26171.1| (AY028703)   211               phosphatase       phosphatidylinositol-3 phosphate                       3-phosphatase adaptor       114-115   CT3509   CG1448 inx3 innexin 3       Q9XYN1|INX2_SCHAM   332                       Innexin Inx2 (Innexin-2) (G-                       Inx2)       116-117   CT34434   CG14656 unknown       NP_542443.1| (NM_080712)   122                       tty-P1 [ Drosophila                           melanogaster ]       118-119   CT34588   CG14778 integral       (AE003604) CG2022 gene   179               peroxisomal membrane       product [ Drosophila                            melanogaster ]       120-121   CT43287   CG14779 EG: 80H7.2   Tubulin-beta mRNA   none               tubulin-beta mRNA   autoregulation signal domain               autoregulation signal               protein       122-123   CT34589   CG14779 EG: 80H7.2   Tubulin-beta mRNA   none               tubulin-beta mRNA   autoregulation signal domain               autoregulation signal               protein       124-125   CT34599   CG14789   AA_TRNA_LIGASE_I   AF455270_1 (AF455270)   261               EG: BACN32G11.6       C21ORF80 [ Mus musculus ]               Aminoacyl-transfer RNA               synthetases class-I               signature protein       126-127   CT34602   CG14792 sta Laminin-   RIBOSOMALS2,   (AB032438) stubarista   410               receptor Stubarista,   RIBOSOMAL_S2_1,   [ Drosophila erecta ]               protein biosynthesis Rp40   RIBOSOMAL_S2_2,                   Ribosomal_S2       128-129   CT34626   CG14813 delta; COP   ATP_GTP_A: ATP/GTP-   NP_001646.2| (NM_001655)   585               coatomer complex COPI   binding site motif A (P-loop)   archain; coatomer protein delta-               delta-COP subunit delta   protein   COP [ Homo sapiens ]       130-131   CT34665   CG14849 unknown       none       132-133   CT3729   CG1489 Pros45 sug1,   AAA, ATP_GTP_A   P54814|PRS8_MANSE 26S   727               multicatalytic       PROTEASE REGULATORY               endopeptidase regulator,       SUBUNIT 8 (18-56 PROTEIN)               multicatalytic               endopeptidase,,               proteasome ATPase,               preoteolysis and               pepitolysis       134-135   CT34842   CG14991 unknown   BAND_41_3, PH_DOMAIN   XP_051693.1| (XM_051693)   635                       mitogen inducible 2 [ Homo                           sapiens ]       136-137   CT34979   CG15104 topoisomerase       NP_055023.1| (NM_014208)   102               I-binding RS protein’       dentin sialophosphoprotein;                       dentin phosphophoryn;       138-139   CT3955   CG1530 unknown   PRO_RICH   XP_092523.1| (XM_092523)   230                       hypothetical protein XP_092523                       [ Homo sapiens ]       140-141   CT35308   CG15321 unknown       none       142-143   CT35676   CG15560 putative cell       NP_499205.1| (NM_066804)   170               membrane-associated       Transmembrane and sushi               mucin       domain [ Caenorhabditis elegans ]       144-145   CT30180   CG15811 Rop rop, ‘Ras   Sec1   NP_037170.1| (NM_013038)   756               opposite       syntaxin binding protein 1                       [ Rattus norvegicus ]       146-147   CT34113   CG15896 unknown       NP_055487.1| (NM_014672)   182                       KIAA0391 gene product [ Homo                           sapiens ]       148-149   CT34115   CG15898 unknown       NP_078828.1| (NM_024552)   47.8                       hypothetical protein FLJ12089                       [ Homo sapiens ]       150-151   CT4708   CG1683 Ant2 Ant2,   ADPTRNSLCASE,   (AF218587) ADP/ATP   485               ADP/ATP translocase.   MITOCARRIER,   translocase [ Lucilia cuprina ]               Adenine nucleotide   MITOCH_CARRIER, mito_carr               translocase 2, ATP/ADP               antiporter       152-153   CT37506   CG16903 EG: 67A9.2       NP_446114.1| (NM_053662)   411               non-specific RNA       cyclin L [ Rattus norvegicus ]               polymerase II               transcription factor       154-155   CT35131   CG16916 Rpt3 p48A, 26S   AAA, CLPPROTEASEA   PRS6_MANSE 26S   681               proteasome regulatory       PROTEASE REGULATORY               complex subunit p48A       SUBUNIT 6B (ATPASE MS73)       156-157   CT4802   CG1696 unknown       NP_056158.1| (NM_015343)   341                       hypothetical protein [ Homo                           sapiens ]       158-159   CT43084   CG1697 rho-4 rho-4 Rho-         Rattus norvegicus  ‘rhomboid-               related [10C6] rhomboid-4       related protein’                       EMBL: Y17258       160-161   CT4810   CG1698 unknown       none       162-163   CT4826   CG1703 ATP-binding   ABC_TRANSPORTER,   (AF293383) ABC50 [ Rattus     802               cassette (ABC) transporter   ABC_tran, ATP_GTP_A,     norvegicus ]                   ATP_GTP_A2, DA_BOX,                   NLS_BP       164-165   CT35402   CG17252 BCL7-like       (NM_001707) B-cell   94.4               BCL7-like       CLL/lymphoma 7B [ Homo                           sapiens ]       166-167   CT21145   CG17309 CSK CSK,   PROTEIN_KINASE_ATP,   AAH18394 (BC018394) c-src   462               involved in protein   PROTEIN_KINASE_DOM,   tyrosine kinase [ Mus musculus ]               phosphorylation   PROTEIN_KINASE_TYR,                   SH2, SH2DOMAIN,                   TYRKINASE, pkinase       168-169   CT5050   CG1740 Ntf-2 NTF-2,   NTF2_DOMAIN   (NM_059921) nuclear transport   127               protein carrier involved in       factor 2 like [ Caenorhabditis                 protein-nucleus import       170-171   CT5086   CG1746 anon-   ATP-synt_C, ATPASEC,   Q9U505|ATPC_MANSE ATP   177               EST: Posey224 hydrogen-   ATPASE_C   synthase subunit C,               transporting ATP       mitochondrial precursor (Lipid-               synthase/enzyme,       binding               hydrogen-transporting               two-sector ATPase       172-173   CT34491   CG17734 unknown       NP_062788.1| (NM_019814)   82.4                       hypoxia induced gene 1 [ Mus                           musculus ]       174-175   CT39345   CG17766 EG: 86E4.3   WD40, WD40_REGION   AF188123_1 (AF188123) TGF-   1160               heterotrimeric G-protein       beta resistance-associated               GTPase       protein TRAG [ Mus musculus ]       176-177   CT39414   CG17791 sqd   RBD, rrm; Eukaryotic putative     Homo sapiens  ‘heterogeneous               heterogeneous-nuclear-   RNA-binding region RNP-1   nuclear ribonucleoprotein D’               ribonucleoprotein-87Fb   signature, RRM-motif protein,   EMBL: AF026126               RNA-binding protein 3,   RRM-motif protein               Squid       178-179   CT39758   CG17871 Or71a tracheal       none               gasfilling mutant1b,               Or71a, odorant receptor       180-181   CT40282   CG18009 Trf2 TATA box       (AB024489) TBP-like protein   210               binding protein-related       [ Gallus gallus ]               factor 2       182-183   CT5456   CG1826 product   BTB, NLS_BP,   (AB067467) KIAA1880 protein   595               involved in developmental   PROTEIN_SPLICING   [ Homo sapiens ]               processes       184-185   CT41472   CG18282 Ubiquitin-like       I45964 polyubiquitin - bovine   431                       (fragment)       186-187   CT42468   CG18578 Ugt86Da UDP-       none               glucuronosyltransferase       188-189   CT13908   CG18734 Fur2 furin       T43251 furin (EC 3.4.21.75) -   1753                       fall armyworm       190-191   CT5890   CG1908 unknown   NLS_BP   none       192-193   CT5932   CG1915 sls sallimus,   AA_TRNA_LIGASE_II_1,     Gallus gallus  ‘connectin/titin’               myosin light chain kinase   ATP_GTP_A, NLS_BP, SH3,   EMBL: D83390                   fn3, ig       194-195   CT6007   CG1937 involved in cell       (AF317634) HRD1 [ Homo     545               growth and maintenance         sapiens ]       196-197   CT5951   CG1938 Dlic2 Dlic2,   ATP_GTP_A   (AF317841) cytoplasmic dynein   399               motor which is a       light-intermediate chain 1               component of the       [ Xenopus                 microtubule associated               protein       198-199   CT6352   CG1994 similar to   ATP_GTP_A   (AB051496) KIAA1709 protein   1013               Achlya ambisexualis       [ Homo sapiens ]               antheridiol steroid               receptor       200-201   CT6373   CG2003 high affinity   transporter     Homo sapiens  ‘Na/PO4               inorganic       cotransporter’ gi: 4885441               phosphate:sodium               symporter       202-203   CT4336   CG2151 Trxr-1 NOT   FADPNR, HGRDTASE,   (U88187) glutathione reductase   753               glutathione reductase   NAD_BINDING,   family member [ Musca                 (NADPH) (EC: 1.6.4.2)   PNDRDTASEI,     domestica ]               involved in thioredoxin   PYRIDINE_REDOX_1,               reduction   pyr_redox       203-205   CT6738   CG2165 BEST: CK01140       (NM_053311) ATPase, Ca++   1262               calcium-transporting       transporting, plasma membrane               ATPase-like       1 [ Rattus         206-207   CT5965   CG2184 Mlc2 muscle-   EF_HAND, EF_HAND_2,   MLR5_FELCA Superfast   130               specific myosin regulatory   efhand   myosin regulatory light chain 2               light chain Mlc2, involved       (MYLC2)               in cell motility       208-209   CT7322   CG2222 unknown       none       210-211   CT7705   CG2309 ERK7 protein       YPC2_CAEEL Putative   392               kinase, protein       serine/threonine-protein kinase               serine/threonine kinase       C05D10.2 in chromosome III       212-213   CT8341   CG2520 lap lap,   ENTH   (AF182339) clathrin assembly   502               chaperone       protein AP180 [ Loligo pealei ]       214-215   CT9021   CG2666 CS-1 CS-1,       (AF221067) chitin synthase 1   2770               enzyme/chitin synthase       [ Lucilia cuprina ]       216-217   CT9593   CG2829   NLS_BP, PFKB_KINASES_1,   (AB004884) PKU-alpha [ Homo     520               BcDNA: GH07910 protein   PROTEIN_KINASE_ATP,     sapiens ]               kinase, protein   PROTEIN_KINASE_DOM,               serine/threonine kinase   PROTEIN_KINASE_ST,                   PRO_RICH, pkinase       218-219   CT9754   CG2849 Rala Ral, RAS   ATP_GTP_A, PRENYLATION,   (XM_035787) similar to Ras-   304               small monomeric GTPase,   RASTRNSFRMNG, ras   related protein RAL-A [ Homo                 regulates developmental         sapiens ]               cell shape changes               through the JNK pathway       220-221   CT9660   CG2829   NLS_BP, PFKB_KINASES_1,   (AB004884) PKU-alpha [ Homo     520               BcDNA: GH07910 protein   PROTEIN_KINASE_ATP,     sapiens ]               kinase, protein   PROTEIN_KINASE_DOM,               serine/threonine kinase   PROTEIN_KINASE_ST,                   PRO_RICH, pkinase       222-223   CT6171   CG2968 hydrogen-       P35434|ATPD_RAT ATP   142               transporting ATP       synthase delta chain,               synthase, coupling factor       mitochondrial precursor               CF(0), delta-chain       224-225   CT10206   CG3034 EG: BACR7A4.6       (Y15172) surfeit protein 5   183               similar to Surf5b [ Homo         [ Takifugu rubripes ]                 sapiens         226-227   CT41361   CG3071 EG: 25E8.3   Trp-Asp (WD) repeats signature   T40471 probable Trp-Asp repeat   273               involved in retrograde   protein   protein - fission yeast               (Golgi to ER) transport               which is putatively a               component of the               coatomer       228-229   CT9947   CG3071 EG: 25E8.3   Trp-Asp (WD) repeats signature   T40471 probable Trp-Asp repeat   273               involved in retrograde   protein   protein - fission yeast               (Golgi to ER) transport               which is putatively a               component of the               coatomer       230-231   CT10723   CG3201 Mlc-c Mlc-c,   EF_HAND, EF_HAND_2,     Homo sapiens  ‘MYOSIN               alkali light chain of non-   efhand   LIGHT CHAIN ALKALI,               muscle myosin-II,       SMOOTH-MUSCLE               cytoskeleton organization       ISOFORM (MLC3SM)               and biogenesis       (LC17B) (LC’ SWP: P24572       232-233   CT11063   CG3313 transcription   NLS_BP, WD40,   (AB067479) KIAA1892 protein   293               factor   WD40_REGION   [ Homo sapiens ]       234-235   CT11487   CG3415 estradiol 17   ADH_SHORT, GDHRDH,   (NM_000414) hydroxysteroid   613               beta-dehydrogenase   THIOL_PROTEASE_HIS,   (17-beta) dehydrogenase 4                   adh_short   [ Homo sapiens ]       236-237   CT11597   CG3446 unknown       (AJ316011) mitochondrial   78.6                       NADH: ubiquinone                       oxidoreductase B16.6       238-239   CT11623   CG3455 Rpt4 Rpt4,         Manduca sex  ‘26S proteasome               endopeptidase,       regulatory ATPase subunit 10b               multicatalytic       (S10b)’ EMBL: AJ223384               endopeptidase regulator,               multicatalytic               endopeptidase,               proteasome ATPase       240-241   CT11966   CG3560 anon-       1BCC|F Chain F, Cytochrome   150               EST: Posey167 NADH       Bc1 Complex From Chicken               dehydrogenase       242-243   CT12417   CG3703       (NM_075735) T19D7.4.p   251               EG: BACR7A4.15       [ Caenorhabditis elegans ]               cytoskeleton organization               and biogenesis       244-245   CT12443   CG3715 Shc dShc, SHC-       S25776 transforming protein   267               adaptor protein, protein       (SHC) - human               kinase putatively involved               in cell growth and               maintenance       246-247   