WO2001038359A2 - Acides nucleiques et polypeptides de recepteurs provenant d'invertebres et methodes d'utilisation - Google Patents

Acides nucleiques et polypeptides de recepteurs provenant d'invertebres et methodes d'utilisation Download PDF

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WO2001038359A2
WO2001038359A2 PCT/US2000/032816 US0032816W WO0138359A2 WO 2001038359 A2 WO2001038359 A2 WO 2001038359A2 US 0032816 W US0032816 W US 0032816W WO 0138359 A2 WO0138359 A2 WO 0138359A2
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protein
nucleic acid
seq
invertebrate
gene
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WO2001038359A3 (fr
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Allen James Ebens, Jr.
Helen Francis-Lang
Kevin Patrick Keegan
Thomas J. Stout
Kathryn A. Kellerman
Justin Torpey
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Genoptera, Llc
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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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)

Definitions

  • This application is in the field of invertebrate genes and proteins, and in particular invertebrate genes encoding receptors.
  • VDCCs Voltage-dependent Ca2 + channels
  • VDCCs are heteromultimers composed of at least three subunits: a pore-forming transmembrane alpha(l) subunit, a hydrophilic intracellular beta subunit, and a membrane-associated alpha(2)/delta subunit.
  • the alpha(2)/delta subunit is derived from a single polypeptide that is cleaved to form disulfide-bridged alpha(2) and delta peptides, both of which are glycosylated (Jay et al, J Biol Chem (1991) 266:3267-3293).
  • the alpha(2)/delta subunit appears to increase alpha(l) currents by both facilitating the assembly of alpha(l) subunits at the cell surface (Brust et al. Neuropharmacology (1993) 32:1089-1102), and by stimulating the peak alpha(l) current (Mikami et al, Nature (1989) 350:398-402).
  • the human malignant hyperthermia susceptibility-3 disorder may result from mutations in the human gene CACNL2A gene that encodes an alpha-2/delta-subunit of human VDCC (Ikes et al, Hum. Molec. Genet. 3: 969-975).
  • Chloride channels perform important roles in the regulation of cellular excitability, transepithelial transport, cell volume regulation, and acidification of intracellular organelles. Many different chloride channels are encoded by genes belonging to several unrelated gene families.
  • the CIC family of chloride channels has nine known members in mammals that show a differential tissue distribution and function both in plasma membranes and intracellular organelles. CIC proteins typically have about 10-12 transmembrane domains. They probably function as dimers and may have one or two pores.
  • the functional expression of channels altered by site-directed mutagenesis has led to important insights into their structure-function relationship.
  • the human inherited diseases Myotonia congenita, Dent's disease and Bartter's syndrome have all been linked to mutations in CIC proteins. Studies of CIC in mice further demonstrate the physiological importance of these proteins (Jentsch et al, Pflugers Arch. (1999) 437(6):783-795).
  • Glutamate receptors are the major excitatory transmitter system in vertebrate brain, playing important roles in learning and memory. They are also important pesticide targets in invertebrates. Based on pharmacological and electrophysiological data, there are three ionotropic subclasses, kainate (KA), AMP A, NMDA, and one apparently unrelated family of G protein-coupled glutamate receptors.
  • the KA receptors can further be classified into two groups, the closely related low-affinity KA receptors (GluR5, GluR6 and GluR7) and the high affinity KA receptors (KA1 and KA2; Hollman and Heinemann, Annu. Rev. Neurosci., (1994) 17:31-108).
  • GluRi and GluR6 Two of the low-affinity KA receptors, GluRi and GluR6, are implicated in human disease.
  • Allelic variants of the GluRi gene are a major genetic determinant in the pathogenesis of juvenile absence epilepsy, a common subtype of idiopathic generalized epilepsy (Sander et al, Am. J. Med. Genet. (1997) 74:416-421).
  • Allelic variants of the GluR6 gene contribute to age of onset of Huntington disease (Rubensztein et al, Proc Nat. Acad. Sci. (1997) 94:3872-3876).
  • Glutamate receptors are molecular targets for pesticides in invertebrates.
  • Avermectins are used against gut parasitic nematodes and the nematodes that cause river blindness in humans (Blaxter and Bird, Ch. 30 in C. elegans II (1997), Riddle et al. eds., Cold Spring Harbor Laboratory Press).
  • nematode Caenorhabditis elegans it has been shown that avermectins act most strongly through glutamate receptors, and populations of treated animals give rise to resistant populations at a high frequency.
  • RNA editing varies with the tissue; this may be a mechanism for regulating channel activity in these tissues.
  • Neuronal nicotinic acetylcholine receptors are pentameric ligand-gated ion channel receptors (Lloyd et al, Life Sciences (1998) 62:1601-1606). Neuronal NAChRs are assembled in a combination of alpha and/or beta subunits which are different in composition from the muscle nicotinic AChR (alphal betal gamma delta) or (alphal betal gamma epsilon).
  • NAChRs exist as multiple subtypes (heteromeric and homomeric combinations) serving different physiological functions in different parts of the brain (Lloyd, et al, supra).
  • the functional diversity of the myriad of combinations includes agonist/antagonist specificity, ion selectivity, ion conductance, and mean channel open time (Boyd, Critical Reviews in Toxicology ( 1997) 27(3):299-318).
  • a neuronal NAChR encodes a protein with an amino terminal extracellular domain of about 200 amino acids, four hydrophobic putative transmembrane domains, and an extracellular C- terminal domain (Boyd, supra). Each alpha and beta subunit in this family contains two cysteines 13 amino acids apart (Boyd, supra). The alpha subunits are the most conserved between vertebrates and invertebrates (Tornoe et al, Toxicon (1995) 33(4):411-424).
  • Neurotoxins such as snake venom bungarotoxin
  • AChRs AChRs
  • Common agonists include nicotine, which demonstrates activity in models for cognition as well as analgesia (Lloyd et al, supra; Girod et al, Annals New York Academy of Science (1999) 868:578-590).
  • Epibatidine a recently discovered neurotoxin from the nose of the South American poison-dart frog, is an agonist with potent analgesic activity and deadly side-effects (Lloyd et al, supra).
  • NAChR agonists are not clinically useful, but they illustrate the broad spectrum of physiological effects elicited by NAChR agonists.
  • the loss of cortical NAChRs is a neurochemical hallmark of Alzheimer's and Parkinson's diseases (Vidal, Molecular and Chemical Neuropathology (1996) 28:3-11). It has been shown that nicotine improves memory and attention in Alzheimer's and Parkinson's diseases, suggesting a potent agonist may alleviate symptoms of the disease.
  • small molecule agonists have potential for the treatment of the motor and cognitive deficits of Parkinson's and Alzheimer's disease (Lloyd et al, supra).
  • 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.
  • the present invention addresses this need by providing novel pesticide targets that are receptor proteins from invertebrates such as the fruit fly Drosophila melanogaster, and by providing methods of identifying compounds that bind to and modulate the activity of such receptors .
  • the invertebrate receptor is an ion channel.
  • the invertebrate ion channel is a chloride ion channel.
  • the invertebrate ion channel is a voltage-dependent calcium channel.
  • the invertebrate receptor is a receptor for a neurotransmitter.
  • the neurotransmitter receptor is a glutamate receptor.
  • One particular glutamate receptor is referred to herein as dmKGLUR.
  • the neurotransmitter receptor is an acetylcholine receptor.
  • dmACHRl and dmACHR4 Particular acetylcholine receptors are referred to herein as dmACHRl and dmACHR4.
  • the invertebrate receptors described herein can be used in genetic screening methods to characterize pathways that such receptors may be involved in, as well as other interacting genetic pathways. It is also an object of the invention to provide methods for screening compounds that interact with an invertebrate receptor of the invention such as those that may have utility as therapeutics or pesticides. These and other objects are provided by the present invention which concerns the identification and characterization of novel invertebrate receptor genes. In particular, invertebrate receptor genes from Drosophila melanogaster are described.
  • Isolated nucleic acid molecules are provided that comprise nucleic acid sequences encoding invertebrate receptor proteins as well as novel fragments and derivatives 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 one or more biological activities of an invertebrate receptor described herein.
  • Vectors and host cells comprising the invertebrate receptor 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 invertebrate receptor protein.
  • novel invertebrate receptor nucleic acids and proteins can be used in screening assays to identify candidate compounds which are potential pesticidal agents or therapeutics that interact with invertebrate receptor proteins.
  • Such assays typically comprise contacting an invertebrate receptor protein or fragment with one or more candidate molecules, and detecting any interaction between the candidate compound and the invertebrate receptor protein.
  • the assays may comprise adding the candidate molecules to cultures of cells genetically engineered to express invertebrate receptor proteins, or alternatively, administering the candidate compound to a metazoan invertebrate organism genetically engineered to express invertebrate receptor protein.
  • the genetically engineered metazoan invertebrate animals of the invention can also be used in methods for studying invertebrate receptor activity. These methods typically involve detecting the phenotype caused by the expression or mis-expression of the invertebrate receptor 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 invertebrate receptor protein.
  • Drosophila melanogaster Drosophila melanogaster
  • An extensive search for acetylcholine receptor nucleic acids and their encoded proteins in Drosophila was conducted in an attempt to identify new and useful tools for probing the function and regulation of acetylcholine receptor genes, and for use as targets in pesticide and drug discovery.
  • Novel invertebrate receptor nucleic acid and its encoded protein are identified herein.
  • the newly identified invertebrate receptor nucleic acid can be used for the generation of mutant phenotypes in animal models or in living cells that can be used to study regulation of invertebrate receptor, and the use of invertebrate receptor as a pesticide or drug target. 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 an invertebrate receptor protein. Thus, the invention provides a superior approach for identifying other components involved in the synthesis, activity, and regulation ofan invertebrate receptor proteins.
