WO2001019857A2 - Cibles insecticides mortelles - Google Patents

Cibles insecticides mortelles Download PDF

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Publication number
WO2001019857A2
WO2001019857A2 PCT/US2000/025224 US0025224W WO0119857A2 WO 2001019857 A2 WO2001019857 A2 WO 2001019857A2 US 0025224 W US0025224 W US 0025224W WO 0119857 A2 WO0119857 A2 WO 0119857A2
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Prior art keywords
protein
seq
nucleic acid
gene
amino acids
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PCT/US2000/025224
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English (en)
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WO2001019857A3 (fr
Inventor
Allen James Ebens, Jr.
Kevin Patrick Keegan
John W. Winslow
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Genoptera, Llc
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Priority to AU74873/00A priority Critical patent/AU7487300A/en
Publication of WO2001019857A2 publication Critical patent/WO2001019857A2/fr
Publication of WO2001019857A3 publication Critical patent/WO2001019857A3/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
    • A01K67/0335—Genetically modified worms
    • A01K67/0336—Genetically modified Nematodes, e.g. Caenorhabditis elegans
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
    • A01K67/0337—Genetically modified Arthropods
    • A01K67/0339—Genetically modified insects, e.g. Drosophila melanogaster, medfly
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out

Definitions

  • Solute and ion uptake in plants, bacteria, fungi, and animals can be an active or passive process.
  • Active transport is mediated by protein carrier proteins that fall into three classes: uniporters, which transport substrates from one side of a cell membrane to the other side; symporters, which carry two substances across a membrane in the same direction (e.g. proton flux is coupled with motion of substrate), and antiporters, which carry one substance in one direction, and a second substance in the opposite direction.
  • tetracycline antiporter-like proteins confer resistance to the antibiotic tetracycline by mediating active efflux of intracellular tetracycline (Tc) in combination with a divalent or monovalent cation in exchange for external H + (Yamaguchi et al, FEBS Lett. (1995) 365:193-197; Cheng et al, J. Bacteriol. (1996) 178:2853-2860).
  • Tc antiporter-like proteins are members of the facilitative transporter superfamily, which conduct the transfer of a wide variety of organic molecules across the plasma membrane including sugars, molecules of intermediary metabolism, neurotransmitters, and amino acids.
  • transporter proteins typically possess 12 transmembrane domains, and appear to be divided into N- and C-terminal halves, each containing 6 transmembrane elements. This 6 + 6 structure appears to be a characteristic feature of the facilitative transporter superfamily which comprises more than 50 related proteins (Marger and Saier, TIBS (1993) 18: 13-20). Transport proteins have been found in humans that share similarity with Tc antiporter-like proteins and may be implicated in human neurological disorders (U.S. Pat. No. 5,538,844; AAB14374 and Duyao MP, et al, Hum. Mol. Genet. (1993) 6:673-6.).
  • Pesticide development has traditionally focused on the chemical and physical properties of the pesticide itself, a relatively time-consuming and expensive process. As a consequence, efforts have been concentrated on the modification of pre-existing, well- validated compounds, rather than on the development of new pesticides A promising alternative is to identify and validate biological targets against which potential hgands can be screened (Margohs and Duyk, Nature Biotech (1998) 16 311) Production of new compounds that are safer, selective, and more efficient can be implemented using target- based discovery approaches Further, upon identification, the molecular diversity inherent m the specific structures of such targets may be exploited via combinatorial chemistry and high-throughput screening High-throughput assays can be run rapidly and inexpensively and, due to their scale, allow access to the structural va ⁇ ety granted by combmatonal chemistry In addition, potential lead compounds can be directly counter-screened on the same target cloned from human or beneficial insect sources to exclude broad spectrum toxins The essential functions of target genes in insects and nematodes may be tested directly using powerful genetic
  • the present invention relates to novel insect genes and gene products that cause lethality when knocked out, thus representing validated insecticide targets
  • the genes are members of the facilitative transporter class of proteins
  • FT1 facilitative transporter
  • FT2 facilitative transporter
  • splice va ⁇ ant of FT2 that can be used in genetic screening methods to characte ⁇ ze pathways that FT may be involved in as well as other interacting genetic pathways
  • methods for screening compounds that interact with FT such as those that may have utility as therapeutics or pesticides
  • Isolated nucleic acid molecules are provided that comp ⁇ se nucleic acid sequences encoding FT protein as well as novel fragments and de ⁇ vatives thereof Methods of using the isolated nucleic acid molecules and fragments of the invention as biopesticides are described, such as use of RNA interference methods that block FT activity.
  • Vectors and host cells comprising the FT nucleic acid molecules are also described, as well as metazoan invertebrate organisms (e.g. insects, coelomates and pseudocoelomates) that are genetically modified to express or mis-express a FT protein.
  • metazoan invertebrate organisms e.g. insects, coelomates and pseudocoelomates
  • FT nucleic acids and proteins can be used in screening assays to identify candidate compounds which are potential pesticidal agents or therapeutics that interact with FT proteins.
  • Such assays typically comprise contacting a FT protein or fragment with one or more candidate molecules, and detecting any interaction between the candidate compound and the FT protein.
  • the assays may comprise adding the candidate molecules to cultures of cells genetically engineered to express FT proteins, or alternatively, administering the candidate compound to a metazoan invertebrate organism genetically engineered to express FT protein.
  • the genetically engineered metazoan invertebrate animals of the invention can also be used in methods for studying FT activity. These methods typically involve detecting the phenotype caused by the expression or mis-expression of the FT protein. The methods may additionally comprise observing a second animal that has the same genetic modification as the first animal and, additionally has a mutation in a gene of interest. Any difference between the phenotypes of the two animals identifies the gene of interest as capable of modifying the function of the gene encoding the FT protein.
  • Novel FT nucleic acids and their encoded proteins are identified herein.
  • the Drosophila FTs presented here were identified via mutation to lethality by P-element transposon insertion, discussed in more detail below.
  • the P-element lethality along with the transport functions, identifies the FT genes as a previously unrecognized insecticidal drug target for antagonist drugs.
  • human homologues of FT may also represent inhibitory targets for drugs which can kill rapidly growing cells of malignant tumors.
  • the newly identified FT nucleic acids can be used for the generation of mutant phenotypes in animal models or in living cells that can be used to study regulation of FT, and the use of FT as a pesticide or drug target.
  • invertebrate model organisms such as Drosophila Due to the ability to rapidly carry out large-scale, systematic genetic screens, the use of invertebrate model organisms such as Drosophila has great utility for analyzing the expression and mis-expression of FT proteins.
  • the invention provides a superior approach for identifying other components involved in the synthesis, activity, and regulation of FT proteins.
  • Systematic genetic analysis of FTs using invertebrate model organisms can lead to the identification and validation of pesticide targets directed to components of the FT pathway.
  • Model organisms or cultured cells that have been genetically engineered to express FT can be used to screen candidate compounds for their ability to modulate FT expression or activity, and thus are useful in the identification of new drug targets, therapeutic agents, diagnostics and prognostics useful in the treatment of disorders associated with facilitative transporters.
  • these invertebrate model organisms can be used for the identification and screening of pesticide targets directed to components of the FT pathway.
  • the invention relates generally to nucleic acid sequences of Facilitative Transporters, and more particularly Facilitative Transporter nucleic acid sequences of Drosophila, hereinafter referred to as FT, and methods of using these sequences.
  • FT1 SEQ ID NO:l
  • FT2 SEQ ID NO:3
  • SEQ ID NO:5 SEQ ID NO:5
  • SEQ ID NO:5 a nucleic acid sequences, FT1 (SEQ ID NO:l), FT2 (SEQ ID NO:3) and its splice variant (SEQ ID NO:5) were isolated from Drosophila that encode facilitative transporter homologues.
  • the invention includes the reverse complements thereof.
  • the subject nucleic acid sequences, derivatives and fragments thereof may be RNA molecules comprising the nucleotide sequences of SEQ ID NOs:l, 3, and 5 (or derivative or fragment thereof) wherein the base U (uracil) is substituted for the base T (thymine).
  • the DNA and RNA sequences of the invention can be single- or double- stranded.
  • isolated nucleic acid sequence includes the reverse complement, RNA equivalent, DNA or RNA single- or double-stranded sequences, and DNA/RNA hybrids of the sequence being described, unless otherwise indicated.
  • FT nucleic acid sequences can be used for a variety of purposes. Interfering RNA (RNAi) fragments, particularly double-stranded (ds) RNAi, can be used to generate loss-of- function phenotypes, or to formulate biopesticides (discussed further below). FT nucleic acid fragments are also useful as nucleic acid hybridization probes and replication amplification primers. Certain "antisense" fragments, i.e. that are reverse complements of portions of the coding sequence of SEQ ID NO:l, 3, or 5, have utility in inhibiting the function of FT proteins. The fragments are of length sufficient to specifically hybridize with the corresponding SEQ ID NOs:l, 3, or 5.
  • the fragments consist of or comprise at least 12, preferably at least 24, more preferably at least 36, and most preferably at least 96 contiguous nucleotides of SEQ ID NO:l, 3, or 5. Further preferred fragments consist or comprise nucleotides 1326-1697 of SEQ ID NO: 1, 1383 to 1480 of SEQ ID NO:3, and 1739 to 1843 of SEQ ID NO:5. When the fragments are flanked by other nucleic acid sequences, the total length of the combined nucleic acid sequence is less than 15 kb, preferably less than 10 kb or less than 5kb, and more preferably less than 2 kb.
  • SEQ ID NO:l encodes a transporter motif, located at approximately nucleotides 190-1471.
  • Other preferred fragments of FT comprise at least 843 contiguous nucleotides, and more preferably at least 848 contiguous nucleotides of SEQ ID NO:3 or 5.
  • Additional preferred fragment of SEQ ID NOs:3 and 5, encode a sugar transporter domain, located at approximately nucleotides 676-1990.
  • the subject nucleic acid sequences may consist solely of SEQ ID NO:l, 3 or 5 or fragments thereof.
  • the subject nucleic acid sequences and fragments thereof may be joined to other components such as labels, peptides, agents that facilitate transport across cell membranes, hybridization- triggered cleavage agents or intercalating agents.
  • the subject nucleic acid sequences and fragments thereof may also be joined to other nucleic acid sequences (i.e. they may comprise part of larger sequences) and are of synthetic/non- natural sequences and/or are isolated and/or are purified, i.e. unaccompanied by at least some of the material with which it is associated in its natural state.
  • the isolated nucleic acids constitute at least about 0.5%, and more preferably at least about 5% by weight of the total nucleic acid present in a given fraction, and are preferably recombinant, meaning that they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome.
  • Derivative nucleic acid sequences of FT include sequences that hybridize to the nucleic acid sequence of SEQ ED NO:l, 3 or 5 under stringency conditions such that the hybridizing derivative nucleic acid is related to the subject nucleic acid by a certain degree of sequence identity.
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule.
  • Stringency of hybridization refers to conditions under which nucleic acids are hybridizable. The degree of stringency can be controlled by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide during hybridization and washing.
  • stringent hybridization conditions are those normally used by one of skill in the art to establish at least a 90% sequence identity between complementary pieces of DNA or DNA and RNA.
  • “Moderately stringent hybridization conditions” are used to find derivatives having at least 70% sequence identity.
  • “low-stringency hybridization conditions” are used to isolate derivative nucleic acid molecules that share at least about 50%o sequence identity with the subject nucleic acid sequence.