CT12517   CG3747 Eaat1 Eaat1,   plasma membrane   (AF330257) glutamate   402               glutamate transporter,       transporter [ Mus musculus ]               Excitatory amino acid               transporter 1       248-249   CT12871   CG3861 citrate (SI)-   CITRATE_SYNTHASE,   P00889|CISY_PIG CITRATE   674               synthase   CITRTSNTHASE, citrate_synt   SYNTHASE,                       MITOCHONDRIAL                       PRECURSOR       250-251   CT12909   CG3874 nucleotide-sugar       (NM_015139) UDP-glucuronic   361               transporter-like       acid/UDP-N-                       acetylgalactosamine dual       252-253   CT13223   CG3981 Unc-76 Dunc-       (NM_005102) zygin 2;   197               76, signal transducer       fasciculation and elongation               involved in axon cargo       protein zeta 2;               transport       254-255   CT4722   CG4013 Smr Smrter   ANTIFREEZEI, myb_DNA-   NCR2_MOUSE NUCLEAR   275               SMRT-related ecdysone   binding   RECEPTOR CO-REPRESSOR               receptor-interacting factor       2 (N-COR2) (SILENCING               SANT domain protein,       MEDIATOR OF               transcription corepressor       256-257   CT13458   CG4094 fumarate   DCRYSTALLIN,   (NM_017005) fumarate   512               hydratase, enzyme   FUMARATE_LYASES,   hydratase [ Rattus norvegicus ]               involved in main   FUMRATELYASE, lyase_1               pathways of carbohydrate               metabolism       258-259   CT13690   CG4129       (XM_043094) KIAA0061   325               BcDNA: LD21623       protein [ Homo sapiens ]               unknown       260-261   CT5938   CG4147 Hsc70-3 Hsc70-   ER_TARGET,   (AB016836) heat shock 70 kD   1159               3, Heat shock protein   HEATSHOCK70, HSP70,   protein cognate [ Bombyx mori ]               cognate 3, involved in   HSP70_1, HSP70_2, HSP70_3               stress response       262-263   CT13852   CG4202 Sas10 Sas10       (NM_023054) disrupter of   259                       silencing SAS10 [ Mus                            musculus ]       264-265   CT14019   CG4300 spermidine   SAM_BIND   (AJ009865) spermine synthase   276               synthase       [ Takifugu rubripes ]       266-267   CT14119   CG4300 spermidine   SAM_BIND   (AJ009865) spermine synthase   276               synthase       [ Takifugu rubripes ]       268-269   CT13914   CG4317 Mipp2 Mipp2,   CYTOCHROME_B_QO     Mus musculus  ‘multiple inositol               multiple inositol-       polyphosphate phosphatase’               polyphosphate       EMBL: AF046908               phosphatase 2       270-271   CT14464   CG4453 transporter, an   ZF_RANBP, zf-RanBP   14578 nucleoporin Nup153   300               endopeptidase involved in       homolog - African clawed frog               behavior which is a       (fragment)               component of the nucleus       272-273   CT14586   CG4481 Glu-RIB ion   ANF_receptor,     Mus musculus  ‘glutamate               channel-alpha-amino-3-   CHANNEL_PORE_K,   receptor channel a3 subunit’               hydroxy-5-methyl-4-   NLS_BP, SBP_GLUR, lig_chan   EMBL: AB022342               isoxazole propionate               selective glutamate               receptor; ionotropic               glutamate receptor       274-275   CT14874   CG4590 inx2 inx2,   Innexin     Schistocerca americana                 neurotransmitter       ‘innexin-2’ EMBL: 115854_1               transporter, Dm-inx pas               related protein 33       276-277   CT15952   CG4974 dally NOT cell   Glypican   (NM_004466) glypican 5   186               adhesion molecule;       [ Homo sapiens ]               heparin sulfate               proteoglycan; Dally       278-279   CT16489   CG5147 unknown       none       280-281   CT16663   CG5208       none               BcDNA: LD27979               unknown       282-283   CT17394   CG5485 high affinity       (AF349043) sulfate anion   340               sulfate permease, sulfate       transporter-1 [ Mus musculus ]               transporter       284-285   CT17382   CG5486 Ubp64E       (NM_063285) ubiquitin   358               Ubiquitin-specific       carboxyl-terminal hydrolase               protease 64E       [ Caenorhabditis         286-287   CT17448   CG5505 endopeptidase,   UCH-1, UCH-2, UCH_2_1,   (XM_027039) KIAA1453   254               ubiquitin-specific   UCH_2_2, UCH_2_3   protein [ Homo sapiens ]               protease, involved in               process of               deubiquitylation       288-289   CT17938   CG5684 non-specific       Q9UIV1|CNO7_HUMAN   376               RNA polymerase II       CCR4-NOT transcription               transcription factor       complex, subunit 7 (CCR4-                       associated factor       290-291   CT17971   CG5722 NPC1 dmNPC1,   5TM_BOX, NLS_BP   (NM_000271) Niemann-Pick   1061               transmembrane receptor       disease, type C1 [ Homo sapiens ]       292-293   CT18192   CG5797 cytoskeletal   PRO_RICH   (AB051482) KIAA1695 protein   541               binding protein       [ Homo sapiens ]       294-295   CT18619   CG5939 Prm Para,   NLS_BP   (AF317670) paramyosin   989               Paramyosin, structural       [ Sarcoptes scabiei ]               protein of muscle, motor       296-297   CT18969   CG6058 Ald fructose-   ALDOLASE_CLASS_I,     Mus musculus  Aldo1               bisphosphate aldolase,   NLS_BP, glycolytic_enzy   MGI: 87994               involved in process of               glycolysis       298-299   CT19788   CG6335 histidine--tRNA   AA_TRNA_LIGASE_II_1,   (NM_008214) histidyl tRNA   641               ligase   AA_TRNA_LIGASE_II_2,   synthetase [ Mus musculus ]                   WHEP-TRS, tRNA-synt_2b       300-301   CT19850   CG6367 serine-type       (AF053921) trypsin-like serine   163               endopeptidase       protease [ Ctenocephalides felis ]       302-303   CT19962   CG6400 unknown   BROMODOMAIN,   Q9NSI6|WDR9_HUMAN WD-   916                   BROMODOMAIN_2,   REPEAT PROTEIN 9                   GPROTEINBRPT, NLS_BP,                   WD40, WD40_REGION,                   WD_REPEATS, bromodomain       304-305   CT20122   CG6470 unknown   ZINC_FINGER_C2H2,   none                   ZINC_FINGER_C2H2_2, zf-                   C2H2       306-307   CT20269   CG6513 signal       (NM_019561) endosulfine   91.3               transduction       alpha; alpha-endosulfine [ Mus                            musculus ]       308-309   CT21021   CG6774 tracheal       (NM_023037) hypothetical   1006               gasfilling mutant       protein CG003 [ Homo sapiens ]       310-311   CT21292   CG6874 unknown       none       312-313   CT43217   CG6928 Sulfate   Sulfate_transp               transporter       314-315   CT21476   CG6930 unknown   NLS_BP,     Caenorhabditis elegans  ‘contains                   ZINC_FINGER_C2H2,   strong similarity to a C2H2-type                   ZINC_FINGER_C2H2_2, zf-   zinc finger’ EMBL: AF000194                   C2H2       316-317   CT21525   CG6946 RNA binding   RBD, rrm     Rattus norvegicus                         ‘ribonucleoprotein F’                       EMBL: AB022209       318-319   CT21704   CG7014 structural   RIBOSOMAL_S7,   (NM_001009) ribosomal protein   347               protein of ribosome,   Ribosomal_S7   S5; 40S ribosomal protein S5               Process protein       [ Homo                 biosynthesis       320-321   CT22195   CG7187 DNA binding       (AY026310) single stranded   351                       DNA binding protein-1 [ Homo                           sapiens ]       322-323   CT22253   CG7215 ubiquitin   UBIQUITIN_2, ubiquitin   P21126|UBLG_MOUSE   75.5                       Ubiquitin-like protein GDX                       (Ubiquitin-like protein 4)       324-325   CT22861   CG7434 RpL22 ribosomal   ANTIFREEZEI   (AF400188) ribosomal protein   165               protein L22       L22 [ Spodoptera frugiperda ]       326-327   CT23083   CG7552 unknown   ATP_GTP_A,     Homo sapiens  ‘65 KD YES-                   WW_DOMAIN_1,   ASSOCIATED PROTEIN                   WW_DOMAIN_2,   (YAP65)’ SWP: P46937                   WW_rsp5_WWP       328-329   CT23596   CG7757 similarity to   NLS_BP   (NM_004698) U4/U6-associated   520               U4/U6-associated RNA       RNA splicing factor [ Homo                 splicing factor         sapiens ]       330-331   CT23626   CG7770 cochaperonin in       (NM_010385) H2-K region   106               process of ‘de novo’       expressed gene 2 [ Mus                 protein folding         musculus ]       332-333   CT23882   CG7901 PP2A-B′ protein   ANTIFREEZEI     Mus musculus  ‘protein               phosphatase, protein       phosphatase 2A B′a3 regulatory               phosphatase type 2A       subunit’ EMBL: U37353               regulator       334-335   CT41698   CG7958 unknown       (AB033050) KIAA1224 protein   427                       [ Homo sapiens ]       336-337   CT23982   CG7958 unknown       (AB033050) KIAA1224 protein   427                       [ Homo sapiens ]       338-339   CT23998   CG7983 guanylate kinase   PRO_RICH   (AF411837) transcription   214                       repressor p66 [ Mus musculus ]       340-341   CT24094   CG8031 unknown       (BC013819) CGI-27 protein   394                       [ Mus musculus ]       342-343   CT24122   CG8037 ELL, DNA-         Gallus gallus  ‘OCCLUDIN’               directed RNA polymerase       SWP: Q91049               III;       344-345   CT24346   CG8148 timeout timeout       (NM_003920) timeless   149                       ( Drosophila ) homolog [ Homo                           sapiens ]       346-347   CT24393   CG8189 ATPsyn-b   Acetyltransf   (AF187862) ATP synthase   213               ATPsyn-b Fo-ATP       subunit B [ Xenopus laevis ]               synthase subunit b       348-349   CT24437   CG8231 T-complex   CHAPERONIN60,   O77622|TCPZ_RABIT T-   754               protein 1, zeta-subunit,   TCOMPLEXTCP1, TCP1_1,   COMPLEX PROTEIN 1, ZETA               chaperone   TCP1_2, TCP1_3, cpn60_TCP1   SUBUNIT (TCP-1-ZETA)                       (CCT-ZETA)       350-351   CT18257   CG8322 ATPCL ATP-   SUCCINYL_COA_LIG_1,   (U18197) ATP: citrate lyase   1555               citrate (pro-S)-lyase   SUCCINYL_COA_LIG_2,   [ Homo sapiens ]                   SUCCINYL_COA_LIG_3,                   ligase-CoA       352-353   CT24731   CG8439 Cct5 Cct5, T-       (XM_052313) chaperonin   791               complex Chaperonin 5,       containing TCP1, subunit 5               tracheal gasfilling mutant       (epsilon) [ Homo         354-355   CT24823   CG8484 Transcription   ZINC_FINGER_C2H2,   (NM_058230) zinc finger   167               factor   ZINC_FINGER_C2H2_2, zf-   protein 354B [ Homo sapiens ]                   C2H2       356-357   CT25072   CG8655 CDC receptor   AA_TRNA_LIGASE_II_2,   (AF005209) HsCdc7 [ Homo     216               signaling protein   PROTEIN_KINASE_DOM,     sapiens ]               serine/threonine kinase   PROTEIN_KINASE_ST,                   pkinase       358-359   CT25274   CG8759 Nacalpha; NAC         Homo sapiens  &amp; agr               protein alpha subunit,       PIR: S49326               component of the nascent               polypeptide-associated               complex       360-361   CT25472   CG8870 endopeptidase,   ANTENNAPEDIA,     Caenorhabditis elegans  ‘similar               monophenol   CHYMOTRYPSIN,   to plasminogen and to trypsin-               monooxygenase activator   TRYPSIN_CATAL,   like serine proteases’                   TRYPSIN_HIS,   EMBL: U29380                   TRYPSIN_SER, trypsin       362-363   CT25624   CG8922 RpS5 Ribosomal   RIBOSOMAL_S7,   (Y12431) 5S ribosomal protein   353               protein S5   Ribosomal_S7   [ Mus musculus ]       364-365   CT8969   CG9165 enzyme,   PORPHBDMNASE,   P08397|HEM3_HUMAN   287               hydroxymethylbilane   Porphobil_deam   PORPHOBILINOGEN               synthase       DEAMINASE                       (HYDROXYMETHYLBILANE                       SYNTHASE) (HMBS)       366-367   CT27084   CG9591 unknown       (XM_043261) KIAA1698   116                       protein [ Homo sapiens ]       368-369   CT27543   CG9748 cap Belle, ATP       1705301A ATP dependent   723               dependent helicase       RNA helicase [ Xenopus laevis ]       370-371   CT27750   CG9821 unknown       none       372-373   CT27796   CG9901 Arp14D Actin-   ACTIN, ACTINS_ACT_LIKE,   P53488|ARP2_CHICK ACTIN-   678               related protein 14D, arp2   actin   LIKE PROTEIN 2 (ACTIN-                       LIKE PROTEIN ACTL)       374-375   CT27906   CG9910 katanin-80       (AF052433) katanin p80 subunit   231               katanin 80, microtubule       [ Strongylocentrotus purpuratus ]               severing which is a               component of the katanin       376-377   CT27940   CG9924 transcription   BTB, MATH   (NM_003563) speckle-type POZ   599               factor       protein [ Homo sapiens ]       378-379   CT27993   CG9946 eIF-2alpha;   NLS_BP, S1   (NM_131800) eIF2 alpha   376               Eukaryotic initiation       subunit [ Danio rerio ]               factor 2A; translation               initiation factor       380-381   CT20536   CG6606 unknown   ATPASE_ALPHA_BETA,   (AB020664) KIAA0857 protein   122                   ATP_GTP_A, C2, NLS_BP,   [ Homo sapiens ]                   RECEPTOR_CYTOKINES_2                  
 
       DEFINITIONS  
       [0021]     For clarity, certain terms used in the specification are defined and used as follows:  
         [0022]     “Associated with/operatively linked” refer to two nucleic acid sequences that are related physically or functionally. For example, a promoter or regulatory DNA sequence is said to be “associated with” a DNA sequence that codes for an RNA or a protein if the two sequences are operatively linked, or situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence.  
