WO1999049036A1 - Lepidopteran gaba gated chloride channel and methods of use thereof - Google Patents

Abstract

The present invention provides isolated nucleic acids encoding lepidopteran gamma aminobutyric acid (GABA) gated chloride channels. In a preferred embodiment, the nucleic acids encoding lepidopteran GABA gated chloride channels are isolatable from Heliothis virescens. The invention further provides recombinant lepidopteran GABA gated chloride channels and methods for identifying agonists and antagonists to a lepidopteran GABA gated chloride channel.

Description

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LEPIDOPTERAN GABA GATED CHLORIDE CHANNEL AND METHODS OF USE THEREOF

FIELD OF THE INVENTION

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).

Adverse toxicology has led to significant restrictions on the use of many insecticides, particularly PCCAs. Further, the selection of resistant pest strains has resulted in the ineffectiveness of some chloride channel blockers and activators as insecticides .

A better understanding of the insect GABA receptor is necessary to develop insecticides that are not subject to resistance, and that exhibit better selectivity and enhanced environmental safety.

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. Low stringency screening with the Drosophila probe was utilized to clone the cyclodiene resistance locus from another diptera, Aedes aegypti. Thompson et. ___1 . (1993) FEBS Letters 325 : 187. To date, an insect GABA receptor has not been cloned from any other insect order.

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. SUMMARY OF THE INVENTION

The present invention is directed to an isolated nucleic acid encoding a lepidopteran GABA gated chloride channel subunit. In a preferred embodiment the nucleic acid is isolatable from Heliothis virescens. In another preferred embodiment 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.

In yet another embodiment, 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. -4-

The present invention further provides a method of identifying agonists and antagonists to a lepidopteran

GABA gated chloride channel .

BRIEF DESCRIPTION OF THE DRAWINGS Figure 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

(Stratagene) . Figure 2 depicts the plasmid pIVY13. The plasmid comprises a BamHI/XhoI fragment of PIVY12 cloned into the baculovirus transfer vector pBacPacδ .

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

Drosophila metallothionein (Mt) promoter, and also carries a hygromycin resistance gene. Figure 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-

DETAILED DESCRIPTION OF THE INVENTION

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. In a preferred embodiment the nucleic acid is isolatable from Heliothis . In a more preferred embodiment 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. In a preferred embodiment 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.

In another preferred embodiment, 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-

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. 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: . It has been discovered in accordance with the present invention that the 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-

Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Moderate stringency conditions, for examples are about 50°C, 2X SSC. The ability of the isolated nucleic acid of the present invention to encode a functional GABA gated chloride channel can be determined by functional assays as described herembelow.

The present invention is further directed to expression vectors comprising the isolated nucleic acids of the present invention. In the expression vectors of the present invention, the nucleic acid encoding a lepidopteran GABA gated chloride channel subunit is operably linked to suitable transcπptional 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. In a preferred embodiment the expression vector comprises a strong constitutive or mducible promoter operably linked to a nucleic acid encoding a -8-

lepidopteran GABA gated chloride channel subunit. 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

Spring Harbor Laboratory Press, Cold Spring Harbor, New York, or any of a myriad of laboratory manuals on recombinant DNA technology that are widely available. Expression vectors into which the nucleic acids of the present invention can be cloned under the control of a suitable promoter are also commercially available.

Another embodiment of the present invention provides host cells containing the expression vectors described above. 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

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such as 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. In a preferred embodiment, the expression vector is designed such that expression of the chloride channel is inducible .

Expression systems utilizing baculovirus vectors and insect host cells 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 Autoσrapha 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 pBacPacδ 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

RECTIFIED SHEET (RULE 91) ISA/EP -10-

cells . In a preferred embodiment the insect cells are Spodoptera . Spodoptera fruσiperda cells including Sf9 are particularly contemplated. During co-transfection, 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-

4_3_8_:371P. 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. Alternatively, 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

RECTIFIED SHEET (RULE 91) ISA/EP -12-

ore preferred embodiment the nucleic acid is isolatable from Heliothis virescens. In another preferred embodiment 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. In another preferred embodiment, 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 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-

subunit results in production of a functional homomeric GABA gated chloride cnannel . 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. In a preferred embodiment, 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. In another preferred embodiment, 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-

been assembled. In a typical 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) . Another embodiment of the present invention provides a composition comprising a recombinant lepidopteran GABA gated chloride channel in a cell membrane. 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-

flux in the cell. 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. In a preferred embodiment, chloride flux is measured by voltage clamp electrophysiology . In another preferred embodiment, the cell is a recombinant baculovirus- infected Sf9 cell or a permanently transformed cell line. In another preferred embodiment, the 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. The recombinant GABA gated chloride

RECTIFIED SHEET (RULE 91) ISA/EP -16-

channel, present m an oocyte, cell, or membrane, (preferably a membrane) is incubated with radiolabeled ligand and unlabeleα 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. Thus 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-

correlated with data from whole insect screening. For example, the level of activity as measured by IC₅₀ for fipronil in oocytes expressing the lepidopteran GABA gated chloride channel is predictive of the activity of fipronil on whole insects as measured by LD₅₀. Further, the effect of fipronil in electrophysiological studies of oocytes expressing the lepidopteran GABA gated chloride channel is correlated with fipronil activity in Heliothis larvae. Thus the discovery of the lepidopteran GABA gated chloride channel and the methods described herein allows the identification of insecticide compounds that are uniquely suited to targeting of lepidopteran pests.

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 chloriαe channel. In a preferred embodiment, the ligand is radiolabeled GABA, muscmol, EBOB, TBPS and BIDN. The 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-

for example as described by Malmquist _____ ___1 . (1994)

Methods : A Companion to Methods in Entomology ϋ:95. 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.

The following examples further illustrate the present invention.

EXAMPLE I

Isolation of a full length cDNA encoding cyclodiene resistant GABA gated chloride channel subunit

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

Mol . Biol . 5_.: 69. The genomic DNA was amplified by polymerase chain reaction (PCR) using degenerate primers vwl21403 and vwl23002 and Amplitaq polymerase (Perkin

Elmer following the manufacturer's instructions. Primer vwl21403 has the sequence 5 ' -GGT CTA GAA CIA CIG TIC TTA

CIA TGA AC-3' (SEQ ID NO:5). 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).

PCR resulted in the amplification of a 70 base pair

(bp) fragment which was cloned into the pKS+ plasmid

(Stratagene) to produce pJWl and sequenced. The amplified fragment had the following sequence: 5 ' -TCT -19-

AGA ACG ACG GTG GTT ACG ATG ACG ACG CTC ATG TCG TCC ACG AAT GCG GCT CTG CCC AAG ATC TCA TAT GTC AAG TCC ATC GAT GTC TAT CTG GGA ACT TGT TTC GTC ATG GTC TTC GCC TCG AG-3' (SEQ ID N0:7) . The technique of rapid amplification of cDNA ends (RACE) according to Frohman et __1 . (1988) Proc. Natl . Acad. Sci. USA 85.: 8998 was used to obtain the 3' end of the Heliothis virescens mRNA. 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. Sequencing indicated that the fragment had the sequence: 5 ' -AGG TCC ATC GAT GTC TAT CTG GGA ACT TGT TTC GTC ATG GTC TTC ACC AGT TTA CTA GAA TAT GCC ACG GTT GGC TAT ATG GCT AAA AGG ATA CAG ATG AGG AAA CAA AGA TTC ACT GCT GTT CAA AAA ATG CAA ATA GAT GGT CCT CCA GGG TCA GCT GAG CCT ATC CCC CCA CCG AGG ACC AGC ACC CTA TCT AGG CCA CCA CCT AGC CGA TTA TCG GAG GTT CGG TTC AAA GTT CAC GAT CCG AAG GCA TAT TCT AAA GGC GGT ACT TTA GAA AAA-3' (SEQ ID NO: 9) .

