[go: up one dir, main page]

WO1999049036A1 - Canaux chlorure a activation gaba de lepidopteres et methodes d'utilisation correspondante - Google Patents

Canaux chlorure a activation gaba de lepidopteres et methodes d'utilisation correspondante Download PDF

Info

Publication number
WO1999049036A1
WO1999049036A1 PCT/US1998/005696 US9805696W WO9949036A1 WO 1999049036 A1 WO1999049036 A1 WO 1999049036A1 US 9805696 W US9805696 W US 9805696W WO 9949036 A1 WO9949036 A1 WO 9949036A1
Authority
WO
WIPO (PCT)
Prior art keywords
lepidopteran
gaba
chloride channel
gated chloride
nucleic acid
Prior art date
Application number
PCT/US1998/005696
Other languages
English (en)
Inventor
Vincent Paul Mary Wingate
Mark Ambroze Wolf
Original Assignee
Rhone-Poulenc Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Rhone-Poulenc Inc. filed Critical Rhone-Poulenc Inc.
Priority to PCT/US1998/005696 priority Critical patent/WO1999049036A1/fr
Priority to AU65795/98A priority patent/AU6579598A/en
Publication of WO1999049036A1 publication Critical patent/WO1999049036A1/fr

Links

Classifications

    • 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

Definitions

  • the gamma-amino butyric acid (GABA) gated chloride channel is a primary target for insecticide action.
  • the present invention provides isolated nucleic acids encoding a lepidopteran GABA gated chloride channel subunit useful for producing a recombinant lepidopteran GABA gated chloride channel.
  • the recombinant GABA gated chloride channel provides a screening system for identifying agonists and antagonists useful as insecticides for lepidopteran pests including Heliothis virescens.
  • BACKGROUND OF THE INVENTION GABA is the major inhibitory neurotransmitter in mammals and insects. In mammals, inhibition is mediated by two types of receptors.
  • Mammalian GABA receptors are coupled to calcium and potassium channels, while GABA A receptors form an integral chloride channel. Insect neuronal GABA receptors exhibit pharmacological similarity to mammalian GABA A receptors, but also exhibit critical differences, for example in the potency order for agonists and antagonists.
  • the insect GABA receptor is a primary target for insecticide action.
  • Physiological and competitive binding studies indicate the presence of at least two insecticide-sensitive binding sites. Binding of insecticides to the noncompetitive blocker site acts to block the chloride channel of the GABA receptor, while binding of insecticides to the other site acts to activate the chloride channel. Insecticides that act to
  • SUBSTITUTE SHEET (RUI-E 26) block the chloride channel include picrotoxinin (PTX) and the polychlorocycloalkanes (PCCAs).
  • PTX picrotoxinin
  • PCCAs polychlorocycloalkanes
  • Vertebrate GABA receptors from a number of sources have been cloned and functionally expressed. Olsen ⁇ i. a . (1990) FASEB J. ⁇ : 1469. Cloning of the insect receptor has proven more difficult due to the lack of a suitable ligand for receptor purification. ffrench- Constant e_£ al. (1991) Proc. Natl. Acad. Sci. 88 : cloned a Drosophila GABA receptor by identifying a locus conferring PTX insensitivity linked to cyclodiene resistance. A single amino acid mutation was identified as responsible for insecticide resistance. ffrench- Constant _____ ___1 . (1993) Nature 363: 449.
  • Insects of the order lepidoptera are significant pests, and in particular the larvae are destructive defoliators. Further, lepidopteran pests are typically harder to control than diptera. Accordingly, there is a need to identify and develop safe and specific insecticides against lepidopteran pests.
  • the present invention addresses this need by providing isolated nucleic acids encoding a lepidopteran GABA gated chloride channel subunit, recombinant lepidopteran GABA gated chloride channels, and a method of identifying lepidopteran GABA receptor agonists and antagonists.
  • the present invention is directed to an isolated nucleic acid encoding a lepidopteran GABA gated chloride channel subunit.
  • the nucleic acid is isolatable from Heliothis virescens.
  • the isolated nucleic acid has a sequence encoding the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • the present invention further provides expression vectors comprising a nucleic acid encoding a lepidopteran GABA gated chloride channel subunit. Host cells comprising the expression vectors are also provided.
  • Another aspect of the present invention provides a recombinant lepidopteran GABA gated chloride channel, and kits and compositions comprising a recombinant lepidopteran GABA gated chloride channel.
  • a method for preparing a lepidopteran GABA gated chloride channel is also provided.
  • the present invention provides a Xenopus oocyte comprising a nucleic acid encoding a lepidopteran GABA gated chloride channel subunit, and a Xenopus oocyte expressing a functional lepidopteran GABA gated chloride channel.
  • the present invention further provides a method of identifying agonists and antagonists to a lepidopteran
  • GABA gated chloride channel GABA gated chloride channel
  • FIG. 1 depicts the plasmid pIVY12.