         [0023]     A “chimeric construct” is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operatively linked to, or associated with, a nucleic acid sequence that codes for an mRNA or which is expressed as a protein, such that the regulatory nucleic acid sequence is able to regulate transcription or expression of the associated nucleic acid sequence. The regulatory nucleic acid sequence of the chimeric construct is not normally operatively linked to the associated nucleic acid sequence as found in nature.  
         [0024]     Co-factor: natural reactant, such as an organic molecule or a metal ion, required in an enzyme-catalyzed reaction. A co-factor is e.g. NAD(P), riboflavin (including FAD and FMN), folate, molybdopterin, thiamin, biotin, lipoic acid, pantothenic acid and coenzyme A, S-adenosylmethionine, pyridoxal phosphate, ubiquinone, menaquinone. Optionally, a co-factor can be regenerated and reused.  
         [0025]     A “coding sequence” is a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably the RNA is then translated in an organism to produce a protein.  
         [0026]     Complementary: “complementary” refers to two nucleotide sequences that comprise antiparallel nucleotide sequences capable of pairing with one another upon formation of hydrogen bonds between the complementary base residues in the antiparallel nucleotide sequences.  
         [0027]     “Conservatively modified variations” of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences, or where the nucleic acid sequence does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are “silent variations” which are one species of “conservatively modified variations.” Every nucleic acid sequence described herein which encodes a protein also describes every possible silent variation, except where otherwise noted. One of skill will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each “silent variation” of a nucleic acid which encodes a protein is implicit in each described sequence.  
         [0028]     Furthermore, one of skill will recognize that individual substitutions deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are “conservatively modified variations,” where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following five groups each contain amino acids that are conservative substitutions for one another: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q). See also, Creighton (1984)  Proteins , W. H. Freeman and Company. In addition, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are also “conservatively modified variations.” 
         [0029]     DNA Shuffling: DNA shuffling is a method to rapidly, easily and efficiently introduce mutations or rearrangements, preferably randomly, in a DNA molecule or to generate exchanges of DNA sequences between two or more DNA molecules, preferably randomly. The DNA molecule resulting from DNA shuffling is a shuffled DNA molecule that is a non-naturally occurring DNA molecule derived from at least one template DNA molecule. The shuffled DNA encodes an enzyme modified with respect to the enzyme encoded by the template DNA, and preferably has an altered biological activity with respect to the enzyme encoded by the template DNA.  
         [0030]     Enzyme/Protein Activity: means herein the ability of an enzyme (or protein) to catalyze the conversion of a substrate into a product. A substrate for the enzyme comprises the natural substrate of the enzyme but also comprises analogues of the natural substrate, which can also be converted, by the enzyme into a product or into an analogue of a product. The activity of the enzyme is measured for example by determining the amount of product in the reaction after a certain period of time, or by determining the amount of substrate remaining in the reaction mixture after a certain period of time. The activity of the enzyme is also measured by determining the amount of an unused co-factor of the reaction remaining in the reaction mixture after a certain period of time or by determining the amount of used co-factor in the reaction mixture after a certain period of time. The activity of the enzyme is also measured by determining the amount of a donor of free energy or energy-rich molecule (e.g. ATP, phosphoenolpyruvate, acetyl phosphate or phosphocreatine) remaining in the reaction mixture after a certain period of time or by determining the amount of a used donor of free energy or energy-rich molecule (e.g. ADP, pyruvate, acetate or creatine) in the reaction mixture after a certain period of time.  
         [0031]     Essential: an “essential”  Drosophila melanogaster  nucleotide sequence is a nucleotide sequence encoding a protein such as e.g. a biosynthetic enzyme, receptor, signal transduction protein, structural gene product, or transport protein that is essential to the growth or survival of the insect.  
         [0032]     Expression Cassette: “Expression cassette” as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operatively linked to the nucleotide sequence of interest which is operatively linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular DNA sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, such as an insect, the promoter can also be specific to a particular tissue or organ or stage of development.  
         [0033]     Gene: the term “gene” is used broadly to refer to any segment of DNA associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.  
         [0034]     Heterologous/exogenous: The terms “heterologous” and “exogenous” when used herein to refer to a nucleic acid sequence (e.g. a DNA sequence) or a gene, refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.  
         [0035]     A “homologous” nucleic acid (e.g. DNA) sequence is a nucleic acid (e.g. DNA) sequence naturally associated with a host cell into which it is introduced.  
         [0036]     The terms “identical” or percent “identity” in the context of two or more nucleic acid or protein sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.  
         [0037]     Inhibitor: a chemical substance that inactivates the enzymatic activity of an enzyme (or protein) of interest The term “insecticide” is used herein to define an inhibitor when applied to an insect at any stage of development  
         [0038]     Insecticide: a chemical substance used to kill or inhibit the growth or viability of insects at any stage of development.  
         [0039]     Interaction: quality or state of mutual action such that the effectiveness or toxicity of one protein or compound on another protein is inhibitory (antagonists) or enhancing (agonists).  
         [0040]     A nucleic acid sequence is “isocoding with” a reference nucleic acid sequence when the nucleic acid sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence.  
         [0041]     An “isolated” nucleic acid molecule or an isolated enzyme is a nucleic acid molecule or enzyme that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule or enzyme may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell.  
         [0042]     Mature Protein: protein that is normally targeted to a cellular organelle and from which the transit peptide has been removed.  
         [0043]     Minimal Promoter: promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation. In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription.  
         [0044]     Modified Enzyme Activity: enzyme activity different from that which naturally occurs in an insect (i.e. enzyme activity that occurs naturally in the absence of direct or indirect manipulation of such activity by man), which is tolerant to inhibitors that inhibit the naturally occurring enzyme activity.  
         [0045]     Native: refers to a gene that is present in the genome of an untransformed insect cell.  
         [0046]     Naturally occurring: the term “naturally occurring” is used to describe an object that can be found in nature as distinct from being artificially produced by man. For example, a protein or nucleotide sequence present in an organism (including a virus), which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally occurring.  
         [0047]     Nucleic acid: the term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al.,  Nucleic Acid Res.  19: 5081 (1991); Ohtsuka et al.,  J. Biol. Chem.  260: 2605-2608 (1985); Rossolini et al.,  Mol. Cell Probes  8: 91-98 (1994)). The terms “nucleic acid” or “nucleic acid sequence” may also be used interchangeably with gene, cDNA, and mRNA encoded by a gene.  
         [0048]     “ORF” means open reading frame.  
         [0049]     Purified: the term “purified,” when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein which is the predominant species present in a preparation is substantially purified. The term “purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least about 50% pure, more preferably at least about 85% pure, and most preferably at least about 99% pure.  
         [0050]     Two nucleic acids are “recombined” when sequences from each of the two nucleic acids are combined in a progeny nucleic acid. Two sequences are “directly” recombined when both of the nucleic acids are substrates for recombination. Two sequences are “indirectly recombined” when the sequences are recombined using an intermediate such as a cross-over oligonucleotide. For indirect recombination, no more than one of the sequences is an actual substrate for recombination, and in some cases, neither sequence is a substrate for recombination.  
         [0051]     “Regulatory elements” refer to sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements comprise a promoter operatively linked to the nucleotide sequence of interest and termination signals. They also typically encompass sequences required for proper translation of the nucleotide sequence.  
         [0052]     Significant Increase: an increase in enzymatic activity that is larger than the margin of error inherent in the measurement technique, preferably an increase by about 2-fold or greater of the activity of the wild-type enzyme in the presence of the inhibitor, more preferably an increase by about 5-fold or greater, and most preferably an increase by about 10-fold or greater.  
         [0053]     Substantially identical: the phrase “substantially identical,” in the context of two nucleic acid or protein sequences, refers to two or more sequences or subsequences that have at least 60%, preferably 80%, more preferably 90, even more preferably 95%, and most preferably at least 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues. In an especially preferred embodiment, the sequences are substantially identical over the entire length of the coding regions. Furthermore, substantially identical nucleic acid or protein sequences perform substantially the same function.  
         [0054]     For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.  
         [0055]     Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith &amp; Waterman,  Adv. Appl. Math.  2: 482 (1981), by the homology alignment algorithm of Needleman &amp; Wunsch,  J. Mol. Biol.  48: 443 (1970), by the search for similarity method of Pearson &amp; Lipman,  Proc. Nat&#39;l. Acad. Sci. USA  85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis,), or by visual inspection (see generally, Ausubel et al., infra).  
         [0056]     One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al.,  J. Mol. Biol.  215: 403410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information on the world wide web at ncbi.nlm.nih.gov/. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always &gt;0) and N (penalty score for mismatching residues; always &lt;0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff &amp; Henikoff,  Proc. Natl. Acad. Sci. USA  89:10915 (1989)).  
         [0057]     In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin &amp; Altschul,  Proc. Nat&#39;l. Acad. Sci. USA  90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.  
         [0058]     Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase “hybridizing specifically to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. “Bind(s) substantially” refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.  
         [0059]     “Stringent hybridization conditions” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993)  Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic  Acid Probes part I chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, N.Y. Generally, highly stringent hybridization and wash conditions are selected to be about 5° C. lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. Typically, under “stringent conditions” a probe will hybridize to its target subsequence, but to no other sequences.  
         [0060]     The T m  is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T m  for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42° C., with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.1 5M NaCl at 72° C. for about 15 minutes. An example of stringent wash conditions is a 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30° C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2× (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.  
         [0061]     The following are examples of sets of hybridization/wash conditions that may be used to clone homologous nucleotide sequences that are substantially identical to reference nucleotide sequences of the present invention: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C., more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C.  
         [0062]     A further indication that two nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross reactive with, or specifically binds to, the protein encoded by the second nucleic acid. Thus, a protein is typically substantially identical to a second protein, for example, where the two proteins differ only by conservative substitutions.  