  • Model organisms or cultured cells that have been genetically engineered to express an invertebrate receptor can be used to screen candidate compounds for their ability to modulate an invertebrate receptor expression or activity, and thus are useful in the identification of new drug targets, therapeutic agents, diagnostics and prognostics useful in the treatment of neurodegenerative disorders. Additionally, these invertebrate model organisms can be used for the identification and screening of pesticide targets directed to components of a invertebrate receptor pathway.
  • the term “isolated” is meant to describe a polynucleotide, a polypeptide, an antibody, or a host cell that is in an environment different from that in which the polynucleotide, the polypeptide, the antibody, or the host cell naturally occurs.
  • the term “substantially purified” refers to a compound (e.g., either a polynucleotide or a polypeptide or an antibody) that is removed from its natural environment and is at least 60% free, preferably 75% free, and most preferably 90% free from other components with which it is naturally associated.
  • a “host cell”, as used herein, denotes microorganisms or eukaryotic cells or cell lines cultured as unicellular entities which can be, or have been, used as recipients for recombinant vectors or other transfer polynucleotides, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. By “transformation” is meant a permanent or transient genetic change induced in a cell following incorporation of new DNA (i.e., DNA exogenous to the cell).
  • Genetic change can be accomplished either by incorporation of the new DNA into the genome of the host cell, or by transient or stable maintenance of the new DNA as an episomal element. Where the cell is a eukaryotic cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell. Isolated nucleic acid molecules of the invention
  • the present invention provides isolated nucleic acid molecules that comprise nucleotide sequences encoding invertebrate receptors.
  • the isolated nucleic acid molecules have a variety of uses, e.g., as hybridization probes, e.g., to identify nucleic acid molecules that share nucleotide sequence identity; in expression vectors to produce the polypeptides encoded by the nucleic acid molecules; and to modify a host cell or animal for use in assays described hereinbelow.
  • 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.
  • polynucleotide and nucleic acid used interchangeably herein, refer to a polymeric forms of nucleotides of any length, either ribonucleotides or deoxynucleotides.
  • this tem includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • the backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups.
  • the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidites and thus can be an oligodeoxynucleoside phosphoramidate or a mixed phosphoramidate-phosphodiester oligomer.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars, and linking groups such as fluororibose and thioate, and nucleotide branches.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support.
  • nucleic acid analogs For hybridization probes, it may be desirable to use nucleic acid analogs, in order to improve the stability and and binding affinity.
  • a number of modifications have been described that alter the chemistry of the phosphodiester backbone, sugars or heterocyclic bases. Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates.
  • Achiral phosphate derivatives include 3'-0'-5'-S- phosphorothioate, 3'-S-5'-0-phosphorothioate, 3'-CH2-5'-0-phosphonate and 3'-NH-5'-0- phosphoroamidate.
  • Peptide nucleic acids replace the entire phosphodiester backbone with a peptide linkage.
  • Sugar modifications are also used to enhance stability and affinity.
  • the a-anomer of deoxyribose may be used, where the base is inverted with respect to the natural b-anomer.
  • the 2'-OH of the ribose sugar may be altered to form 2'-0-methyl or 2'-0-allyl sugars, which provides resistance to degradation without comprising affinity.
  • Modification of the heterocyclic bases must maintain proper base pairing.
  • Some useful substitutions include deoxyuridine for deoxythymidine; 5-methyl-2'- deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine.
  • 5- propynyl-2'-deoxyuridine and 5- propynyl-2'-deoxycytidine have been shown to increase affinity and biological activity when substituted for deoxythymidine and deoxycytidine, respectively.
  • Derivative nucleic acid sequences of the subject nucleic acid molecules include sequences that hybridize to the nucleic acid sequence of any one of SEQ ID NOS: 1, 3, 5, 7, or 9 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.
  • Modely 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% 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 Protocols in Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)).
  • a preferred derivative nucleic acid is capable of hybridizing to SEQ ID NO:l 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% SDS (sodium dodecyl sulfate). Fragments of the subject nucleic acid molecules can be used for a variety of purposes. Interfering
  • RNA (RNAi) fragments can be used to generate loss-of-function phenotypes, or to formulate biopesticides (discussed further below). Fragments of the subject nucleic acid molecules 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 the subject nucleic acid sequences have utility in inhibiting the function of proteins encoded by the subject nucleic acid molecules. The fragments are of length sufficient to specifically hybridize with the corresponding subject nucleic acid molecule.
  • the fragments generally consist of or comprise at least 12, preferably at least 24, more preferably at least 36, and more preferably at least 96 contiguous nucleotides of a subject nucleic acid molecule.
  • the total length of the combined nucleic acid sequence is less than 15 kb, preferably less than 10 kb or less than 5kb, more preferably less than 2 kb, and in some cases, preferably less than 500 bases.
  • 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 that have at least about 70% sequence identity with one of SEQ ID NOS: 1, 3, 5, 7, or 9 are capable of hybridizing to one of SEQ ID NOS: 1, 3, 5, 7, or 9 under moderately stringent conditions that comprise: pretreatment of filters containing nucleic acid for 6 hours 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% BSA, 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
  • Other preferred derivative nucleic acid sequences are capable of hybridizing to one of SEQ ID NOS: 1, 3, 5, 7, or 9 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.
  • Another type of derivative of the subj ect nucleic acid sequences includes corresponding humanized sequences.
  • a humanized nucleic acid sequence is one in which one or more codons has 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.
  • nucleic acid derivatives can be generated with codon usage optimized for expression in other organisms, such as yeasts, bacteria, and plants, where it is desired to engineer the expression of receptor proteins by using specific codons chosen according to the preferred codons used in highly expressed genes in each organism.
  • a dmACHRl 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 dmACHRl nucleic acid sequence.
  • a detailed discussion of the humanization of nucleic acid sequences is provided in U.S. Pat. No. 5,874,304 to Zolotukhin et al.
  • a derivative invertebrate receptor nucleic acid sequence, or fragment thereof may comprise 100% sequence identity with any one of SEQ ID NOS: 1, 3, 5, 7, or 9 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.
  • the invention provides nucleic acid sequences of acetylcholine receptors, and more particularly acetylcholine receptor nucleic acid sequences of Drosophila, hereinafter referred to as dmACHRl, and methods of using these sequences.
  • dmACHRl acetylcholine receptor nucleic acid sequences of Drosophila
  • SEQ ID NO: 1 a nucleic acid sequence (SEQ ID NO: 1) was isolated from Drosophila that encodes an acetylcholine receptor homolog.
  • the invention includes the reverse complements thereof.
  • the subject nucleic acid sequences, derivatives and fragments thereof may be RNA molecules comprising the nucleotide sequence of SEQ ID NO: 1 (or derivative or fragment thereof) wherein the base U (uracil) is substituted for the base T (thymine).
  • a dmACHRl nucleic acid molecule comprises at least about 375, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, or at least about 1500 contiguous nucleotides of the sequence set forth in SEQ ID NO: 1.
  • a dmACHRl nucleic acid molecule comprises a nucleotide sequence that encodes at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 325, at least about 350, or at least about 375 contiguous amino acids of the sequence set forth in SEQ ID NO:2, up to the entire sequence of SEQ ID NO:2.
  • SEQ ID NO: 1 encodes an extracellular domain at approximately nucleotides 81 to 314; and 4 transmembrane domains which are located at approximately nucleotides 315- 363, 396-446, 498-549, and 1095-1134, respectively.
  • Derivative dmACHRl nucleic acid sequences usually have at least 70% sequence identity, preferably at least 80% 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 : 1 , or domain-encoding regions thereof.
  • the derivative nucleic acid encodes a polypeptide comprising the dmACHRl amino acid sequence of SEQ ID NO:2, or a fragment or derivative thereof as described further below under the subheading "dmACHRl proteins”.
  • dmACHRl protein fragments and derivatives are discussed further below in connection with specific dmACHRl proteins.
  • the invention provides nucleic acid sequences of acetylcholine receptors, and more particularly acetylcholine receptor nucleic acid sequences oi Drosophila, hereinafter referred to as dmACHR4, and methods of using these sequences.
  • dmACHR4 acetylcholine receptor nucleic acid sequences oi Drosophila
  • dmACHR4 acetylcholine receptor nucleic acid sequences oi Drosophila
  • the subject nucleic acid sequences, derivatives and fragments thereof may be RNA molecules comprising the nucleotide sequence of SEQ ID NO:3 (or derivative or fragment thereof) wherein the base U (uracil) is substituted for the base T (thymine).
  • a dmACHR4 nucleic acid molecule comprises at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, or at least about 1900 contiguous nucleotides of the sequence set forth in SEQ ID NO:3, up to the entire sequence of SEQ ID NO:3.
  • a dmACHR4 nucleic acid molecule comprises a nucleotide sequence that encodes at least about 25, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 325, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, or at least about 575 contiguous amino acids of the sequence set forth in SEQ ID NO:4, up to the entire sequence of SEQ ID NO:4. Additional preferred fragments of SEQ ID NO : 3 encode extracellular or intracellular domains, which are located at approximately nucleotides 126-1355, 1407-1427, 1479-1526, 1578-1730, and 1781- 1799.
  • Derivative dmACHR4 nucleic acid sequences usually have at least 70% sequence identity, preferably at least 80% 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:3, or domain-encoding regions thereof.
  • the derivative nucleic acid encodes a polypeptide comprising a dmACHR4 amino acid sequence of SEQ ID NO:4, or a fragment or derivative thereof as described further below under the subheading "dmACHR4 proteins".
  • the invention provides kinate glutamate receptors nucleic acids, and more particularly kinate glutamate receptor nucleic acid sequences of Drosophila, and methods of using these sequences.