Because the anchor primer had annealed to an A-rich region in the Heliothis virescens coding region, 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). The -20-

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

ATTCTCTTTAACTATACCGGACTTGTTTTAACTATACCGGACTTGTTTTAACTTTA GGGTGCTTATGATCAACCATCCATCAAGTCTCGGTAAAGTTCTTTAAGTCTAGAAC GCTCAGTAAAATAATAGCGTTCTTTGTGTTTATAAATATAATTATAGTACAGATCA CTATGTTTATTATAGATAAGTGTCGTGTATATTGGCACTGGTAATATTAATTCTTT AGAAAATAAAGATAATATGAATTCAAAAAAAAAAAAAAAAAAAAAAAAAAA-3 ' (SEQ ID NO: 11) . 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-

according to the manufacturer' s instructions and then cloned into the lambda ZAP expression vector cloning system (Stratagene) and packaged with Gigapack II packaging system (Stratagene) following the manufacturer's instructions. Thus a non-amplified

5 lliibbrraarryy oolf 5x10 recombinants was made and then amplified, Part of the amplified library was screened with 32P labeled random primer derived probes of the 3'RACE inserts of plasmids pIVY7 and pIVYlO using standard techniques. One positive clone was identified and plaque purified. The phagemid pBK-CMV containing the insert was excised following the manufacturer's instructions (Stratagene). The 6.6 kb phagemid clone, designated pIVY12, consists of the Heliothis sequence cloned into the EcoRI/XhoI sites of pBK-CMV and is depicted in Figure 1. 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,

Clal, BstXI, Smal and Kpnl . Double stranded sequencing was accomplished with the Sequenase system (Amersham) . The following DNA sequence (SEQ ID NO:l) was determined. 10 20 30 40 50 GAATTCGGCACGAGGACGCCTGAGGGCCTGTAAGAACACGCCAGTCCGGC

60 70 80 90 100

CGCCACGGTGATACGCGGCTGCCGGCAGCCAGCGTCCGCAAGGGCGCACG

110 120 130 140 150

CGGACCTGCAAAACATGCATACGAGCCGTCCGCGCGGCGTGCACAGCATC 160 170 180 190 200

GCGCTAGTGCTGTCTCTCGCGATTGCCTGGTTACCTCATGCTGACCATGC -22-

210 220 230 240 250 CGCGGGAGCGGGAGGAGGGGGAATGTTTGGTGACGTCAATATCTCAGCCA

260 270 280 290 300

TTTTGGATTCGCTAAGTGTAAGCTACGACAAAAGAGTGAGGCCGAACTAT 310 320 330 340 350

GGAGGACCGCCAGTGGATGTGGGAGTCAACATGTACGTGCTCTCCATCAG

360 370 380 390 400 CTCCTTATCTGAAGTGAAAATGGATTTCACCCTGGATTTCTACTTCAGAC

410 420 430 440 450 AATTTTGGACAGACCCCAGGCTTGCTTACAAAAAAAGCACGGGTGTGGAG

460 470 480 490 500 ACTCTGTCCGTCGGCTCGGAATTTATTAGAAACATATGGGTACCCGACAC

510 520 530 540 550

CTTCTTTGTTAACGAAAAACAGTCATATTTCCACATAGCTACTACAAGCA 560 570 580 590 600

ACGAATTCATACGCATTCATCATTCTGGATCTATTACTAGGAGTATAAGA

610 620 630 640 650 CTGACTATCACCGCTTCTTGTCCGATGGATTTGCAGTATTTTCCGATGGA

660 670 680 690 700 CCGTCAATTATGCAATATTGAAATCGAAAGTTTTGGCTACACCATGCGGG

710 720 730 740 750 ACATCCGATACAAGTGGAATGAGGGGCCCAACTCAGTGGGTGTGTCGAGC

760 770 780 790 800

GAAGTGTCTTTGCCGCAATTCAAGGTGCTGGGCCACCGGCAGCGGGCCAT 810 820 830 840 850

GGAGATTTCTCTTACGACAGGAAACTACTCTCGTCTGGCATGTGAAATTC

860 870 880 890 900 AATTTGTAGGCTCGATGGGATACTATTTAATTCAGATTTATATTCCGTCT

910 920 930 940 950 GGCCTAATTGTCATTATATCTTGGGTATCATTTTGGTTGAATCGAAATGC

960 970 980 990 1000 GACACCTGCAAGGGTATCACTAGGTGTCACAACTGTATTGACGATGACGA -23-

1010 1020 1030 1040 1050

CGCTCATGTCGTCCACGAATGCGGCTCTGCCCAAGATCTCATATGTCAAG

1060 1070 1080 1090 1100

TCCATCGATGTCTATCTGGGAACTTGTTTCGTCATGGTCTTCACCAGTTT 1110 1120 1130 1140 1150

ACTAGAATATGCCACGGTTGGCTATATGGCGAAAAGGATACAGATGAGGA

1160 1170 1180 1190 1200

AACAAAGATTCACTGCTGTTCAAAAAATGCAAATAGATGGTCCTCCAGGG

1210 1220 1230 1240 1250 TCAGCTGAGCCTATCCCCCCACCGAGGACCAGCACCCTATCTAGGCCACC

1260 1270 1280 1290 1300

ACCTAGCCGATTATCGGAGGTTCGGTTCAAAGTTCACGATCCGAAGGCAT

1310 1320 1330 1340 1350

ATTCTAAAGGCGGTACTTTAGAAAACACTATCAATGGGGCTCGGGGCCAG 1360 1370 1380 1390 1400

CCAGGACCTGCTCCACCGGCAGACGAAGAAGCTGGACCACCTCCGCATCT

1410 1420 1430 1440 1450

CGTTCATGCTTTCCAAGGTATCAACAAACTGCTCGGCACGACCCCCTCGG

1460 1470 1480 1490 1500 ACATCGACAAGTACTCGCGCATCGTGTTCCCCGTCTGCTGCGTTTGCTTT

1510 1520 1530 1540 1550

AACCTTATGTACTGGATCATTTACCTTCACGTGTCTGACGTCGTGGCTGA

1560 1570 1580 1590 1600

TGACTTGGTACTACTAGGCGAAGAAAATTGAATTCTCTTTAACTATACCG 1610 1620 1630 1640 1650

GACTTGTTTTAACTTAGGGTGCTTATGATCAACCATCCATCAGGTTTCGG

1657 TAAAGTT

Sequencing indicated that pIVY12 encoded a full length Heliothis virescens Rdl clone directionally cloned into the EcoRI and Xhol sites of phagemid pBK- -24-