         [0063]     The phrase “specifically (or selectively) binds to an antibody,” or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised to the protein with the amino acid sequence encoded by any of the nucleic acid sequences of the invention can be selected to obtain antibodies specifically immunoreactive with that protein and not with other proteins except for polymorphic variants. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988)  Antibodies, A Laboratory Manual , Cold Spring Harbor Publications, New York “Harlow and Lane”), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.  
         [0064]     A “subsequence” refers to a sequence of nucleic acids or amino acids that comprise a part of a longer sequence of nucleic acids or amino acids (e.g., protein) respectively.  
         [0065]     “Synthetic” refers to a nucleotide sequence comprising structural characters that are not present in the natural sequence. For example, an artificial sequence that resembles more closely the G+C content and the normal codon distribution of dicot and/or monocot genes is said to be synthetic.  
         [0066]     Substrate: a substrate is the molecule that an enzyme naturally recognizes and converts to a product in the biochemical pathway in which the enzyme naturally carries out its function, or is a modified version of the molecule, which is also recognized by the enzyme and is converted by the enzyme to a product in an enzymatic reaction similar to the naturally-occurring reaction.  
         [0067]     Target gene: A “target gene” is any gene in an insect cell. For example, a target gene is a gene of known function or is a gene whose function is unknown, but whose total or partial nucleotide sequence is known. Alternatively, the function of a target gene and its nucleotide sequence are both unknown. A target gene is a native gene of the insect cell or is a heterologous gene that had previously been introduced into the insect cell or a parent cell of said insect cell, for example by genetic transformation. A heterologous target gene is stably integrated in the genome of the insect cell or is present in the insect cell as an extrachromosomal molecule, e.g. as an autonomously replicating extrachromosomal molecule.  
         [0068]     Transformation: a process for introducing heterologous DNA into a cell, tissue, or insect Transformed cells, tissues, or insects are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.  
         [0069]     “Transformed,” “transgenic,” and “recombinant” refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A “non-transformed,” “non-transgenic,” or “non-recombinant” host refers to a wild-type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.  
         [0070]     Viability: “viability” as used herein refers to a fitness parameter of an insect. Insects are assayed for their homozygous performance of  Drosophila  development, indicating which proteins are indispensable to maintain life in  Drosophila.   
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0000]     I. Identification Of Essential  Drosophila melanogaster  Nucleotide Sequences Using Transposable Element Insertion Mutagenesis  
         [0071]     As shown in Table 2 and the examples below, the identification of novel nucleotide sequences, as well as the essentiality of the nucleotide sequences for normal insect viability, have been demonstrated in  Drosophila  using P-element transposable insertion mutagenesis. Having established the essentiality of the function of the encoded proteins in Drosophila and having identified the nucleotide sequences encoding these essential proteins, the inventors thereby provide an important and sought-after tool for new insecticide development.  
         [0072]     A lethal phenotype caused by insertion of a P-element indicates that the affected nucleotide sequence codes for an essential protein in the insect. The characterization of the insertion site using flanking sequence DNA is needed to associate an individual lethal line with specific nucleotide sequences. Genomic DNA adjacent to the 5′ and/or 3′ end of the P-element from the insertion line is generated using inverse PCR.  
                                   TABLE 2                           Method of validation of nucleic acid sequences as essential            SEQ ID           NO   validation method                    14   dsRNA and p-element disruption       16   p-element disruption       18   p-element disruption       20   p-element disruption       22   p-element disruption       24   p-element disruption       26   p-element disruption       28   p-element disruption       30   dsRNA       32   p-element disruption       34   p-element disruption       36   p-element disruption       38   p-element disruption       40   p-element disruption       42   dsRNA       44   p-element disruption       46   p-element disruption       48   p-element disruption       50   p-element disruption       52   DsRNA       54   p-element disruption       56   p-element disruption       58   p-element disruption       60   p-element disruption       62   p-element disruption       64   p-element disruption       66   p-element disruption       68   DsRNA       70   DsRNA       72   DsRNA       74   p-element disruption       76   p-element disruption       78   p-element disruption       80   p-element disruption       82   p-element disruption       84   p-element disruption       86   DsRNA       88   p-element disruption       90   p-element disruption       92   p-element disruption       94   p-element disruption       96   p-element disruption       98   p-element disruption       100   p-element disruption       102   p-element disruption       104   p-element disruption       106   dsRNA and p-element disruption       108   p-element disruption       110   DsRNA       112   p-element disruption       114   DsRNA       116   p-element disruption       118   p-element disruption       120   p-element disruption       122   p-element disruption       124   p-element disruption       126   p-element disruption       128   p-element disruption       130   p-element disruption       132   p-element disruption       134   p-element disruption       136   p-element disruption       138   p-element disruption       140   p-element disruption       142   p-element disruption       144   p-element disruption       146   p-element disruption       148   p-element disruption       150   p-element disruption       152   p-element disruption       154   p-element disruption       156   p-element disruption       158   p-element disruption       160   DsRNA       162   p-element disruption       164   p-element disruption       166   p-element disruption       168   p-element disruption       170   p-element disruption       172   p-element disruption       174   p-element disruption       176   p-element disruption       178   p-element disruption       180   p-element disruption       182   p-element disruption       184   p-element disruption       186   p-element disruption       188   p-element disruption       190   p-element disruption       192   p-element disruption       194   DsRNA       196   p-element disruption       198   p-element disruption       200   p-element disruption       202   p-element disruption       204   DsRNA       206   p-element disruption       208   p-element disruption       210   p-element disruption       212   p-element disruption       214   DsRNA       216   p-element disruption       218   p-element disruption       220   p-element disruption       222   DsRNA       224   p-element disruption       226   p-element disruption       227   p-element disruption       228   p-element disruption       230   p-element disruption       232   p-element disruption       234   p-element disruption       236   p-element disruption       238   p-element disruption       240   p-element disruption       242   p-element disruption       244   DsRNA       246   p-element disruption       248   p-element disruption       250   p-element disruption       252   p-element disruption       254   p-element disruption       256   p-element disruption       258   p-element disruption       260   p-element disruption       262   p-element disruption       264   p-element disruption       266   p-element disruption       268   p-element disruption       270   p-element disruption       272   dsRNA and p-element disruption       274   p-element disruption       276   p-element disruption       278   p-element disruption       280   p-element disruption       282   p-element disruption       284   p-element disruption       286   DsRNA       288   DsRNA       290   p-element disruption       292   p-element disruption       294   p-element disruption       296   DsRNA       298   DsRNA       300   p-element disruption       302   p-element disruption       304   p-element disruption       306   p-element disruption       308   p-element disruption       310   p-element disruption       312   p-element disruption       314   p-element disruption       316   p-element disruption       318   p-element disruption       320   p-element disruption       322   p-element disruption       324   p-element disruption       326   p-element disruption       328   p-element disruption       330   p-element disruption       332   p-element disruption       334   p-element disruption       336   p-element disruption       338   p-element disruption       340   p-element disruption       342   dsRNA and p-element disruption       344   DsRNA       346   p-element disruption       348   dsRNA and p-element disruption       350   DsRNA       352   dsRNA and p-element disruption       354   p-element disruption       356   p-element disruption       358   dsRNA and p-element disruption       360   p-element disruption       362   p-element disruption       364   p-element disruption       366   p-element disruption       368   DsRNA       370   p-element disruption       372   p-element disruption       374   p-element disruption       376   p-element disruption       378   p-element disruption       380   p-element disruption                  
 
 I. Determining the Complete Coding Sequences of the Essential  Drosophila  Nucleotide Sequences 
 
         [0073]     The essential  Drosophila  nucleotide sequences are identified by isolating nucleotide sequences flanking the P-element insertion and aligning that sequence with genomic  Drosophila  sequence obtained from the Celera  Drosophila  database. The protein prediction for each genomic region is obtained by use of an exon algorithm program such as GeneMark. All exon algorithm programs currently used for prediction of proteins are susceptible to inaccuracies, including incomplete predictions of coding sequences, missing alternative splice variants, combining of nearby exons of adjacent genes, and mistranslation at intron-exon borders. The prediction of a complete coding sequence can be confirmed by several methods including polymerase chain reaction (PCR) amplification using the 5′ and 3′ sequence to verify the message, reverse transcription PCR (rtPCR) using an oligonucleotide internal sequence to identify the 5′ and/or 3′ end, and screening of cDNA libraries from insect tissues with probes made from a particular sequence to isolate a true full-length clone. To confirm that the message size is accurate, a Northern blot can be hybridized with a probe from the nucleotide sequence. In addition, matches to the  Drosophila  EST database helps to confirm existence of message and gives information about the temporal and spatial pattern of expression. Mutation-causing P elements are known to preferentially cluster in the 5′ region of affected genes (Spradling et al,  Proc. Natl. Acad. Sci. USA  92: 10824-10830 (1995)), a tendency that increases the chance of recovering overlaps between short flanking sequences and 5′ ESTs. The present invention therefore provides a number of essential nucleotide sequences as well as the amino acid sequences encoded thereby. cDNA clone sequences are set forth in even numbered SEQ ID NOs:14-380. The corresponding encoded amino acid sequences are set forth in odd numbered SEQ ID NOs:15-381.  
         [0074]     The isolated gene sequences disclosed herein may be manipulated according to standard genetic engineering techniques to suit any desired purpose. For example, an entire  Drosophila  gene sequence or portions thereof may be used as a probe capable of specifically hybridizing to coding sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include, e.g. sequences that are unique among insect nucleotide sequences for a particular protein of interest and are at least 10 nucleotides in length, preferably at least 20 nucleotides in length, and most preferably at least 50 nucleotides in length. Such probes are used to amplify and analyze related nucleotide sequences from a chosen organism via PCR. This technique is useful to isolate additional insect nucleotide sequences from a desired organism or as a diagnostic assay to determine the presence of particular nucleotide sequences in an organism. This technique also is used to detect the presence of altered nucleotide sequences associated with a particular condition of interest such as insecticide tolerance, poor health, etc.  
         [0075]     Gene-specific hybridization probes also are used to quantify levels of a particular gene mRNA in an insect using standard techniques such as Northern blot analysis. This technique is useful as a diagnostic assay to detect altered levels of gene expression that are associated with particular conditions such as enhanced tolerance to insecticides that target a particular gene.  
         [0076]     I.A. Identification of Essential  Drosophila melannogaster  Nucleotide Sequences Using RNAi  
         [0077]     RNA-mediated interference (RNAi) is a recently discovered method to determine gene function in a number of organisms, wherein double-stranded RNA (dsRNA) directs gene-specific, post-transcriptional silencing. See, e.g., Kuwabara &amp; Olson (2000) Parasitol Today 16(8):347-349; Bass (2000) Cell 101(3):235-238; Hunter (2000) Curr Biol 10(4):R137-140; Bosher &amp; Labouesse (2000) Nat Cell Biol 2(2):E31-36; Sharp (1999) Genes Dev 13(2):139-141. The double-stranded RNA molecule can be synthesized in vitro and then introduced into the organism by injection or other methods. Alternatively, a heritable transgene exhibiting dyad symmetry can provide a transcript that folds as a hairpin structure. Methods for examining gene functions using dsRNAi in  Drosophila  are disclosed in Example 4a and further in Kennerdell &amp; Carthew (2000) Nat Biotech 18(8):896-898; Lam &amp; Thummel (2000) Curr Biol 10(16):957-963; Misquitta &amp; Paterson (1999) Proc Natl Acad Sci USA 96 (4):1451-1456. The present invention describes RNA-mediated interference of sequences listed in Table 2 and Table 6. Double-stranded RNA complementary to each sequence was synthesized in vitro and injected into early  Drosophila  embryos, as described in Example 4a. Development of injected embryos was assessed by scoring: (a) morphological criteria using a light microscope (Campos-Ortega &amp; Hartenstein (1985) The Embryonic Development of  Drosophila melanogaster , Springer-Verlag, Berlin), (b) embryo hatching to become a larvae, (c) puparium formation, and (d) eclosion of the pupae as an adult fly, as indicated in Table 6 herein below. Buffer-injected embryos were injected and monitored in parallel as a control. The percentage of embryos injected with dsRNA that survive to the adult stage is depicted in set forth in Table 6.  
         [0078]     Essential genes were identified as those resulting in a percent viable adults below 38% when disrupted by RNAi. This threshold was determined by comparison to multiple buffer-injected controls.  