  • the plasmid comprises the Heliothis sequence (SEQ ID N0:1) encoding the cyclodiene resistant GABA gated chloride channel subunit cloned into the EcoRI/XhoI sites of pBK-CMV
  • Figure 2 depicts the plasmid pIVY13.
  • the plasmid comprises a BamHI/XhoI fragment of PIVY12 cloned into the baculovirus transfer vector pBacPac ⁇ .
  • Figure 3 presents the results of whole cell voltage clamp recordings made 30 hours after infection of Sf9 cells with a baculovirus vector containing cDNA encoding a lepidopteran GABA gated chloride channel .
  • Figure 4 presents electrophysiological recordings demonstrating the effect of GABA and fipronil on a lepidopteran GABA gated chloride channel expressed in oocytes.
  • Figure 5 depicts the plasmid pMK33.
  • the vector is capable of expressing proteins under the control of the
  • FIG. 6 depicts the plasmid pIVY17.
  • the plasmic comprises the Heliothis sequence encoding the GABA gated chloride channel subunit under the control of the metallothionein promoter.
  • Figure 7 presents electrophysiological recordings demonstrating a functional GABA gated chloride channel expressed in a permanently transformed cell line. -5-
  • the present invention is directed to isolated nucleic acids encoding lepidopteran GABA gated chloride channel subunits .
  • An isolated nucleic acid encoding a lepidopteran GABA gated chloride channel subunit is defined herein as an nucleic acid isolatable from an insect of the order lepidoptera and capable of encoding, under appropriate conditions, a functional GABA gated chloride channel.
  • the nucleic acid is isolatable from Heliothis .
  • the nucleic acid is isolatable from Heliothis virescens .
  • a functional GABA gated chloride channel is defined herein as a protein or polypeptide multimer having the ability to bind GABA and thereby mediate chloride flux in a cell expressing the GABA gated chloride channel.
  • the isolated nucleic acid may be DNA or RNA, including cDNA and mRNA.
  • the isolated nucleic acid has a sequence encoding the amino acid seguence set forth in SEQ ID N0:2 or SEQ ID NO: .
  • the ordinarily skilled artisan with the knowledge of the degeneracy of the genetic code, can determine DNA and RNA sequences that encode the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4. Further, the sequence can be selected to optimize expression in a particular host organism by utilizing known preferred codons for the host organism of choice.
  • the isolated nucleic acid has the nucleotide sequence set forth in SEQ ID NO:l or SEQ ID NO: 3. In another embodiment, the isolated nucleic acid has the sequence of nucleotides 115 to 1581 of SEQ ID NO:l or SEQ ID NO:3. Fragments of -6-
  • SEQ ID NO:l contains the codon TCA at nucleotides 967-969 encoding the amino acid serine at amino acid position 285 in SEQ ID NO:2.
  • SEQ ID NO:3 differs from SEQ ID NO:l by the substitution of the codon GCA at nucleotides 967-969 encoding alanine at amino acid position 285 in SEQ ID NO: .
  • nucleic acid having the sequence of SEQ ID NO:l encodes a lepidopteran GABA gated chloride channel subunit and, when expressed in a host cell, provides a functional homomeric lepidopteran GABA gated chloride channel conferring cyclodiene resistance.
  • the nucleic acid having the sequence of SEQ ID NO: 3 encodes a lepidopteran GABA gated chloride channel subunit and, when expressed in a host cell, provides a functional homomeric lepidopteran GABA gated chloride channel that is less cyclodiene insensitive than the channel encoded by SEQ ID NO:l.
  • the present invention further encompasses nucleic acids isolatable from lepidoptera and capable of hybridizing under moderate or high stringency conditions to the complement of an isolated nucleic acid having the sequence of SEQ ID NO:l or SEQ ID NO: 3, and further capable of encoding, under appropriate conditions, a functional GABA gated chloride channel subunit.
  • Moderate and high stringency hybridization conditions are known to the skilled artisan and described, for example, in Sambrook e£ ___1 . (1989) Molecular Cloning: A -7-
  • the present invention is further directed to expression vectors comprising the isolated nucleic acids of the present invention.
  • the nucleic acid encoding a lepidopteran GABA gated chloride channel subunit is operably linked to suitable transc ⁇ ptional and/or translational regulatory elements to effect expression of the GABA gated chloride channel receptor in a suitable host cell.
  • the regulatory elements may be derived from mammalian, microbial, viral or insect genes, and include, for example, promoters, enhancers, transcription and translation initiation sequences, termination sequences, origins of replication, and sequences encoding leader and transport sequences. Suitable regulatory elements are selected for optimal expression in a desired host cell.
  • Useful expression vectors can be constructed by methods known to one of ordinary skill in the art, and vectors into which the nucleic acid of the invention can be inserted are also commercially available. Recombinant viral vectors, including retrovirus, baculovirus, parvovirus and densovirus vectors are particularly preferred.
  • the expression vector comprises a strong constitutive or mducible promoter operably linked to a nucleic acid encoding a -8-