  • the ultimate hybridization stringency reflects both the actual hybridization conditions as well as the washing conditions following the hybridization, and it is well known in the art how to vary the conditions to obtain the desired result. Conditions routinely used are set out in readily available procedure texts (e.g., Current Protocol in
  • a preferred derivative nucleic acid is capable of hybridizing to SEQ ID NO:l, 3 or 5, under stringent hybridization conditions that comprise: prehybridization of filters containing nucleic acid for 8 hours to overnight at 65° C in a solution comprising 6X single strength citrate (SSC) (IX SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5X Denhardt's solution, 0.05% sodium pyrophosphate and 100 ⁇ g/ml herring sperm DNA; hybridization for 18-20 hours at 65° C in a solution containing 6X SSC, IX Denhardt's solution, 100 ⁇ g/ml yeast tRNA and 0.05%> sodium pyrophosphate; and washing of filters at 65° C for 1 h in a solution containing 0.2X SSC and 0.1%
  • Derivative nucleic acid sequences that have at least about 70% sequence identity with SEQ ID NO:l, 3, or 5 are capable of hybridizing to the FT sequences under moderately stringent conditions that comprise: pretreatment of filters containing nucleic acid for 6 h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1 % Ficoll, 1 % BS A, and 500 ⁇ g/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ⁇ g/ml salmon sperm DNA, and 10%> (wt/vol) dextran sulfate; followed by washing twice for 1 hour at 55° C in
  • Other preferred derivative nucleic acid sequences are capable of hybridizing to SEQ ID NOs:l, 3, or 5, under low stringency conditions that comprise: incubation for 8 hours to overnight at 37° C in a solution comprising 20%> formamide, 5 x SSC, 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10%) dextran sulfate, and 20 ⁇ g/ml denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to 20 hours; and washing of filters in 1 x SSC at about 37° C for 1 hour.
  • percent (%) nucleic acid sequence identity with respect to a subject sequence, or a specified portion of a subject sequence, is defined as the percentage of nucleotides in the candidate derivative nucleic acid sequence identical with the nucleotides in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2.0al9 (Altschul et al, J. Mol. Biol. (1997) 215:403-410; http://blast.wustl.edu/blast/README.html; hereinafter referred to generally as "BLAST") with all the search parameters set to default values.
  • the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched.
  • a percent (%) nucleic acid sequence identity value is determined by the number of matching identical nucleotides divided by the sequence length for which the percent identity is being reported.
  • Derivative FT nucleic acid sequences usually have at least 50%) sequence identity, preferably at least 60%>, more preferably at least 70%, more preferably at least 80%o sequence identity, more preferably at least 85%> sequence identity, still more preferably at least 90%> sequence identity, and most preferably at least 95%) sequence identity with SEQ ID NO:l, 3, or 5, a coding region thereof (e.g. nucleotides 375-2190 of SEQ ID NO:3), or a domain-encoding regions thereof (e.g. a sugar transporter or transmembrane domain).
  • the derivative nucleic acid encodes a polypeptide comprising a FT amino acid sequence of SEQ ID NO:2, 4 or 6, or a fragment or derivative thereof as described further below under the subheading "FT proteins".
  • a derivative FT nucleic acid sequence, or fragment thereof may comprise 100% sequence identity with SEQ ID NO:l, 3, or 5, but be a derivative thereof in the sense that it has one or more modifications at the base or sugar moiety, or phosphate backbone. Examples of modifications are well known in the art (Bailey, Ullmann's Encyclopedia of Industrial Chemistry (1998), 6th ed. Wiley and Sons). Such derivatives may be used to provide modified stability or any other desired property.
  • a humanized nucleic acid sequence is one in which one or more codons have been substituted with a codon that is more commonly used in human genes. Preferably, a sufficient number of codons have been substituted such that a higher level expression is achieved in mammalian cells than what would otherwise be achieved without the substitutions.
  • Tables are available in the art that show, for each amino acid, the calculated codon frequency in humans genes for 1000 codons (Wada et al., Nucleic Acids Research (1990) 18(Suppl.):2367-2411).
  • FT nucleic acid sequence in which the glutamic acid codon, GAA has been replaced with the codon GAG, which is more commonly used in human genes is an example of a humanized FT nucleic acid sequence.
  • GAA glutamic acid codon
  • GAG codon GAG
  • Nucleic acid encoding the amino acid sequence of SEQ ID NO:2, 4 or 6, or fragment or derivative thereof may be obtained from an appropriate cDNA library prepared from any eukaryotic species that encodes FT proteins such as vertebrates, preferably mammalian (e.g. primate, porcine, bovine, feline, equine, and canine species, etc.) and invertebrates, such as arthropods, particularly insects species (preferably Drosophila), acarids, Crustacea, molluscs, nematodes, and other worms.
  • An expression library can be constructed using known methods.
  • mRNA can be isolated to make cDNA which is ligated into a suitable expression vector for expression in a host cell into which it is introduced.
  • Various screening assays can then be used to select for the gene or gene product (e.g. oligonucleotides of at least about 20 to 80 bases designed to identify the gene of interest, or labeled antibodies that specifically bind to the gene product).
  • the gene and/or gene product can then be recovered from the host cell using known techniques.
  • PCR Polym erase chain reaction
  • oligonucleotide primers representing fragmentary sequences of interest amplify RNA or DNA sequences from a source such as a genomic or cDNA library (as described by Sambrook et al, supra). Additionally, degenerate primers for amplifying homologs from any species of interest may be used.
  • a PCR product of appropriate size and sequence is obtained, it may be cloned and sequenced by standard techniques, and utilized as a probe to isolate a complete cDNA or genomic clone.
  • Fragmentary sequences of FT nucleic acids and derivatives may be synthesized by known methods.
  • oligonucleotides may be synthesized using an automated DNA synthesizer available from commercial suppliers (e.g. Biosearch, Novato, CA; Perkin- Elmer Applied Biosystems, Foster City, CA).
  • Antisense RNA sequences can be produced intracellularly by transcription from an exogenous sequence, e.g. from vectors that contain antisense FT nucleic acid sequences. Newly generated sequences may be identified and isolated using standard methods.
  • An isolated FT nucleic acid sequence can be inserted into any appropriate cloning vector, for example bacteriophages such as lambda derivatives, or plasmids such as PBR322, pUC plasmid derivatives and the Bluescript vector (Stratagene, San Diego, CA). Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., or into a transgenic animal such as a fly. The transformed cells can be cultured to generate large quantities of the FT nucleic acid. Suitable methods for isolating and producing the subject nucleic acid sequences are well known in the art (Sambrook et al, supra; DNA Cloning: A Practical Approach, Vol. 1, 2, 3, 4, (1995) Glover, ed., MRL Press, Ltd., Oxford, U.K.).
  • the nucleotide sequence encoding a FT protein or fragment or derivative thereof can be inserted into any appropriate expression vector for the transcription and translation of the inserted protein-coding sequence.
  • the necessary transcriptional and translational signals can be supplied by the native FT gene and/or its flanking regions.
  • a variety of host-vector systems may be utilized to express the protein-coding sequence such as mammalian cell systems infected with virus (e.g. vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g. baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.
  • Expression of a FT protein may be controlled by a suitable promoter/enhancer element.
  • a host cell strain may be selected which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired.
  • the expression vector can comprise a promoter operably linked to a FT gene nucleic acid, one or more origins of replication, and, one or more selectable markers (e.g. thymidine kinase activity, resistance to antibiotics, etc.).
  • selectable markers e.g. thymidine kinase activity, resistance to antibiotics, etc.
  • recombinant expression vectors can be identified by assaying for the expression of the FT gene product based on the physical or functional properties of the FT protein in in vitro assay systems (e.g. immunoassays).
  • the FT protein, fragment, or derivative may be optionally expressed as a fusion, or chimeric protein product (i.e. it is joined via a peptide bond to a heterologous protein sequence of a different protein).
  • a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other in the proper coding frame using standard methods and expressing the chimeric product.
  • a chimeric product may also be made by protein synthetic techniques, e.g. by use of a peptide synthesizer.
  • the gene product can be isolated and purified using standard methods (e.g. ion exchange, affinity, and gel exclusion chromatography; centrifugation; differential solubility; electrophoresis).
  • the amino acid sequence of the protein can be deduced from the nucleotide sequence of the chimeric gene contained in the recombinant and can thus be synthesized by standard chemical methods (Hunkapiller et al, Nature (1984) 310:105-111).
  • native FT proteins can be purified from natural sources, by standard methods (e.g. immunoaffinity purification).
  • FT proteins of the invention comprise or consist of an amino acid sequence of SEQ ID NO:2, 4 or 6, or fragments or derivatives thereof.
  • Compositions comprising these proteins may consist essentially of the FT protein, fragments, or derivatives, or may comprise additional components (e.g. pharmaceutically acceptable carriers or excipients, culture media, carriers used in pesticide formulations, etc.).
  • FT protein derivatives typically share a certain degree of sequence identity or sequence similarity with SEQ ID NO:2, 4,or 6, or a fragment thereof.
  • percent (%>) amino acid sequence identity with respect to a subject sequence, or a specified portion of a subject sequence, is defined as the percentage of amino acids in the candidate derivative amino acid sequence identical with the amino acid in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by BLAST (Altschul et al, supra) using the same parameters discussed above for derivative nucleic acid sequences.
  • a % amino acid sequence identity value is determined by the number of matching identical amino acids divided by the sequence length for which the percent identity is being reported.
  • Percent (%>) amino acid sequence similarity is determined by doing the same calculation as for determining % amino acid sequence identity, but including conservative amino acid substitutions in addition to identical amino acids in the computation.
  • a conservative amino acid substitution is one in which an amino acid is substituted for another amino acid having similar properties such that the folding or activity of the protein is not significantly affected.
  • Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino acids are leucine, isoleucine, methionine and valine; interchangeable polar amino acids are glutamine and asparagine; interchangeable basic amino acids are arginine, lysine and histidine; interchangeable acidic amino acids are aspartic acid and glutamic acid; and interchangeable small amino acids are alanine, serine, threonine, cysteine, and glycine.
  • a FT protein derivative shares at least 50 % sequence identity or similarity, preferably at least 60%o, more preferably at least 70%, more preferably at least 80%), more preferably at least 85%, still more preferably at least 90%, and most preferably at least 95% sequence identity or similarity with a contiguous stretch of at least 25 amino acids, preferably at least 50 amino acids, more preferably at least 100 amino acids, and in some cases, the entire length of SEQ ID NO:2, 4 or 6.
  • the FT protein derivative has transporter activity.
  • the FT1 protein derivative may consist of or comprise a sequence that shares 100% similarity with any contiguous stretch of at least 13 amino acids, preferably at least 15 amino acids, more preferably at least 18 amino acids, and most preferably at least 23 amino acids of SEQ ID NO:2.
  • Preferred derivatives of FT1 consist of or comprise an amino acid sequence that has at least 80%o, preferably at least 85%o, more preferably at least 90%, and most preferably at least 95% sequence identity or sequence similarity with any of amino acid residues 24-451 , which represent the likely transporter motif; with any of amino acid residues 1-50 and 257-271, which represent intracellular domains; and with any of amino acid residues 75-106 and 415-426, which represent extracellular domains.
  • Preferred fragments of FT1 proteins consist or comprise at least 10, preferably at least 12, more preferably at least 15, and most preferably at least 20 contiguous amino acids of SEQ ID NO:2.
  • Preferred FT2 protein derivatives may consist of or comprise a sequence that shares 100%
  • FT2 consist of or comprise an amino acid sequence that has at least 80%>, preferably at least 85%o, more preferably at least 90%), and most preferably at least 95% sequence identity or sequence similarity with any of amino acid residues 100-538 of SEQ ID NO:4 or 6, encoding a putative sugar transporter domain.