         [0000]     II. Recombinant Production of Protein and Uses Thereof  
         [0079]     For recombinant production of a protein of the invention in a host organism, a nucleotide sequence encoding the protein is inserted into an expression cassette designed for the chosen host and introduced into the host where it is recombinantly produced. The choice of the specific regulatory sequences such as promoter, signal sequence, 5′ and 3′ untranslated sequence, and enhancer appropriate for the chosen host is within the level of the skill of the routineer in the art. The resultant molecule, containing the individual elements linking in the proper reading frame, is inserted into a vector capable of being transformed into the host cell. Suitable expression vectors and methods for recombinant production of proteins are well known for host organisms such as  E. coli , yeast, and insect cells (see, e.g., Lucknow and Summers, Bio/Technol. 6:47 (1988)). Additional suitable expression vectors are baculovirus expression vectors, e.g., those derived from the genome of Autographica californica nuclear polyhedrosis virus (AcMNPV). A preferred baculovirus/insect system is PVL1392(3) used to transfect  Spodoptera frugiperda  SF9 cells (ATCC) in the presence of linear Autographica californica baculovirus DNA (Phramingen, San Diego, Calif.). The resulting virus is used to infect HighFive Tricoplusia ni cells (Invitrogen, La Jolla, Calif.).  
         [0080]     Recombinantly produced proteins are isolated and purified using a variety of standard techniques. The actual techniques used vary depending upon the host organism used, whether the protein is designed for secretion, and other such factors. Such techniques are well known to the skilled artisan (see, e.g. chapter 16 of Ausubel, F. et al., “Current Protocols in Molecular Biology”, pub. by John Wiley &amp; Sons, Inc. (1994).  
         [0000]     IV. Assays for Characterizing the Proteins  
         [0081]     Recombinantly produced proteins are useful for a variety of purposes. For example, they can be used in in vitro assays to screen known insecticidal chemicals whose target has not been identified to determine if they inhibit protein activity. Such in vitro assays may also be used as more general screens to identity chemicals that inhibit such protein activity and that are therefore novel insecticide candidates. Recombinantly produced proteins may also be used to elucidate the complex structure of these molecules and to further characterize their association with known inhibitors in order to rationally design new inhibitory insecticides. Alternatively, the recombinant protein can be used to isolate antibodies or peptides that modulate the activity and are useful in transgenic solutions.  
         [0000]     V. In Vivo Inhibitor Assay: Discovery of Small Molecule Ligands that Interact with Proteins of Unknown Function.  
         [0082]     Having identified a protein as a potential insecticide target based on its essentiality for insect viability, a next step is to develop an assay that allows screening large numbers of chemicals to determine which ones interact with the protein. Although it is straightforward to develop assays for proteins of known function, developing assays with proteins of unknown functions can be more difficult.  
         [0083]     To address this issue, novel technologies are used that can detect interactions between a protein and a ligand without knowing the biological function of the protein. A short description of three methods is presented, including fluorescence correlation spectroscopy, surface-enhanced laser desorption/ionization, and biacore technologies. In addition to those descibed here, there are additional methods that are currently being developed that are also amenable to automated, large-scale screening.  
         [0084]     Fluorescence Correlation Spectroscopy (FCS) theory was developed in 1972 but it is only in recent years that the technology to perform FCS became available (Madge et al. (1972)  Phys. Rev. Lett.,  29: 705-708; Maiti et al. (1997)  Proc. Natl. Acad. Sci. USA,  94: 11753-11757). FCS measures the average diffusion rate of a fluorescent molecule within a small sample volume. The sample size can be as low as 10 3  fluorescent molecules and the sample volume as low as the cytoplasm of a single bacterium. The diffusion rate is a function of the mass of the molecule and decreases as the mass increases. FCS can therefore be applied to protein-ligand interaction analysis by measuring the change in mass and therefore in diffusion rate of a molecule upon binding. In a typical experiment, the target to be analyzed is expressed as a recombinant protein with a sequence tag, such as a poly-histidine sequence, inserted at the N- or C-terminus. The expression takes place in  E. coli , yeast or insect cells. The protein is purified by chromatography. For example, the poly-histidine tag can be used to bind the expressed protein to a metal chelate column such as Ni2+ chelated on iminodiacetic acid agarose. The protein is then labeled with a fluorescent tag such as carboxytetramethylrhodamine or BODIPY® (Molecular Probes, Eugene, Oreg.). The protein is then exposed in solution to the potential ligand, and its diffusion rate is determined by FCS using instrumentation available from Carl Zeiss, Inc. (Thornwood, N.Y.). Ligand binding is determined by changes in the diffusion rate of the protein.  
         [0085]     Surface-Enhanced Laser Desorption/Ionization (SELDI) was invented by Hutchens and Yip during the late 1980&#39;s (Hutchens and Yip (1993)  Rapid Commun. Mass Spectrom.  7: 576-580). When coupled to a time-of-flight mass spectrometer (TOF), SELDI provides means to rapidly analyze molecules retained on a chip. It can be applied to ligand-protein interaction analysis by covalently binding the target protein on the chip and analyze by MS the small molecules that bind to this protein (Worrall et al. (1998)  Anal. Biochem.  70: 750-756). In a typical experiment, the target to be analyzed is expressed as described for FCS. The purified protein is then used in the assay without further preparation. It is bound to the SELDI chip either by utilizing the poly-histidine tag or by other interaction such as ion exchange or hydrophobic interaction. The chip thus prepared is then exposed to the potential ligand via, for example, a delivery system able to pipet the ligands in a sequential manner (autosampler). The chip is then submitted to washes of increasing stringency, for example a series of washes with buffer solutions containing an increasing ionic strength. After each wash, the bound material is analyzed by submitting the chip to SELDI-TOF. Ligands that specifically bind the target will be identified by the stringency of the wash needed to elute them.  
         [0086]     Biacore relies on changes in the refractive index at the surface layer upon binding of a ligand to a protein immobilized on the layer. In this system, a collection of small ligands is injected sequentially in a 2-5 microlitre cell with the immobilized protein. Binding is detected by surface plasmon resonance (SPR) by recording laser light refracting from the surface. In general, the refractive index change for a given change of mass concentration at the surface layer is practically the same for all proteins and peptides, allowing a single method to be applicable for any protein (Liedberg et al. (1983)  Sensors Actuators  4: 299-304; Malmquist (1993)  Nature  361: 186-187). In a typical experiment, the target to be analyzed is expressed as described for FCS. The purified protein is then used in the assay without further preparation. It is bound to the Biacore chip either by utilizing the poly-histidine tag or by other interaction such as ion exchange or hydrophobic interaction. The chip thus prepared is then exposed to the potential ligand via the delivery system incorporated in the instruments sold by Biacore (Uppsala, Sweden) to pipet the ligands in a sequential manner (autosampler). The SPR signal on the chip is recorded and changes in the refractive index indicate an interaction between the immobilized target and the ligand. Analysis of the signal kinetics on rate and off rate allows the discrimination between non-specific and specific interaction.  
         [0087]     The compounds that are active in the methods disclosed herein may be used to combat agricultural pests such as aphids, locusts, spider mites, and boll weavils as well as such insect pests which attack stored grains and against immature stages of insects living on plant tissue. The compounds are also useful as a nematodicide for the control of agriculturally important soil nematodes and plant parasites.  
         [0000]     VI. Production of Peptides  
         [0088]     Phage particles displaying diverse peptide libraries permits rapid library construction, affinity selection, amplification and selection of ligands directed against an essential protein (H. B. Lowman,  Annu. Rev. Biophys. Biomol. Struct.  26, 401-424 (1997)). Structural analysis of these selectants can provide new information about ligand-target molecule interactions and then in the process also provide a novel molecule that can enable the development of new insecticides based upon these peptides as leads.  
         [0089]     The invention will be further described by reference to the following detailed examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified.  
       EXAMPLES  
       [0090]     Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, et al.,  Molecular Cloning , eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and by T. J. Silhavy, M. L. Berman, and L. W. Enquist,  Experiments with Gene Fusions , Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al.,  Current Protocols in Molecular Biology , pub. by Greene Publishing Assoc. and Wiley-Interscience (1987). Well known  Drosophila  molecular genetics techniques can be found, for example, in Robert, D. B.,  Drosophila, A Practical Approach  (IRL Press, Washington, D.C., 1986).  
       Example 1  
     Identification of Lethal Lines  
       [0091]     Essential nucleotide sequences are identified through the isolation of lethal mutants defective in development The genetic scheme for mobilization of P-lacW is as performed in Deak et. al,  Genetics  147: 1697-1722 (1997). Additional lethal lines are identified and disclosed in Braun, A., B. Lemaitre, et al.,  Genetics  147: 623-634 (1997); Galloni, M. and B. A. Edgar,  Development  126: 2365-2375 (1999); Gateff, E.,  Int. J. Dev. Biol.  38(4): 565-590 (1994); Mechler, B. M. J. Biosci.,  Bangalore  19(5): 537-556 (1994); Roch, F., F. Serras, et al.,  Mol. Gen. Genet.  257: 103-112 (1998); Russell, M. A., L. Ostafichuk, et al.,  Genome  41: 7-13 (1998); and in Torok, T., G. Tick et al.  Genetics  135: 71-80 (1993), Schaefer et al., Aug. 8, 1999 Personal communication to FlyBase. Furthermore, the BDGP gene disruption project of single P-element insertions reveals lethal lines mutating 25% of vital  Drosophila  genes Spradling, A. C., D. Stern, et al.,  Genetics  153: 135-177 (1999).  
         [0092]     Males carrying the transposase source P(Δ2-3) are crossed en masse to yellow white females homozygous for a P-lacW insertion on the X chromosome. Males carrying the PlacW insertion on the X and Δ2-3 on the third chromosome are collected from this cross. The F0 “jumpstart” males are crossed in groups of 10-15 to 20-25 females of w spl; Sb/TM3, Ser genetype. Male F1 progeny with pigmented eyes indicate that the P-lacW has jumped to an autosome. An average of 10-15 males from each F0 cross lacking Δ2-3 are crossed individually to y w, DTS4/TM3, Sb Ser females, that all third chromosomal insertions result in balanced F2 stocks. Insertions on other autosomes yield white-eyed flies in the F2 generation and are eliminated. The balanced third chromosome insertions are tested for lethality in the next generation by placing four to six pairs of y w; P-lacW/TM3, Sb Ser flies in a vial and examining their progeny for the presence of homozygous P-lacW flies. To analyze the lethal phase, the TM3, Sb Ser balancer is replaced by the TM6C, TB Sb chromosome. In such a genetic background, homozygous mutants can be identified by their wild-type body-length. An average of 10-15 pairs of flies are placed in vials supplemented with yeast paste, and the eggs are collected from each line for 1 day. The development of 50-100 progeny is monitored, and the presence of homozygotes are recorded in all developmental stages. Lethal phase is assigned to a developmental stage in which homozygote animals last appear. Lethal lines are identified and maintained.  