  • Suitable promoters are well known and readily available to one of ordinary skill in the art, and include for example, the polyhedrin promoter (Kitts _____ ______ , 1993, BioTechniques. 14.: 810), heat shock promoter (Stellar _____ al . , 1985, EMBO J.. 4.: 167) and metallothionein promoter (Kaufman _____ _____. , 1989, Cell 5_9_:359) .
  • Expression vectors can be constructed by well known molecular biological methods as described for example in Sambrook _____ al . (1989) Molecular Cloning: A Laboratory Manual Cold
  • the host cell may be procaryotic or eukaryotic, including bacterial, yeast, insect or mammalian. Insect and mammalian cells are preferred. Particularly preferred host cells include insect cell lines, including for example Spodoptera frug perda and Trichoplusia ni cells.
  • the host cells may be transformed, transfected or infected with the expression vectors of the present invention by methods well-known to one of ordinary skill in the art. Transfection may be accomplished by known methods, such as liposome mediated transfection, calcium phosphate mediated transfection, micromjection and electroporation. Permanently transformed insect cell lines are particularly preferred. For example, insect cell lines 18.0299
  • Drosophila cell line SHI can be transformed with the expression vectors of the present invention by commercially available lipofectin (GIBCO-BRL) to provide permanently transformed cell lines expressing a functional lepidopteran GABA gated chloride channel.
  • GEBCO-BRL lipofectin
  • the expression vector is designed such that expression of the chloride channel is inducible .
  • baculovirus vectors are also preferred.
  • the use of baculoviruses as recombinant expression vectors to infect lepidopteran insect cells is known in the art and described for example by Luckow e_t ⁇ 1. (1988) Bio/Technology _S : 47-55 and Miller (1988) Ann. Rev. Microbiol . 12:177-199.
  • the baculovirus vectors generally contain a strong baculovirus promoter operably linked to a nucleic acid of the present invention such that the promoter directs expression of the lepidopteran GABA gated chloride channel.
  • Baculovirus polyhedrin promoters such as the Auto ⁇ rapha californica nuclear polyhedrosis virus polyhedrin promoter are preferred.
  • the baculovirus expression vectors of the present invention are made by inserting the nucleic acid encoding the lepidopteran GABA gated chloride channel subunit downstream of the polyhedrin promoter in a baculovirus transfer vector, for example pBacPac ⁇ available from Clontech.
  • Baculovirus transfer vectors further contain flanking baculovirus sequences that allow homologous recombination between the transfer vector and baculovirus DNA during co-transfection .
  • the transfer vector containing the nucleic acid of the invention and viral DNA are used to co-transfect insect
  • insect cells are Spodoptera .
  • Spodoptera fru ⁇ iperda cells including Sf9 are particularly contemplated.
  • homologous recombination results in the transfer of an expression cassette containing the polyhedrin promoter and the nucleic acid of the present invention to the polyhedrin locus of the viral DNA.
  • the resulting recombinant virus is used to generate viral stocks by standard methods.
  • Insect host cells are infected with the recombinant virus to produce insect cells expressing the GABA gated chloride channel subunit.
  • the present invention further provides a Xenopus oocyte comprising a nucleic acid encoding a lepidopteran GABA gated chloride channel subunit, and a Xenopus oocyte expressing a functional lepidopteran GABA gated chloride channel.
  • the Xenopus oocyte expression system is useful to identify nucleic acids capable of encoding functional lepidopteran GABA gated chloride channels.
  • Xenopus oocytes expressing functional lepidopteran GABA gated chloride channels also provide a system for screening potential insecticides useful against insects of the order lepidoptera.
  • Xenopus oocytes comprising a nucleic acid encoding a lepidopteran GABA gated chloride channel subunit, and Xenopus oocytes expressing a functional lepidopteran GABA gated chloride channel can be made using the nucleic acids of the present invention and methods known to one of ordinary skill in the art.
  • the use of Xenopus oocytes for the expression of exogenous nucleic acids coding for functional receptors is described, for example, by Barnard e_£ ⁇ ___. , (1982) Proc. R. Soc. London EL.215_:241-246 and Fleming e_t al. (1991) J. Phvsiol. ⁇ 11-
  • Expression vectors containing cDNA encoding the GABA gated chloride channel subunit under the control of a strong promoter can be injected into the nuclei of oocytes, after which oocytes are incubated for from one to several days, followed by assessment for presence of functional GABA gated chloride channels.
  • mRNA can be synthesized in vitro from cDNA encoding the GABA gated chloride channel subunit, and injected into oocytes, followed by assessment for presence of functional GABA gated chloride channels as described hereinbelow.
  • the present invention is further directed to recombinant lepidopteran GABA gated chloride channel.