  • a nucleic acid sequence (SEQ ID NO:5) was isolated from Drosophila that encodes a kinate glutamate receptor homolog, hereinafter referred to as dmKGLUR.
  • the invention includes the reverse complements thereof.
  • the subject nucleic acid sequences, derivatives and fragments thereof may be RNA molecules comprising the nucleotide sequence of SEQ ID NO: 1 (or derivative or fragment thereof) wherein the base U (uracil) is substituted for the base T (thymine).
  • a dmKGLUR nucleic acid molecule comprises at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, at least about 2100, at least about 2200, at least about 2300, at least about 2400, at least about 2500, at least about 2600, at least about 2700, at least about 2800, at least about 2900, at least about 3000, at least about 3100, at least
  • a dmKGLUR nucleic acid molecule comprises a nucleotide sequence that encodes at least about 25, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 325, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, or at least about 800 contiguous amino acids of the sequence set forth in SEQ ID NO:6, up to the entire sequence of SEQ ID NO:6.
  • Additional preferred fragments of SEQ ID NO:5 encode extracellular or intracellular domains, which are located at approximately nucleotides 221-1630, 1709-1759, 1823-1885, 1949-1978, and 2042- 2600.
  • Derivative dmKGLUR nucleic acid sequences usually have at least 70% sequence identity, preferably at least 80% 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:5.
  • the derivative nucleic acid encodes a polypeptide comprising a dmKGLUR amino acid sequence of SEQ ID NO: 6, or a fragment or derivative thereof as described further below under the subheading "dmKGLUR proteins”.
  • the invention provides nucleic acid sequences of chloride channels, and more particularly chloride channel nucleic acid sequences of Drosophila, and methods of using these sequences.
  • a nucleic acid sequence SEQ ID NO:7 was isolated from Drosophila that encodes a chloride channel homolog, hereinafter referred to as dmCLC.
  • the invention includes the reverse complements thereof.
  • the subject nucleic acid sequences, derivatives and fragments thereof may be RNA molecules comprising the nucleotide sequence of SEQ ID NO:7 (or derivative or fragment thereof) wherein the base U (uracil) is substituted for the base T (thymine).
  • a dmCLC nucleic acid molecule comprises at least about 50, at least about
  • a dmCLC nucleic acid molecule comprises a nucleotide sequence that encodes at least about 25, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 325, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, or at least about 850 contiguous amino acids of the sequence set forth in SEQ ID
  • Additional preferred fragments of SEQ ID NO:7 encode extracellular or intracellular domains, which are located at approximately nucleotides 1-336, 400-552, 616-792, 856-951, 1015-1019, 1084- 1122, 1186-1224, 1288-1335, 1399-1452, 1516-1641, 1735-1740, 1904-1869, 1933-1959, 2023-2331, 2395-2622.
  • Derivative dmCLC nucleic acid sequences usually have at least 70% sequence identity, preferably at least 80% 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:7, or domain-encoding regions thereof.
  • the derivative nucleic acid encodes a polypeptide comprising a dmCLC amino acid sequence of SEQ ID NO: 8, or a fragment or derivative thereof as described further below under the subheading "dmCLC proteins".
  • the invention provides nucleic acid sequences of calcium channels, and more particularly calcium channels nucleic acid sequences of Drosophila, and methods of using these sequences.
  • a nucleic acid sequence SEQ ID NO:9 was isolated from Drosophila that encodes a calcium channel alpha(2)/delta subunit homolog, hereinafter referred to as dmCACH.
  • the invention includes the reverse complements thereof.
  • the subject nucleic acid sequences, derivatives and fragments thereof may be RNA molecules comprising the nucleotide sequence of SEQ ID NO:9 (or derivative or fragment thereof) wherein the base U (uracil) is substituted for the base T (thymine).
  • a dmCACH nucleic acid molecule comprises at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, at least about 2100, at least about 2200, at least about 2300, at least about 2400, at least about 2500, at least about 2600, at least about 2700, at least about 2800, at least about 2900, at least about 3000, at least about 3100, at least about
  • a dmCACH nucleic acid molecule comprises a nucleotide sequence that encodes at least about 25, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 325, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, at least about 1000, at least about 1050, at least about 1100, at least about 1150, or at least about 1200 contiguous amino acids of the sequence set forth in SEQ ID NO: 10, up to the entire sequence of SEQ ID NO: 10.
  • VWA von Willebrand factor type A
  • Derivative dmCACH nucleic acid sequences usually have at least 70% sequence identity, preferably at least 80% 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 : 9, or domain-encoding regions thereof.
  • the derivative nucleic acid encodes a polypeptide comprising a dmCACH amino acid sequence of SEQ ID NO: 10, or a fragment or derivative thereof as described further below under the subheading "dmCACH proteins".
  • the present invention further provides recombinant vectors ("constructs") comprising polynucleotides of the invention.
  • Recombinant vectors are useful for propagation of the subject polynucleotides (cloning vectors). They are also useful for effecting expression of a polynucleotide in a cell (expression vectors). Some vectors accomplish both cloning and expression functions. The choice of appropriate vector is well within the skill of the art. Many such vectors are available commercially.
  • host-vector systems may be utilized to propagate and/or express a polynucleotide of the invention.
  • Such host-vector systems represent vehicles by which coding sequences of interest may be produced and subsequently purified, and also represent cells that may, when transformed or transfected with the appropriate nucleotide coding sequences, produce a polypeptide of the invention.
  • These include, but are not limited to, microorganisms (e.g., E. coli, B.
  • subtilis transformed with recombinant bacteriophage vectors, plasmid DNA, or cosmid DNA vectors comprising a subject polynucleotide; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast vectors comprising a subject polynucleotide); insect cell systems (e.g., Spodoptera frugiperd ⁇ ) infected with recombinant virus expression vectors (e.g., baculovirus vectors, many of which are commercially available.
  • yeast e.g., Saccharomyces, Pichia
  • insect cell systems e.g., Spodoptera frugiperd ⁇
  • virus expression vectors e.g., baculovirus vectors, many of which are commercially available.
  • the present invention further provides host cells, which may be isolated host cells, comprising a polynucleotide or a vector of the invention.
  • Suitable host cells include prokaryotes such as E. coli, B. subtilis, eukaryotes, including insect cells in combination with baculovirus vectors, yeast cells, such as Saccharomyces cerevisiae, nematode cells, worm cells, or cells of a higher organism such as vertebrates, and the like, may be used as the expression host cells.
  • Host cells are described in more detail hereinbelow.
  • a subject nucleic acid molecule or fragment or derivative thereof may be obtained from an appropriate cDNA library prepared from any eukaryotic species that encodes a subject 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. For example, 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 Polymerase 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 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 the subject 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 dmACHRl nucleic acid sequences. Newly generated sequences may be identified and isolated using standard methods.
  • a subject nucleic acid molecule 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 dmACHRl 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.
  • the nucleotide sequence encoding a subject 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 dmACHRl gene and/or its flanking regions.
  • 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.
  • the expression vector can comprise a promoter operably linked to a subject gene nucleic acid, one or more origins of replication, and, one or more selectable markers (e.g. thymidine kinase activity, resistance to antibiotics, etc.).
  • recombinant expression vectors can be identified by assaying for the expression of the subject gene product based on the physical or functional properties of the subject protein in in vitro assay systems (e.g. immunoassays).
  • a subject 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. Once a recombinant cell that expresses a subject gene sequence is identified, the gene product can be isolated and purified using standard methods (e.g.
  • 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 o/., Nature (1984) 310:105- 111).
  • native protems can be purified from natural sources, by standard methods (e.g. immunoaffinity purification).
  • polypeptides of the invention provides isolated invertebrate receptor polypeptides.
  • the invertebrate receptor polypeptides are neuronal receptor polypeptides.
  • the subject polypeptides are useful in various assays, e.g., assays to detect compounds that interact with the subject polypeptides.
  • compositions comprising a subject polypeptide.
  • Compositions comprising these proteins may consist essentially of a subject protein, fragments, or derivatives, or may comprise additional components (e.g. pharmaceutically acceptable carriers or excipients, culture media, carriers used in pesticide formulations, etc.).
  • “An invertebrate receptor polypeptide” encompasses polypeptides that share a certain degree of sequence identity or sequence similarity with any one of SEQ ID NOS:2, 4, 6, 8, or 10, 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 aspartic acid and glutamic acid; and interchangeable small amino acids alanine, serine, threonine, cysteine, and glycine.
  • the fragment or derivative of a subject protein is preferably "functionally active" meaning that the subject protein derivative or fragment exhibits one or more functional activities associated with a full- length, wild-type subject protein comprising the amino acid sequence of one of SEQ ID NOS:2, 4, 6, 8, or 10.
  • a fragment or derivative may have antigenicity such that it can be used in immunoassays, for immunization, for inhibition of activity of a subject protein, etc, as discussed further below regarding generation of antibodies to a subject protein.
  • a functionally active fragment or derivative of a subject protein is one that displays one or more biological activities associated with a subject protein, such as receptor activity.
  • functionally active fragments also include those fragments that exhibit one or more structural features of a subject protein, such as transmembrane domains.
  • the functional activity of the subject 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).
  • 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 of a subject protein).
  • Derivatives of the subject proteins can be produced by various methods known in the art.
  • a cloned gene sequence comprising a nucleotide sequence encoding a subject protein 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.
  • a 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 variety 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 carried out by known technique (e.g. specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBEL t , acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.).
  • Derivative 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 subject protein sequence.
  • Chimeric or fusion proteins can be made comprising a subject protein or fragment thereof (preferably comprising one or more structural or functional domains of the subject protein) joined at its amino- or carboxy-terminus 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 protein (comprising a coding sequence encoding a subject protein, which coding sequence is joined in-frame to a coding sequence for a different protein); ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other in the proper coding frame, and expressing the chimeric product; and protein synthetic techniques, e.g. by use of a peptide synthesizer.