                                                   TABLE 3                           P-element location                    Inverse           seq ID   p-element line   PCR   df cross                    14   l(1)G0335   516M3h-f09   Df(2L)Dwee[wo5]       16   l(3)064301   979H5h-b01   Previously verified       18   l(3)092416   1022H5h-   Previously verified               c03       20   l(1)G0384   449M3h-b09   Df(1)JC70/Dp(1; Y)dx[+]5, y[+]/C(1)M5       22   l(1)G0449   267M3h-d07   Df(1)JC70/Dp(1; Y)dx[+]5, y[+]/C(1)M5       24   l(3)s126215   1082H5h-   GN50(63E; 64B)               f05       26   l(1)G0435   661m3h   C(1; Y)1, Df(1)g, y[1] f[1] B[1]/C(1)A, y[1]/Dp(1; f)LJ9, y[+] g[+] na[+] Ste[+]       28   l(3)079101   798H5h-e01   df 084D04-06; 085B06       32   l(3)s147104   1108H5h-   6-7(82D; 82F)by62(85D; 85F)               h06       34   l(3)047418   957H5h-a05   Previously verified       36   l(1)G0425   619M5h-b-   Dp(1; Y)619, y[+] B[S]/w[1] otd[9]/C(1)DX, y[1] w[1] f[1]               e10       38   l(3)122404   1079H5h-   Previously verified               f02       40   l(1)G0105   360H5hA   Df(1)svr, N[spl-1] ras[2] fw[1]/Dp(1; Y)y[2]67g19.1/C(1)DX, y[1] f[1]       44   l(3)057809   971M5h-e06   Previously Verified       46   l(1)G0127   373M3h-f03   Previously Verified       48   l(1)G0469   629H3h-f   C(1; Y)1, Df(1)g, y[1] f[1] B[1]/C(1)A, y[1]/Dp(1; f)LJ9, y[+] g[+] na[+] Ste[+]       50   l(3)S070103   788M5h-h03   091F01-02; 092D03-06 BL#3012       54   l(3)S104104   1057M5h-   Previously Verified               g08       56   l(3)s090609   1017H5h-   emc5(61C; 62A)               a03       58   l(3)093909   1026H5h-   Previously Verified               a11       60   l(1)G0095   354M3h-e10   Df(1)GE202/Y; Dp(1; 2)sn[+]72d/Dp(?; 2)bw[D], bw[D]       62   l(1)G0031   577M3h-h06   BL3219 C(1; Y)1, Df(1)g, y[1] f[1] B[1]/C(1)A, y[1]/Dp(1; f)LJ9, y[+] g[+]                   na[+] Ste[+]       64   l(1)G0354   524M3h-g04   BL1319 Tp(1; 2)w-ec, ec[64d] cm[1] ct[6] sn[3]/C(1)DX, y[1] w[1] f[1]       66   l(1)G0062   333H5h-b02   Df(1)R20, y[1?]/C(1)DX, y[1] w[1] f[1]/Dp(1; Y)y[+]mal[+]       74   l(2)k00237   AQ034169   BL3219 C(1; Y)1, Df(1)g, y[1] f[1] B[1]/C(1)A, y[1]/Dp(1; f)LJ9, y[+] g[+]                   na[+] Ste[+]       76   l(1)G0181   492H3h-f   BL936 Df(1)64c18, g[1] sd[1]/Dp(1; 2; Y)w[+]/C(1)DX, y[1] w[1] f[1]       78   l(3)078514   797H5h-d12   def. 087D01-02; 088E05-06       80   l(3)s112110   1069H5h-   ry506(88B; 88D)               e04       82   l(3)024120   930H5h-e06   Previously verified       84   l(1)G0150   442M3h-b02   Df(1)R20, y[1?]/C(1)DX, y[1] w[1] f[1]/Dp(1; Y)y[+]mal[+]       88   l(3)054211   968H5h-a09   Previously verified       90   l(1)G0399   659m3h   BL 901Df(1)svr, N[spl-1] ras[2] fw[1]/Dp(1; Y)y[2]67g19.1/C(1)DX, y[1] f[1]       92   l(1)G0399   659m3h   BL 901Df(1)svr, N[spl-1] ras[2] fw[1]/Dp(1; Y)y[2]67g19.1/C(1)DX, y[1] f[1]       94   l(3)S104002   1061H5h-   W4(75B; 75C)by62(85D; 85F)               d08       96   l(3)S133705   1092M5h-   Previously verified               f09       98   l(3)041706   949H5h-g10   Previously verified       100   l(1)G0251   392M3h-f11   Df(1)64c18, g[1] sd[1]/Dp(1; 2; Y)w[+]/C(1)DX, y[1] w[1] f[1]       102   l(3)100409   1050H5h-   crb87-5(95F; 96A)               c09       104   l(1)G0491   643M5h-b-   BL3219 C(1; Y)1, Df(1)g, y[1] f[1] B[1]/C(1)A, y[1]/Dp(1; f)LJ9, y[+] g[+]               g11   na[+] Ste[+]       108   l(1)G0306   603m3h   BL1879 Df(1)GE202/Y; Dp(1; 2)sn[+]72d/Dp(?; 2)bw[D], bw[D]       112   l(1)G0344   609H5hA   BL3219 C(1; Y)1, Df(1)g, y[1] f[1] B[1]/C(1)A, y[1]/Dp(1; f)LJ9, y[+] g[+]                   na[+] Ste[+]       116   l(3)s083705   1006H5h-   2-2(81F; 82F)               h07       118   l(1)G0044   319M3h-c02   Df(1)svr, N[spl-1] ras[2] fw[1]/Dp(1; Y)y[2]67g19.1/C(1)DX, y[1] f[1]       120   l(1)G0012   300M5h-b-   Df(1)svr, N[spl-1] ras[2] fw[1]/Dp(1; Y)y[2]67g19.1/C(1)DX, y[1] f[1]               e08       122   l(1)G0012   300M5h-b-   Df(1)svr, N[spl-1] ras[2] fw[1]/Dp(1; Y)y[2]67g19.1/C(1)DX, y[1] f[1]               e08       124   l(1)G0431   566H3h-f   BL 901 Df(1)svr, N[spl-1] ras[2] fw[1]/Dp(1; Y)y[2]67g19.1/C(1)DX, y[1] f[1]       126   l(1)G0130   376H3h-f-   Df(1)svr, N[spl-1] ras[2] fw[1]/Dp(1; Y)y[2]67g19.1/C(1)DX, y[1] f[1]               e10       128   l(1)G0010   576M3h-c07   BL5279 Df(1)JC70/Dp(1; Y)dx[+]5, y[+]/C(1)M5       130   l(3)s118602   1076H5h-   ZP1(66A; 66C)G28(66B; 66C)ry506(88B; 88D)red1(88B; 88D)               e11       132   l(1)G0285   508H3h-f-   BL3033 Df(1)R20, y[1?]/C(1)DX, y[1] w[1] f[1]/Dp(1; Y)y[+]mal[+]               e03       134   l(3)s137212   1094H5h-   GN50(63E; 64B)               g05       136   P{GawB}c338   F49 (13m3h       138   l(1)G0334   515M3h-g09   BL5279 Df(1)JC70/Dp(1; Y)dx[+]5, y[+]/C(1)M5       140   l(1)G0464   627M3h-d   BL5292 (008C-D; 009B + 001A01; 001B02)       142   l(3)099013   1044H5h-   Previously Verified               c04       144   l(3)144912   1103H5h-   Previously verified               h01       146   l(1)G0345   471M3h-d03   BL5279 Df(1)JC70/Dp(1; Y)dx[+]5, y[+]/C(1)M5       148   l(1)G0453   663M3h-d03   BL5292 y[1] nej[Q7] v[1] f[1]/Dp(1; Y)FF1, y[+]/C(1)DX, y[1] w[1] f[1]       150   l(1)G038   616H5hB   BL 929 Df(1)v-L15, y[1]/C(1)DX, y[1] w[1] f[1]; Dp(1; 2)v[+]75d/+       152   l(1)G0492   666M3h-d06   Previously verified       154   l(1)G0052   325M5h-b-   Df(1)v-N48, f[*]/Dp(1; Y)y[+]v[+]#3/C(1)DX, y[1] f[1]               f01       156   l(1)G0269   653M5h-b   BL3033 Df(1)R20, y[1?]/C(1)DX, y[1] w[1] f[1]/Dp(1; Y)y[+]mal[+]       158   l(1)G0241   422H3h-f-   Dp(1; Y)BSC1, y[+]/w[67c23] P{lacW]l(1)G0060[G0060]/C(1)RM, y[1] v[1]               d02       162   l(1)G0141   277M5h-b-   Dp(1; Y)BSC1, y[+]/w[67c23] P{lacW]l(1)G0060[G0060]/C(1)RM, y[1] v[1]               b08       164   l(1)G0250   468H5h-e02   BL5292 y[1] nej[Q7] v[1] f[1]/Dp(1; Y)FF1, y[+]/C(1)DX, y[1] w[1] f[1]       166   l(3)sS030003   943H5h-e09   M-Kx1(86C; 87B)T-61(86E; 87A)T32(86E; 87C)       168   l(1)G0428   456M3h-c04   BL1538 Df(1)os[UE69]/C(1)DX, y[1] f[1]/Dp(1; Y)W39, y[+] ! = fcl[+]Y       170   l(3)072603   996H5h-h02   previously verified       172   l(3)S094310   1029H5h-   previously verified               c08       174   l(1)G0220   467M3h-d02   M19 BL1527 Df(1)svr, N[spl-1] ras[2] fw[1]/Dp(1; Y)y[2]67g19.1/C(1)DX,                   y[1] f[1]       176   l(3)090417   811H5h-e11   def. 087D01-02; 088E05-06       178   l(3)s2172   AQ034107   gasfilling screen       180   l(1)G0025   310M3h-d09   Df(1)JC70/Dp(1; Y)dx[+]5, y[+]/C(1)M5       182   l(1)G0076   343M34-d11   Previously verified       184   l(1)G0151   482M3h-g04   BL1527 Df(1)svr, N[spl-1] ras[2] fw[1]/Dp(1; Y)y[2]67g19.1/C(1)DX, y[1] f[1]       186   l(3)S069605   990M5h-f06   previously verified       188   l(1)G0221   434H3h-f-   Df(1)19, f[1]/C(1)RM, y[1] shi[1] f[1]; Dp(1; Y)shi[+]3, y[+]               f02       190   l(1)G0075   342M3h-d12   Df(1)v-N48, f[*]/DP(1; Y)y[+]#3/C(1)DX, y[1] f[1]       192   l(3)s002001   886H5h-c09   R-G5(62A; 62D)R-G7(62B; 62F)       196   l(1)G0046   321M3h-c04   Df(1)64c18, g[1] sd[1]/Dp(1; 2; Y)w[+]/C(1)DX, y[1] w[1] f[1]       198   l(1)G0020   303M5h-b-   Dp(1; Y)619, y[+] B[S]/w[1] otd[9]/C(1)DX, y[1] w[1]f[1]               f06       200   l(3)s095214   1032H5h-   faf-BP(100D; 100F)               b05       202   l(1)G0481   275H5bB   Dp(1; Y)619, y[+] B[S]/w[1] otd[9]/C(1)DX, y[1] w[1] f[1]       206   l(3)s119608   1077H5h-   B81(99C; 100F)               e12       208   l(1)G0172   650H3h-f-   BL5292 y[1] nej[Q7] v[1] f[1]/DP(1; Y)FF1, y[+]/C(1)DX, y[1] w[1] f[1]               c12       210   l(1)G0429   564M3h-b11   BL5459 C(1; Y)6, y[1] w[*] P{white-un4}BE1305 mew[023]/C(1)RM, Y[1]                   pn[1] v[1]; Dp(1; f)y[+]       212   l(3)005028   892H5h-a04   Previously verified       216   l(1)G0343   520M5h-b   BL5594 Df(1)dhd81, w[1118]/C(1)DX, y[1] f[1]; Dp(1; 2)4FRDup/+       218   l(1)G0343   520M5h-b   BL5594 Df(1)dhd81, w[1118]/C(1)DX, y[1] f[1]; Dp(1; 2)4FRDup/+       220   l(1)G0174   463M3h-c10   Df(1)dhd81, w[1118]/C(1)DX, y[1] f[1]; Dp(1; 2)4FRDup/+       224   l(1)G0132   377H3h-f-   Df(1)svr, N[spl-1] ras[2] fw[1]/DP(1; Y)y[2]67g19.1/C(1)DX, y[1] f[1]               f10       226   l(1)G0144   387M3h-f06   Df(1)64c18, g[1] sd[1]/Dp(1; 2; Y)w[+]/C(1)DX, y[1] w[1] f[1]       228   l(1)G0144   387M3h-f06   Df(1)64c18, g[1] sd[1]/Dp(1; 2; Y)w[+]/C(1)DX, y[1] w[1] f[1]       230   l(1)G0312   291M5h-b-   Df(1)64c18, g[1] sd[1]/Dp(1; 2; Y)w[+]/C(1)DX, y[1] w[1] f[1]               g08       232   l(3)S044402   954M5h-b06   Previously Verified       234   l(1)G0375   534M5h-b-   BL936 Df(1)64c18, g[1] sd[1]/Dp(1; 2; Y)w[+]/C(1)DX, y[1] w[1] f[1]               h03       236   l(1)G0159   486M3h-d09   BL5279 Df(1)JC70/Dp(1; Y)dx[+]5, y[+]/C(1)M5       238   l(1)G0227   651H3h-f   BL5279 Df(1)JC70/Dp(1; Y)dx[+]5, y[+]/C(1)M5       240   l(1)G0212   433M3h-a06   Df(1)19, f[1]/C(1)RM, y[1] shi[1] f[1]; Dp(1; Y)shi[+]3, y[+]       242   l(1)G0296   383H5hA   Df(1)svr, N[spl-1] ras[2] fw[1]/Dp(1; Y)y[2]67g19.1/C(1)DX, y[1]0 f[1]       244   l(3)j2B9   AQ026304   gasfilling screen       248   l(1)G0007   298M3h-a08   Previously verified       250   l(3)070006   991H5h-b08   Previously verified       252   l(1)G0423   454M3h-c02   Df(1)svr, N[spl-1] ras[2] fw[1]/Dp(1; Y)y[2]67g19.1/C(1)DX, y[1] f[1]       254   l(1)G0361   527H3h-f   BL556 Dp(1; Y)BSC1, y[+]/w[67c23] P{lacW]l(1)G0060[G0060]/C(1)RM,                   y[1] v[1]       256   l(1)G0290   285H5hA   Df(1)JC70/Dp(1; Y)dx[+]5, y[+]/C(1)M5       258   l(1)G0436   570M3h-c03   BL 929 Df(1)v-L15, y[1]/C(1)DX, y[1] w[1] f[1]; Dp(1; 2)v[+]75d/+       260   l(1)G0111   362M5hA   Dp(1; Y)BSC1, y[+]/w[67c23] P{lacW]1(1)G0060[G0060]/C(1)RM, y[1] v[1]       262   l(1)G0183   264H3h-f-   Df(1)svr, N[spl-1] ras[2] fw[1]/Dp(1; Y)y[2]67g19.1/C(1)DX, y[1] f[1]               e07       264   l(3)S100209   1049H5h-   Previously verified               d08       266   l(3)S100209   1049H5h-   Previously verified               d08       268   l(1)G0438   572M3h-c05   BL5270 Df(1)19, f[1]/C(1)RM, y[1] shi[1] f[1]; Dp(1; Y)shi[+]3, y[+]       270   l(1)G0116   366M5h-b-   Df(1)19, f[1]/C(1)RM, y[1] shi[1] f[1]; Dp(1; Y) shi[+]3, y[+]               f09       272   l(3)S025007   934M5h-g05   Previously verified       274   l(1)G0419   561M3h-b09   BL 929 Df(1)v-L15, y[1]/C(1)DX, y[1] w[1] f[1]; Dp(1; 2)v[+]75d/+       276   l(3)S008418   900H5h-a05   Previously verified       278   l(3)141110   1098H5h-   Previously