  • the recombinant lepidopteran GABA gated chloride channel may be isolated in a membrane preparation or present in the cell membrane of the host cell or oocyte in which it has been recombinantly produced. Whole cells and membrane preparations comprising the recombinant lepidopteran GABA gated chloride channel are particularly contemplated. Recombinant lepidopteran
  • GABA gated chloride channel is useful, for example, to screen potential insecticides by specific binding assays or functional assays.
  • the GABA gated chloride channel is defined herein as a protein or polypeptide or multimer thereof having the ability to bind GABA and thereby mediate chloride flux is a cell expressing the GABA gated chloride channel.
  • the recombinant lepidopteran GABA gated chloride channel is the expression product of an isolated nucleic acid of the present invention. In a preferred embodiment the nucleic acid is isolatable from Heliothis genus. In a
  • the nucleic acid is isolatable from Heliothis virescens.
  • the isolated nucleic acid has a sequence encoding the ammo acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.
  • the ordinarily skilled artisan with the knowledge of the degeneracy of the genetic code, can determine DNA and RNA sequences that encode the amino acid sequence set forth in SEQ ID NO: 2 and SEQ ID NO: . Further, the sequence can be selected to optimize expression in a particular host organism by utilizing known preferred codons for the host organism of choice.
  • the isolated nucleic acid has the nucleotide sequence set forth in SEQ ID NO:l or SEQ ID NO: 3.
  • the isolated nucleic acid has the sequence of nucleotides 115 to 1581 of SEQ ID N0:1 or SEQ ID NO: 3. Fragments of a nucleic acid having the sequence of SEQ ID NO:l or SEQ ID NO: 3 that maintain the ability to encode a functional lepidopteran GABA gated chloride channel are also encompassed by the present invention.
  • the present invention further encompasses nucleic acids isolatable from lepidoptera and capable of hybridizing under moderate or high stringency conditions to the complement of an isolated nucleic acid having the sequence of SEQ ID NO:l or SEQ ID NO: 3, and further capable of encoding, under appropriate conditions, a functional GABA gated chloride channel.
  • the present invention further provides a method of making a recombinant lepidopteran GABA gated chloride channel. It has been discovered in accordance with the present invention that expression of a nucleic acid encoding a lepidopteran GABA gated chloride channel -13-
  • Recombinant lepidopteran GABA gated chloride channel is made by transforming, transfect g or infecting a suitable host cell with an expression vector comprising a nucleic acid encoding a lepidopteran GABA gated chloride channel subunit, culturing the host cell under conditions suitable for expression, and optionally recovering the recombinant lepidopteran GABA gated chloride channel.
  • the recombinant lepidopteran GABA gated chloride channel is made in insect cells, preferably Spodoptera frugiperda 9, by infecting the insect cells with a recombinant virus in which the nucleic acid of the invention is under the control of a polyhedrin promoter, and culturing the cells under conditions suitable for expression of the recombinant lepidopteran GABA gated chloride channel.
  • the recombinant lepidopteran GABA gated chloride channel is made in permanently transformed cell lines as described hereinabove .
  • a functional lepidopteran GABA gated chloride channel can be identified by one of ordinary skill in the art by functional or binding assays. An exhaustive review of techniques and protocols is provided in Rudy _____ al., eds. (1992) Methods in Enzymology 207 , Academic Press, Inc., San Diego, CA. For example, in a functional assay, whole cell voltage clamp recording of host cells or oocytes expressing the lepidopteran GABA gated chloride channel subunit can be used to assess chloride flux in response to application of GABA. Dose- dependent GABA-evoked currents indicate that a functional lepidopteran GABA gated chloride cnannel has -14-
  • ligand-bmding assay potential agonists and antagonists are identified by their ability to displace radiolabeled ligands known to act as sites on the GABA receptor.
  • Suitable ligands include GABA, uscmol, EBOB (n- propylethynylbicycloorthobenzoate) , and TBPS (t- butylbicyclophosphorothionate) , and BIDN (3,3- bis (trifluoromethyl) bicyclo [2,2, 1] heptane-2, 2- dicarbonitrile) .
  • GABA GABA
  • uscmol EBOB (n- propylethynylbicycloorthobenzoate)
  • TBPS t- butylbicyclophosphorothionate
  • BIDN 3,3- bis (trifluoromethyl) bicyclo [2,2, 1] heptane-2, 2- dicarbonitrile
  • the composition may be a membrane preparation, including a freeze dried membrane preparation, or an intact cell or oocyte expressing the functional lepidopteran GABA gated chloride channel.
  • the composition is useful, for example, to screen potential insecticides by functional or specific binding assays and may further comprise radiolabeled compounds.
  • the composition may further comprise appropriate carriers or diluents, including, for example, physiological buffers.