  • Preferred fragments of FT2 proteins consist or comprise at least 14, preferably at least 16, more preferably at least 19, and most preferably at least 24 contiguous amino acids of SEQ ID NO:2 or 4.
  • the fragment or derivative of the FT protein is preferably "functionally active" meaning that the FT protein derivative or fragment exhibits one or more functional activities associated with a full-length, wild-type FT protein comprising the amino acid sequence of SEQ ID NO:2, 4 or 6.
  • a fragment or derivative may have antigenicity such that it can be used in immunoassays, for immunization, for inhibition of FT activity, etc, as discussed further below regarding generation of antibodies to FT proteins.
  • a functionally active FT fragment or derivative is one that displays one or more biological activities associated with FT proteins such as transport activity.
  • FT proteins, derivatives and fragments can be assayed by various methods known to one skilled in the art (Current Protocols in Protein Science (1998) Coligan et al., eds., John Wiley & Sons, Inc., Somerset, New Jersey). In a preferred method, which is described in detail below, a model organism, such as Drosophila, is used in genetic studies to assess the phenotypic effect of a fragment or derivative (i.e. a mutant FT protein).
  • FT derivatives can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, a cloned FT gene sequence can be cleaved at appropriate sites with restriction endonuclease(s) (Wells et al, Philos. Trans. R. Soc. London SerA (1986) 317:415), followed by further enzymatic modification if desired, isolated, and ligated in vitro, and expressed to produce the desired derivative.
  • restriction endonuclease(s) Wells et al, Philos. Trans. R. Soc. London SerA (1986) 317:415), followed by further enzymatic modification if desired, isolated, and ligated in vitro, and expressed to produce the desired derivative.
  • a FT gene can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or to form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification
  • a va ⁇ ety of mutagenesis techniques are known in the art such as chemical mutagenesis, in vitro site-directed mutagenesis (Carter et al , Nucl Acids Res (1986) 13 4331), use of TAB ® linkers (available from Pharmacia and Upjohn, Kalamazoo, MI), etc
  • manipulations include post translational modification, e g glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc Any of numerous chemical modifications may be earned out by known technique (e g specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papam, V8 protease, NaBH , acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tumcamycm, etc )
  • Denvative proteins can also be chemically synthesized by use of a peptide synthesizer, for example to introduce nonclassical amino acids or chemical amino acid analogs as substitutions or additions into the FT protem sequence
  • Chimenc or fusion proteins can be made comprising a FT protein or fragment thereof (preferably composing one or more structural or functional domains of the FT protem) joined at its ammo- or carboxy-termmus via a peptide bond to an amino acid sequence of a different protein
  • Chimeric proteins can be produced by any known method, including recombinant expression of a nucleic acid encoding the protem (composing a FT- codmg sequence joined in-frame to a coding sequence for a different protein), ligating the appropnate nucleic acid sequences encoding the desired ammo acid sequences to each other in the proper coding frame, and expressing the chimeoc product, and protem synthetic techniques, e g by use of a peptide synthesizer
  • FT gene regulatory DNA elements such as enhancers or promoters that reside within nucleotides 1 to 376 for SEQ ID NO 3 or nucleotides 1 to 374 for SEQ ID NO 5 can be used to identify tissues, cells, genes and factors that specifically control FT protein production
  • Analyzing components that are specific to FT protein function can lead to an understanding of how to manipulate these regulatory processes, especially for pesticide and therapeutic applications, as well as an understanding of how to diagnose dysfunction in these processes.
  • Gene fusions with the FT regulatory elements can be made.
  • compact genes that have relatively few and small intervening sequences such as those described herein for Drosophila
  • regulatory elements that control spatial and temporal expression patterns are found in the DNA immediately upstream of the coding region, extending to the nearest neighboring gene.
  • Regulatory regions can be used to construct gene fusions where the regulatory DNAs are operably fused to a coding region for a reporter protein whose expression is easily detected, and these constructs are introduced as transgenes into the animal of choice.
  • An entire regulatory DNA region can be used, or the regulatory region can be divided into smaller segments to identify sub-elements that might be specific for controlling expression a given cell type or stage of development.
  • Reporter proteins that can be used for construction of these gene fusions include E. coli beta- galactosidase and green fluorescent protein (GFP). These can be detected readily in situ, and thus are useful for histological studies and can be used to sort cells that express FT proteins (O'Kane and Gehring PNAS (1987) 84(24):9123-9127; Chalfie et al, Science (1994) 263:802-805; and Cumberledge and Krasnow (1994) Methods in Cell Biology 44:143-159).
  • GFP green fluorescent protein
  • Recombinase proteins such as FLP or ere
  • Recombinase proteins can be used in controlling gene expression through site-specific recombination (Golic and Lindquist (1989) Cell 59(3):499- 509; White et al, Science (1996) 271 :805-807).
  • Toxic proteins such as the reaper and hid cell death proteins, are useful to specifically ablate cells that normally express FT proteins in order to assess the physiological function of the cells (Kingston, In Current Protocols in Molecular Biology (1998) Ausubel et al, John Wiley & Sons, Inc. sections 12.0.3-12.10) or any other protein where it is desired to examine the function this particular protein specifically in cells that synthesize FT proteins.
  • a binary reporter system can be used, similar to that described further below, where the FT regulatory element is operably fused to the coding region of an exogenous transcriptional activator protein, such as the GAL4 or tTA activators described below, to create a FT regulatory element "driver gene".
  • the exogenous activator controls a separate "target gene” containing a coding region of a reporter protein operably fused to a cognate regulatory element for the exogenous activator protein, such as UAS G or a tTA-response element, respectively.
  • FT regulatory element-reporter gene fusions are also useful for tests of genetic interactions, where the objective is to identify those genes that have a specific role in controlling the expression of FT genes, or promoting the growth and differentiation of the tissues that expresses FT proteins
  • FT gene regulatory DNA elements are also useful in protein-DNA binding assays to identify gene regulatory proteins that control the expression of FT genes
  • the gene regulatory proteins can be detected using a vanety of methods that probe specific protem-DNA interactions well known to those skilled in the art (Kingston, supra) including in vivo footpnnting assays based on protection of DNA sequences from chemical and enzymatic modification withm living or permeabihzed cells, and in vitro footpnnting assays based on protection of DNA sequences from chemical or enzymatic modification using protem extracts, nitrocellulose filter-binding assays and gel electrophoresis mobility shift assays using radioactively labeled regulatory DNA elements mixed with protein extracts
  • yeast "one-hybrid” system can be used (Li and Herskowitz, Science (1993) 262 1870-1874, Luo et al , Biotechmques (1996) 20(4) 564-568, Vidal et al , PNAS (1996) 93(19) 10315-10320)
  • a va ⁇ ety of methods can be used to identify or screen for molecules, such as proteins or other molecules, that interact with FT proteins, or denvatives or fragments thereof
  • the assays may employ punfied FT proteins, or cell lines or model organisms such as Drosophila and C elegans, that have been genetically engineered to express FT proteins Suitable screening methodologies are well known in the art to test for proteins and other molecules that mteract with FT genes and proteins (see e g , PCT International Publication No WO 96/34099)
  • the newly identified interacting molecules may provide new targets for pharmaceutical or pesticidal agents Any of a vanety of exogenous molecules, both naturally occurring and/or synthetic (e.g., libraries of small molecules or peptides, or phage display libraries) may be screened for binding capacity.
  • the FT protein or fragment is mixed with candidate molecules under conditions conducive to binding, sufficient time is allowed for any binding to occur, and assays are performed to test for bound complexes.
  • Assays to find interacting proteins can be performed by any method known in the art, for example, immunoprecipitation with an antibody that binds to the protein in a complex followed by analysis by size fractionation of the immunoprecipitated proteins (e.g. by denaturing or nondenaturing polyacrylamide gel electrophoresis), Western analysis, non-denaturing gel electrophoresis, etc.
  • a preferred method for identifying interacting proteins is a two-hybrid assay system or variation thereof (Fields and Song, Nature (1989) 340:245-246; U.S. Pat. No. 5,283,173; for review see Brent and Finley, Annu. Rev. Genet. (1997) 31 :663-704).
  • the most commonly used two-hybrid screen system is performed using yeast. All systems share three elements: 1) a gene that directs the synthesis of a "bait" protein fused to a DNA binding domain; 2) one or more "reporter” genes having an upstream binding site for the bait, and 3) a gene that directs the synthesis of a "prey” protein fused to an activation domain that activates transcription of the reporter gene.
  • the "bait” is preferably a FT protein, expressed as a fusion protein to a DNA binding domain; and the "prey” protein is a protein to be tested for ability to interact with the bait, and is expressed as a fusion protein to a transcription activation domain.
  • the prey proteins can be obtained from recombinant biological libraries expressing random peptides.
  • the bait fusion protein can be constructed using any suitable DNA binding domain, such as the E. coli LexA repressor protein, or the yeast GAL4 protein (Bartel et al,
  • the prey fusion protein can be constructed using any suitable activation domain such as GAL4, VP-16, etc.
  • the preys may contain useful moieties such as nuclear localization signals (Ylikomi et al, ⁇ MBO J. (1992) 11 :3681-3694; Dingwall and Laskey, Trends Biochem. Sci. Trends Biochem. Sci. (1991) 16:479-481) or epitope tags (Allen et al, Trends Biochem. Sci. Trends Biochem. Sci. (1995) 20:511-516) to facilitate isolation of the encoded proteins.
  • Any reporter gene can be used that has a detectable phenotype such as reporter genes that allow cells expressing them to be selected by growth on appropriate medium (e.g.
  • HIS3, LEU2 described by Chien et al, PNAS (1991) 88:9572-9582; and Gyuris et al, Cell (1993) 75:791-803).
  • Other reporter genes such as LacZ and GFP, allow cells expressing them to be visually screened (Chien et al, supra).
  • the host cell in which the interaction assay and transcription of the reporter gene occurs can be any cell, such as mammalian (e.g. monkey, mouse, rat, human, bovine), chicken, bacterial, or insect cells.
  • mammalian e.g. monkey, mouse, rat, human, bovine
  • chicken e.g. bacterial, or insect cells.
  • Various vectors and host strains for expression of the two fusion protein populations in yeast can be used (U.S. Pat. No. 5,468,614; Bartel et al, Cellular Interactions in
  • interaction of VP16-tagged derivatives with GAL4-derived baits drives the synthesis of SV40 T antigen, which in turn promotes the replication of the prey plasmid, which carries an SV40 origin (Vasavada et al, PNAS (1991) 88:10686-10690).
  • the bait FT gene and the prey library of chimeric genes are combined by mating the two yeast strains on solid or liquid media for a period of approximately 6-8 hours.
  • the resulting diploids contain both kinds of chimeric genes, i.e., the DNA-binding domain fusion and the activation domain fusion.
  • Transcription of the reporter gene can be detected by a linked replication assay in the case of SV40 T antigen (described by Vasavada et al, supra) or using immunoassay methods, preferably as described in Alam and Cook (Anal. Biochem. (1990)188:245-254).
  • the activation of other reporter genes like URA3, HIS3, LYS2, or LEU2 enables the cells to grow in the absence of uracil, histidine, lysine, or leucine, respectively, and hence serves as a selectable marker.
  • Other types of reporters are monitored by measuring a detectable signal. For example, GFP and lacZ have gene products that are fluorescent and chromogenic, respectively.
  • the DNA sequences encoding the proteins can be isolated.