verified               g08       280   l(3)S148011   1110H5h-   P115(89B; 89E)C4(89E; 90A)               g08       282   l(3)S023204   923M5h-f05   Previously verified       284   l(3)S096404   1037H5h-   Previously verified               a08       286   l(3)145511   1104H5h-   Previously verified               h02       292   l(3)S110013   1066H5h-   Previously verified               h08       294   l(3)010605   904H5h-d11   Previously verified       296   l(3)100604   1051H5h-   Previously verified               c10       302   l(3)001604   883H5h-c06   Previously verified       304   l(1)G0358   526M3h-g06   BL1538 Df(1)os[UE69]/C(1)DX, y[1] f[1]/Dp(1; Y)W39, y[+] ! = fcl[+]Y       306   l(3)067006   984H5h-g07   Previously Verified       308   l(1)G0070   338M3h-d08   Df(1)os[UE69]/C(1)DX, y[1] f[1]/Dp(1; Y)W39, y[+] ! = fcl[+]Y       310   l(3)02240   G00700   Df(3L)AC1       312   l(3)088205   1013H5h-   Previously Verified               c01       314   l(3)S042228   951H5h-f01   vin2(67F; 68D)vin5(68A; 69A)       316   l(3)S050407   964H5h-a07   M-Kx1(86C; 87B)T-61(86E; 87A)T32(86E; 87C)       318   l(3)011046   908H5h-d09   Previously verified       320   l(3)S094204   1028H5h-   ea(88E; 89A)               b01       322   l(3)001917   738H5h-a03   def. 089E01-F04; 091B01-B02       324   l(3)131602   858H5h-h10   def. 089E01-F04; 091B01-B02       326   l(1)G0451   624M3h-a10   BL 901 Df(1)svr, N[spl-1] ras[2] fw[1]/Dp(1; Y)y[2]67g19.1/C(1)DX, y[1] f[1]       328   l(3)S022231   920H5h-g04   Previously verified       330   l(3)S085401   225M3d   Df(3L-Xs-533/TM6B Sb[1]Ser[1] (76B4-77B)       332   l(3)075515   794H5h-d09   def. 076B04; 077B       334   l(3)131602   858H5h-h10   def. 089E01-F04; 091B01-B02       336   l(3)058302   972H5h-a11   Previously verified       338   l(3)058302   972H5h-a11   Previously verified       340   l(3)S005916   895H5h-d01   lxd6(67F; 68D)P14(90C; 91A)       342   l(3)025616   752H5h-b02   def. 087D01-02; 088E05-06       348   l(3)S089302   1014H5h-   AC1(67A; 67D)               a01       354   l(2)06444   AQ025653   In(2R)vg[W]       356   l(3)026115   938H5h-e07   Previousyl verified       358   l(1)G0461   626M3h-a12   BL5279 Df(1)JC70/Dp(1; Y)dx[+]5, y[+]/C(1)M5       360   l(2)04329   G00564   Df(2R)vg135 Df(2R)CX1       362   l(3)113105   1070H5h-   Previously verified               e05       364   l(1)G0213   495M5h-b   BL1537 Dp(1; Y)W73, y[31d] B[1], f[+], B[S]/C(1)DX, y[1] f[1]/y[1]                   baz[EH171]       366   l(3)003606   888H5h-d06   Previously verified       368   l(3)S005042   893H5h-c01   eN19(93B; 94)eR1(93B; 93D)       372   l(3)S075101   1002H5h-   pXT103(85A; 85C)               h04       374   l(1)G0455   269H5h-a01   BL5678 duplication       376   l(1)G0260   432M3h-a05   Df(1)19, f[1]/C(1)RM, y[1] shi[1] f[1]; Dp(1; Y)shi[+]3, y[+]       378   l(3)S086909   806H5h-b04   087D01-02; 088E05-06 BL1534       380   l(1)G0272   435H3h-f-   M26 BL5270 Df(1)19, f[1]/C(1)RM, y[1] shi[1] f[1]; Dp(1; Y)shi[+]3, y[+]               g02                  
 
       Example 2  
     Sequence Determination  
       [0093]     Inverse PCR: To determine the flanking sequence of the lethal lines, the “Inverse PCR and Cycle Sequencing Protocol for Recovery of Sequences Flanking PZ, PlacW; and PEP elements” of E. Jay Rehm, Berkeley  Drosophila  Genome Project on the world wide web at fruitfly.org/methods/ is used with slight modifications. These modifications include the following: genomic DNA is obtained from 10 flies, rather than 30 flies, with adjustments for final concentrations; all DNA precipitations are performed using glycogen; for some reactions, the digest volume is used in the appropriate ligations; the number of cycles in PCR reactions was increased to 40; Pry1 and Pry2 were used to sequence the PEP line flanking sequences.  
         [0094]     Genomic DNA isolation: Flies are collected and frozen at −20° C. until ready for use. Genomic DNA is prepared by grinding flies in 200 μl Buffer A with a disposable grinder 30× (Buffer A is composed of 100 mM Tris-Cl, pH7.5, 100 mM EDTA, 100 mM NaCl, 0.5% SDS). Add 200 μl additional Buffer A; grind another 15×. Keep on ice until finished. Incubate at 65° C. for 30 minutes. Vortex to mix. Add 800 μl freshly made LiCl/KAc Solution (LiCl/Kac Solution is comprised of 1 part 5 M KAc and 2.5 parts 6 M LiCl). Vortex. Incubate −20° C. for 20 minutes. Spin at maximum speed at room temperature 15+ minutes. Transfer 1 ml supernatant to a clean tube avoiding floating debris. Add 600 μl room temperature isopropanol to supernatant. Mix well by tipping. Add 0.5 μl glycogen. Vortex. Incubate at room temperature for 5 minutes. Spin 15 minutes at room temperature, maximum speed. Aspirate away the supernatant Wash 2× with 500 μl 70% room temperature ethanol; vortex between washes. Spin for 10 minutes at room temperature, maximum speed. Aspirate away supernatant. Dry in a speed vacuum for 10 minutes. Resuspend in 50 μl TE+0.1 mg/ml RNAse A {for 1 ml TE/RNAse A Solution, add 990 μl TE+10 μl RNAse A (10 mg/ml)). Check 5 μl on 0.8% gel.  
         [0095]     Digest Genomic DNA (Sau3A I, HinP1I, or Msp I—done separately): Set up digests in 96 well tray. Per reaction, add 10 μl genomic DNA, 5 μl 10× Buffer, 2 μl 0.1 mg/ml RNAase A stock, 30.5 μl dH 2 O, 10 units of enzyme (8 units for Sau 3AI), 0.5 μl of 100×BSA (for Sau 3AI only). Incubate at 37° C. for 2.5 hours. Check on 0.8% gel before heat-inactivating at 65° C. for 20 minutes.  
         [0096]     Ligate P Element and Flanking DNA: Set-up ligation tube with 400 μl of ligation mixture then add 30-50 μl of the digest: Per reaction, add 30 μl of digested genomic DNA, 43 μl of 10× ligation buffer (NEB), 375 μl of dH 2 O, and 2 μl of ligase (2 Weiss units). Incubate overnight at 4° C. Total reaction volume is adjusted as appropriate.  
         [0097]     Precipitate Ligated DNA: To ligation tube, add 40 μl 3M NaAc pH5.2+1 ml 100% room temperature ethanol+1 μl glycogen. Mix by tipping. Incubate −20° C. for 15+ minutes. Spin 15 minutes, 4° C. Aspirate away supernatant Wash with 500 μl room temperature 70% ethanol. Vortex. Spin room at temperature for 10 minutes. Aspirate away supernatant. Dry in speed vacuum for 10 minutes. Resuspend in 50μl TE. Vortex to mix. Transfer to 96 well plate.  
         [0098]     PCR: Set up PCR reactions in 96 well plates (Applied Biosystems). Set up PCR reactions with primers appropriate for the type of P element and the end of the element from which genomic sequence is to be recovered.  
         [0099]     Primers for PCR: (type of P element 5′ or 3′ end forward primer reverse primer annealing temperature):  
                                                       PZ P-element5′ endPlac4Plac1   60°           PZ P-element3′ endPry4Pry1   55°           PZ P-element3′ endPry2Pry1   60°           PlacW P-element5′ endPlac4Plac1   60°           PlacW P-element3′ endPry4Plw3-1   55°           PlacW P-element3′ endPry2Pry1   60°           PEP P-element5′ endPwht1Plac1   60°           PEP P-element3′ endPry4Pry1   55°           PEP P-element3′ endPry2Pry1   60°                      
 
         [0100]     The Pry2/Pry1 combination has a higher annealing temperature than the Pry4/Pry1 and Pry4/Plw3-1 combinations, but the resulting PCR products do not allow sequencing directly off the 3′ end of the P-element. The latter primer combinations are therefore used in all initial experiments; the Pry2/Pry1 combination can be used in those cases where strong and unique bands do not result.  
         [0101]     Per reaction: 10 μl of ligated genomic DNA, 1 μl of 10 mM dNT mix, 1 μl of 10 μM forward primer stock, 1 μl of 10 μl reverse primer stock, 5 μl of 10× Qiagen Taq buffer, 31.5 μl of dH 2 O, 0.5 μl of Qiagen Taq.  
         [0102]     Cycles: 1×95° C. for 5 minutes; 40×(95° C. for 30 seconds; 60° C. (high temp) or 55° C. (low temp) for 30 seconds; 68° C. for 2 minutes); 1×72° C. for 10 minutes; hold at 4° C.; run 10 μl on 1.5% gel to check. Rearray positive wells to 96 well plate for sequencing clean-up. The primer sets for PCR are as shown in the table below:  
                             TABLE 4                           PCR Primers            Digest, End, Temperature   Forward PCR Primer   Reverse PCR Primer               H5h   Plac4   Plac1       H3h   Pry2   Pry1       H3l   Pry4   Plw3-1       M5h   Plac4   Plac1       M3h   Pry2   Pry1       M3l   Pry4   Plw3-1       S5h   Plac4   Plac1       S3h   Pry2   Pry1       S3l   Pry4   Plw3-1                  
 
         [0103]     PCR Primer Sequences (5′ to 3′):  
                               Plac4 (27)               -act gtg cgt tag gtc ctg ttc att gtt   SEQ ID NO:1               Plac1 (24)       -cac cca agg ctc tgc tcc cac aat   SEQ ID NO:2               Pry4 (23)       -caa tca tat cgc tgt ctc act ca   SEQ ID NO:3               Pry1 (26)       -cct tag cat gtc cgt ggg gtt tga at   SEQ ID NO:4               Pry2 (28)       -ctt gcc gac ggg acc acc tta tgt tat t   SEQ ID NO:5               Plw3-1 (19)       -tgt cgg cgt cat caa ctc c   SEQ ID NO:6               Pwht1 (19)       -gta acg cta atc act ccg aac agg tca ca   SEQ ID NO:7          
 
         [0104]     Enzymatic Clean-Up for Sequencing: To 40 μl PCR reaction, add 4 μl of enzyme mix. Incubate at 37° C. for 1 hour. Inactivate at 70° C. for 10 minutes. (Enzyme Mix consists of 2.5 U/μl Exonuclease I (Amersham E700732), 0.5 U/μl Shrimp Alkaline Phosphatase (Amersham E70183), 1× Amplitaq PCR buffer, add dH 2 O to final volume.)  
       Example 3  
     Sequence Analysis  
       [0105]     Sequence of the flanking sequence generated by inverse PCR is performed on an ABI 3700 sequencer (Perkin Elmer) using BIG DYE sequencing reaction.  
         [0106]     Primer sets for sequencing are as shown in the table below:  
                                 TABLE 5                           PCR Primers for Flanking Sequences                Digest, End, Temperature   Forward Primer   Reverse Primer                       H5h   Splac2   Sp1           H3h   Pry2   Sp5           H3l   Spep1   Sp5           M5h   Splac2   Sp1           M3h   Pry2   Sp5           M3l   Spep1   Sp5           S5h   Splac2   Sp1           S3h   Pry2   Sp6           S3l   Spep1   Sp6                      
 
         [0107]     The following primer sets are designed to sequence both ends of PCR products recovered from PlacW and PZ strains:  
         [0108]     Splac2 and Sp1—for use with the Plac4/Plac1 5′ PCR primer combination with either PZ or PlacW P-elements; allows sequencing of both ends of the PCR fragment.  