  • proteins of the invention comprise or consist of an amino acid sequence of SEQ ID NO:2, or fragments or derivatives thereof
  • a dmACHRl protein is a polypeptide comprising at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 325, at least about 350, or at least about 375 contiguous amino acids of the sequence set forth in SEQ ID NO:2, up to the entire sequence of SEQ ID NO:2.
  • a dmACHRl protein derivative shares at least 80% sequence identity or similarity, preferably at least 85%, 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.
  • the dmACHRl protein derivative may consist of or comprise a sequence that shares 100% similarity with any contiguous stretch of at least 125 amino acids, preferably at least 127 amino acids, more preferably at least 130 amino acids, and most preferably at least 140 amino acids of SEQ ID NO:2.
  • Preferred derivatives of dmACHRl consist of or comprise an amino acid sequence that has at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity or sequence similarity with amino acid residues 1 to 78, which is an extracellular domain.
  • the derivative dmACHRl protein has the biological activity of a dmACHRl protein.
  • Preferred fragments of dmACHRl proteins consist or comprise at least 125, preferably at least 127, more preferably at least 130, and most preferably at least 135 contiguous amino acids of SEQ ID NO:2.
  • proteins of the invention comprise or consist of an amino acid sequence of
  • compositions comprising these proteins may consist essentially of the dmACHR4 protein, fragments, or derivatives, or may comprise additional components (e.g. pharmaceutically acceptable carriers or excipients, culture media, carriers used in pesticide formulations, etc.).
  • a protein of the invention comprises an amino acid sequence of at least about 25, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about
  • a dmACHR4 protein derivative shares at least 80% sequence identity or similarity, preferably at least 85%, 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:4.
  • the dmACHR4 protein derivative or fragment may consist of or comprise a sequence that shares 100% similarity or identity with any contiguous stretch of at least 6 amino acids, preferably at least 8 amino acids, more preferably at least 10 amino acids, still more preferably at least 13 amino acids, and most preferably at least 18 amino acids of SEQ ID NO:4.
  • Preferred derivatives of dmACHR4 consist of or comprise an amino acid sequence that has at least 75%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity or sequence similarity with any of amino acid residues 28-437, 455-461, 479-494, 512-562, and 580-585, which are likely extracellular or intracellular domains.
  • proteins of the invention comprise or consist of an amino acid sequence of
  • compositions comprising these proteins may consist essentially of the dmKGLUR protein, fragments, or derivatives, or may comprise additional components (e.g. pharmaceutically acceptable carriers or excipients, culture media, carriers used in pesticide formulations, etc.).
  • a protein of the invention comprises an amino acid sequence of at least about 25, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 325, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, or at least about 800 contiguous amino acids of the sequence set forth in SEQ ID NO: 6, up to the entire sequence of
  • a dmKGLUR protein derivative shares at least 70% sequence identity or similarity, more preferably at least 80%, still more preferably at least 85%, 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:6.
  • regions of SEQ ID NO:6 from which the dmKGLUR protein derivative shares this homology reside in residues 30-504, 166-504 and 637-822 of SEQ ID NO:5, or any other domain-encoding regions thereof.
  • the dmKGLUR protein derivative may consist of or comprise a sequence that shares 100% similarity with any contiguous stretch of at least 19 amino acids, preferably at least 21 amino acids, more preferably at least 24amino acids, and most preferably at least 29 amino acids of SEQ ID NO:6, or residues 166 to 822 thereof.
  • Preferred fragments of dmKGLUR 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:6, or residuses 166 to 822 thereof.
  • proteins of the invention comprise or consist of an amino acid sequence of SEQ ID NO:2, or fragments or derivatives thereof.
  • Compositions comprising these proteins may consist essentially of the dmCLC protein, fragments, or derivatives, or may comprise additional components (e.g. pharmaceutically acceptable carriers or excipients, culture media, carriers used in pesticide formulations, etc.).
  • a protein of the invention comprises an amino acid sequence of at least about 25, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 325, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about
  • a dmCLC protein derivative shares at least 75% sequence identity or similarity, 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, still more preferably at least 200 amino acids, and in some cases, the entire length of SEQ ID NO:8.
  • Preferred derivatives of dmCLC consist of or comprise an amino acid sequence that has the above-listed % sequence identity or similarity with any of amino acid residues 1-112, 134-184, 206-264, 286-317, 506-557, 675-777, and 799-874, which are extracellular or intracellular domains.
  • the dmCLC protein derivative may consist of or comprise a sequence that shares 100% similarity with any contiguous stretch of at least 39 amino acids, preferably at least 41 amino acids, more preferably at least 44 amino acids, and most preferably at least 49 amino acids of SEQ ID NO:8.
  • Another preferred derivative of dmCLC protein consists of or comprises a sequence of at least 22 amino acids that share 100% similarity with an equivalent number of contiguous amino acids of residues 713-873 of SEQ ID NO:8.
  • Preferred fragments of dmCLC proteins consist or comprise at least 34, preferably at least 36, more preferably at least 39, and most preferably at least 44 contiguous amino acids of SEQ ID NO:8.
  • Other preferred fragments include any 12 contiguous amino acids, preferably any 22 contiguous amino acids, and more preferably any 62 contiguous amino acids of residues 713-873 of SEQ ID NO:8.
  • proteins of the invention comprise or consist of an amino acid sequence of
  • compositions comprising these proteins may consist essentially of the dmCACH protein, fragments, or derivatives, or may comprise additional components (e.g. pharmaceutically acceptable carriers or excipients, culture media, carriers used in pesticide formulations, etc.).
  • a polypeptide of the invention comprises an amino acid sequence of at least about 25, at least about 50, at least about 75, at least about 100, at least about 125, at least about
  • a dmCACH protein derivative shares at least 70% sequence identity or similarity, preferably at least 75%, more preferably at least 80%, still more preferably at least 85% or 90% sequence identity or similarity 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, more preferably at least 200 amino acids, still more preferably at least 500 amino acids, and in some cases, the entire length of SEQ ID NO: 10.
  • Further preferred derivatives of dmCACH consist of or comprise an amino acid sequence that has the above-listed sequence identities or similarities with amino acid residues 281 -467, which is the VWA protein fold domain.
  • the dmCACH 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: 10.
  • Preferred fragments of dmCACH proteins consist or comprise at least 8, preferably at least 10, more preferably at least 13, and most preferably at least 18 contiguous amino acids of SEQ ID NO: 10.
  • the invention further provides regulatory DNA elements that control expression of a subject nucleic acid molecule.
  • Gene regulatory DNA elements such as enhancers or promoters that reside within the subject nucleic acids, can be used to identify tissues, cells, genes and factors that specifically control production of the encoded protein. Analyzing components that are specific to 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.
  • a regulatory element that controls dmACHRl gene expression resides within nucleotides 1 to 80 of SEQ ID NO: 1. In these embodiments, preferably at least 20, more preferably at least 25, and most preferably at least 50 contiguous nucleotides within nucleotides 1 to 80 of SEQ ID NO: 1 are used. In other embodiments, a regulatory element that controls dmACHR4 gene expression resides within nucleotides 1 to 44 of SEQ ID NO:3. In these embodiments, preferably at least 20, more preferably at least 25, and most preferably at least 40 contiguous nucleotides within nucleotides 1 to 44 of SEQ ID NO: 3 are used.
  • a regulatory element that controls dmKGLUR gene expression resides within nucleotides 1 to 133 of SEQ ID NO:5. In these embodiments, preferably at least 20, more preferably at least 25, and most preferably at least 50 contiguous nucleotides within nucleotides 1 to 133 of SEQ ID NO:5 are used. In other embodiments, a regulatory element that controls dmCACH gene expression resides within nucleotides 1 to 588 of SEQ ID NO:9. In these embodiments, preferably at least 20, more preferably at least 25, and most preferably at least 50 contiguous nucleotides within nucleotides 1 to 588 of SEQ ID NO:9 are used.
  • Gene fusions with the subject regulatory elements can be made. For compact genes that have relatively few and small intervening sequences, such as those described herein for Drosophila, it is typically the case that the 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 protems 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 a subject protein (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).
  • 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 a subject protein (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-
  • Recombinase proteins such as FLP or cre
  • 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 a subject protein 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 a subject protein.
  • a binary reporter system can be used, similar to that described further below, where the subject 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 regulatory element "driver gene".
  • an exogenous transcriptional activator protein such as the GAL4 or tTA activators described below
  • 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.
  • the subject 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 the subject genes, or promoting the growth and differentiation of the tissues that expresses a subject protein.
  • the subject gene regulatory DNA elements are also useful in protein-DNA binding assays to identify gene regulatory proteins that control the expression of the subject genes.
  • the gene regulatory proteins can be detected using a variety of methods that probe specific protein-DNA interactions well known to those skilled in the art (Kingston, supra) including in vivo footprinting assays based on protection of DNA sequences from chemical and enzymatic modification within living or permeabilized cells; and in vitro footprinting assays based on protection of DNA sequences from chemical or enzymatic modification using protein extracts, nitrocellulose filter-binding assays and gel electrophoresis mobility shift assays using radioactively labeled regulatory DNA elements mixed with protein extracts.
  • Candidate gene regulatory proteins can be purified using a combination of conventional and DNA-affinity purification techniques. Molecular cloning strategies can also be used to identify proteins that specifically bind the subject gene regulatory DNA elements.
  • a Drosophila cDNA library in an expression vector can be screened for cDNAs that encode the subject gene regulatory element DNA- binding activity.
  • yeast "one-hybrid" system can be used (Li and Herskowitz, Science (1993) 262:1870-1874; Luo et al, Biotechniques (1996) 20(4):564-568; Vidal et al, PNAS (1996) 93(19):10315-10320).