  • the activation domain sequences or DNA-binding domain sequences (depending on the prey hybnd used) are amplified, for example, by PCR using pairs of ohgonucleotide p ⁇ mers specific for the coding region of the DNA binding domain or activation domain
  • Other known amplification methods can be used, such as hgase chain reaction, use of Q rep case, or va ⁇ ous other methods descnbed (see K ⁇ cka et al , Molecular Probing, Blotting, and Sequencing (1995) Academic Press, New York, Chapter 1 and Table IX)
  • the DNA sequences encoding the proteins can be isolated by transformation of E coli using the yeast DNA and recovenng the plasmids from E coli
  • the yeast vector can be isolated, and the insert encoding the fusion protein subcloned into a bacterial expression vector, for growth of the plasmid in E coli
  • a limitation of the two-hybnd system occurs when transmembrane portions of proteins in the bait or the prey fusions are used This occurs because most two-hybnd systems are designed to function by formation of a functional transcription activator complex withm the nucleus, and use of transmembrane portions of the protein can interfere with proper association, folding, and nuclear transport of bait or prey segments (Ausubel et al , supra, Allen et al , supra) Since the FT proteins are transmembrane proteins, it is preferred that intracellular or extracellular domains be used for bait in a two-hybnd scheme
  • FT proteins encoded by S ⁇ Q ID NO 2, 4 or 6, and derivatives and fragments thereof, such as those discussed above, may be used as an immunogen to generate monoclonal or polyclonal antibodies and antibody fragments or derivatives (e g chimenc, single chain, Fab fragments)
  • fragments of a FT protem preferably those identified as hydrophilic, are used as immunogens for antibody production using art-known methods such as by hybndomas, production of monoclonal antibodies m germ-free animals (PCT/US90/02545), the use of human hybndomas (Cole et al , PNAS (1983) 80 2026- 2030, Cole et al , in Monoclonal Antibodies and Cancer Therapy (1985) Alan R Liss, pp 77-96), and production of humanized antibodies (Jones et al , Nature (1986) 321 522-525, U S Pat 5,530,101)
  • FT polypeptidet al e
  • binding affinity may be assayed by determination of equilibrium constants of antigen-antibody association (usually at least about 10 7 M " ', preferably at least about 10 8 M "1 , more preferably at least about 10 M "1 ).
  • Immunoassays can be used to identify proteins that interact with or bind to FT proteins.
  • Various assays are available for testing the ability of a protein to bind to or compete with binding to a wild-type FT protein or for binding to an anti-FT protein antibody.
  • Suitable assays include radioimmunoassays, ELISA (enzyme linked immunosorbent assay), immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, immunoelectrophoresis assays, etc.
  • radioimmunoassays e.g., ELISA (enzyme linked immunosorbent assay), immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels), western blots, precipitation reactions, agglutin
  • FT genes or FT interacting genes can be assessed as potential pesticide or drug targets, or as potential biopesticides. Further, transgenic plants that express FT proteins can be tested for activity against insect pests (Estruch et al, Nat. Biotechnol (1997) 15(2):137-141).
  • FT1 and FT2 are validated pesticide targets since disruption of the Drosophila FT1 and FT2 genes result in lethality when homozygous, indicating that the FT proteins likely transports across the plasma membrane a small molecule critical for cell function and survival of the insects.
  • the FT gene products may be involved in a variety of physiological functions such as the function of the digestive tract of the insect (including motility and absorption of the gut or entire alimentry canal), neuronal function, and neuroendocrine regulation.
  • the mutation to lethality of these genes indicates that drugs that agonize or antagonize an FT gene product may be effective pesticidal agents.
  • pesticide refers generally to chemicals, biological agents, and other compounds that kill, paralyze, sterilize or otherwise disable pest species in the areas of agricultural crop protection, human and animal health.
  • exemplary pest species include parasites and disease vectors such as mosquitoes, fleas, ticks, parasitic nematodes, chiggers, mites, etc.
  • Pest species also include those that are eradicated for aesthetic and hygienic purposes (e.g. ants, cockroaches, clothes moths, flour beetles, etc.), home and garden applications, and protection of structures (including wood boring pests such as termites, and marine surface fouling organisms).
  • Pesticidal compounds can include traditional small organic molecule pesticides (typified by compound classes such as the organophosphates, pyrethroids, carbamates, and organochlorines, benzoylureas, etc).
  • Other pesticides include proteinaceous toxins such as the Bacillus thuringiensis Crytoxins (Gill et al, Annu Rev Entomol (1992) 37:615-636) and Photorabdus luminescens toxins (Bowden et al, Science (1998) 280:2129-2132); and nucleic acids such as FT dsRNA or antisense nucleic acids that interferes with FT activity.
  • Pesticides can be delivered by a variety of means including direct application to pests or to their food source.
  • toxic proteins and pesticidal nucleic acids can be administered using biopesticidal methods, for example, by viral infection with nucleic acid or by transgenic plants that have been engineered to produce interfering nucleic acid sequences or encode the toxic protein, which are ingested by plant- eating pests.
  • Putative pesticides, drugs, and molecules can be applied onto whole insects, nematodes, and other small invertebrate metazoans, and the ability of the compounds to modulate (e.g. block or enhance) FT activity can be observed.
  • the effect of various compounds on FT can be assayed using cells that have been engineered to express one or more FT genes and associated proteins.
  • the compounds to be tested are dissolved in DMSO or other organic solvent, mixed with a bacterial suspension at various test concentrations, preferably OP50 strain of bacteria (Brenner, Genetics (1974) 110:421-440), and supplied as food to the worms.
  • the population of worms to be treated can be synchronized larvae (Sulston and Hodgkin, in The nematode C. elegans (1988), supra) or adults or a mixed-stage population of animals.
  • Ratios are treated with different concentrations of compounds, typically ranging from 1 mg/ml to 0.001 mg/ml. Behavioral aberrations, such as a decrease in motility and growth, and morphological aberrations, sterility, and death are examined in both acutely and chronically treated adult and larval worms.
  • larval and adult worms are examined immediately after application of the compound and re-examined periodically (every 30 minutes) for 5-6 hours.
  • Chronic or long-term assays are performed on worms and the behavior of the treated worms is examined every 8-12 hours for 4-5 days. In some circumstances, it is necessary to reapply the pesticide to the treated worms every 24 hours for maximal effect.
  • Potential insecticidal compounds can be administered to insects in a variety of ways, including orally (including addition to synthetic diet, application to plants or prey to be consumed by the test organism), topically (including spraying, direct application of compound to animal, allowing animal to contact a treated surface), or by injection.
  • Insecticides are typically very hydrophobic molecules and must commonly be dissolved in organic solvents, which are allowed to evaporate in the case of methanol or acetone, or at low concentrations can be included to facilitate uptake (ethanol, dimethyl sulfoxide).
  • the first step in an insect assay is usually the determination of the minimal lethal dose (MLD) on the insects after a chronic exposure to the compounds.
  • the compounds are usually diluted in DMSO, and applied to the food surface bearing 0-48 hour old embryos and larvae.
  • MLD minimal lethal dose
  • this step allows the determination of the fraction of eggs that hatch, behavior of the larvae, such as how they move /feed compared to untreated larvae, the fraction that survive to pupate, and the fraction that eclose (emergence of the adult insect from puparium). Based on these results more detailed assays with shorter exposure times may be designed, and larvae might be dissected to look for obvious morphological defects. Once the MLD is determined, more specific acute and chronic assays can be designed.
  • a typical acute assay compounds are applied to the food surface for embryos, larvae, or adults, and the animals are observed after 2 hours and after an overnight incubation.
  • embryos defects in development and the percent that survive to adulthood are determined.
  • larvae defects in behavior, locomotion, and molting may be observed.
  • behavior and neurological defects are observed, and effects on fertility are noted.
  • a chronic exposure assay adults are placed on vials containing the compounds for 48 hours, then transfened to a clean container and observed for fertility, neurological defects, and death.
  • Compounds that modulate (e.g. block or enhance) FT activity may also be assayed using cell culture.
  • the effect of exogenously added compounds cells expressing FT may be screened for their ability to modulate the activity of FT genes based upon measurements of neurotransmitter or facilitative transport across membranes.
  • Assays for changes facilitative transport and uptake can be performed on cultured cells expressing endogenous normal or mutant FTs. Such studies also can be performed on cells transfected with vectors capable of expressing the FTs, or functional domains of one of the FTs, in normal or mutant form.
  • cells may be cotransfected with genes encoding FT proteins.
  • Xenopus oocytes may be injected with normal or mutant FT sequences.
  • Changes in FT-related or FT-mediated transport activity can be measured by two-microelectrode voltage-clamp recordings in oocytes and/or by rate of uptake of radioactive tetracycline molecules (Arriza et al, J. Neurosci.(1994) 14:5559-5569; Arriza et al, J. Biol. Chem. (1993) 268:15329-15332; Mbungu et al, Archives of Biochemistry and Biophysics (1995) 318:489-497). These procedures may be used to screen a battery of compounds, particularly potential pesticides or drugs.
  • the selectivity of a material for FT may be determined by testing the effect of the compound using cells expressing FT and comparing the results with that obtained using cells not expressing FT (see US Patent Nos. 5,670,335 and 5,882,873).
  • Compounds that selectively modulate the FT are identified as potential pesticide and drug candidates having FT specificity.
  • FT nucleic acids and fragments thereof can be used to inhibit FT function, and thus can be used as biopesticides.
  • dsRNA double- stranded RNA
  • the biopesticides may comprise the nucleic acid molecule itself, an expression construct capable of expressing the nucleic acid, or organisms transfected with the expression construct.
  • the biopesticides may be applied directly to plant parts or to soil surrounding the plants (e.g. to access plant parts growing beneath ground level), or directly onto the pest.
  • Biopesticides comprising FT nucleic acids may be prepared in a suitable vector for delivery to a plant or animal.
  • suitable vectors include Agrobacterium tumefaciens Ti plasmid-based vectors (Horsch et al, Science (1984) 233:496-89; Fraley et al, Proc. NafL Acad. Sci. USA (1983) 80:4803), and recombinant cauliflower mosaic virus (Hohn et al, 1982, In Molecular Biology of Plant Tumors, Academic Press, New York, pp 549-560; U.S. Patent No. 4,407,956 to Howell). Retrovirus based vectors are useful for the introduction of genes into vertebrate animals (Burns et al, Proc. Natl. Acad. Sci. USA (1993) 90:8033-37).
  • Transgenic insects can be generated using a transgene comprising FT genes operably fused to an appropriate inducible promoter.
  • a tTA-responsive promoter may be used in order to direct expression of FT proteins at an appropriate time in the life cycle of the insect.
  • vectors for the introduction of genes into insects include P element (Rubin and Spradling, Science (1982) 218:348-53; U.S. Pat. No. 4,670,388), "hermes” (O'Brochta et al, Genetics (1996) 142:907-914), "minos” (U.S. Pat.
  • mis-expressed FT pathway protein may be one having an amino acid sequence that differs from wild-type (i.e. it is a derivative of the normal protein).
  • a mis-expressed FT pathway protein may also be one in which one or more amino acids have been deleted, and thus is a "fragment" of the normal protein.
  • mis-expression also includes ectopic expression (e.g. by altering the normal spatial or temporal expression), over-expression (e.g. by multiple gene copies), underexpression, non-expression (e.g. by gene knockout or blocking expression that would otherwise normally occur), and further, expression in ectopic tissues.
  • the term “gene of interest” refers to a FT pathway gene, or any other gene involved in regulation or modulation, or downstream effector of the FT pathway.
  • the in vivo and in vitro models may be genetically engineered or modified so that they 1) have deletions and/or insertions of one or more FT pathway genes, 2) harbor interfering RNA sequences derived from FT pathway genes, 3) have had one or more endogenous FT pathway genes mutated (e.g. contain deletions, insertions, rearrangements, or point mutations in FT gene or other genes in the pathway), and or 4) contain transgenes for mis-expression of wild-type or mutant forms of such genes.