         [0109]     Spep1 and Sp3—for use with the Pry4/Pry1 3′ PCR primer combination with PZ P-elements; allows sequencing of both ends of the PCR fragment.  
         [0110]     Spep1 and Sp6—for use with the Pry4/Plw3-1 3′ PCR primer combination with PlacW P-elements where Sau3a digestion is performed; allows sequencing of both ends of the PCR fragment.  
         [0111]     Spep1 and Sp5—for use with the Pry4/Plw3-1 3′ PCR primer combination where HinP1 digestion is performed; allows sequencing of both ends of the PCR fragment.  
         [0112]     Pry1 and Pry2—for use with the Pry1/Pry2 3′ PCR primer combination; allows sequencing of both ends of the PCR fragment.  
         [0113]     The PCR products recovered from PEP strains are sequenced with the following primers: Sp1—for use with the Pwht1/Plac1 5′ PCR primer combination with the PEP element; Spep1—for use with the Pry4/Pry1 3′ PCR primer combination with the PEP element; Pry1 and Pry2 for use with the Pry1/Pry2 3′ PCR primer combination with the PEP element.  
         [0114]     Primer Sequences (5′ to 3′):  
                               Splac2 (25)               -gaa ttc act ggc cgt cgt ttt aca a   SEQ ID NO:8               Sp1 (22)       -aca caa cct ttc ctc tca aca a   SEQ ID NO:9               Sp3 (24)       -gag tac gca aag ctt taa cta tgt   SEQ ID NO:10               Sp6 (23)       -tga cca cat cca aac atc ctc tt   SEQ ID NO:11               Sp5 (25)       -gca tca caa aaa tcg acg ctc aag t   SEQ ID NO:12               Spep1 (19)       -gac act cag aat act att c   SEQ ID NO:13          
 
         [0115]     Melting temperatures of sequencing primers: 
        Splac2—60.1° C.     Sp1—50.6° C.     Sp3—49.3° C.     Sp6—54.9° C.     Sp5—60.3° C.     Spep1—44.8° C.        
 
       Example 4  
     Secondary Confirmation of Lethality  
       [0122]     The lethality of the chromosome carrying the P-element insertion is demonstrated genetically as described in Example 1. The essential  Drosophila  nucleotide sequences are identified by isolating nucleotide sequences flanking the P-element insertion and aligning those sequences with genomic  Drosophila  sequence obtained from the Celera  Drosophila  database. However, in some instances, a second site mutation exists on the chromosome that is responsible for the lethality. In other instances, the location of the flanking sequence is such that determination of which gene(s) are affected by the P-element insertion is rendered difficult or impossible. Thus, to provide secondary confirmation that the gene indicated is essential, there are many methods that one skilled in the art can use, e.g., rescue of the lethality using transformation technology, perturbation of the gene in a targeted manner, or failure to complement a deficiency.  
         [0123]     To provide secondary confirmation, lethal lines are crossed to a line containing a deficiency. This creates a hemizygous condition in that particular region and reveals the recessive phenotype of the P-element. Complementation with deficiencies that unequivocally remove the P-element insertion site is taken as proof that the P-element does not cause the associated phenotype. Failure to complement indicates that the strain is verified. This method is as performed in Spradling, A. C., D. Stern, et al.,  Genetics  153: 135-177 (1999). If the insert is present on the X chromosome, which is present in two copies in females but only one copy in males, then the recessive phenotype of the P-element insert is revealed by this hemizygous condition in males. A rescue cross is performed to a stock containing a duplication spanning the region of the insert on the X chromosome on one of the autosomes. If the males survive then the presence of an essential gene disrupted by the P-element but rescued by the duplication is confirmed. While lines with secondary mutations closely linked to the P insertion might be erroneously verified by these procedures, further molecular and genetic analyses suggest that the frequency of such errors is small. RNA interference, described in Fire, A., S. Xu, et al.,  Nature  391, 806-811 (1998) and Kennerdell, J. R. and Carthew, R. W.,  Cell  95, 1017-1026 (1998), is used as a method to target a gene of interest and demonstrate that the perturbation of the identified gene produces a lethal phenotype.  
       Example 4  
     Double-Stranded RNA Interference  
       [0124]     Preparation of dsRNA for Injection. Sequences to be expressed as dsRNA were cloned into Bluescript KS(+) (Stratagene of La Jolla, Calif.), linearized with the appropriate restriction enzymes, and transcribed in vitro with the Ambion T3 and T7 Megascript kits following the manufacturer&#39;s instructions (Ambion Inc. of Austin, Tex.). Transcripts were annealed in injection buffer (0.1 mM NaPO 4  pH 7.8, 5mM KCl) after heating to 85° C. and cooling to room temperature over a 1- to 24 hr period. All annealed transcripts were analyzed on agarose gels with DNA markers to confirm the size of the annealed RNA and quantitated as described previously (Fire et al. (1998)  Nature  391(6669):806-811). Injected RNA was not gel-purified. Injection of 0.1 nl of a 0.1- to 1.0-mg/ml solution of a 1-kb dsRNA corresponds to roughly 10 7  molecules/injection.  
         [0125]     Injection of  Drosophila melanogaster  Embryos. Fly cages were set up using 2- to 4day flies. Agar-grape juice plates were replaced every hour to synchronize the egg collection for 1-2 days. The eggs were collected over a 30-to 60-min period for subsequent injection. The eggs were washed into a nylon mesh basket with tap water. The chorion was removed by brief soaking in a dilute bleach solution. Eggs were positioned on a glass slide such that each egg was in a same orientation. Double-stranded RNA was injected into middle of each egg using an Eppendorf transjector (Eppendorf Scientific, Inc. of Westbury, N.Y.). Following injection, slides were stored in a moist chamber to prevent dessication of the embryos. Embryos were monitored for development and transferred as first intar larvae to vials containing  Drosophila  medium. Methods for rearing  Drosophila  staging and common genetic techniques can be found, for example, in Roberts (1986)  Drosophila melanogaster, A Practical Approach , IRL Press, Washington, D.C.; Ashburner (1989a)  Drosophila: A Laboratory Handbook , Cold Spring Harbor Laboratory Press, New York, N.Y.; Ashburner (1989b)  Drosophila: A Laboratory Manual , Cold Spring Harbor Laboratory Press, New York, N.Y.; Goldstein &amp; Fyrberg, eds (1994) in  Methods in Cell Biology . Vol. 44, Academic Press, San Diego, Calif.  
         [0126]     The data in Table 6 demonstrates the lethal effect of disrupting the production of protein from the message of the specified gene through RNAi. Based on data from postitve and negative controls, a reduction in survival (% viable adults from developed eggs) below 38% represents a significant lethal effect. Many genes show a complete loss of survivability (with 0% viable). Others show a range of phenotypic penetrance, which is most likely due to the variability of the RNAi technique, but are still considered lethals because they are significantly below controls.  
                                                                                   TABLE 6                           Data for dsRNA Interference                        # eggs               % viable                   showing               adults from           Inventor&#39;s   # eggs   morphological   # hatched           developed       seq ID   reference   Injected   development   larvae   # pupae   # adults   eggs                        none, buffer only   941   806   580   500   433   53.72       14   GIN00231, CT28483   163   148   107   28   26   17.57       30   GIN00961, CT31117   472   386   170   8   1   0.26       42   GIN01243, CT36241   107   99   81   9   7   7.07       52   GIN01682, CT1465   140   127   87   23   15   11.81       68   GIN01885, CT13424   170   154   78   17   8   5.19       70   GIN01896, CT14932   164   140   78   44   38   27.14       72   GIN01977, CT23511   79   70   18   17   15   21.43       86   GIN02340, CT28931   190   159   0   0   0   0.00       106   GIN03775, CT33819   172   148   16   0   0   0.00       110   GIN03797, CT33841   136   127   12   0   0   0.00       114   GIN04053, CT3509   168   145   106   1   1   0.69       160   GIN05757, CT4810   159   144   109   37   32   22.22       194   GIN07111, CT6007   159   140   94   0   0   0.00       204   GIN07278, CT6738   174   166   7   3   1   0.60       214   GIN07446, CT9021   125   119   1   0   0   0.00       222   GIN07609, CT6171   372   316   119   0   0   0.00       246   GIN08205, CT12517   717   569   433   26   25   4.39       274   GIN08858, CT14874   177   161   13   3   3   1.86       288   GIN09788, CT17938   100   83   71   5   2   2.41       290   GIN09819, CT17971   181   142   107   7   1   0.70       298   GIN10338, CT19788   170   137   88   5   1   0.73       300   GIN10364, CT19850   58   55   47   14   6   10.91       344   GIN11831, CT24122   103   87   0   0   0   0.00       346   GIN11918, CT24346   469   408   301   257   88   21.57       350   GIN11993, CT24437   145   130   93   0   0   0.00       352   GIN12074, CT18257   104   93   80   3   3   3.23       354   GIN12174, CT24731   168   145   122   1   1   0.69       360   GIN12437, CT25274   473   424   334   237   63   14.86       370   GIN13270, CT27543   101   92   78   2   2   2.17                  
 
       Example 5  
     Isolation of Full Length cDNA  
       [0127]     A cDNA screen is performed using a  Drosophila melanogaster  cDNA library probed with a portion of each nucleotide sequence disclosed in the Sequence Listing. Positive colonies are selected, a subset sequenced, and a clone corresponding to the full-length cDNA is recovered. Alternatively, primers from the predicted 5′ and 3′ end are used in polymerase chain reaction with either a  Drosophila  cDNA library or first strand cDNAs obtained by reverse transcription of  Drosophila  mRNAs as template to amplify a fragment representing the full-length clone.  
       Example 6  
     Expression of Recombinant Protein in Insect Cells  
       [0128]     Baculovirus vectors, which are derived from the genome of AcNPV virus, are designed to provide high levels of expression of cDNA in the SF9 line of insect cells (ATCC CRL#1711). Recombinant baculovirus expressing the cDNA of the present invention is produced by the following standard methods (InVitrogen MaxBac Manual): cDNA constructs are ligated into the polyhedrin gene in a variety of baclovirus transfer vectors, including the pAC360 and the BleAc vector (InVitrogen). Recombinant baculoviruses are generated by homologous recombination following co-transfection of the baculovirus transfer vector and linearized AcNPV genomic DNA (Kitts, P. A.,  Nucleic Acid Res.  18: 5667 (1990)) into SF9 cells. Recombinant pAC360 viruses are identified by the absence of inclusion bodies in infected cells and recombinant pBlueBac viruses are identified on the basis of B-galactosidase expression (Summers, M. D. and Smith, G. E., Texas Agriculture Exp. Station Bulletin No. 1555). Following plaque purification, the  Drosophila  cDNA expression is measured.  
         [0129]     The cDNA encoding the entire open reading frame for the  Drosophila  cDNA is inserted into the BamHI site of pBlueBacII. Constucts in the positive orientation, which are identified by sequence analysis, are used to transfect SF9 cells in the presence of linear AcNPV wild type DNA. Authentic, active  Drosophila  cDNA is found in the cytoplasm of infected cells. Active  Drosophila  cDNA is extracted from infected cells by hypotonic or detergent lysis.  
       Example 7  
     Expression of Recombinant Protein in  E. coli    
       [0130]     A cDNA clone of the present invention is subcloned into an appropriate expression vector and transformed into  E. coli  using the manufacturer&#39;s conditions. Specific examples include plasmids such as pBluescript (Stratagene, La Jolla, Calif.), pFLAG (International Biotechnologies, Inc., New Haven, Conn.), and pTrcHis (Invitrogen, La Jolla, Calif.).  E. coli  is cultured, and expression of the recombinant protein is confirmed. Recombinant protein is then isolated using standard techniques.  
       Example 8  
     In Vitro Binding Assays  
       [0131]     Recombinant protein is obtained, for example according to Example 6 or Example 7. The protein is immobilized on chips appropriate for ligand binding assays. The protein immobilized on the chip is exposed to sample compound in solution according to methods well know in the art. While the sample compound is in contact with the immobilized protein measurements capable of detecting protein-ligand interactions are conducted. Examples of such measurements are SEDLI, biacore and FCS, described above. Compounds found to bind the protein are readily discovered in this fashion and are subjected to further characterization.  
         [0132]     The above disclosed embodiments are illustrative. This disclosure of the invention will place one skilled in the art in possession of many variations of the invention. All such obvious and foreseeable variations are intended to be encompassed by the appended claims.  
         [0133]     The numerous publications and patents referred to in this document are hereby incorporated by reference, in their entirety.

Technology Classification (CPC): 2