  • a variety of methods can be used to identify or screen for molecules, such as proteins or other molecules, that interact with a subject protein, or derivatives or fragments thereof.
  • the assays may employ purified subject protein, or cell lines or model organisms such as Drosophila and C. elegans, that have been genetically engineered to express a subject protein. Suitable screening methodologies are well known in the art to test for proteins and other molecules that interact with a subject gene and or protein (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 variety of exogenous molecules may be screened for binding capacity.
  • a subjectprotein 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 subject 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, BioTechniques (1993) 14:920-924, Chasman et al, Mol. Cell. Biol. (1989) 9:4746-4749; Ma et al, Cell (1987) 48:847-853; Ptashne et al, Nature (1990) 346:329-331).
  • any suitable DNA binding domain such as the E. coli LexA repressor protein, or the yeast GAL4 protein (Bartel et al, BioTechniques (1993) 14:920-924, Chasman et al, Mol. Cell. Biol. (1989) 9:4746-4749; Ma et al, Cell (1987) 48:847-853; Ptashne et al, Nature (1990) 346:329-331).
  • 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, EMBO 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 preferred host for two-hybrid screening is the yeast
  • 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.
  • interaction of activation tagged VP16 derivatives with a GAL4-derived bait drives expression of reporters that direct the synthesis of hygromycin B phosphotransferase, chloramphenicol acetyltransferase, or CD4 cell surface antigen (Fearon et al, PNAS (1992) 89:7958-7962).
  • 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 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 hybrid used) are amplified, for example, by PCR using pairs of oligonucleotide primers specific for the coding region of the DNA binding domain or activation domain.
  • Other known amplification methods can be used, such as ligase chain reaction, use of Q replicase, or various other methods described (see Kricka 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 recovering 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-hybrid system occurs when transmembrane portions of proteins in the bait or the prey fusions are used. This occurs because most two-hybrid systems are designed to function by formation of a functional transcription activator complex within 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). Where the subject protein is a transmembrane protein, it is preferred that intracellular or extracellular domains be used for bait in a two-hybrid scheme.
  • the subject proteins, 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. chimeric, single chain, Fab fragments).
  • fragments of a subject protein preferably those identified as hydrophilic, are used as immunogens for antibody production using art- known methods such as by hybridomas; production of monoclonal antibodies in germ-free animals (PCT/US90/02545); the use of human hybridomas (Cole et al, PNAS (1983) 80:2026-2030; Cole et al, in Monoclonal Antibodies and Cancer Therapy (1985) Alan R.
  • the subject polypeptide fragments provide specific antigens and/or immunogens, especially when coupled to carrier proteins.
  • peptides are covalently coupled to keyhole limpet hemocyanin antigen (KLH) and the conjugate is emulsified in Freund's complete adjuvant.
  • KLH keyhole limpet hemocyanin antigen
  • Laboratory rabbits are immunized according to conventional protocol and bled. The presence of specific antibodies is assayed by solid phase immunosorbent assays using immobilized corresponding polypeptide.
  • Binding affinity may be assayed by determination of equilibrium constants of antigen-antibody association (usually at least about 10 7 M "1 , preferably at least about 10 8 M '1 , more preferably at least about 10 9 M "1 ).
  • Immunoassays can be used to identify proteins that interact with or bind to a subject protein.
  • Various assays are available for testing the ability of a protein to bind to or compete with binding to a wild-type subject protein or for binding to an anti-subject 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
  • invertebrate receptor genes or invertebrate receptor interacting genes can be assessed as potential pesticide or drug targets, or as potential biopesticides. Further, transgenic plants that express invertebrate receptor proteins can be tested for activity against insect pests (Estruch et al, Nat. Biotechnol (1997) 15(2): 137-141).
  • 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).
  • aesthetic and hygienic purposes e.g. ants, cockroaches, clothes moths, flour beetles, etc.
  • home and garden applications e.g. ants, cockroaches, clothes moths, flour beetles, etc.
  • 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 subject dsRNA or antisense nucleic acids that interferes with activity of a subject protein-coding nucleic acid molecule.
  • 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 e.g. dsRNA
  • 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) activity of a subject protein can be observed.
  • the effect of various compounds on a subject protein can be assayed using cells that have been engineered to express one or more subject proteins 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.
  • 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 sulf oxide).
  • 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.
  • MLD minimal lethal dose
  • the compounds are usually diluted in DMSO, and applied to the food surface bearing 0-48 hour old embryos and larvae.
  • 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
  • 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.
  • Compounds that modulate (e.g. block or enhance) activity of a subject protein may also be assayed using cell culture. For example, various compounds added to cells expressing a subject protein may be screened for their ability to modulate the activity of a gene encoding a subject protein based upon measurements of receptor activity. Assays for changes in receptor function can be performed on cultured cells expressing endogenous normal or mutant subject proteins. Such studies also can be performed on cells transfected with vectors capable of expressing a subject protein, or functional domains of one of the subject protein, in normal or mutant form. In addition, to enhance the signal measured in such assays, cells may be cotransfected with genes encoding a subject protein.
  • AChR ion channels e.g. ⁇ -conotoxin, ⁇ -bungarotoxin.
  • AChR ion channels e.g. ⁇ -conotoxin, ⁇ -bungarotoxin.
  • the affinity of these antagonists or agonist analogs can be assayed employing radioactive compounds (Buller and White, Methods in Enzymol. (1992) 207, 368-75).
  • binding of ligand to the receptors in presence or absence of compounds may be assayed by radioligand binding assays as described (Swanson GT, et al., Mol Pharmacol, 1998 53:942-949).
  • Electrophysiological recordings may be performed on whole cells expressing recombinant dmKGLUR in presence or absence of various compounds (Donevan SD, et al., J Pharmacol Exp Ther 1998 285:539-545; Swanson GT, supra).
  • the compounds may be screened for their ability to modulate dmCLC-related chloride ion transport by measurements of ion channel fluxes and/or transmembrane voltage or current fluxes using patch clamp, voltage clamp and fluorescent dyes sensitive to intracellular chloride or transmembrane voltage.
  • Ion channel function can also be assayed using transcriptional reporters, altered localization of protein, or by measurements of activation of second messengers such as cyclic AMP, cGMP tyrosine kinases, phosphates, increases in intracellular chloride ion levels, etc.
  • second messengers such as cyclic AMP, cGMP tyrosine kinases, phosphates, increases in intracellular chloride ion levels, etc.
  • Recombinantly made proteins may also be reconstructed in artificial membrane systems to study ion channel conductance and, therefore, the cells employed in such assays may comprise an artificial membrane or cell.
  • Assays for changes in ion regulation or metabolism can be performed on cultured cells expressing endogenous normal or mutant dmCLCs.
  • Such studies also can be performed on cells transfected with vectors capable of expressing one of the dmCLCs, or functional domains of one of the dmCLCs, in normal or mutant form.
  • cells may be cotransfected with genes encoding ion channel protems.
  • Xenopus oocytes or human embryonic kidney cells (HEK293) may be injected with normal or mutant dmCLC sequences.
  • Changes in dmCLC -related or dmCLC-mediated ion channel activity can be measured by two-microelectrode voltage-clamp recordings in oocytes or by whole-cell patch-clamp recordings in HEK293 cells.
  • dmCACH is a membrane protein and a subunit of a calcium channel
  • the compounds may be screened for their ability to modulate dmCACH-related calcium ion transport. Measurements may be taken of calcium channel fluxes, of transmembrane voltage, and/or of current fluxes using patch clamp, voltage clamp, and flourescent dyes sensitive to intracellular calcium or tansmembrane voltage. Ion channel function can also be assayed using transcriptional reporters, altered localization of protein, or by measurements of activation of second messengers such as cyclic AMP, cGMP tyrosine kinases, phosphates, increases in intracellular calcium ion levels, etc.
  • second messengers such as cyclic AMP, cGMP tyrosine kinases, phosphates, increases in intracellular calcium ion levels, etc.
  • Recombinantly made proteins may also be reconstructed in artificial membrane systems to study ion channel conductance and, therefore, the cells employed in such assays may comprise an artificial membrane or cell.
  • Assays for changes in ion regulation or metabolism can be performed on cultured cells expressing endogenous normal or mutant dmCACH. Such studies also can be performed on cells transfected with vectors capable of expressing dmCACH, or functional domains of dmCACH, in normal or mutant form.
  • cells may be cotransfected with genes encoding ion channel proteins. For example, Xenopus oocytes or human embryonic kidney cells (HEK293) may be injected with normal or* mutant dmCACH sequences.
  • Changes in dmCACH-related or dmCACH-mediated ion channel activity can be measured by two-microelectrode voltage-clamp recordings in oocytes or by whole-cell patch-clamp recordings in HEK293 cells. Methods for performing the experiments described here are detailed by Rudi and Iverson (Rudi B., and Iverson, L. in: Methods in Enzymology. Vol.207: Ion Channels. 1997, Publisher: Academic Press, New York). These procedures may be used to screen a battery of compounds, particularly potential pesticides or drugs.
  • the selectivity of a material for a dmCACH may be determined by testing the effect of the compound using cells expressing dmCACHs and comparing the results with that obtained using cells not expressing dmCACHs (see US Patent Nos. 5,670,335 and 5,882,873).
  • Invertebrate receptor Nucleic Acids as Biopesticides The subject nucleic acids and fragments thereof, such as antisense sequences or double-stranded
  • RNA can be used to inhibit function of a subject nucleic acid, and thus can be used as biopesticides. Methods of using dsRNA interference are described in published PCT application WO 99/32619.
  • 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 a subject nucleic acid 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. Natl. 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 an invertebrate receptor gene operably fused to an appropriate inducible promoter.
  • a tTA-responsive promoter may be used in order to direct expression of a subject protein at an appropriate time in the life cycle of the insect. In this way, one may test efficacy as an insecticide in, for example, the larval phase of the life cycle (i.e. when feeding does the greatest damage to crops).