  • Such genetically modified in vivo and in vitro models are useful for identification of genes and proteins that are involved in the synthesis, activation, control, etc.
  • the model systems can also be used for testing potential pesticidal or pharmaceutical compounds that interact with the FT pathway, for example by administering the compound to the model system using any suitable method (e.g. direct contact, ingestion, injection, etc.) and observing any changes in phenotype, for example defective movement, lethality, etc.
  • suitable method e.g. direct contact, ingestion, injection, etc.
  • Various genetic engineering and expression modification methods which can be used are well-known in the art, including chemical mutagenesis, transposon mutagenesis, antisense RNAi, dsRNAi, and transgene-mediated mis-expression.
  • Loss-of-function mutations in an invertebrate metazoan FT gene can be generated by any of several mutagenesis methods known in the art (Ashburner, In Drosophila melanogaster: A Laboratory Manual (1989) , Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press: pp. 299-418; Fly pushing: The Theory and Practice of Drosophila melanogaster Genetics (1997) Cold Spring Harbor Press, Plainview, NY; The nematode C. elegans (1988) Wood, Ed., Cold Spring Harbor Laboratory Press, Cold Spring harbor, New York).
  • Techniques for producing mutations in a gene or genome include use of radiation (e.g., X-ray, UV, or gamma ray); chemicals (e.g., EMS, MMS, ENU, formaldehyde, etc.); and insertional mutagenesis by mobile elements including dysgenesis induced by transposon insertions, or transposon-mediated deletions, for example, male recombination, as described below.
  • radiation e.g., X-ray, UV, or gamma ray
  • chemicals e.g., EMS, MMS, ENU, formaldehyde, etc.
  • insertional mutagenesis by mobile elements including dysgenesis induced by transposon insertions, or transposon-mediated deletions, for example, male recombination, as described below.
  • transposons e.g., P element, EP-type "overexpression trap” element, mariner element, piggyBac transposon, hermes, minos, sleeping beauty, etc.
  • transposons e.g., P element, EP-type "overexpression trap” element, mariner element, piggyBac transposon, hermes, minos, sleeping beauty, etc.
  • Transposon insertions lying adjacent to a gene of interest can be used to generate deletions of flanking genomic DNA, which if induced in the germline, are stably propagated in subsequent generations.
  • the utility of this technique in generating deletions has been demonstrated and is well-known in the art.
  • One version of the technique using collections of P element transposon induced recessive lethal mutations (P lethals) is particularly suitable for rapid identification of novel, essential genes in Drosophila (Cooley et al, Science (1988) 239:1121-1128; Spralding et al, PNAS (1995) 92:0824-10830).
  • the genomic sequence flanking each transposon insert is determined either by plasmid rescue (Hamilton et al, PNAS (1991) 88:2731-2735) or by inverse polymerase chain reaction (Rehm, http://www.fruitfly.org/methods/).
  • the FT1 and FT2 genes were identified from a P lethal screen. Disruption of the Drosophila FT genes result in lethality when homozygous, indicating that the protein is critical for cell function and the survival of insects.
  • FT genes may be identified and/or characterized by generating loss-of-function phenotypes in animals of interest through RNA-based methods, such as antisense RNA (Schubiger and Edgar, Methods in Cell Biology (1994) 44:697-713).
  • RNA-based methods such as antisense RNA (Schubiger and Edgar, Methods in Cell Biology (1994) 44:697-713).
  • One form of the antisense RNA method involves the injection of embryos with an antisense RNA that is partially homologous to the gene of interest (in this case a FT gene).
  • Another form of the antisense RNA method involves expression of an antisense RNA partially homologous to the gene of interest by operably joining a portion of the gene of interest in the antisense orientation to a powerful promoter that can drive the expression of large quantities of antisense RNA, either generally throughout the animal or in specific tissues.
  • RNA-generated loss-of-function phenotypes have been reported previously for several Drosophila genes including cactus, pecanex, and Kr ⁇ ppel (LaBonne et al, Dev. Biol. (1989) 136(1):1-16; Schuh and Jackie, Genome (1989) 31(l):422-425; Geisler et al, Cell (1992) 71(4):613-621).
  • Loss-of-function phenotypes can also be generated by cosuppression methods (Bingham Cell (1997) 90(3):385-387; Smyth, Curr. Biol. (1997) 7(12):793-795; Que and Jorgensen, Dev. Genet. (1998) 22(1):100-109).
  • Cosuppression is a phenomenon of reduced gene expression produced by expression or injection of a sense strand RNA corresponding to a partial segment of the gene of interest. Cosuppression effects have been employed extensively in plants and C.
  • dsRNAi double-stranded RNA interference
  • This method is based on the interfering properties of double- stranded RNA derived from the coding regions of gene, and has proven to be of great utility in genetic studies of C. elegans (Fire et al, Nature (1998) 391 :806-811), and can also be used to generate loss-of-function phenotypes in Drosophila (Kennerdell and Carthew, Cell (1998) 95:1017-1026; Misquitta and Patterson PNAS (1999) 96:1451-1456).
  • dsRNAi double-stranded RNA interference
  • complementary sense and antisense RNAs derived from a substantial portion of a gene of interest are synthesized in vitro.
  • the resulting sense and antisense RNAs are annealed in an injection buffer, and the double- stranded RNA injected or otherwise introduced into animals (such as in their food or by soaking in the buffer containing the RNA). Progeny of the injected animals are then inspected for phenotypes of interest (PCT publication no. WO99/32619).
  • the dsRNA can be delivered to the animal by bathing the animal in a solution containing a sufficient concentration of the dsRNA.
  • dsRNA derived from a FT gene can be generated in vivo by simultaneous expression of both sense and antisense RNA from appropriately positioned promoters operably fused to FT sequences in both sense and antisense orientations.
  • the dsRNA can be delivered to the animal by engineering expression of dsRNA within cells of a second organism that serves as food for the animal, for example engineering expression of dsRNA in E. coli bacteria which are fed to C. elegans, or engineering expression of dsRNA in baker's yeast which are fed to Drosophila, or engineering expression of dsRNA in transgenic plants which are fed to plant eating insects such as Leptinotarsa or Heliothis.
  • RNAi has been successfully used in cultured Drosophila cells to inhibit expression of targeted proteins (Clemens et al. PNAS, June 6, 2000, vol. 97, no. 12, pp. 6499-6503).
  • cell lines in culture can be manipulated using RNAi both to perturb and study the function of FT pathway components and to validate the efficacy of therapeutic or pesticidal strategies that involve the manipulation of this pathway.
  • peptide aptamers are peptides or small polypeptides that act as dominant inhibitors of protein function.
  • Peptide aptamers specifically bind to target proteins, blocking their function ability (Kolonin and Finley, PNAS (1998) 95:14266-14271). Due to the highly selective nature of peptide aptamers, they may be used not only to target a specific protein, but also to target specific functions of a given protein (e.g. transport function). Further, peptide aptamers may be expressed in a controlled fashion by use of promoters which regulate expression in a temporal, spatial or inducible manner. Peptide aptamers act dominantly; therefore, they can be used to analyze proteins for which loss-of-function mutants are not available.
  • Peptide aptamers that bind with high affinity and specificity to a target protein may be isolated by a variety of techniques known in the art. In one method, they are isolated from random peptide libraries by yeast two-hybrid screens (Xu et al, PNAS (1997) 94:12473-12478). They can also be isolated from phage libraries (Hoogenboom et al, Immunotechnology (1998) 4:1-20) or chemically generated peptides/libraries.
  • RNA aptamers are specific RNA ligands for proteins, that can specifically inhibit protein function of the gene (Good et al, Gene Therapy (1997) 4:45-54; Ellington, et al, Biotechnol. Annu. Rev. (1995) 1 : 185-214). In vitro selection methods can be used to identify RNA aptamers having a selected specificity (Bell et al, J. Biol. Chem. (1998) 273:14309-14314). It has been demonstrated that RNA aptamers can inhibit protein function in Drosophila (Shi et al, Proc. Natl. Acad. Sci USA (19999) 96:10033-10038). Accordingly, RNA aptamers can be used to decrease the expression of a FT protein or derivative thereof, or a protein that interacts with a FT proteins. Transgenic animals can be generated to test peptide or RNA aptamers in vivo
  • transgenic Drosophila lines expressing the desired aptamers may be generated by P element mediated transformation (discussed below). The phenotypes of the progeny expressing the aptamers can then be characterized.
  • Intracellularly expressed antibodies, or intrabodies are single-chain antibody molecules designed to specifically bind and inactivate target molecules inside cells. Intrabodies have been used in cell assays and in whole organisms such as Drosophila (Chen et al, Hum. Gen. Ther. (1994) 5:595-601 ; Hassanzadeh et al, Febs Lett. (1998) 16(1, 2):75- 80 and 81-86). Inducible expression vectors can be constructed with intrabodies that react specifically with a FT protein. These vectors can be introduced into model organisms and studied in the same manner as described above for aptamers.
  • transgenic animals are created that contain gene fusions of the coding regions of the FT genes (from either genomic DNA or cDNA) or genes engineered to encode antisense RNAs, cosuppression RNAs, interfering dsRNA, RNA aptamers, peptide aptamers, or intrabodies operably joined to a specific promoter and transcriptional enhancer whose regulation has been well characterized, preferably heterologous promoters/enhancers (i.e. promoters/enhancers that are non-native to the FT pathway genes being expressed).
  • Methods are well known for incorporating exogenous nucleic acid sequences into the genome of animals or cultured cells to create transgenic animals or recombinant cell lines.
  • transposable elements For invertebrate animal models, the most common methods involve the use of transposable elements. There are several suitable transposable elements that can be used to incorporate nucleic acid sequences into the genome of model organisms. Transposable elements are particularly useful for inserting sequences into a gene of interest so that the encoded protein is not properly expressed, creating a "knock-out" animal having a loss-of- function phenotype. Techniques are well-established for the use of P element in Drosophila (Rubin and Spradling, Science (1982) 218:348-53; U.S. Pat. No. 4,670,388) and Tel in C. elegans (Zwaal et al, Proc. Natl. Acad. Sci. U.S.A.
  • transposable elements Modern Biological Analysis of an Organism (1995) Epstein and Shakes, Eds.).
  • Tel -like transposable elements can be used such as minos, mariner and sleeping beauty.
  • transposable elements that function in a variety of species have been identified, such as PiggyBac (Thibault et al, Insect Mol Biol (1999) 8(1):119-23), hobo, and hermes.
  • P elements or marked P elements, are preferred for the isolation of loss-of-function mutations in Drosophila FT genes because of the precise molecular mapping of these genes, depending on the availability and proximity of preexisting P element insertions for use as a localized transposon source (Hamilton and Zinn, Methods in Cell Biology (1994) 44:81-94; and Wolfher and Goldberg, Methods in Cell Biology (1994) 44:33-80).
  • modified P elements are used which contain one or more elements that allow detection of animals containing the P element.
  • marker genes are used that affect the eye color of Drosophila, such as derivatives of the Drosophila white or rosy genes (Rubin and Spradling, Science (1982) 218(4570):348-353; and Klemenz et al, Nucleic Acids Res. (1987) 15(10):3947-3959).
  • any gene can be used as a marker that causes a reliable and easily scored phenotypic change in transgenic animals.
  • markers include bacterial plasmid sequences having selectable markers such as ampicillin resistance (Steller and Pirrotta, EMBO. J.
  • a preferred method of transposon mutagenesis in Drosophila employs the "local hopping" method described by Tower et al. (Genetics (1993) 133:347-359).