CMV. The coding sequence starts at 115 bp and ends at 1581 bp, and encodes a polypeptide of 488 amino acids having the predicted sequence:

MHTSRPRGVHSIALVLSLAIAWLPHADHAAGAGGGGMFGDVNISAILDSLSVSYDK RVRPNYGGPPVDVGVNMYVLSISSLSEVKMDFTLDFYFRQFWTDPRLAYKKSTGVE TLSVGSEFIRNIWVPDTFFVNEKQSYFHIATTSNEFIRIHHSGSITRSIRLTITAS CPMDLQYFPMDRQLCNIEIESFGYTMRDIRYKWNEGPNSVGVSSEVSLPQFKVLGH RQRAMEISLTTGNYSRLACEIQFVGSMGYYLIQIYIPSGLIVIISWVSFWLNRNAT PARVSLGVTTVLTMTTLMSSTNAALPKISYVKSIDVYLGTCFVMVFTSLLEYATVG YMAKRIQMRKQRFTAVQKMQIDGPPGSAEPIPPPRTSTLSRPPPSRLSEVRFKVHD PKAYSKGGTLENTINGARGQPGPAPPADEEAGPPPHLVHAFQGINKLLGTTPSDID KYSRIVFPVCCVCFNLMYWIIYLHVSDVVADDLVLLGEEN (SEQ ID NO: 2) .

EXAMPLE II

Site-directed mutagenesis of cDNA encoding cyclodiene resistant GABA gated chloride channel subunit

The serine residue encoded by nucleotide TCA at positions 967-969 in plasmid pIVY12 was mutated to alanine (encoded by nucleotides GCA) by site-directed mutagenesis by overlap extension using the polymerase chain reaction as described by Ho e_t al. (1989) Gene 77 :

51-59 using the Pfu polymerase (Stratagene) . The mutation was verified by DNA sequence analysis from a number of full length clones. The cDNA insert of a representative plasmid, pIVY16, has the following sequence (SEQ ID N0:3) :

10 20 30 40 50

GAATTCGGCACGAGGACGCCTGAGGGCCTGTAAGAACACGCCAGTCCGGC

60 70 80 90 100 CGGCACGGTGATACGCGGCTGCCGGCAGCCAGCGTCCGCAAGGGCGCACG -25-

110 120 130 140 150 CGGACCTGCAAAACATGCATACGAGCCGTCCGCGCGGCGTGCACAGCATC

160 170 180 190 200

GCGCTAGTGCTGTCTCTCGCGATTGCCTGGTTACCTCATGCTGACCATGC 210 220 230 240 250

CGCGGGAGCGGGAGGAGGGGGAATGTTTGGTGACGTCAATATCTCAGCCA

260 270 280 290 300 TTTTGGATTCGCTAAGTGTAAGCTACGACAAAAGAGTGAGGCCGAACTAT

310 320 330 340 350 GGAGGACCGCCAGTGGATGTGGGAGTCAACATGTACGTGCTCTCCATCAG

360 370 380 390 400 CTCCTTATCTGAAGTGAAAATGGATTTCACCCTGGATTTCTACTTCAGAC

410 420 430 440 450

AATTTTGGACAGACCCCAGGCTTGCTTACAAAAAAAGCACGGGTGTGGAG 460 470 480 490 500

ACTCTGTCCGTCGGCTCGGAATTTATTAGAAACATATGGGTACCCGACAC

510 520 530 540 550 CTTCTTTGTTAACGAAAAACAGTCATATTTCCACATAGCTACTACAAGCA

560 570 580 590 600 ACGAATTCATACGCATTCATCATTCTGGATCTATTACTAGGAGTATAAGA

610 620 630 640 650 CTGACTATCACCGCTTCTTGTCCGATGGATTTGCAGTATTTTCCGATGGA

660 670 680 690 700

CCGTCAATTATGCAATATTGAAATCGAAAGTTTTGGCTACACCATGCGGG 710 720 730 740 750

ACATCCGATACAAGTGGAATGAGGGGCCCAACTCAGTGGGTGTGTCGAGC

760 770 780 790 800 GAAGTGTCTTTGCCGCAATTCAAGGTGCTGGGCCACCGGCAGCGGGCCAT

810 820 830 840 850 GGAGATTTCTCTTACGACAGGAAACTACTCTCGTCTGGCATGTGAAATTC

860 870 880 890 900 AATTTGTAGGCTCGATGGGATACTATTTAATTCAGATTTATATTCCGTCT -26-

910 290 930 940 950

GGCCTAATTGTCATTATATCTTGGGTATCATTTTGGTTGAATCGAAATGC

960 970 980 990 1000

GACACCTGCAAGGGTAGCACTAGGTGTCACAACTGTATTGACGATGACGA 1010 1020 1030 1040 1050

CGCTCATGTCGTCCACGAATGCGGCTCTGCCCAAGATCTCATATGTCAAG

1060 1070 1080 1090 1100

TCCATCGATGTCTATCTGGGAACTTGTTTCGTCATGGTCTTCACCAGTTT

1110 1120 1130 1140 1150 ACTAGAATATGCCACGGTTGGCTATATGGCGAAAAGGATACAGATGAGGA

1160 1170 1180 1190 1200

AACAAAGATTCACTGCTGTTCAAAAAATGCAAATAGATGGTCCTCCAGGG

1210 1220 1230 1240 1250

TCAGCTGAGCCTATCCCCCCACCGAGGACCAGCACCCTATCTAGGCCACC 1260 1270 1280 1290 1300

ACCTAGCCGATTATCGGAGGTTCGGTTCAAAGTTCACGATCCGAAGGCAT

1310 1320 1330 1340 1350

ATTCTAAAGGCGGTACTTTAGAAAACACTATCAATGGGGCTCGGGGCCAG

1360 1370 1380 1390 1400 CCAGGACCTGCTCCACCGGCAGACGAAGAAGCTGGACCACCTCCGCATCT

1410 1420 1430 1440 1450

CGTTCATGCTTTCCAAGGTATCAACAAACTGCTCGGCACGACCCCCTCGG

1460 1470 1480 1490 1500

ACATCGACAAGTACTCGCGCATCGTGTTCCCCGTCTGCTGCGTTTGCTTT 1510 1520 1530 1540 1550

AACCTTATGTACTGGATCATTTACCTTCACGTGTCTGACGTCGTGGCTGA

1560 1570 1580 1590 1600

TGACTTGGTACTACTAGGCGAAGAAAATTGAATTCTCTTTAACTATACCG

1610 1620 1630 1640 1650 GACTTGTTTTAACTTAGGGTGCTTATGATCAACCATCCATCAGGTTTCGG

1657

TAAAGTT -27-

The coding sequence starts at 115 bp and ends at 1581 bp, and encodes a polypeptide of 488 amino acids having the predicted sequence:

MHTSRPRGVHSIALVLSLAIAWLPHADHAAGAGGGGMFGDVNISAILDSLSVSYDK RVRPNYGGPPVDVGVNMYVLSISSLSEVKMDFTLDFYFRQFWTDPRLAYKKSTGVE TLSVGSEFIRNIWVPDTFFVNEKQSYFHIATTSNEFIRIHHSGSITRSIRLTITAS CPMDLQYFPMDRQLCNIEIESFGYTMRDIRYKWNEGPNSVGVSSEVSLPQFKVLGH RQRAMEISLTTGNYSRLACEIQFVGSMGYYLIQIYIPSGLIVIISWVSFWLNRNAT PARVALGVTTVLTMTTLMSSTNAALPKISYVKSIDVYLGTCFVMVFTSLLEYATVG YMAKRIQMRKQRFTAVQKMQIDGPPGSAEPIPPPRTSTLSRPPPSRLSEVRFKVHD PKAYSKGGTLENTINGARGQPGPAPPADEEAGPPPHLVHAFQGINKLLGTTPSDID KYSRIVFPVCCVCFNLMYWIIYLHVSDVVADDLVLLGEEN (SEQ ID NO : 2 ) . In vitro transcribed mRNA was made from the mutated cDNA encoded by plasmid pIVY16 and used for oocyte physiological studies.

-28-

EXAMPLE III

Expression of cDNA encoding cyclodiene resistant GABA gated chloride channel subunit in baculovirus expression system A BamHI/XhoI DNA fragment from plasmid pIVY12 described in Example I containing the complete coding sequence of the Heliothis virescens cyclodiene resistant

GABA gated chloride channel subunit was cloned into the baculovirus transfer vector pBacPacδ (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 pBacPacδ and is depicted in Figure 2. Spodoptera fruαiperda 9 (Sf9) cells were cotransfected by pIVY13 and wild type Autoαrapha californica nuclear polyhedrosis virus (AcMNPV) using the lipofectin procedure. In transfer vector pIVY13, 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. -29-

To verify that expression of cDNA encoding the GABA gated chloride channel subunit results in production of a functional GABA gated chloride channel, whole cell voltage clamp recordings were made at thirty hours post infection. Results of the whole cell voltage clamp recordings are presented in Figure 3.

-₃ Application of 1x10 M GABA is indicated by the arrow marked "on." The data in Figure 3 indicate that GABA exhibits the gross functional effect on the GABA gated chloride channel, i.e. flux of chloride ion. The results presented in this example indicate that expression of DNA encoding a lepidopteran GABA gated chloride channel subunit in Sf9 cells results in a functional GABA gated chloride channel.

EXAMPLE IV

Expression of nucleic acid encoding lepidopteran GABA gated chloride channel in Xenopus oocytes

Messenger 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 Kit™ (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-

Recordings were made using a Dagan TEV200 voltage clamp interfaced with a MacLab4 data acquisition system running the MacLab Chart data acquisition/analysis software. Oocytes were positioned under a dissecting scope under constant perfusion with ND-96 standard saline using a Razel syringe perfusion pump, model A99- FY at 93.9 cc/hr. Glass electrodes (A-M Systems, Inc., 1.5mm x 0.86mm) were filled with 3M KCl and resistance (a function of the diameter of the channel opening) was measured to be between 0.7 and 1.5 mega ohms. Both electrodes were inserted into the oocyte at opposite sides, the resting potential was recorded and the voltage clamp turned on. 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. Further evidence that expression of the GABA gated chloride channel subunit in oocytes results in a functional GABA gated chloride channel was provided by similar assays with muscimol, a known GABA agonist, and picrotoxinin, a known GABA antagonist, both of which exhibited the predicted gross effects. Although known agonists and antagonists exhibited the predicted gross effects, the discrete -31-

pharmacology of the lepidopteran GABA gated chloride channel was determined to be unique, particularly with regard to chloride channel blockers (antagonists). In particular, 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. In particular, 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. In particular, 10 micromoles of picrotoxinin will result in nearly complete block of Drosophila Rdl (ffrench- Constant _____ al. , 1993, Nature 363: 449-451), while the same dose applied to the Heliothis channel will result in about a fifty percent block. This example demonstrates that the expression of mRNA corresponding to the cDNA in pIVYl2 results in a functional GABA gated chloride channel in oocytes, and further that the channel exhibits gross pharmacology similar to other chloride channels, but discrete pharmacology that distinguishes it from other characterized insect chloride channels. -32-

EXAMPLE V

Production of permanently transformed cell lines expressing a functional GABA gated chloride channel

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

Rdl cDNA. The 2.2 kbp fragment was cloned into the Spel and BamHI sites of pMK33. Plasmid pMK33 is depicted in

Figure 5 and was obtained from David S. Hogness, Departments of Biochemistry and Developmental Biology, Stanford University Medical Center, Stanford, CA. 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.

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, also designated pMtHy, 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 (Krasnow _____ al . , Cell 57:1031-1043, 1989) containing the actin 5C 3' end, is first cloned into the Bluescript+KS (Stratagene) Xbal site, filling in the ends. The XhoI-NotI fragment of the resulting plasmid is then inserted into Xhol-, NotI-

RECTIFIED SHEET (RULE 91) ISA/EP -33-

cleaved pMt/Hyl, to create pMt/Hy. Xhol, EcoRV, BamHI, and Spel are unique, Clal, Sail, EcoRi, PstI, and Hindlll are not unique.

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 μg/ml. The Heliothis GABA gated chloride channel expression was induced by 10 μM 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.

- 34 - SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) ^'APPLICANT: RHONE-POULENC INC. (all designated states except U.S. WINGATE, Vincent Paul Mary (U.S. only) WOLFF, Mark Ambroze (U.S. only)

(ii) TITLE OF INVENTION: LEPIDOPTERAN GABA GATED CHLORIDE CHANNEL AND METHODS OF USE THEREOF

(iii) NUMBER OF SEQUENCES: 12

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: BURNS, DOANE, SWECKER & MATHIS, L.L.P.