  • Vectors for the introduction of genes into insects include P element (Rubin and Spradling, Science (1982) 218:348-53; U.S. Pat. No.
  • Model systems that display detectable phenotypes, can be used for the identification and characterization of a subject invertebrate receptor pathway gene or other genes of interest and/or phenotypes associated with the mutation or mis-expression of a subject invertebrate receptor pathway protein.
  • the term "mis-expression” as used herein encompasses mis-expression due to gene mutations.
  • a mis-expressed a subject invertebrate receptor 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 subject invertebrate receptor 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 subject invertebrate receptor pathway gene, or any other gene involved in regulation or modulation, or downstream effector of the subject invertebrate receptor 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 subject invertebrate receptor pathway genes, 2) harbor interfering RNA sequences derived from subject invertebrate receptor pathway genes, 3) have had one or more endogenous subject invertebrate receptor pathway genes mutated (e.g. contain deletions, insertions, rearrangements, or point mutations in subject invertebrate receptor 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 subject invertebrate receptor 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 that 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 a subject invertebrate receptor 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
  • antisense double-stranded RNA interference
  • peptide and RNA aptamers directed deletions
  • homologous recombination dominant negative alleles
  • intrabodies e.g., a 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).
  • Invertebrate receptor 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 an invertebrate receptor 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 Kriippel (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
  • 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. elegans to generate loss-of-function phenotypes, and there is a single report of cosuppression in Drosophila, where reduced expression of the Adh gene was induced from a white-Adh transgene using cosuppression methods (Pal-Bhadra et al, Cell (1997) 90(3):479-490).
  • 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. W099/32619).
  • 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 subject gene can be generated in vivo by simultaneous expression of both sense and antisense RNA from appropriately positioned promoters operably fused to a subject nucleotide sequence 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. (2000) Proc. Natl. Acad. Sci. USA 97:6499-6503).
  • cell lines in culture can be manipulated using RNAi both to perturb and study the function of pathway components of a subject protein and to validate the efficacy of therapeutic or pesticidal strategies that involve 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. receptor 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
  • RNA aptamers can inhibit protein function in Drosophila (Shi et al, Proc. Natl. Acad. Sci USA (19999) 96: 10033-10038).
  • RNA aptamers can be used to decrease the expression of dmACHRl protein or derivative thereof, or a protein that interacts with a subject protein.
  • Transgenic animals can be generated to test peptide or RNA aptamers in vivo (Kolonin, MG, and
  • 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 dmACHRl 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 dmACHRl gene (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 dmACHRl pathway genes being expressed).
  • 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 dmACHRl pathway genes being expressed).
  • 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.
  • Tcl-like transposable elements can be used such as minos, mariner and sleeping beauty. Additionally, 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 dmACHRl 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 Wolfrier 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 oi 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 Alocal 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. dmACHRl) by assays based on PCR.
  • dmACHRl 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 oi 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).
  • Examples of well-characterized heterologous promoters that may be used to create the transgenic animals include heat shock promoters/enhancers, which are useful for temperature induced mis-expression. In Drosophila, these include the hsp 70 and hsp83 genes, and in C.
  • elegans include hsp 16-2 and hsp 16- 41.
  • Tissue specific promoters/enhancers are also useful, a in Drosophila, include eyeless (Mozer and Benzer, Development (1994) 120:1049-1058), sevenless (Bowtell et al, PNAS (1991) 88(15):6853-
  • 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 invertebrate receptor 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 an invertebrate receptor 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.
  • Two examples of 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 fromE. 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 UAS G 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 dmACHRl gene or other invertebrate receptor gene disclosed herein 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.
  • activity of a multmeric complex can be decreased by expression of genes coding exogenous protein fragments that bind to the association domains of the wild type proteins and prevent multimer formation.
  • over-expression of an inactive protein unit can sequester wild-type active units in inactive multimers, and thereby decrease multimeric activity (Nocka et al, EMBO J. (1990) 9:1805-1813).
  • the DNA binding domain can be deleted, or the activation domain deleted.
  • the DNA binding domain unit can be expressed without the activation domain causing sequestering of the target DNA.
  • DNA binding sites are tied up without any possible activation of expression.
  • expression of a rigid unit can also inactivate resultant complexes. It is also possible to replace an activation domain with a transcriptional repression domain and thus change a transcriptional activator into a transcriptional repressor. Transcriptional repression domains from the engrailed and Kruppel proteins have been used for such a purpose (Jaynes and O'Ferrell, EMBO J. (1991) 10:1427-1433; Lic t et al, PNAS (1993) 90:11361-11365).
  • Various expression analysis techniques may be used to identify genes which are differentially expressed between a cell line or an animal expressing a wild type invertebrate receptor gene compared to another cell line or animal expressing a mutant invertebrate receptor gene.
  • 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 an invertebrate receptor gene.
  • Gene expression profiling can also be used to identify other genes (or proteins) that may have a functional relation to an invertebrate receptor (e.g. may participate in a signaling pathway with the invertebrate receptor gene).
  • 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 the invertebrate receptor gene.
  • invertebrate receptor 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 invertebrate receptor 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 imagin
  • 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 an invertebrate receptor pathway gene can be used to confirm whether an existing mutant insect or worm line corresponds to a mutation in one or more invertebrate receptor pathway genes, by rescuing the mutant phenotype. Briefly, a genomic fragment containing the invertebrate receptor 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. For Drosophila, an appropriate helper plasmid is used in the injections to supply transposase for transposon-based vectors.
  • any appropriate insect such as Drosophila
  • worm such as C. elegans transformation vector
  • Resulting germline transformants are crossed for complementation testing to an existing or newly created panel oi Drosophila or C. elegans lines whose mutations have been mapped to the vicinity of the gene of interest (Fly Pushing: The Theory and Practice oi 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 invertebrate receptor pathway gene. This prediction can be further confirmed by sequencing the invertebrate receptor pathway gene from the mutant line to identify the lesion in the invertebrate receptor pathway gene.
  • a genetic modifier screen using invertebrate model organisms is a particularly preferred method for identifying genes that interact with invertebrate receptor 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 invertebrate receptor 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 aberrations (Fly Pushing: Theory and Practice oi 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 surrounding 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 invertebrate receptor 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 oi Drosophila Genetics, supra). Generally, individual complementation groups defined in this way correspond to individual genes.
  • invertebrate receptor modifier genes When invertebrate receptor modifier genes are identified, 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. For therapeutic applications related to the function of invertebrate receptor genes, human and rodent homologs of the modifier genes are of particular interest. For pesticide and other agricultural applications, 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, sn ⁇ 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.;
  • Drosophila genetic modifier screens are quite powerful and sensitive, some genes that interact with invertebrate receptor 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 invertebrate receptor gene can be performed by crossing several thousand Drosophila EP lines individually into a genetic background containing a mutant or mis-expressed invertebrate receptor 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 invertebrate receptor 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 invertebrate receptor 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.
  • Example 1 Preparation of Drosophila cDNA Library
  • EST expressed sequence tag
  • 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.
  • 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, Stiatagene) and used to transform E. coli (XL 1 blue).
  • the final complexity of the library was 6 X 10 6 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 (XL 1 blue or DH10B), plated at moderate density, and the colonies picked into a 384-well master plate containing bacterial growth media using a Qbot robot (Genetix, Wales, UK). 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 carried out using ABI377 automated sequencers and used either ABI FS, dirhodamine or BigDye chemistries (Applied Biosystems, Inc., Foster City, CA).
  • the PCR conditions used for cloning the invertebrate receptor nucleic acid sequence were as follows: 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.
  • 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 athttp://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 transposon-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.
  • dmACHRl nucleic acid comprises a contiguous nucleotide sequence of 1540 bases in length, encompassing an open reading frame (ORF) of 1152 nucleotides encoding a predicted protein of 384 amino acids.
  • ORF open reading frame
  • dmACHR4 nucleic acid dmACHR4 nucleic acid comprises a contiguous nucleotide sequence of 1903 bases in length, encompassing an open reading frame (ORF) of 1755 nucleotides encoding a predicted protein of 585 amino acids.
  • the ORF extends from base 45-1802 of SEQ ID NO:3.
  • dmKGLUR nucleic acid dmKGLUR nucleic acid comprises a nucleotide sequence of 3631 bases in length, encompassing an open reading frame (ORF) of 2466 nucleotides encoding a predicted protein of 822 amino acids.
  • the ORF extends from base 134-2602 of SEQ ID NO:5.
  • dmCLC nucleic acid dmCLC nucleic acid comprises a nucleotide sequence of 3366 bases in length, encompassing an open reading frame (ORF) of 2622 nucleotides encoding a predicted protein of 873 amino acids.
  • the ORF extends from base 1-2622 of SEQ ID NO:7.
  • dmCACH nucleic acid dmCACH nucleic acid comprises a nucleotide sequence of 5.862 kilobases in length, encompassing an open reading frame (ORF) of 3645 nucleotides encoding a predicted protein of 1215 amino acids.
  • the ORF extends from base 589-4233 of SEQ ID NO:9.
  • the closest homologue predicted by BLAST analysis is a nicotinic acetylcholine receptor from Locusta migratoria with 71% identity and 73% similarity.
  • the BLAST analysis also revealed several other nicotinic acetylcholine receptor proteins from various species which share significant amino acid homology with dmACHRl .
  • BLAST results for the dmACHRl amino acid sequence indicate 125 amino acid residues as the shortest stretch of contiguous amino acids that is novel and for which there are no sequences sharing 100% sequence similarity with respect to sequences in public databases.