  • Each new P insertion line can be tested molecularly for transposition of the P element into the gene of interest (e.g. FT1 or FT2) by assays based on PCR.
  • FT1 or FT2 the gene of interest
  • Products of the PCR reactions are detected by agarose gel electrophoresis. The sizes of the resulting DNA fragments reveal the site of P element insertion relative to the gene of interest.
  • Southern blotting and restriction mapping using DNA probes derived from genomic DNA or cDNAs of the gene of interest can be used to detect transposition events that rearrange the genomic DNA of the gene.
  • P transposition events that map to the gene of interest can be assessed for phenotypic effects in heterozygous or homozygous mutant Drosophila.
  • Drosophila lines carrying P insertions in the gene of interest can be used to generate localized deletions using known methods (Kaiser, Bioassays (1990) 12(6):297-301 ; Harnessing the power of Drosophila genetics, In Drosophila melanogaster: Practical Uses in Cell and Molecular Biology, Goldstein and Fyrberg, Eds., Academic Press, Inc. San Diego, California). This is particularly useful if no P element transpositions are found that disrupt the gene of interest. Briefly, flies containing P elements inserted near the gene of interest are exposed to a further round of transposase to induce excision of the element.
  • Progeny in which the transposon has excised are typically identified by loss of the eye color marker associated with the transposable element.
  • the resulting progeny will include flies with either precise or imprecise excision of the P element, where the imprecise excision events often result in deletion of genomic DNA neighboring the site of P insertion.
  • Such progeny are screened by molecular techniques to identify deletion events that remove genomic sequence from the gene of interest, and assessed for phenotypic effects in heterozygous and homozygous mutant Drosophila.
  • Tel transposable element can be used for directed mutagenesis of a gene of interest.
  • a Tel library is prepared by the methods of Zwaal et al, supra and Plasterk, supra, using a strain in which the Tel transposable element is highly mobile and present in a high copy number.
  • the library is screened for Tel insertions in the region of interest using PCR with one set of primers specific for Tel sequence and one set of gene-specific primers and C. elegans strains that contain Tel transposon insertions within the gene of interest are isolated.
  • transposable elements can be used to incorporate the gene of interest, or mutant or derivative thereof, as an additional gene into any region of an animal's genome resulting in mis-expression (including over- expression) of the gene.
  • a preferred vector designed specifically for misexpression of genes in transgenic Drosophila is derived from pGMR (Hay et al, Development (1994) 120:2121-2129), is 9Kb long, and contains: an origin of replication for E.
  • the expression unit contains a first multiple cloning site (MCS) designed for insertion of an enhancer and a second MCS located 500 bases downstream, designed for the insertion of a gene of interest.
  • MCS multiple cloning site
  • homologous recombination or gene targeting techniques can be used to substitute a gene of interest for one or both copies of the animal's homologous gene.
  • the transgene can be under the regulation of either an exogenous or an endogenous promoter element, and be inserted as either a minigene or a large genomic fragment.
  • gene function can be analyzed by ectopic expression, using, for example, Drosophila (Brand et al, Methods in Cell Biology (1994) 44:635- 654) or C. elegans (Mello and Fire, Methods in Cell Biology (1995) 48:451-482).
  • heterologous promoters examples include heat shock promoters/enhancers, which are useful for temperature induced mis-expression.
  • heat shock promoters/enhancers include the hsp70 and hsp83 genes, and in C. elegans, include hsp 16-2 and hsp 16-41.
  • Tissue specific promoters/enhancers are also useful, and in Drosophila, include eyeless (Mozer and Benzer, Development (1994) 120:1049-1058), sevenless (Bowtell et al, PNAS (1991) 88(15):6853- 6857), and g/ass-responsive promoters/enhancers (Quiring et al, Science (1994) 265:785- 789) which are useful for expression in the eye; and enhancers/promoters derived from the dpp or vestigal genes which are useful for expression in the wing (Staehling-Hampton et al, Cell Growth Differ.
  • tissue specific promoters/enhancers include the myo-2 gene promoter, useful for pharyngeal muscle-specific expression; the hlh-1 gene promoter, useful for body- muscle-specific expression; and the gene promoter, useful for touch-neuron-specific gene expression.
  • gene fusions for directing the mis-expression of FT pathway genes are incorporated into a transformation vector which is injected into nematodes along with a plasmid containing a dominant selectable marker, such as rol-6.
  • Transgenic animals are identified as those exhibiting a roller phenotype, and the transgenic animals are inspected for additional phenotypes of interest created by mis-expression of the FT pathway gene.
  • binary control systems that employ exogenous DNA are useful when testing the mis-expression of genes in a wide variety of developmental stage-specific and tissue-specific patterns.
  • binary exogenous regulatory systems include the UAS/GAL4 system from yeast (Hay et al, PNAS (1997) 94(10):5195-5200; Ellis et al, Development (1993) 119(3):855-865), and the "Tet system” derived from E. coli (Bello et al., Development (1998) 125:2193-2202).
  • the UAS/GAL4 system is a well-established and powerful method of mis-expression in Drosophila which employs the UASQ upstream regulatory sequence for control of promoters by the yeast GAL4 transcriptional activator protein (Brand and Perrimon, Development (1993) 118(2):401-15).
  • transgenic Drosophila termed "target” lines
  • driver lines transgenic Drosophila strains
  • GAL4 coding region is operably fused to promoters/enhancers that direct the expression of the GAL4 activator protein in specific tissues, such as the eye, wing, nervous system, gut, or musculature.
  • the gene of interest is not expressed in the target lines for lack of a transcriptional activator to drive transcription from the promoter joined to the gene of interest.
  • transgenic Drosophila driver lines are generated where the coding region for a tetracycline-controlled transcriptional activator (tTA) is operably fused to promoters/enhancers that direct the expression of tTA in a tissue-specific and/or developmental stage-specific manner.
  • the driver lines are crossed with transgenic Drosophila target lines where the coding region for the gene of interest to be mis-expressed is operably fused to a promoter that possesses a tTA-responsive regulatory element.
  • Expression of the gene of interest can be induced at will simply by removal of tetracycline from the food. Also, the level of expression of the gene of interest can be adjusted by varying the level of tetracycline in the food.
  • Tet system as a binary control mechanism for mis-expression has the advantage of providing a means to control the amplitude and timing of mis-expression of the gene of interest, in addition to spatial control. Consequently, if a gene of interest (e.g.
  • a FT gene has lethal or deleterious effects when mis-expressed at an early stage in development, such as the embryonic or larval stages, the function of the gene of interest in the adult can still be assessed by adding tetracycline to the food during early stages of development and removing tetracycline later so as to induce mis-expression only at the adult stage.
  • Dominant negative mutations by which the mutation causes a protein to interfere with the normal function of a wild-type copy of the protein, and which can result in loss-of- function or reduced- function phenotypes in the presence of a normal copy of the gene, can be made using known methods (Hershkowitz, Nature (1987) 329:219-222).
  • overexpression of an inactive form achieved, for example, by linking the mutant gene to a highly active promoter, can cause competition for natural substrates or ligands sufficient to significantly reduce net activity of the normal protein.
  • changes to active site residues can be made to create a virtually irreversible association with a target.
  • Various expression analysis techniques may be used to identify genes which are differentially expressed between a cell line or an animal expressing wild type FT genes compared to another cell line or animal expressing mutant FT genes.
  • Such expression profiling techniques include differential display, serial analysis of gene expression (SAGE), transcript profiling coupled to a gene database query, nucleic acid array technology, subtractive hybridization, and proteome analysis (e.g. mass-spectrometry and two- dimensional protein gels).
  • Nucleic acid array technology may be used to determine a global (i.e., genome-wide) gene expression pattern in a normal animal for comparison with an animal having a mutation in a FT gene.
  • Gene expression profiling can also be used to identify other genes (or proteins) that may have a functional relation to FT (e.g.
  • the genes are identified by detecting changes in their expression levels following mutation, i.e., insertion, deletion or substitution in, or over-expression, under-expression, mis-expression or knock-out, of FT genes.
  • FT pathway genes that have been mutated (i.e. deletions, insertions, and/or point mutations) animal models that are both homozygous and heterozygous for the altered FT pathway gene are analyzed.
  • specific phenotypes that may be investigated include lethality; sterility; feeding behavior, perturbations in neuromuscular function including alterations in motility, and alterations in sensitivity to pesticides and pharmaceuticals.
  • Some phenotypes more specific to flies include alterations in: adult behavior such as, flight ability, walking, grooming, phototaxis, mating or egg-laying; alterations in the responses of sensory organs, changes in the morphology, size or number of adult tissues such as, eyes, wings, legs, bristles, antennae, gut, fat body, gonads, and musculature; larval tissues such as mouth parts, cuticles, internal tissues or imaginal discs; or larval behavior such as feeding, molting, crawling, or puparian formation; or developmental defects in any germline or embryonic tissues.
  • adult behavior such as, flight ability, walking, grooming, phototaxis, mating or egg-laying
  • alterations in the responses of sensory organs changes in the morphology, size or number of adult tissues such as, eyes, wings, legs, bristles, antennae, gut, fat body, gonads, and musculature
  • larval tissues such as mouth parts, cuticles, internal tissues or imaginal disc
  • phenotypes more specific to nematodes include: locomotory, egg laying, chemosensation, male mating, and intestinal expulsion defects.
  • locomotory egg laying, chemosensation, male mating, and intestinal expulsion defects.
  • single phenotypes or a combination of specific phenotypes in model organisms might point to specific genes or a specific pathway of genes, which facilitate the cloning process.
  • Genomic sequences containing a FT pathway gene can be used to confirm whether an existing mutant insect or worm line corresponds to a mutation in one or more FT pathway genes, by rescuing the mutant phenotype.
  • a genomic fragment containing the FT pathway gene of interest and potential flanking regulatory regions can be subcloned into any appropriate insect (such as Drosophila) or worm (such as C. elegans) transformation vector, and injected into the animals.
  • any appropriate insect such as Drosophila
  • worm such as C. elegans transformation vector
  • Vox Drosophila an appropriate helper plasmid is used in the injections to supply transposase for transposon-based vectors. Resulting germline transformants are crossed for complementation testing to an existing or newly created panel o ⁇ Drosophila or C.
  • elegans lines whose mutations have been mapped to the vicinity of the gene of interest (Fly Pushing: The Theory and Practice o ⁇ Drosophila Genetics, supra; and Caenorhabditis elegans: Modern Biological Analysis of an Organism (1995), Epstein and Shakes, eds.). If a mutant line is discovered to be rescued by this genomic fragment, as judged by complementation of the mutant phenotype, then the mutant line likely harbors a mutation in the FT pathway gene. This prediction can be further confirmed by sequencing the FT pathway gene from the mutant line to identify the lesion in the FT pathway gene.
  • RNAi methods can be used to simulate loss- of-function mutations in the genes being analyzed. It is of particular interest to investigate whether there are any interactions of FT genes with other well-characterized genes, particularly genes involved in sugar or tetracycline transport.
  • a genetic modifier screen using invertebrate model organisms is a particularly preferred method for identifying genes that interact with FT genes, because large numbers of animals can be systematically screened making it more possible that interacting genes will be identified.
  • a screen of up to about 10,000 animals is considered to be a pilot-scale screen.
  • Moderate-scale screens usually employ about 10,000 to about 50,000 flies, and large-scale screens employ greater than about 50,000 flies.
  • animals having a mutant phenotype due to a mutation in or misexpression of one or more FT genes are further mutagenized, for example by chemical mutagenesis or transposon mutagenesis.