(B) STREET: P.O. Box 1404

(C) CITY: Alexandria

(D) STATE: Virginia

(E) COUNTRY: United States

(F) ZIP: 22313-1404

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

( i) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: WO PCT/US98/05696

(B) FILING DATE: 24-MAR-1998

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Baumeister , Mary Katherine

(B) REGISTRATION NUMBER: 26,254

(C) REFERENCE/DOCKET NUMBER: 022650-462

( ix) TELECOMMUNICATION INFORMATION :

(A) TELEPHONE: (703) 836-6620

(B) TELEFAX: (703) 836-2021

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1657 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

RECTIFIED SHEET (RULE 91) ISA/EP 35

(A) NAME/KEY: CDS

(B) LOCATION: 115..1581

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GAATTCGGCA CGAGGACGCC TGAGGGCCTG TAAGAACACG CCAGTCCGGC CGGCACGGTG 60

ATACGCGGCT GCCGGCAGCC AGCGTCCGCA AGGGCGCACG CGGACCTGCA AAAC ATG 117

Met

1

CAT ACG AGC CGT CCG CGC GGC GTG CAC AGC ATC GCG CTA GTG CTG TCT 165 His Thr Ser Arg Pro Arg Gly Val His Ser lie Ala Leu Val Leu Ser 5 10 IS

CTC GCG ATT GCC TGG TTA CCT CAT GCT GAC CAT GCC GCG GGA GCG GGA 213 Leu Ala lie Ala Trp Leu Pro His Ala Asp His Ala Ala Gly Ala Gly 20 25 30

GGA GGG GGA ATG TTT GGT GAC GTC AAT ATC TCA GCC ATT TTG GAT TCG 261 Gly Gly Gly Met Phe Gly Asp Val Asn lie Ser Ala lie Leu Asp Ser 35 40 45

CTA AGT GTA AGC TAC GAC AAA AGA GTG AGG CCG AAC TAT GGA GGA CCG 309 Leu Ser Val Ser Tyr Asp Lys Arg Val Arg Pro Asn Tyr Gly Gly Pro 50 55 60 65

CCA GTG .GAT GTG GGA GTC AAC ATG TAC GTG CTC TCC ATC AGC TCC TTA 357 Pro Val Asp Val Gly Val Asn Met Tyr Val Leu Ser lie Ser Ser Leu 70 75 80

TCT GAA GTG AAA ATG GAT TTC ACC CTG GAT TTC TAC TTC AGA CAA TTT 405 Ser Glu Val Lys Met Asp Phe Thr Leu Asp Phe Tyr Phe Arg Gin Phe 85 90 95

TGG ACA GAC CCC AGG CTT GCT TAC AAA AAA AGC ACG GGT GTG GAG ACT 453 Trp Thr Asp Pro Arg Leu Ala Tyr Lys Lys Ser Thr Gly Val Glu Thr 100 105 110

CTG TCC GTC GGC TCG GAA TTT ATT AGA AAC ATA TGG GTA CCC GAC ACC 501 Leu Ser Val Gly Ser Glu Phe lie Arg Asn lie Trp Val Pro Asp Thr 115 120 125

TTC TTT GTT AAC GAA AAA CAG TCA TAT TTC CAC ATA GCT ACT ACA AGC 5 9 Phe Phe Val Asn Glu Lys Gin Ser Tyr Phe His He Ala Thr Thr Ser 130 135 140 145

AAC GAA TTC ATA CGC ATT CAT CAT TCT GGA TCT ATT ACT AGG AGT ATA 597 Asn Glu Phe He Arg He His His Ser Gly Ser He Thr Arg Ser He 150 155 160

AGA CTG ACT ATC ACC GCT TCT TGT CCG ATG GAT TTG CAG TAT TTT CCG 645 Arg Leu Thr He Thr Ala Ser Cys Pro Met Asp Leu Gin Tyr Phe Pro 165 170 175 - 36 -

ATG GAC CGT CAA TTA TGC AAT ATT GAA ATC GAA AGT TTT GGC TAC ACC 693 Met Asp Arg Gin Leu Cys Asn He Glu He Glu Ser Phe Gly Tyr Thr 180 185 190

ATG CGG GAC ATC CGA TAC AAG TGG AAT GAG GGG CCC AAC TCA GTG GGT 741 Met Arg Asp He Arg Tyr Lys Trp Asn Glu Gly Pro Asn Ser Val Gly 195 200 205

GTG TCG AGC GAA GTG TCT TTG CCG CAA TTC AAG GTG CTG GGC CAC CGG 789 Val Ser Ser Glu Val Ser Leu Pro Gin Phe Lys Val Leu Gly His Arg 210 215 220 225

CAG CGG GCC ATG GAG ATT TCT CTT ACG ACA GGA AAC TAC TCT CGT CTG 837 Gin Arg Ala Met Glu He Ser Leu Thr Thr Gly Asn Tyr Ser Arg Leu 230 235 240

GCA TGT GAA ATT CAA TTT GTA GGC TCG ATG GGA TAC TAT TTA ATT CAG 885 Ala Cys Glu He Gin Phe Val Gly Ser Met Gly Tyr Tyr Leu He Gin 245 250 255

ATT TAT ATT CCG TCT GGC CTA ATT GTC ATT ATA TCT TGG GTA TCA TTT 933 He Tyr He Pro Ser Gly Leu He Val He He Ser Trp Val Ser Phe 260 265 270

TGG TTG AAT CGA AAT GCG ACA CCT GCA AGG GTA TCA CTA GGT GTC ACA 981 Trp Leu Asn Arg Asn Ala Thr Pro Ala Arg Val Ser Leu Gly Val Thr 275 280 285

ACT GTA TTG ACG ATG ACG ACG CTC ATG TCG TCC ACG AAT GCG GCT CTG 1029 Thr Val Leu Thr Met Thr Thr Leu Met Ser Ser Thr Asn Ala Ala Leu 290 295 300 305

CCC AAG ATC TCA TAT GTC AAG TCC ATC GAT GTC TAT CTG GGA ACT TGT 1077 Pro Lys He Ser Tyr Val Lys Ser He Asp Val Tyr Leu Gly Thr Cys 310 315 320

TTC GTC ATG GTC TTC ACC AGT TTA CTA GAA TAT GCC ACG -GTT GGC TAT 1125 Phe Val Met Val Phe Thr Ser Leu Leu Glu Tyr Ala Thr Val Gly "Tyr 325 330 335

ATG GCG AAA AGG ATA CAG ATG AGG AAA CAA AGA TTC ACT GCT GTT CAA 1173 Met Ala Lys Arg He Gin Met Arg Lys Gin Arg Phe Thr Ala Val Gin 340 345 350

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

CCG AGG ACC AGC ACC CTA TCT AGG CCA CCA CCT AGC CGA TTA TCG GAG ^' 1269 Pro Arg Thr Ser Thr Leu Ser Arg Pro Pro Pro Ser Arg Leu Ser Glu 370 375 380 385

GTT CGG TTC AAA GTT CAC GAT CCG AAG GCA TAT TCT AAA GGC GGT ACT 1317 Val Arg Phe Lys Val His Asp Pro Lys Ala Tyr Ser Lys Gly Gly Thr 390 395 400 - 37 -

TTA GAA AAC ACT ATC AAT GGG GCT CGG GGC CAG CCA GGA CCT GCT CCA 1365 Leu Glu Asn Thr He Asn Gly Ala Arg Gly Gin Pro Gly Pro Ala Pro 405 410 415

CCG GCA GAC GAA GAA GCT GGA CCA CCT CCG CAT CTC GTT CAT GCT TTC 1413 Pro Ala Asp Glu Glu Ala Gly Pro Pro Pro His Leu Val His Ala Phe 420 425 430

CAA GGT ATC AAC AAA CTG CTC GGC ACG ACC CCC TCG GAC ATC GAC AAG 1461 Gin Gly He Asn Lys Leu Leu Gly Thr Thr Pro Ser Asp He Asp Lys 435 440 445

