(en)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.

1.ApplicationNumber: US-52757105-A
1.PublishNumber: US-2007003926-A1
2.Date Publish: 20070104
3.Inventor: STAM LYNN
KAMDAR KIM
SPANA ERIC
BACHMANN JANE

4.Inventor Harmonized: STAM LYNN(US)
KAMDAR KIM(US)
SPANA ERIC(US)
BACHMANN JANE(CH)

5.Country: US
6.Claims:
(en)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.

7.Description:
(en)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.


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
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
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.
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.
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.
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.
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' activity. The novel inhibitors discovered through such screens may then be used as insecticides to control undesirable insect infestation.
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
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.
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.
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.
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.
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.
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.
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).
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
SEQ ID NOs:1-13 are PCR primers.
Even numbered SEQ ID NOs:14-380 are nucleotide sequences described in the table below.
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'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 '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 & 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
For clarity, certain terms used in the specification are defined and used as follows:
“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.
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.
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.
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.
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.
“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.
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.”
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.
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.
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.
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.
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.
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.
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.
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.
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
Insecticide: a chemical substance used to kill or inhibit the growth or viability of insects at any stage of development.
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).
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.
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.
Mature Protein: protein that is normally targeted to a cellular organelle and from which the transit peptide has been removed.
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.
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.
Native: refers to a gene that is present in the genome of an untransformed insect cell.
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.
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.
“ORF” means open reading frame.
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.
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.
“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.
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.
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.
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.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'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).
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 >0) and N (penalty score for mismatching residues; always <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 & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
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 & Altschul, Proc. Nat'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.
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.
“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.
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.
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.
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.
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.
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.
“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.
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.
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.
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.
“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.
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
I. Identification Of Essential Drosophila melanogaster Nucleotide Sequences Using Transposable Element Insertion Mutagenesis
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.
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

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.
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.
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.
I.A. Identification of Essential Drosophila melannogaster Nucleotide Sequences Using RNAi
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 & Olson (2000) Parasitol Today 16(8):347-349; Bass (2000) Cell 101(3):235-238; Hunter (2000) Curr Biol 10(4):R137-140; Bosher & 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 & Carthew (2000) Nat Biotech 18(8):896-898; Lam & Thummel (2000) Curr Biol 10(16):957-963; Misquitta & 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 & 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.
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.
II. Recombinant Production of Protein and Uses Thereof
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.).
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 & Sons, Inc. (1994).
IV. Assays for Characterizing the Proteins
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.
V. In Vivo Inhibitor Assay: Discovery of Small Molecule Ligands that Interact with Proteins of Unknown Function.
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.
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.
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.
Surface-Enhanced Laser Desorption/Ionization (SELDI) was invented by Hutchens and Yip during the late 1980'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.
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.
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.
VI. Production of Peptides
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.
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
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
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).
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
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.
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.
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.
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.
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.
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.
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°

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.
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.
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

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

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
Sequence of the flanking sequence generated by inverse PCR is performed on an ABI 3700 sequencer (Perkin Elmer) using BIG DYE sequencing reaction.
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

The following primer sets are designed to sequence both ends of PCR products recovered from PlacW and PZ strains:
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.
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.
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.
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.
Pry1 and Pry2—for use with the Pry1/Pry2 3′ PCR primer combination; allows sequencing of both ends of the PCR fragment.
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.
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

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
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.
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
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'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.
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 & Fyrberg, eds (1994) in Methods in Cell Biology . Vol. 44, Academic Press, San Diego, Calif.
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'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
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
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.
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
A cDNA clone of the present invention is subcloned into an appropriate expression vector and transformed into E. coli using the manufacturer'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
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.
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.
The numerous publications and patents referred to in this document are hereby incorporated by reference, in their entirety.