  • the present invention further provides methods of identifying agonists and antagonists to a lepidopteran GABA gated chloride channel.
  • Agonists to a lepidopteran GABA gated chloride channel are defined as compounds that, like GABA, when applied to the chloride channel result in opening of the channel as measured by flux of chloride ions into or out of the cell.
  • Antagonists to a lepidopteran GABA gated chloride channel are defined as compounds that block the chloride channel, as measured for example by a decrease in GABA mediated chloride ion -15-
  • a method of identifying an agonist comprises applying the putative agonist to a Xenopus oocyte, a cell or a membrane expressing the lepidopteran GABA gated chloride channel in the presence of chloride ions, and measuring chloride flux, wherein flux of chloride is indicative of an agonist.
  • a method of identifying an antagonist comprises applying the putative antagonist to a Xenopus oocyte or a cell or membrane expressing the lepidopteran GABA gated chloride channel in the presence of chloride ions and measuring chloride flux, followed by applying the putative antagonist and GABA to the cell or membrane and measuring chloride flux; and comparing the chloride flux obtained in the presence of the putative antagonist and GABA to the flux obtained under similar conditions in the presence of GABA only, wherein a decrease in flux of chloride observed in the presence of the putative antagonist relative the flux observed in the presence of GABA alone is indicative of an antagonist.
  • chloride flux is measured by voltage clamp electrophysiology .
  • the cell is a recombinant baculovirus- infected Sf9 cell or a permanently transformed cell line.
  • concentrations of agonists, antagonists and GABA are from about 0. InM to about l.OmM.
  • Agonists and antagonists against the lepidopteran GABA gated chloride channel can also be identified by ligand binding assays. Agonists and antagonists are identified by their ability to displace radiolabeled ligands known to act as agonists or antagonists, respectively.
  • an oocyte, cell, or membrane (preferably a membrane) is incubated with radiolabeled ligand and unlabele ⁇ candidate agonist or antagonist. After incubation, the incubation mixture is filtered, and radioactivity retained on the filters is measured by methods known the art, for example liquid scintillation counting. The ability of the candidate compound to inhibit specific binding of the radiolabeled ligand provides a measure of the compound's agonist or antagonist activity.
  • Agonists and antagonists against the lepidopteran GABA gated chloride channel are useful as insecticides against lepidoptera . It has been discovered in accordance with the present invention that the lepidopteran GABA gated chloride channel exhibits distinct pharmacology relative to GABA gated chloride channels of mammals and other insects. Thus, the present recombinant lepidopteran GABA gated chloride channel allows identification of lepidopteran-specific insecticides heretofore not possible even with other insect channels. Lepidopteran pests are typically harder to control than diptera, perhaps because Lepidoptera feed on alkaloid-containing plant tissue, such as cotton.
  • lepidopteran pest control is unique, as reflected by the distinct pharmacology of the lepidopteran GABA gated chloride channel described herein. Accordingly, the present invention solves a need in the art by providing methods of identifying insecticides against lepidoptera. It has further been discovered that the functional assays described hereinabove for identification of agonists and antagonists are particularly well- -17-
  • the level of activity as measured by IC 50 for fipronil in oocytes expressing the lepidopteran GABA gated chloride channel is predictive of the activity of fipronil on whole insects as measured by LD 50 .
  • the effect of fipronil in electrophysiological studies of oocytes expressing the lepidopteran GABA gated chloride channel is correlated with fipronil activity in Heliothis larvae.
  • the present invention further provides a kit for identifying agonists and antagonists to a lepidopteran GABA gated chloride channel.
  • the kit contains a first container containing a recombinant lepidopteran GABA receptor in a cell membrane.
  • the membrane may be in the form of a membrane preparation, including a freeze dried membrane preparation, or an intact cell or oocyte expressing the functional lepidopteran GABA gated chloride channel.
  • the kit of the present invention optionally further comprises radiolabeled binding ligands known to act at sites on the GABA gated chlori ⁇ e channel.
  • the ligand is radiolabeled GABA, muscmol, EBOB, TBPS and BIDN.
  • compositions and kits of the present invention are useful for identifying lepidopteran insecticides by functional assays, binding assays, immunoassays, scintillation proximity assays, and biomolecular interaction analysis using surface plasmon resonance, -18-
  • the present invention also provides a kit for identifying agonists and antagonists to a lepidopteran GABA chloride channel wherein the GABA receptor is incorporated into a reconstituted native and/or synthetic lipid membrane by which chloride conductance may be measured.