  • mutant allele is genetically recessive, as is commonly the situation for a loss-of-function allele, then most typically males, or in some cases females, which carry one copy of the mutant allele are exposed to an effective mutagen, such as EMS, MMS, ENU, triethylamine, diepoxyalkanes, ICR-170, formaldehyde, X-rays, gamma rays, or ultraviolet radiation.
  • the mutagenized animals are crossed to animals of the opposite sex that also carry the mutant allele to be modified.
  • wild type males are mutagenized and crossed to females carrying the mutant allele to be modified.
  • progeny of the mutagenized and crossed flies that exhibit either enhancement or suppression of the original phenotype are presumed to have mutations in other genes, called "modifier genes", that participate in the same phenotype-generating pathway.
  • modify genes mutations in other genes, called "modifier genes", that participate in the same phenotype-generating pathway.
  • These progeny are immediately crossed to adults containing balancer chromosomes and used as founders of a stable genetic line.
  • progeny of the founder adult are retested under the original screening conditions to ensure stability and reproducibility of the phenotype. Additional secondary screens may be employed, as appropriate, to confirm the suitability of each new modifier mutant line for further analysis.
  • Standard techniques used for the mapping of modifiers that come from a genetic screen in Drosophila include meiotic mapping with visible or molecular genetic markers; male-specific recombination mapping relative to P-element insertions; complementation analysis with deficiencies, duplications, and lethal P-element insertions; and cytological analysis of chromosomal abenations (Fly Pushing: Theory and Practice o ⁇ Drosophila Genetics, supra; Drosophila: A Laboratory Handbook, supra).
  • Genes corresponding to modifier mutations that fail to complement a lethal P-element may be cloned by plasmid rescue of the genomic sequence sunounding that P-element.
  • modifier genes may be mapped by phenotype rescue and positional cloning (Sambrook et al, supra).
  • Newly identified modifier mutations can be tested directly for interaction with other genes of interest known to be involved or implicated with FT genes using methods described above. Also, the new modifier mutations can be tested for interactions with genes in other pathways that are not believed to be related to neuronal signaling (e.g. nanos in Drosophila). New modifier mutations that exhibit specific genetic interactions with other genes implicated in neuronal signaling, but not interactions with genes in unrelated pathways, are of particular interest.
  • the modifier mutations may also be used to identify "complementation groups". Two modifier mutations are considered to fall within the same complementation group if animals carrying both mutations in trans exhibit essentially the same phenotype as animals that are homozygous for each mutation individually and, generally are lethal when in trans to each other (Fly Pushing: The Theory and Practice o ⁇ Drosophila Genetics, supra). Generally, individual complementation groups defined in this way co ⁇ espond to individual genes.
  • homologous genes in other species can be isolated using procedures based on cross-hybridization with modifier gene DNA probes, PCR-based strategies with primer sequences derived from the modifier genes, and/or computer searches of sequence databases.
  • human and rodent homologs of the modifier genes are of particular interest.
  • homologs of modifier genes in insects and arachnids are of particular interest.
  • Insects, arachnids, and other organisms of interest include, among others, Isopoda; Diplopoda; Chilopoda; Symphyla; Thysanura; Collembola; Orthoptera, such as Scistocerca spp; Blattoidea, such as Blattella germanica; Dermaptera; Isoptera; Anoplura; Mallophaga; Thysanoptera; Heteroptera; Homoptera, including Bemisia tabaci, and Myzus spp.; Lepidoptera including Plodia interpunctella, Pectinophora gossypiella, Plutella spp., Heliothis spp., and Spodoptera species; Coleoptera such as Leptinotarsa, Diabrotica spp., Anthonomus spp., and Tribolium spp.; Hymenoptera; Diptera, including Anopheles spp.; Siphonaptera
  • Drosophila genetic modifier screens are quite powerful and sensitive, some genes that interact with FT genes may be missed in this approach, particularly if there is functional redundancy of those genes. This is because the vast majority of the mutations generated in the standard mutagenesis methods will be loss- of-function mutations, whereas gain-of- function mutations that could reveal genes with functional redundancy will be relatively rare.
  • Another method of genetic screening in Drosophila has been developed that focuses specifically on systematic gain-of- function genetic screens (Rorth et al, Development (1998) 125: 1049-1057).
  • This method is based on a modular mis-expression system utilizing components of the GAL4/UAS system (described above) where a modified P element, termed an "enhanced P” (EP) element, is genetically engineered to contain a GAL4-responsive UAS element and promoter. Any other transposons can also be used for this system.
  • the resulting transposon is used to randomly tag genes by insertional mutagenesis (similar to the method of P element mutagenesis described above).
  • Thousands of transgenic Drosophila strains, termed EP lines can be generated, each containing a specific UAS-tagged gene. This approach takes advantage of the preference of P elements to insert at the 5'-ends of genes. Consequently, many of the genes that are tagged by insertion of EP elements become operably fused to a GAL4-regulated promoter, and increased expression or mis-expression of the randomly tagged gene can be induced by crossing in a GAL4 driver gene.
  • Systematic gain-of- function genetic screens for modifiers of phenotypes induced by mutation or mis-expression of a FT gene can be performed by crossing several thousand Drosophila EP lines individually into a genetic background containing a mutant or mis- expressed FT gene, and further containing an appropriate GAL4 driver transgene. It is also possible to remobilize the EP elements to obtain novel insertions. The progeny of these crosses are then analyzed for enhancement or suppression of the original mutant phenotype as described above. Those identified as having mutations that interact with the FT gene can be tested further to verify the reproducibility and specificity of this genetic interaction.
  • EP insertions that demonstrate a specific genetic interaction with a mutant or mis-expressed FT gene have a physically tagged new gene which can be identified and sequenced using PCR or hybridization screening methods, allowing the isolation of the genomic DNA adjacent to the position of the EP element insertion.
  • a Drosophila expressed sequence tag (EST) cDNA library was prepared as follows. Tissue from mixed stage embryos (0-20 hour), imaginal disks and adult fly heads were collected and total RNA was prepared. Mitochondrial rRNA was removed from the total RNA by hybridization with biotinylated rRNA specific oligonucleotides and the resulting RNA was selected for polyadenylated mRNA. The resulting material was then used to construct a random primed library. First strand cDNA synthesis was primed using a six nucleotide random primer. The first strand cDNA was then tailed with terminal transferase to add approximately 15 dGTP molecules.
  • EST Drosophila expressed sequence tag
  • the second strand was primed using a primer which contained a Notl site followed by a 13 nucleotide C-tail to hybridize to the G-tailed first strand cDNA.
  • the double stranded cDNA was ligated with BstXl adaptors and digested with Notl .
  • the cDNA was then fractionated by size by electrophoresis on an agarose gel and the cDNA greater than 700 bp was purified.
  • the cDNA was ligated with Notl, BstXl digested pCDNA-sk+ vector (a derivative of pBluescript, Stratagene) and used to transform E. coli (XLlblue).
  • the final complexity of the library was 6 X 10° independent clones.
  • the cDNA library was normalized using a modification of the method described by Bonaldo et al. (Genome Research (1996) 6:791-806).
  • Biotinylated driver was prepared from the cDNA by PCR amplification of the inserts and allowed to hybridize with single stranded plasmids of the same library.
  • the resulting double-stranded forms were removed using strepavidin magnetic beads, the remaining single stranded plasmids were converted to double stranded molecules using Sequenase (Amersham, Arlington Hills, IL), and the plasmid DNA stored at -20°C prior to transformation. Aliquots of the normalized plasmid library were used to transform E.
  • coli XLlblue or DH10B
  • the clones were allowed to grow for 24 hours at 37° C then the master plates were frozen at -80° C for storage.
  • the total number of colonies picked for sequencing from the normalized library was 240,000.
  • the master plates were used to inoculate media for growth and preparation of DNA for use as template in sequencing reactions. The reactions were primarily carried out with primer that initiated at the 5' end of the cDNA inserts. However, a minor percentage of the clones were also sequenced from the 3' end.
  • Clones were selected for 3' end sequencing based on either further biological interest or the selection of clones that could extend assemblies of contiguous sequences ("contigs") as discussed below.
  • DNA sequencing was earned out using ABI377 automated sequencers and used either ABI FS, dirhodamine or BigDye chemistries (Applied Biosystems, Inc., Foster City, CA).
  • FT1 and FT2 were discovered from a screen using collections of P element transposon induced recessive lethal mutations (P lethals) to identify novel genes. Briefly, genomic sequence surrounding transposable elements disclosed by the Berkeley Drosophila Genome Project (http://www.fruitfly.org) was retrieved by inverse PCR, and blasted against the FlyTagTM database, which resulted in identification of pertinent clones for full-length cloning.
  • P lethals P element transposon induced recessive lethal mutations
  • Example 3 Cloning of FT Nucleic Acid Sequence
  • the PCR conditions used for cloning the FT nucleic acid sequences comprised a denaturation step of 94° C, 5 min; followed by 35 cycles of: 94° C 1 min, 55° C 1 min 72° C 1 min; then, a final extension at 72° C 10 min. All DNA sequencing reactions were performed using standard protocols for the BigDye sequencing reagents (Applied Biosystems, Inc.) and products were analyzed using ABI 377 DNA sequencers. Trace data obtained from the ABI 377 DNA sequencers was analyzed and assembled into contigs using the Phred-Phrap programs.
  • primers were designed to the known DNA sequences in the clones, using the Primer-3 software (Steve Rozen, Helen J. Skaletsky (1998) Primer3. Code available at http://www-genome.wi.mit.edu/genome_software/other/primer3.html.). These primers were then used in sequencing reactions to extend the sequence until the full sequence of the insert was determined.
  • the GPS-1 Genome Priming System in vitro transposon kit (New England Biolabs, Inc., Beverly, MA) was used for transpo son-based sequencing, following manufacturer's protocols. Briefly, multiple DNA templates with randomly interspersed primer-binding sites were generated. These clones were prepared by picking 24 colonies/clone into a Qiagen REAL Prep to purify DNA and sequenced by using supplied primers to perform bidirectional sequencing from both ends of transposon insertion.
  • Sequences were then assembled using Phred/Phrap and analyzed using Consed. Ambiguities in the sequence were resolved by resequencing several clones. For FT2, this effort resulted in 2 contiguous nucleotide sequences: 1) a contiguous nucleotide sequence of 3205 bases in length (SEQ ED NO:3), encompassing an open reading frame (ORF) of 1818 nucleotides encoding a predicted protein of 605 amino acids (SEQ ID NO:4).
  • SEQ ED NO:3 a contiguous nucleotide sequence of 3205 bases in length
  • ORF open reading frame
  • the ORF extends from base 376-2196 of SEQ ED NO:3; a contiguous nucleotide sequence of 2996 bases in length (SEQ ED NO:5), which is a splice variant of SEQ ED NO:3, also encompassing an open reading frame (ORF) of 1818 nucleotides encoding a predicted protein of 656 amino acids (SEQ ED NO:6).
  • the ORF extends from base 374-2194 of SEQ ED NO:5.
  • the contiguous nucleotide sequence of 1.7 kilobases in length encompasses an ORF of 1431 nucleotides.
  • the ORF extends from base 119-1550 of SEQ ED NO:l and encodes a predicted protein of 477 amino acids (SEQ ED NO:2).
  • Table 1 is a summary of the features of FT2.
  • Columns 2 and 4 show the regions of the protein sequences that correspond to the domains listed in column 1 ; and columns 3 and 5 show the corresponding nucleic acid coding regions.
  • Nucleotide and amino acid sequences for each of the FT2 nucleic acid sequences and their encoded proteins were searched against all available nucleotide and amino acid sequences in the public databases, using BLAST (Altschul et al, supra). Table 2 below summarizes the results. The 5 most similar sequences are listed. Results are the same for FT2 and its splice variant.