TAC TCG CGC ATC GTG TTC CCC GTC TGC TGC GTT TGC TTT AAC CTT ATG 1509 Tyr Ser Arg He Val Phe Pro Val Cys Cys Val Cys Phe Asn Leu Met 450 455 460 465

TAC TGG ATC ATT TAC CTT CAC GTG TCT GAC GTC GTG GCT GAT GAC TTG 1557 Tyr Trp He He Tyr Leu His Val Ser Asp Val Val Ala Asp Asp Leu 470 475 480

GTA CTA CTA GGC GAA GAA AAT TGA ATTCTCTTTA ACTATACCGG ACTTGTTTTA 1611 Val Leu Leu Gly Glu Glu Asn * 485

ACTTAGGGTG CTTATGATCA ACCATCCATC AGGTTTCGGT AAAGTT 1657

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 489 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met His Thr Ser Arg Pro Arg Gly Val His Ser He Ala Leu Val Leu 1 5 10 15

Ser Leu Ala He Ala Trp Leu Pro His Ala Asp His Ala Ala Gly Ala 20 25 30

Gly Gly Gly Gly Met Phe Gly Asp Val Asn He Ser Ala He Leu Asp 35 40 45

Ser Leu Ser Val Ser Tyr Asp Lys Arg Val Arg Pro Asn Tyr Gly Gly 50 55 60

Pro Pro Val Asp Val Gly Val Asn Met Tyr Val Leu Ser He Ser Ser 65 70 75 80

Leu Ser Glu Val Lys Met Asp Phe Thr Leu Asp Phe Tyr Phe Arg Gin 85 90 95 - 38 -

Phe Trp Thr Asp Pro Arg Leu Ala Tyr Lys Lys Ser Thr Gly Val Glu 100 105 110

Thr Leu Ser Val Gly Ser Glu Phe He Arg Asn He Trp Val Pro Asp 115 120 125

Thr Phe Phe Val Asn Glu Lys Gin Ser Tyr Phe His He Ala Thr Thr 130 135 140

Ser Asn Glu Phe He Arg He His His Ser Gly Ser He Thr Arg Ser 145 150 155 160

He Arg Leu Thr He Thr Ala Ser Cys Pro Met Asp Leu Gin Tyr Phe 165 170 175

Pro Met Asp Arg Gin Leu Cys Asn He Glu He Glu Ser Phe Gly Tyr 180 185 190

Thr Met Arg Asp He Arg Tyr Lys Trp Asn Glu Gly Pro Asn Ser Val 195 200 205

Gly Val Ser Ser Glu Val Ser Leu Pro Gin Phe Lys Val Leu Gly His 210 215 220

Arg Gin Arg Ala Met Glu He Ser Leu Thr Thr Gly Asn Tyr Ser Arg 225 230 235 240

Leu Ala Cys Glu He Gin Phe Val Gly Ser Met Gly Tyr Tyr Leu He 245 250 255

Gin He Tyr He Pro Ser Gly Leu He Val He He Ser Trp Val Ser 260 265 270

Phe Trp Leu Asn Arg Asn Ala Thr Pro Ala Arg Val Ser Leu Gly Val 275 280 285

Thr Thr Val Leu Thr Met Thr Thr Leu Met Ser Ser Thr. Asn Ala Ala 290 295 300

Leu Pro Lys He Ser Tyr Val Lys Ser He Asp Val Tyr Leu Gly Thr 305 310 315 320

Cys Phe Val Met Val Phe Thr Ser Leu Leu Glu Tyr Ala Thr Val Gly 325 330 335

Tyr Met Ala Lys Arg He Gin Met Arg Lys Gin Arg Phe Thr Ala Val 340 345 350

Gin Lys Met Gin He Asp Gly Pro Pro Gly Ser Ala Glu Pro He Pro 355 360 365

Pro Pro Arg Thr Ser Thr Leu Ser Arg Pro Pro Pro Ser Arg Leu Ser 370 375 380

Glu Val Arg Phe Lys Val His Asp Pro Lys Ala Tyr Ser Lys Gly Gly - 39 -

385 390 395 400

Thr Leu Glu Asn Thr He Asn Gly Ala Arg Gly Gin Pro Gly Pro Ala 405 410 415

Pro Pro Ala Asp Glu Glu Ala Gly Pro Pro Pro His Leu Val His Ala 420 425 430

Phe Gin Gly He Asn Lys Leu Leu Gly Thr Thr Pro Ser Asp He Asp 435 440 445

Lys Tyr Ser Arg He Val Phe Pro Val Cys Cys Val Cys Phe Asn Leu 450 455 460

Met Tyr Trp He He Tyr Leu His Val Ser Asp Val Val Ala Asp Asp 465 470 ' 475 480

Leu Val Leu Leu Gly Glu Glu Asn * 485

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1657 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 115..1581

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

GAATTCGGCA CGAGGACGCC TGAGGGCCTG TAAGAACACG CCAGTCCGGC CGGCACGGTG 60

ATACGCGGCT GCCGGCAGCC AGCGTCCGCA AGGGCGCACG CGGACCTGCA AAAC ATG 117

Met 490

CAT ACG AGC CGT CCG CGC GGC GTG CAC AGC ATC GCG CTA GTG CTG TCT 165 His Thr Ser Arg Pro Arg Gly Val His Ser He Ala Leu Val Leu Ser 495 500 505

CTC GCG ATT GCC TGG TTA CCT CAT GCT GAC CAT GCC GCG GGA GCG GGA 213 Leu Ala He Ala Trp Leu Pro His Ala Asp His Ala Ala Gly Ala Gly 510 515 520

GGA GGG GGA ATG TTT GGT GAC GTC AAT ATC TCA GCC ATT TTG GAT TCG 261 Gly Gly Gly Met Phe Gly Asp Val Asn He Ser Ala He Leu Asp Ser 525 530 535 - 40 -

CTA AGT GTA AGC TAC GAC AAA AGA GTG AGG CCG AAC TAT GGA GGA CCG 309 Leu Ser Val Ser Tyr Asp Lys Arg Val Arg Pro Asn Tyr Gly Gly Pro 540 545 550

CCA GTG GAT GTG GGA GTC AAC ATG TAC GTG CTC TCC ATC AGC TCC TTA 357 Pro Val Asp Val Gly Val Asn Met Tyr Val Leu Ser He Ser Ser Leu 555 560 565 570

TCT GAA GTG AAA ATG GAT TTC ACC CTG GAT TTC TAC TTC AGA CAA TTT 405 Ser Glu Val Lys Met Asp Phe Thr Leu Asp Phe Tyr Phe Arg Gin Phe 575 580 585

TGG ACA GAC CCC AGG CTT GCT TAC AAA AAA AGC ACG GGT GTG GAG ACT 453 Trp Thr Asp Pro Arg Leu Ala Tyr Lys Lys Ser Thr Gly Val Glu Thr 590 595 600

CTG TCC GTC GGC TCG GAA TTT ATT AGA AAC ATA TGG GTA CCC GAC ACC 501 Leu Ser Val Gly Ser Glu Phe He Arg Asn He Trp Val Pro Asp Thr 605 610 615