  • Genomic DNA was obtained from a Rhone Poulenc in- house population of Heliothis virescens obtained from a field strain by the modified CTAB (cetyltrimethylammonium bromide) method described by Rogers _____ al- (1985) Plant
  • genomic DNA was amplified by polymerase chain reaction (PCR) using degenerate primers vwl21403 and vwl23002 and Amplitaq polymerase (Perkin
  • Primer vwl21403 has the sequence 5 ' -GGT CTA GAA CIA CIG TIC TTA
  • Primer vwl23002 has the sequence 5 ' -GGC TCG AGG C(A/G)A AIA CCA TIA C(A/G)A (A/G)CC A-3' (SEQ. ID. NO:6).
  • PolyA mRNA was isolated from a mixed population of developing Heliothis virescens embryos obtained from a Rhone Poulenc m-house population.
  • One microgram of polyA mRNA was used with a 3'RACE kit (GIBCO-BRL) following the manufacturer's instructions and utilizing as the specific primer vw0528O2, derived from the 70bp fragment and having the sequence 5 ' -AGG TCC ATC GAT GTC TAT CTG GGA A-3' (SEQ ID NO:8).
  • the amplified fragment from the first 3'RACE was cloned into the plasmid pKS+ to produce a plasmid designated pIVY7.
  • a second 3'RACE was performed utilizing as the specific primer vwll2293-301, derived from the amplified fragment of the first 3 ' RACE and having the sequence 5 ' -GTT CAC GAT CCG AAG GCA TAT TCT-3 ' (SEQ ID NO:10).
  • amplified product was cloned into pKS+ to generate plasmid pIVYlO. Sequencing indicated that the amplified product had the sequence: GTT CAC GAT CCG AAG GCA TAT TCT AAA GGC GGT ACT TTA GAA AAC ACT ATC AAT GGG GCT CGG GGC CAG CCA GGA CCT GCT CCA CCG GCA GAC GAA GAA GCT GGA CCA CCT CCG CAT CTC GTT CAT GCT TTC CAA GGT ATC AAC AAA CTG CTC GGC ACG ACC CCC TCG GAC ATC GAC AAG TAC TCG CGC ATC GTG TTC CCC GTC TGC TTC GTT TGC TTT AAC CTT ATG TAC TGG ATC ATT TAC CTT CAC GTG TCT GAC GTC GTG GCT GAT GAC TTG GTA CTA CTA GGC GAA GAA AAT TGA
  • Nucleotides in groups of three represent coding sequences and the unbroken nucleotide sequence represents non-translated mRNA sequence. The non-translated sequence ends with 27 consecutive A residues and is downstream from a translation stop codon .
  • a specific primer designated vw040401 was synthesized based upon the non-translated 3' end of the Heliothis virescens mRNA identified by 3' RACE.
  • Primer vw040401 has the sequence 5'-AACTTGCTCGAGACTTGATGGAT-3' (SEQ ID NO: 12) and was used to construct a Heliothis virescens cDNA library. Five micrograms of polyA mRNA isolated from a mixed Heliothis virescens embryo population was used to make the library.
  • Primer vw040401 was engineered to contain an Xhol site and was then substituted for the first strand primer in the Zap cDNA synthesis kit (Stratagene) . The cDNA was made -21-
  • Polylinker 1 at position 0.0 contains recognition sites for Sad, BssHI, PstI, SPEI, BamHI and EcoRI .
  • Polylinker 2 at position 2.10 contains recognition sites for Xhol, Seal, Xbal, NotI, Apal,
  • the coding sequence starts at 115 bp and ends at 1581 bp, and encodes a polypeptide of 488 amino acids having the predicted sequence:
  • the cDNA insert of a representative plasmid, pIVY16 has the following sequence (SEQ ID N0:3) :
  • the coding sequence starts at 115 bp and ends at 1581 bp, and encodes a polypeptide of 488 amino acids having the predicted sequence:
  • GABA gated chloride channel subunit was cloned into the baculovirus transfer vector pBacPac ⁇ (Clontech) .
  • the resulting vector, pIVY13 contains cDNA encoding the Heliothis virescens cyclodiene resistant GABA gated chloride channel subunit under the control of the polyhedrin promoter.
  • the 8.0 kb vector, pIVY13 consists of the Heliothis sequence of pIVY12 cloned into the BamHI/XHoI site of pBacPac ⁇ and is depicted in Figure 2.
  • Sf9 Spodoptera fru ⁇ iperda 9 (Sf9) cells were cotransfected by pIVY13 and wild type Auto ⁇ rapha californica nuclear polyhedrosis virus (AcMNPV) using the lipofectin procedure.
  • AdMNPV Auto ⁇ rapha californica nuclear polyhedrosis virus
  • flanking AcMNPV sequences allowed recombination with the viral DNA, thus resulting in the transfer of the expression cassette of the Heliothis cDNA and the polyhedrin promoter to the polyhedrin locus of the viral DNA.
  • the recombinant virus thus generated was designated Elisel3.
  • Viral stocks of Elisel3 were generated by standard procedures described by O'Reilly et al . (1992) Baculovirus Expression Vectors, A Laboratory Manual, W.H. Freeman and Company.
  • Sf9 cells were infected with Elisel3 at a multiplicity of infection of 5 plaque forming units (PFU) per cell by standard procedures.
  • RNA was produced from the cDNA template of pIVY12 described in Example I by in vitro transcription with the Ambion mMessage mMachine In Vitro Transcription KitTM (Ambion, Inc.) The mRNA was injected into oocytes by the following procedure.
  • Frogs were anesthetized in a 2 gram/liter solution of 3-amino benzoic acid ethyl ester for thirty minutes, after which oocytes were surgically removed from the abdominal cavity. Follicles were digested by collagenase treatment under sterile conditions by standard methods. Oocytes were injected with 50 nl of mRNA by glass electrodes. Following a 24 to 48 hour incubation, two- electrode voltage clamp recordings were made. -30-