  • BLAST results for the FT2 amino acid sequences indicate 15 amino acid residues as the shortest stretch of contiguous amino acids that is novel with respect to public sequences and 34 amino acids as the shortest stretch of contiguous amino acids for which there are no sequences contained within public sequences sharing 100% sequence similarity.
  • sequence having closest identity and similarity is disclosed in WO9839448-A2 (a human secreted protein encoded by gene 77 clone HOEAS24 deposited as clone ATCC 97900 and ATCC 209046).
  • the two FT2 cDNAs (SEQ ID NO:3 and 5) are nearly identical and encode putative transporter proteins. They differ within a short segment of DNA and translated protein amino acid sequences, and part of the 3' untranslated sequence near the polyadenylation signal. Comparison of the two cDNAs with the intron/exon structure predicted for the corcesponding genomic sequence, suggests alternative ex on usage within the same encoding gene, and resulting expression as separate transcripts. A comparison of the open reading frame of both cDNA clones with the GenBank database, using BLAST, identifies the strongest identity with a C.
  • Pfam analysis predicts the presence of the a sugar transporter structural and functional motif from amino acids 100-538, nucleic acids 676-1990 for both SEQ ID NO:3 and 5.
  • cDNAs represent a novel class of glucose or tetracycline transporters is also supported by differences with the G X8 G X3 GP X2 G G' amino acid motif found in 31 antiporters including the bacterial TetA(C) gene, a tetracycline/H+antiporter.
  • Transmembrane domains were predicted using the TopPred2 algorithm (Stockholm Univeristy) and the amino acid segments are described in Table 1. Eleven transmembrane domains were predicted for both cDNAs which contrasts with the 12 transmembrane domains typically predicted for the small solute transporter proteins such as the glucose or tetracycline transporter.
  • the physiological solute substrate and role of the tetracycline resistant transporter is not clearly defined.
  • the facilitator transporter superfamily conducts the transfer across plasma membranes of a wide variety of organic molecules including sugars, molecules of intermediary metabolism, neurotransmitters, and amino acids.
  • the observation that the highest homology of the Drosophila gene is to C. elegans and bacterial genes suggest a critical solute transport role in these organisms as well.
  • the transporter process may be physiologically relevant to the function of the digestive tract of the insect or nematode, including motility and absorption of the gut or entire alimentary canal, and neuronal and neuroendocrine regulation.
  • the mutation to lethality indicates that drugs which agonize or antagonize the glucose-tetracycline resistance-like transporter may be effective insecticidal agents and this class of transporters represent protein targets for drug screening and discovery.
  • Embryonic RNA in situ expression data shows widespread expression including the central nervous system, strong maternal expression, and strong esophageal and salivary gland expression.
  • nucleic acid and protein sequences were analyzed using the Pfam and Prosite programs This identified a transporter motif at ammo acid residues 24-451 (nucleotides 190-1471), and 12 transmembrane domains at ammo acid residues 50-73, 109-133, 143-161 , 164-186, 203- 223, 233-254, 270-289, 301-319, 341-357, 368-386, 394-413, and 427-450, which conespond to nucleotide residues 445-517, 547-601, 610-676, 727-787, 817-880, 928-985, 1021-1075, 1141-1189, 1222-1276, 1300-1357, and 1399-1468, respectively
  • nucleotide and amino acid sequences of the FT1 nucleic acid sequence and its encoded protem were searched against all available nucleotide and amino acid sequences in the public databases, using BLAST (Altschul et al , supra) Table 3 below summanzes the results In each case, the 5 most similar sequences are listed
  • BLAST results for FT1 ammo acid sequence indicate 10 amino acid residues as the longest stretch of contiguous ammo acids that is novel with respect to public sequences, and 13 amino acids as the longest stretch of contiguous ammo acids for which there are no sequences contained withm public databases sharing 100% sequence similanty Drosophila EST sequence encoding incomplete stretches of the tetracycline transporter-like cDNA open reading frame appeared m public databases, but without functional annotation These include nucleotide residues 2-628 (GenBank AI512438 1), 215-785 (GenBank AA392114), and 723-1325 (GenBank AI513464) which encompass nucleotides 2-1325 of the total 1- 1697 predicted for the full open reading frame.
  • Example 6 Testing of Pesticide Compounds for Activity against Channel Complexes
  • cDNAs encoding FT are cloned into mammalian cell culture-compatible vectors (e.g. pCDNA, Invitrogen, Carlsbad, CA), and the resultant constructs are transiently transfected into mammalian cells.
  • the transiently transfected cell lines are typically used 24 to 48 hours following transfection for electrophysiology studies. Whole cell recordings, using the voltage clamp technique, are taken on the transfected cells versus cells transfected with vector only.
  • Cells are voltage-clamped at B60 mV and continuously superfused with ND96 (96mM NaCl, 2mM KC1, 1.8mM CaCl 2 lmM MgCl 2 , 5mM HEPES, pH 7.5) containing varying concentrations of compounds. Current and fluxes are then measured. Also, cell lines transiently transfected with FT can be assayed for uptake of radioactive or fluorescent tetracycline. In case of radioactive compounds, cells are incubated in 0.5 ⁇ m radioactive ( 3 H-, or 14 C-) tetracycline for 1 hour, washed with saline, and then assayed for compound uptake using a scintillation counter. Appropriate controls are comparison of this uptake to uptake in cells injected with water, or non-injected cells.
  • ND96 96mM NaCl, 2mM KC1, 1.8mM CaCl 2 lmM MgCl 2 , 5m

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Abstract

L'invention concerne des acides nucléiques et des protéines utilisés comme vecteurs de transport (FT), qui ont été isolés de Drosophila melanogaster et sont appelés FT1 et FT2. Les acides nucléiques et les protéines FT peuvent être utilisés pour modifier génétiquement des organismes invertébrés métazoaires, tels que les insectes et les vers, ou des cellules cultivées, ce qui donne lieu à une expression ou une expression erronée de FT. Ces organismes ou cellules modifiés génétiquement peuvent être utilisés dans des essais de criblage pour identifier des composés candidats en tant que agents pesticides ou thérapeutiques potentiels interagissant avec la protéine FT. Ils peuvent également être utilisés dans des méthodes destinées à étudier l'activité de FT et à identifier d'autres gènes modulant la fonction du gène FT ou interagissant avec celui-ci.
PCT/US2000/025224 1999-09-16 2000-09-15 Cibles insecticides mortelles WO2001019857A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1414959A1 (fr) * 2001-07-06 2004-05-06 Commonwealth Scientific And Industrial Research Organisation Administration d'arn bicatenaires a des arthropodes
US9029527B2 (en) 1998-03-20 2015-05-12 Commonwealth Scientific And Industrial Research Organisation Synthetic genes and genetic constructs
US9708621B2 (en) 1999-08-13 2017-07-18 Commonwealth Scientific And Industrial Research Organisation Methods and means for obtaining modified phenotypes
US9963698B2 (en) 1998-03-20 2018-05-08 Commonwealth Scientific And Industrial Research Organisation Control of gene expression

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991013167A1 (fr) * 1990-02-26 1991-09-05 The Board Of Trustees Of The Leland Stanford Junior University Identification et expression de sequences d'adn de recepteurs de steroides d'insectes
WO1994016071A2 (fr) * 1993-01-15 1994-07-21 Massachusetts Institute Of Technology Dosages et genes relatifs a la mort cellulaire
EP0617125A2 (fr) * 1993-03-23 1994-09-28 The General Hospital Corporation Gène pour la protéine de transport provenant de la région de la maladie de Huntinton
WO1996022307A1 (fr) * 1995-01-19 1996-07-25 The Research Foundation Of State University Of New York Genes codant le canal calcique d'un insecte

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991013167A1 (fr) * 1990-02-26 1991-09-05 The Board Of Trustees Of The Leland Stanford Junior University Identification et expression de sequences d'adn de recepteurs de steroides d'insectes
WO1994016071A2 (fr) * 1993-01-15 1994-07-21 Massachusetts Institute Of Technology Dosages et genes relatifs a la mort cellulaire
EP0617125A2 (fr) * 1993-03-23 1994-09-28 The General Hospital Corporation Gène pour la protéine de transport provenant de la région de la maladie de Huntinton
WO1996022307A1 (fr) * 1995-01-19 1996-07-25 The Research Foundation Of State University Of New York Genes codant le canal calcique d'un insecte

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ADAMS M D ET AL: " InterPro; IPR003662; sub_transporter. SUGAR_TRANSPORT_1;" SWALL EMBL :Q9V395, 1 May 2000 (2000-05-01), XP002177185 http://srs6.ebi.ac.uk/srs6bin/cgi-bin/wget z?-e+Äswall-id:Q9V395Ü & ADAMS MD ET AL.: "The genome sequence of Drosophila melanogaster." SCIENCE 2000 MAR 24;287(5461):2185-95, *
BATEMAN A, BIRNEY E, DURBIN R, EDDY SR, FINN RD, SONNHAMMER EL.: "Pfam 3.1: 1313 multiple alignments and profile HMMs match the majority of proteins." NUCLEIC ACIDS RES. 1999 JAN 1;27(1):260-2., XP001023651 cited in the application *
ESCHER SA, RASMUSON-LESTANDER A.: "The Drosophila glucose transporter gene: cDNA sequence, phylogenetic comparisons, analysis of functional sites and secondary structures." HEREDITAS., vol. 130, no. 2, May 1999 (1999-05), pages 95-103, XP001023730 lund, sweden *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9029527B2 (en) 1998-03-20 2015-05-12 Commonwealth Scientific And Industrial Research Organisation Synthetic genes and genetic constructs
US9963698B2 (en) 1998-03-20 2018-05-08 Commonwealth Scientific And Industrial Research Organisation Control of gene expression
US10190127B2 (en) 1999-08-13 2019-01-29 Commonwealth Scientific And Industrial Research Organisation Methods and means for obtaining modified phenotypes
US9708621B2 (en) 1999-08-13 2017-07-18 Commonwealth Scientific And Industrial Research Organisation Methods and means for obtaining modified phenotypes
US8877727B2 (en) 2001-07-06 2014-11-04 Commonwealth Scientific And Industrial Research Organisation Delivery of dsRNA to arthropods
US8415320B2 (en) 2001-07-06 2013-04-09 Commonwealth Scientific And Industrial Research Organisation Delivery of dsRNA to arthropods
EP1414959A1 (fr) * 2001-07-06 2004-05-06 Commonwealth Scientific And Industrial Research Organisation Administration d'arn bicatenaires a des arthropodes
US8263573B2 (en) 2001-07-06 2012-09-11 Commonwealth Scientific And Industrial Research Organisation Delivery of dsRNA to arthropods
US9085770B2 (en) 2001-07-06 2015-07-21 Commonwealth Scientific And Industrial Research Organisation Delivery of dsRNA to arthropods
US9663786B2 (en) 2001-07-06 2017-05-30 Commonwealth Scientific And Industrial Research Organisation Delivery of dsRNA to arthropods
US8101343B2 (en) 2001-07-06 2012-01-24 Commonwealth Scientific And Industrial Research Organisation Delivery of dsRNA to arthropods
EP2333061A1 (fr) * 2001-07-06 2011-06-15 Commonwealth Scientific and Industrial Research Organization Administration d'ARN double brin a des arthropodes
EP1414959A4 (fr) * 2001-07-06 2006-06-14 Commw Scient Ind Res Org Administration d'arn bicatenaires a des arthropodes
US10323245B2 (en) 2001-07-06 2019-06-18 Commonwealth Scientific And Industrial Research Organisation Delivery of dsRNA to arthropods

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