TTC TTT GTT AAC GAA AAA CAG TCA TAT TTC CAC ATA GCT ACT ACA AGC 549 Phe Phe Val Asn Glu Lys Gin Ser Tyr Phe His He Ala Thr Thr Ser 620 625 630

AAC GAA TTC ATA CGC ATT CAT CAT TCT GGA TCT ATT ACT AGG AGT ATA 597 Asn Glu Phe He Arg He His His Ser Gly Ser He Thr Arg Ser He 635 640 645 650

AGA CTG ACT ATC ACC GCT TCT TGT CCG ATG GAT TTG CAG TAT TTT CCG 645 Arg Leu Thr He Thr Ala Ser Cys Pro Met Asp Leu Gin Tyr Phe Pro 655 660 665

ATG GAC CGT CAA TTA TGC AAT ATT GAA ATC GAA AGT TTT GGC TAC ACC 693 Met Asp Arg Gin Leu Cys Asn He Glu He Glu Ser Phe Gly Tyr Thr 670 675 680

ATG CGG GAC ATC CGA TAC AAG TGG AAT GAG GGG CCC AAC- TCA GTG GGT 741 Met Arg Asp He Arg Tyr Lys Trp Asn Glu Gly Pro Asn Ser Val Gly 685 690 695

GTG TCG AGC GAA GTG TCT TTG CCG CAA TTC AAG GTG CTG GGC CAC CGG 789 Val Ser Ser Glu Val Ser Leu Pro Gin Phe Lys Val Leu Gly His Arg 700 705 710

CAG CGG GCC ATG GAG ATT TCT CTT ACG ACA GGA AAC TAC TCT CGT CTG 837 Gin Arg Ala Met Glu He Ser Leu Thr Thr Gly Asn Tyr Ser Arg Leu 715 720 725 730

GCA TGT GAA ATT CAA TTT GTA GGC TCG ATG GGA TAC TAT TTA ATT CAG 885 Ala Cys Glu He Gin Phe Val Gly Ser Met Gly Tyr Tyr Leu He Gin 735 740 745

ATT TAT ATT CCG TCT GGC CTA ATT GTC ATT ATA TCT TGG GTA TCA TTT 933 He Tyr He Pro Ser Gly Leu He Val He He Ser Trp Val Ser Phe 750 755 760 - 41 -

TGG TTG AAT CGA AAT GCG ACA CCT GCA AGG GTA GCA CTA GGT GTC ACA 981 Trp Leu Asn Arg Asn Ala Thr Pro Ala Arg Val Ala Leu Gly Val Thr 765 770 775

ACT GTA TTG ACG ATG ACG ACG CTC ATG TCG TCC ACG AAT GCG GCT CTG 1029 Thr Val Leu Thr Met Thr Thr Leu Met Ser Ser Thr Asn Ala Ala Leu 780 785 790

CCC AAG ATC TCA TAT GTC AAG TCC ATC GAT GTC TAT CTG GGA ACT TGT 1077 Pro Lys He Ser Tyr Val Lys Ser He Asp Val Tyr Leu Gly Thr Cys 795 800 805 810

TTC GTC ATG GTC TTC ACC AGT TTA CTA GAA TAT GCC ACG GTT GGC TAT 1125 Phe Val Met Val Phe Thr Ser Leu Leu Glu Tyr Ala Thr Val Gly Tyr 815 820 825

ATG GCG AAA AGG ATA CAG ATG AGG AAA CAA AGA TTC ACT GCT GTT CAA 1173 Met Ala Lys Arg He Gin Met Arg Lys Gin Arg Phe Thr Ala Val Gin 830 835 840

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

CCG AGG ACC AGC ACC CTA TCT AGG CCA CCA CCT AGC CGA TTA TCG GAG 1269 Pro Arg Thr Ser Thr Leu Ser Arg Pro Pro Pro Ser Arg Leu Ser Glu 860 865 870

GTT CGG TTC AAA GTT CAC GAT CCG AAG GCA TAT TCT AAA GGC GGT ACT 1317 Val Arg Phe Lys Val His Asp Pro Lys Ala Tyr Ser Lys Gly Gly Thr 875 880 885 890

TTA GAA AAC ACT ATC AAT GGG GCT CGG GGC CAG CCA GGA CCT GCT CCA 1365 Leu Glu Asn Thr He Asn Gly Ala Arg Gly Gin Pro Gly Pro Ala Pro 895 900 905

CCG GCA GAC GAA GAA GCT GGA CCA CCT CCG CAT CTC GTT CAT GCT TTC 1413 Pro Ala Asp Glu Glu Ala Gly Pro Pro Pro His Leu Val His Ala Phe 910 915 920

CAA GGT ATC AAC AAA CTG CTC GGC ACG ACC CCC TCG GAC ATC GAC AAG 1461 Gin Gly He Asn Lys Leu Leu Gly Thr Thr Pro Ser Asp He Asp Lys 925 930 935

TAC TCG CGC ATC GTG TTC CCC GTC TGC TGC GTT TGC TTT AAC CTT ATG 1509 Tyr Ser Arg He Val Phe Pro Val Cys Cys Val Cys Phe Asn Leu Met 940 945 950

TAC TGG ATC ATT TAC CTT CAC GTG TCT GAC GTC GTG GCT GAT GAC TTG 1557 Tyr Trp He He Tyr Leu His Val Ser Asp Val Val Ala Asp Asp Leu 955 960 965 970

GTA CTA CTA GGC GAA GAA AAT TGA ATTCTCTTTA ACTATACCGG ACTTGTTTTA 1611 Val Leu Leu Gly Glu Glu Asn * 975 - 42 - ACTTAGGGTG CTTATGATCA ACCATCCATC AGGTTTCGGT AAAGTT 1657

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 489 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :

Met His Thr Ser Arg Pro Arg Gly Val His Ser He Ala Leu Val Leu 1 5 10 15

Ser Leu Ala He Ala Trp Leu Pro His Ala Asp His Ala Ala Gly Ala 20 25 30

Gly Gly Gly Gly Met Phe Gly Asp Val Asn He Ser Ala He Leu Asp 35 40 45

Ser Leu Ser Val Ser Tyr Asp Lys Arg Val Arg Pro Asn Tyr Gly Gly 50 55 60

Pro Pro Val Asp Val Gly Val Asn Met Tyr Val Leu Ser He Ser Ser 65 70 75 80

Leu Ser Glu Val Lys Met Asp Phe Thr Leu Asp Phe Tyr Phe Arg Gin 85 90 95

Phe Trp Thr Asp Pro Arg Leu Ala Tyr Lys Lys Ser Thr Gly Val Glu 100 105 110

Thr Leu Ser Val Gly Ser Glu Phe He Arg Asn He Trp Val Pro Asp 115 120 125-

Thr Phe Phe Val Asn Glu Lys Gin Ser Tyr Phe His He Ala Thr Thr 130 135 140

Ser Asn Glu Phe He Arg He His His Ser Gly Ser He Thr Arg Ser 145 150 155 160

He Arg Leu Thr He Thr Ala Ser Cys Pro Met Asp Leu Gin Tyr Phe 165 170 175

Pro Met Asp Arg Gin Leu Cys Asn He Glu He Glu Ser Phe Gly Tyr 180 185 190

Thr Met Arg Asp He Arg Tyr Lys Trp Asn Glu Gly Pro Asn Ser Val 195 200 205

Gly Val Ser Ser Glu Val Ser Leu Pro Gin Phe Lys Val Leu Gly His 210 215 220 - 43 -