  • Neuronal NAChRs encode a protein with an amino terminal extracellular domain of about 200 amino acids, four hydrophobic putative transmembrane domains, and an extracellular C- terminal domain (Boyd RT, supra). Each alpha and beta subunit in this family contains two cysteines 13 amino acids apart (Boyd RT, supra).
  • the DmACHRl sequence contains cysteines (CYS248 & CYS260) which are 13 amino acids apart, suggesting dmACHRl is neuronal of either the alpha or beta subfamily.
  • the alpha subunits are the most conserved between vertebrates and invertebrates, with one conserved structural motif being 'YXCC starting at Y190 (Torpedo numbering) (Tornoe et al, supra). This motif seems to be the defining criterion for classification of invertebrate subunits as alpha-like or non-alpha-like (Tornoe et al, supra). Based on this analysis, dmACHRl sequence is alpha-like with the sequence YTCC corresponding to amino acids 52-55. This is important because alpha subunits are thought to be the ligand binding domains (Boyd, supra).
  • this pair of cysteine residues are a putative agonist binding site (Boyd, supra).
  • the lophotoxin antagonists which are unique among NAChR antagonists because they contain no nitrogen and thus no positive charge, react to form a covalent bond with the tyrosine of the 'YXCC motif (Tornoe, et al, supra). This data cumulatively suggests that this subunit is a critical component in determining small molecule binding modes and therefore is a key therapeutic target.
  • dmACHR4 Sequence analysis predicted the following features: a signal peptide at amino acids 1-27
  • nucleotides 45-125 a likely signal peptide cleavage site at amino acids 27-28 (nucleotides 123-128); 4 transmembrane domains at amino acids 438-454, 462-478, 495-511, and 563-579 (nucleotides 1356- 1406, 1428-1478, 1527-1577, and 1731-1781); and a Na + /K + ATPase C-terminus (PF00689) at amino acids 374-531 (nucleotides 1164-1637).
  • nucleotide and amino acid sequences for the dmACHR4 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. TABLE 2
  • TopPred predicted a signal peptide at amino acids 9-29 (nucleotides 158-220), followed by four transmembrane domains at amino acids 505-525, 543-563, 585-605, and 616-636 (nucleotides 1631- 1708, 1760-1822, 1886-1948, and 1979-2041, respectively).
  • PFAM predicted a ligand gated ion channel domain (PF00060) at amino acids 5-783 (nucleotides 148-2485).
  • Nucleotide and amino acid sequences for the dmKGLUR nucleic acid sequence and encoded protein were searched against all available nucleotide and amino acid sequences in the public databases, using BLAST (Altschul et al, supra). Table 3 below summarizes the results. The 5 most similar sequences are listed.
  • the closest homologue predicted by BLAST analysis is a rat glutamate receptor, with 40% identity and 60% homology to the dmKGLUR protein.
  • the same level of identity and homology is seen in a number of vertebrate glutamate receptors, particularly the low affinity kinate receptors GLUR5, GLUR6, and GLUR7.
  • BLAST results for the dmKGLUR amino acid sequence indicate 14 amino acid residues as the shortest stretch of contiguous amino acids that is novel with respect to published sequences and 19 amino acids as the shortest stretch of contiguous amino acids for which there are no sequences contained within public database sharing 100% sequence similarity. dmCLC
  • transmembrane domains Fourteen transmembrane domains were predicted, at amino acids 113-133, 185-205, 265-285, 318-338, 341-361, 375-395, 409-429, 446-466, 485-505, 558-578, 581-601, 624-644, 654-674, and 778-798 (corresponding to nucleotides 337-399, 553-615, 793-855, 952-1014, 1020-1083, 1123-1185, 1225-1287, 1336-1398, 1453-1515, 1642-1734, 1741-1803, 1870-1932, 1960-2022, and 2332-2394).
  • nucleotide and amino acid sequences for the dmCLC nucleic acid sequence and encoded protein were searched against all available nucleotide and amino acid sequences in the public databases, using
  • the closest homolog predicted by BLAST analysis is a human chloride channel, with 58% identity and 72% homology with dmCLC.
  • the BLAST analysis also revealed several other chloride channels from various organisms which share significant amino acid homology 55-58% identity and 70- 72% homology with dmCLC.
  • BLAST results for the dmCLC amino acid sequence indicate 34 amino acid residues as the shortest stretch of contiguous amino acids that is novel with respect to published sequences and 39 amino acids as the shortest stretch of contiguous amino acids for which there are no sequences contained within public database sharing 100% sequence similarity.
  • PSORT predicted a transmembrane domain at amino acids 1192-1208.
  • Pfam predicted a von Willebrand factor type A (VWA) domain (PF00092), which is predicted to create a protein fold that is found in dihydropyridine-sensitive calcium channels, as well as other proteins.
  • VWA von Willebrand factor type A
  • the closest homolog predicted by BLAST analysis is a calcium channel alpha-2-delta-C subunit from mouse, with 39% identity and 59% homology.
  • the BLAST analysis also revealed significant homologies to calcium channel alpha/delta subunits from other vertebrate species.
  • BLAST results for the dmCACH amino acid sequence indicate 8 amino acid residues as the shortest stretch of contiguous amino acids that is novel with respect to published sequences and 13 amino acids as the shortest stretch of contiguous amino acids for which there are no sequences contained within public database sharing 100% sequence similarity.
  • Equilibrium binding of tritiated compounds with cells expressing dmACHRs is measured by using a filtration assay. Briefly, 60 nM membrane-bound receptor is incubated with increasing concentrations of tritiated compounds in BC3H1 extracellular buffer (145 mM NaCl/5.3 mM KC1/1.8 mM CaCl 2 «2H20/1.7 mM MgCl 2 «6H20/25 mM Hepes, pH 7.4), to give a final volume of 30 ⁇ l, for 40 min at 25°C.
  • BC3H1 extracellular buffer 145 mM NaCl/5.3 mM KC1/1.8 mM CaCl 2 «2H20/1.7 mM MgCl 2 «6H20/25 mM Hepes, pH 7.4
  • GF/F glass fiber filters (1.3 cm diameter) (Whatman) are presoaked in 1% Sigmacote in BC3H1 buffer (Sigma) for 3 h, then aligned in a 96-well Minifold Filtration Apparatus (Schleicher & Schuell) and placed on top of one 11 X 14 cm GB002 gel blotting paper sheet (Schleicher & Schuell). Thirty-five microliters of each reaction mixture is spotted per well and washed twice with 200 ⁇ l ice-cold BC3H1 buffer. The filter-bound radioactivity is quantified by scintillation counting. Saturation curves are constructed by varying the tritiated compound concentration from 50 nM to 10 ⁇ M. The amount of nonspecific binding is determined in the presence of non-radioactive analogs of the tritiated compounds.
  • HEK 293 cells are maintained in Dulbecco's modified Eagle's medium supplemented with 100 ⁇ g/ml penicillin, 100 ⁇ g/ml streptomycin, and 10% fetal calf serum.
  • Dulbecco's modified Eagle's medium supplemented with 100 ⁇ g/ml penicillin, 100 ⁇ g/ml streptomycin, and 10% fetal calf serum.
  • cells are split to low density on glass coverslips coated with 100 ⁇ g/ml poly-D-lysine and collagen.
  • Transfection of dmKGLUR is by standard calcium-phosphate precipitation with 0.5-1 ⁇ g of cDNA for 5-12 hr at 37° and 5% C02.
  • membranes are prepared as described previously (Swanson et al, Neuron (1997) 19:913-926). Samples are incubated in 10 mM HEPES, pH 8.0, containing [ 3 H]kainate (58 Ci/mmol; New England Nuclear, Wilmington, DE) in a final volume of 0.5 ml for 1 hr at 0°C. Nonspecific binding is defined as that not displaced by 100 ⁇ M kainate. For competition studies, 10 nM [ 3 H]kainate is used.
  • Bound and unbound radioligand are separated by vacuum filtration onto GF/C or GF/B filters (Whatman, Maidestone, UK), presoaked for 1 hr in 0.1% polyethyleneimine (RBI, Natick, MA), followed by two 4-ml washes in ice-cold HEPES. All assays are performed in triplicate. Results from competition curves are fitted to the Hill equation.
  • Equilibrium binding of tritiated compounds with cells expressing dmCLC is measured by using a filtration assay. Briefly, 60 nM membrane-bound receptor is incubated with increasing concentrations of tritiated compounds in BC3H1 extracellular buffer (145 mM NaCl 5.3 mM KC1 1.8 mM CaCl 2 « 2H20/1.7 mM MgCl 2 '6H20/25 mM Hepes, pH 7.4), to give a final volume of 30 ⁇ l, for 40 min at 25°C.
  • BC3H1 extracellular buffer 145 mM NaCl 5.3 mM KC1 1.8 mM CaCl 2 « 2H20/1.7 mM MgCl 2 '6H20/25 mM Hepes, pH 7.4
  • GF/F glass fiber filters (1.3 cm diameter) (Whatman) are presoaked in 1% Sigmacote in BC3H1 buffer (Sigma) for 3 h, then aligned in a 96-well Minifold Filtration Apparatus (Schleicher & Schuell) and placed on top of one 11 X 14 cm GB002 gel blotting paper sheet (Schleicher & Schuell). Thirty-five microliters of each reaction mixture is spotted per well and washed twice with 200 ⁇ l ice-cold BC3H1 buffer. The filter- bound radioactivity is quantified by scintillation counting. Saturation curves are constructed by varying the tritiated compound concentration from 50 nM to 10 ⁇ M. The amount of nonspecific binding is determined in the presence of non-radioactive analogs of the tritiated compounds.
  • Example 7 Elect rophysiological studies using HEK293 cells
  • Electrophysiological recordings on HEK293 cells are made 1-3 days after transfection. Patch clamp recordings are made using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Patch electrodes are thick-walled borosilicate glass (Warner Instruments, Hamden, CT) and have a final resistance of 2-4 M ⁇ after fire-polishing.