  • Oocytes were held at a resting potential between -70 and -50 mV.
  • Control responses of GABA were obtained by stopping the perfusion of saline and perfusing with a known concentration Of GABA, in ND- 96 with 0.1% DMSO.
  • the average of several GABA applications was taken as the maximal chloride current for that particular GABA dose.
  • the effect of 10 micromolar GABA on the lepidopteran GABA gated chloride channel expressed in Xenopus oocytes is depicted in Figure .
  • GABA application is indicated by the arrow marked "on.”
  • the data in Figure 4 indicate that GABA exhibits the predicted gross functional effect on the GABA gated chloride channel.
  • the pharmacology of the lepidopteran GABA gated chloride channel was determined to be unique, particularly with regard to chloride channel blockers (antagonists).
  • the channels consistently exhibited a decreased sensitivity to the block of chloride ion efflux by inhibitors such as picrotoxinin and fipronil, when agonized by GABA, as shown in Figure 4.
  • the Heliothis virescens cyclodiene resistant chloride channel is about ten times less sensitive to block by fipronil than the Drosophila Rdl chloride channel. Sensitivity to picrotoxinin is also reduced by more than 100 times relative to Drosophila Rdl.
  • Plasmid pIVY17 was constructed by digesting pIVY12 with Xbal and BamHI, releasing a 2.2 kbp DNA fragment containing the coding region of the Heliothis
  • the expression vector pMK33 is designed to express proteins under the control of the Drosophila metallothionein (Mt) promoter, and also carries a hygromycin resistance gene, allowing selection of stable cell lines carrying the plasmid.
  • Mc Drosophila metallothionein
  • the resulting plasmid pIVY17 contains the Heliothis Rdl cDNA under the control of the metallothionein promoter.
  • the 12.50 kb plasmid pIVY17 is depicted in Figure 6.
  • the 8.0 kb plasmid pMK33 can also be obtained by inserting the EcoRI-NotI fragment of pHSX-MT (Kaufman e£ al. , Cell 5_2: 359-371, 1989) , containing the Drosophila Mt promoter, into BamHI-, Notl-cleaved pcopnyg (Rio et. _____. , Cell 44:21-32, 1986) filling in the EcoRI and BamHI ends.
  • the 3' end of the Actin 5C gene is then inserted into pMT/Hyl in two steps.
  • the Sail fragment of pPac Kerman e£ al. , Cell 5_2: 359-371, 1989
  • Drosophila cell line SHI was transformed with plasmid pIVY17 using lipofectin obtained from GIBCO-BRL following the manufacturer's instructions. Selection of insect cell transformants was accomplished with hygromycin at a concentration of 200 ⁇ g/ml. The Heliothis GABA gated chloride channel expression was induced by 10 ⁇ M cadmium chloride added to the growth medium for 48 hours prior to electrophysiological readings. Whole cell voltage patch clamping recordings were performed on a number of cell lines as described in Example III. Typical results are presented in Figure 7 and demonstrate that GABA exhibits the predicted gross functional effect on the GABA gated chloride channel, i.e., flux of chloride ion. This example demonstrates that expression of DNA encoding a lepidopteran GABA gated chloride channel subunit in permanently transformed cell lines results in a functional GABA gated chloride channel.
  • ADDRESSEE BURNS, DOANE, SWECKER & MATHIS, L.L.P.
  • MOLECULE TYPE DNA (genomic)
  • AAA ATG CAA ATA GAT GGT CCT CCA GGG TCA GCT GAG CCT ATC CCC CCA 1221 Lys Met Gin He Asp Gly Pro Pro Gly Ser Ala Glu Pro He Pro Pro 355 360 365
  • MOLECULE TYPE DNA (genomic)
  • AAA ATG CAA ATA GAT GGT CCT CCA GGG TCA GCT GAG CCT ATC CCC CCA 1221 Lys Met Gin He Asp Gly Pro Pro Gly Ser Ala Glu Pro He Pro Pro 845 850 855
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • AAGATCTCAT ATGTCAAGTC CATCGATGTC TATCTGGGAA CTTGTTTCGT CATGGTCTTC 120
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Neurology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention se rapporte à des acides nucléiques isolés codant pour des canaux chlorure activés par l'acide η-amino-butirique (GABA) chez des lépidoptères. Dans une réalisation préférée, on isole les acides nucléiques codant pour les canaux chlorure à activation GABA de lépidoptères à partir de Heliothis virescens. L'invention se rapporte en outre à des canaux chlorure recombinés à activation GABA de lépidoptères ainsi qu'à des procédés d'identification d'agonistes et d'antagonistes d'un canal chlorure à activation GABA de lépidoptères.
PCT/US1998/005696 1998-03-24 1998-03-24 Canaux chlorure a activation gaba de lepidopteres et methodes d'utilisation correspondante WO1999049036A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/US1998/005696 WO1999049036A1 (fr) 1998-03-24 1998-03-24 Canaux chlorure a activation gaba de lepidopteres et methodes d'utilisation correspondante
AU65795/98A AU6579598A (en) 1998-03-24 1998-03-24 Lepidopteran gaba gated chloride channel and methods of use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1998/005696 WO1999049036A1 (fr) 1998-03-24 1998-03-24 Canaux chlorure a activation gaba de lepidopteres et methodes d'utilisation correspondante

Publications (1)

Publication Number Publication Date
WO1999049036A1 true WO1999049036A1 (fr) 1999-09-30

Family

ID=22266658

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/005696 WO1999049036A1 (fr) 1998-03-24 1998-03-24 Canaux chlorure a activation gaba de lepidopteres et methodes d'utilisation correspondante

Country Status (2)

Country Link
AU (1) AU6579598A (fr)
WO (1) WO1999049036A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002044374A3 (fr) * 2000-12-01 2003-03-27 Heska Corp Molecules d'acide nucleique et proteines de sous-unite de recepteur gaba de puce, et utilisation de celles-ci

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5767261A (en) * 1995-11-08 1998-06-16 Rhone -Poulenc Ag Company Lepidopteran GABA gated chloride channel and nucleic acids encoding subunits thereof
WO1998049185A1 (fr) * 1997-04-28 1998-11-05 Fmc Corporation Canaux chlorure actives par gaba chez des lepidopteres

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5767261A (en) * 1995-11-08 1998-06-16 Rhone -Poulenc Ag Company Lepidopteran GABA gated chloride channel and nucleic acids encoding subunits thereof
WO1998049185A1 (fr) * 1997-04-28 1998-11-05 Fmc Corporation Canaux chlorure actives par gaba chez des lepidopteres

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN R ET AL: "Cloning and functional expression of a Drosophila gamma-aminobutyric acid receptor [published erratum appears in Proc Natl Acad Sci U S A 1994 Oct 11;91(21):10242]", PROC NATL ACAD SCI U S A, JUN 21 1994, 91 (13) P6069-73, UNITED STATES, XP002095037 *
EMINV DATABASE Accession number AF006189 Yuhas D.A ET AL. Heliothis virescens GABA-gated chloride channel isoform a2 mRNA, *
TRINV DATABASE Accession number O18468 01-JAN-1998 (TREMBLREL. 05, CREATED) YUHAS D.A. ET AL. GABA-GATED CHLORIDE CHANNEL ISOFORM A2. HELIOTHIS VIRESCENS (NOCTUID MOTH) (OWLET MOTH). *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002044374A3 (fr) * 2000-12-01 2003-03-27 Heska Corp Molecules d'acide nucleique et proteines de sous-unite de recepteur gaba de puce, et utilisation de celles-ci

Also Published As

Publication number Publication date
AU6579598A (en) 1999-10-18

Similar Documents

Publication Publication Date Title
Atkinson et al. A component of calcium-activated potassium channels encoded by the Drosophila slo locus
AU680852B2 (en) Stably transfected cell lines expressing GABA-A receptors
US5674747A (en) Viral vector coding for juvenile hormone esterase
EP0789712A1 (fr) Genes codant une famille de canaux de potassium
US5767261A (en) Lepidopteran GABA gated chloride channel and nucleic acids encoding subunits thereof
CA2095490A1 (fr) Recepteur humain du glutamate metabotropique et composes d'adn connexes
WO1995001996A1 (fr) Toxine de l'araignee a effet insecticide
JP2805448B2 (ja) 殺虫に有効なペプチド
WO1999049036A1 (fr) Canaux chlorure a activation gaba de lepidopteres et methodes d'utilisation correspondante
Sonoda et al. Genomic organization of the para‐sodium channel α‐subunit genes from the pyrethroid‐resistant and‐susceptible strains of the diamondback moth
US5854002A (en) Method of identifying compounds that bind to the insect gaba receptor
US7232658B2 (en) Heliothis glutamate receptor
CA2094987A1 (fr) Proteines humaines receptrices de glutamate et composes d'adn
US6800435B2 (en) Insect sodium channels from insecticide-susceptible and insecticide-resistant house flies
Nguyen et al. Isolation and heterologous expression of two genomic clones encoding Shaker‐related potassium channels of trout CNS
US20030165806A1 (en) Potassium channels, nucleotide sequences encoding them, and methods of using same
AU747253B2 (en) Genes encoding a family of potassium channels
US20020037556A1 (en) Heliothis virescens ultraspiracle (USP) protein
CA2151236C (fr) Lignees cellulaires transfectees stables exprimant des recepteurs du gaba-a
CA2307470A1 (fr) Gene viral et gene d'insecte qui interrompent la metamorphose de l'insecte
US20050032165A1 (en) Potassium channels, nucleotide sequences encoding them, and methods of using same
AU2775502A (en) Insect sodium channels from insecticide-susceptible and insecticide-resistant house flies
WO2002000864A2 (fr) Inhibiteur de l'histone acetyltransferase et leur utilisation en tant qu'insecticides
CA2202878A1 (fr) Genes codant une famille de canaux de potassium

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM GW HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA

122 Ep: pct application non-entry in european phase