  • the internal solution is composed of 110 mM CsF, 30 mM CsCl, 4 mM NaCl, 0.5 mM CaCl 2 , 10 mM HEPES, and 5 mM EGTA (adjusted to pH 7.3 with CsOH).
  • the external bath solution contains 150 mM NaCl, 2.8 mM KC1, 1.8 mM CaCl 2 , 1.0 mM MgCl 2 , and 10 mM HEPES (pH is adjusted to 7.3 with NaOH).
  • Compounds are applied through three-barrel glass tubing (Vitro Dynamics, Rockaway, NJ), which is pulled to a internal barrel diameter of ⁇ 100 ⁇ m and mounted on a piezo-ceramic bimorph.
  • the piezo bimorph is driven by voltage pulses from pClamp v. 6.03 software (Axon Instruments) fed through a stimulation-isolation unit (S-100; Winston Electronic, Millbrae, CA).
  • cells are lifted off the coverslip after a whole-cell patch is obtained.
  • Data are acquired directly to a computer and are analyzed off-line using pClamp software. Exponential decays are fitted with the Chebyshev or Simplex least-squares algorithms in Clampfit. Dose-response curves are fitted to the Hill equation using Origin software (Microcal Software, Northampton, MA).
  • Example 8 Electrophysiological studies using Xenopus oocytes
  • Oocytes are removed iromXenopus laevis frogs anesthetized by immersion in 0.2% tricaine for 15 to 30 min.
  • Oocytes are injected with recombinant dmKGLUR 24 hr later. Experiments are either carried out with RNA injections into the cytoplasm, or DNA injections into the nucleus.
  • RNA injections glass capillary tubes (World Precision Instruments, Sarasota, FL) are pulled to a fine tip on a vertical micropipette puller and broken back to an outside diameter of 21 ⁇ m.
  • RNA stocks are diluted to a final concentration of 1 to 2 ⁇ g/ ⁇ l and injected into the oocytes (23-50 nl) with a microinjector (World Precision Instruments).
  • the nucleus is extruded by gentle centrifugation (1600 rpm, 15 min), and 27.6 nl of DNA (1 ⁇ g/ ⁇ l) is injected into the nucleus.
  • Two electrode voltage clamp recordings are obtained with a Geneclamp amplifier (Axon Instruments) using 3 mM KCl-filled microelectrodes (1-5 M ⁇ ). Recordings are carried out at a holding potential of -60 mV.
  • Compounds that modulate (e.g. block or enhance) ion channels can be assayed on cultured cells.
  • compounds that modulate (e.g. block or enhance) calcium channel permeability to extracellular calcium can be assayed using cultured cells.
  • Cultured mammalian cells e.g. HEK 293
  • HEK 293 can be either transiently or stably transfected with DNA vectors containing the calcium channel gene (Margolskee et al, Biotechn (1993) 15:906-911; Seisenberger et al, Naunyn Schmiedebergs Arch Pharmacol (1995) 352 (6):662-669).
  • Ionic currents that pass through expressed calcium channels are carried by either calcium or barium ions and can be recorded by patch-clamp technique (Hamill et al, Pflugers Arch. (1981) 391 [2]:85-100). Solutions containing compounds of interest can then be screened by passing them through the recording cell and monitoring the current changes.
  • Example 10 Assay of compounds on Xenopus Laevis oocytes using electrophvsiology
  • RNA messenger RNA
  • mRNA messenger RNA
  • Soreq and Seidman Methods in Enzymol. (1992) 207:225-256. After 1-5 days of incubation, calcium channel proteins are produced on the oocyte's plasma membrane.
  • Ionic currents that pass through these expressed calcium channels are carried by either calcium or barium ions and can be recorded by two-electrode recording and/or patch clamp techniques (St ⁇ hmer, Methods in Enzymol. (1992) 207:319-339). Solutions containing the compounds of interest are then screened by passing them through the recording oocyte and monitoring the ionic current changes.
  • Example 11 Cell-based assay employing imaging techniques
  • Fluorescent dyes that sense intracellular calcium can be employed to detect calcium concentration changes caused by calcium channel activity.
  • Dyes like fura-2 and indo-1 are UV light-excitable, ratiometric Ca2+ indicators (Silver, Meth Cell Biol (1998) 56:237-251). These dyes change their molecular configurations and emission spectrums when calcium binds to them. Cells microinjected with the dyes can be monitored under UV excitation. The emission fluorescence spectrum can be used as a reporter of intracellular free calcium and, hence, calcium channel activity.
  • GFP-FRET- Green Fluorescence Protein - Fluorescent Resonance Energy
  • Transfer based peptide molecules
  • free calcium or calcium-calmodulin complex binds to the linker regions of these double GFP molecules, the distance and/or orientation between the two GFP molecules changes, and a difference of FRET occurs.
  • a calibrated calcium or calcium-calmodulin concentration is then read as an indicator of underlying calcium channel activity.
  • Fluorescent membrane potential dyes can be used to monitor cell membrane potential changes induced by calcium channel activity. Membrane-bound, charged fluorescent molecules are added to the cell membrane. As membrane potential changes, the position of this molecule's fluorophore is affected. A change in the fluorophore 's quenching environment produces a fluorescent signal, which can be used to calibrate the membrane potentials.
  • FRET fluorescent resonant energy transfer
  • Example 12 Cell based ligand-binding assay A ligand-binding assay can also measure activity of calcium channels expressed in either mammalian cells or Xenopus oocytes. A number of small molecules have been shown to interact reversibly and with high affinity with various ion channels. Dihydropyridines (DHP) and ⁇ -conotoxins interact specifically with certain types of calcium channels. Radioactively-labeled ligands can thus be applied to intact cells or to purified membranes containing receptor ion channels. Using this method, the total number of ion channel receptors on a particular cell or membrane, as well as the affinity of a ligand to its receptor, can be determined by liquid scintillation or ⁇ -counting. (Buller and White, Methods in Enzymol. (1992) 207:368-375).
  • Example 13 Assay of compounds that modulate ion channels using Xenopus laevis oocytes Compounds that modulate ion channels can also be assayed using Xenopus laevis oocytes expression system.
  • Messenger RNA mRNA
  • mRNA messenger RNA
  • invertebrate ion channel- encoding nucleic acid can be in vitro transcribed from invertebrate ion channel- encoding nucleic acid and microinjected into Xenopus laevis oocytes using a glass micropipette (Soreq and Seidman, Methods in Enzymol. (1992) 207:225-56). After 1 to 5 days incubation, ion channel proteins are produced on the oocyte's plasma membrane.
  • Ionic current through these expressed channel and carried by cation can be recorded by two-electrode recording and/or patch clamp techniques (Stiihmer, Methods in Enzymol. (1992) 207:319-39). Solutions containing interesting compounds can be screened by passing through the recording oocyte and monitoring the ionic current changes.
  • Example 14 Cell-based assay employing imaging techniques Fluorescent membrane potential dyes can be used in monitoring cell membrane potential changes induced by invertebrate receptor activity. Membrane-bound charged fluorescent molecules are added to the cell membrane. As membrane potential changes, the position of the fluorophore is affected. A change of the fluorophore's quenching environment gives a fluorescent signal, which, can be used to calibrate the membrane potentials. Two-component dye systems in which changes in transmembrane potential are detected via fluorescent resonant energy transfer (FRET) between a membrane-bound fluorophore and a charged, membrane-mobile fluorophore have also been developed recently. (Gonzalez et al, Chem Biol.
  • FRET fluorescent resonant energy transfer

Abstract

Cette invention concerne des acides nucléiques récepteurs provenant d'invertébrés et isolés à partir de ∫i⊃Drosophila melanogaster∫/i⊃. L'acide nucléique et la protéine provenant d'invertébrés peuvent s'utiliser pour modifier génétiquement des organismes invertébrés metazoaires tels que des insectes et des vers, ou bien des cellules cultivées, avec pour résultat l'expression ou l'expression erronée de la protéine réceptrice. Les organismes ou cellules génétiquement modifiés peuvent être utilisés pour des dosages de criblage destinés à identifier des composés candidats sous forme d'agents pesticides potentiels ou de substances thérapeutiques qui interagissent avec la protéine réceptrice. Ils peuvent également intervenir dans des méthodes visant à étudier l'activité de récepteur provenant d'invertébrés et à identifier d'autres gènes qui modulent la fonction du gène récepteur ou interagissent avec ce gène.
PCT/US2000/032816 1999-11-29 2000-11-29 Acides nucleiques et polypeptides de recepteurs provenant d'invertebres et methodes d'utilisation WO2001038359A2 (fr)

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EP1414959A1 (fr) * 2001-07-06 2004-05-06 Commonwealth Scientific And Industrial Research Organisation Administration d'arn bicatenaires a des arthropodes
EP1597272A2 (fr) * 2002-11-08 2005-11-23 Fmc Corporation Canaux calciques de lepidoptera dependants d'un potentiel d'action
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

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Cited By (16)

* 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
EP2333061A1 (fr) * 2001-07-06 2011-06-15 Commonwealth Scientific and Industrial Research Organization Administration d'ARN double brin a des arthropodes
US8101343B2 (en) 2001-07-06 2012-01-24 Commonwealth Scientific And Industrial Research Organisation Delivery of dsRNA to arthropods
US8263573B2 (en) 2001-07-06 2012-09-11 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
US8877727B2 (en) 2001-07-06 2014-11-04 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
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
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
EP1597272A4 (fr) * 2002-11-08 2007-10-24 Fmc Corp Canaux calciques de lepidoptera dependants d'un potentiel d'action
EP1597272A2 (fr) * 2002-11-08 2005-11-23 Fmc Corporation Canaux calciques de lepidoptera dependants d'un potentiel d'action

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