Arg Gin Arg Ala Met Glu He Ser Leu Thr Thr Gly Asn Tyr Ser Arg 225 230 235 240

Leu Ala Cys Glu He Gin Phe Val Gly Ser Met Gly Tyr Tyr Leu He 245 250 255

Gin He Tyr He Pro Ser Gly Leu He Val He He Ser Trp Val Ser 260 265 270

Phe Trp Leu Asn Arg Asn Ala Thr Pro Ala Arg Val Ala Leu Gly Val 275 280 285

Thr Thr Val Leu Thr Met Thr Thr Leu Met Ser Ser Thr Asn Ala Ala 290 295 300

Leu Pro Lys He Ser Tyr Val Lys Ser He Asp Val Tyr Leu Gly Thr 305 310 315 320

Cys Phe Val Met Val Phe Thr Ser Leu Leu Glu Tyr Ala Thr Val Gly 325 330 335

Tyr Met Ala Lys Arg He Gin Met Arg Lys Gin Arg Phe Thr Ala Val 340 345 350

Gin Lys Met Gin He Asp Gly Pro Pro Gly Ser Ala Glu Pro He Pro 355 360 365

Pro Pro Arg Thr Ser Thr Leu Ser Arg Pro Pro Pro Ser Arg Leu Ser 370 375 380

Glu Val Arg Phe Lys Val His Asp Pro Lys Ala Tyr Ser Lys Gly Gly 385 390 395 400

Thr Leu Glu Asn Thr He Asn Gly Ala Arg Gly Gin Pro Gly Pro Ala 405 410 415

Pro Pro Ala Asp Glu Glu Ala Gly Pro Pro Pro His Leu "Val His Ala 420 425 430

Phe Gin Gly He Asn Lys Leu Leu Gly Thr Thr Pro Ser Asp He Asp 435 440 445

Lys Tyr Ser Arg He Val Phe Pro Val Cys Cys Val Cys Phe Asn Leu 450 455 460

Met Tyr Trp He He Tyr Leu His Val Ser Asp Val Val Ala Asp Asp 465 470 475 480

Leu Val Leu Leu Gly Glu Glu Asn * 485

(2) INFORMATION FOR SEQ ID NO : 5 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs 44

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GGTCTAGAAC NACNGTNCTT ACNATGAAC 29

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GGCTCGAGGC RAANACCATN ACRARCCA 28

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 128 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

TCTAGAACGA CGGTGGTTAC GATGACGACG CTCATGTCGT CCACGAATGC GGCTCTGCCC 60

AAGATCTCAT ATGTCAAGTC CATCGATGTC TATCTGGGAA CTTGTTTCGT CATGGTCTTC 120

GCCTCGAG 128

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs 45 -

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 : AGGTCCATCG ATGTCTATCT GGGAA 25 (2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 279 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 : AGGTCCATCG ATGTCTATCT GGGAACTTGT TTCGTCATGG TCTTCACCAG TTTACTAGAA 60 TATGCCACGG TTGGCTATAT GGCTAAAAGG ATACAGATGA GGAAACAAAG ATTCACTGCT 120 GTTCAAAAAA TGCAAATAGA TGGTCCTCCA GGGTCAGCTG AGCCTATCCC CCCACCGAGG 180 ACCAGCACCC TATCTAGGCC ACCACCTAGC CGATTATCGG AGGTTCGGTT CAAAGTTCAC 240 GATCCGAAGG CATATTCTAA AGGCGGTACT TTAGAAAAA 279 (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GTTCACGATC CGAAGGCATA TTCT 24 - 46 ( 2 ) INFORMATION FOR SEQ ID NO : 11 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 575 base pairs

(B) TYPE : nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY : linear

( ii ) MOLECULE TYPE : DNA (genomic )

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

GTTCACGATC CGAAGGCATA TTCTAAAGGC GGTACTTTAG AAAACACTAT CAATGGGGCT 60

CGGGGCCAGC CAGGACCTGC TCCACCGGCA GACGAAGAAG CTGGACCACC TCCGCATCTC 120

GTTCATGCTT TCCAAGGTAT CAACAAACTG CTCGGCACGA CCCCCTCGGA CATCGACAAG 180

TACTCGCGCA TCGTGTTCCC CGTCTGCTTC GTTTGCTTTA ACCTTATGTA CTGGATCATT 240

TACCTTCACG TGTCTGACGT CGTGGCTGAT GACTTGGTAC TACTAGGCGA AGAAAATTGA 300

ATTCTCTTTA ACTATACCGG ACTTGTTTTA ACTATACCGG ACTTGTTTTA ACTTTAGGGT 360

GCTTATGATC AACCATCCAT CAAGTCTCGG TAAAGTTCTT TAAGTCTAGA ACGCTCAGTA 420

AAATAATAGC GTTCTTTGTG TTTATAAATA TAATTATAGT ACAGATCACT ATGTTTATTA 480

TAGATAAGTG TCGTGTATAT TGGCACTGGT AATATTAATT CTTTAGAAAA TAAAGATAAT 540

ATGAATTCAA AAAAAAAAAA AAAAAAAAAA AAAAA 575 (2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: AACTTGCTCG AGACTTGATG GAT 23

Claims

-47-What s Claimed is:

1. An isolated nucleic acid encoding a lepidopteran GABA gated chloride channel subunit.

2. The isolated nucleic acid of Claim 1 wherein said nucleic acid is isolatable from Heliothis virescens .

3. The isolated nucleic acid of Claim 2 having the sequence of SEQ ID No: 1 or SEQ ID NO: 3.

4. The isolated nucleic acid of Claim 2 having the sequence of nucleotides 115 to 1581 of SEQ ID NO: 1 or

SEQ ID NO: 3.

5. An isolated nucleic acid having a sequence encoding the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

6. An isolated nucleic acid capable of hybridizing to the complement of the nucleic acid of Claim 5 wherein said isolated nucleic acid encodes a lepidopteran GABA gated chloride channel subunit.

7. An isolated nucleic acid capable of hybridizing to the complement of the nucleic acid of SEQ ID NO: 1 or

SEQ ID NO: 3 wherein said isolated nucleic acid encodes a lepidopteran GABA gated chloride channel subunit.

8. An expression vector comprising the isolated nucleic acid of any one of Claims 1-7. -48-

9. A host cell comprising an expression vector comprising the isolated nucleic acid of any one of Claims 1-7.

10. The host cell of Claim 9 wherein said host cell is a bacterial cell, a yeast cell, an insect cell or a mammalian cell.

11. A Xenopus oocyte comprising the isolated nucleic acid of any one of Claims 1-7.

12. Recombinant lepidopteran GABA gated chloride channel.

13. The recombinant lepidopteran GABA gated chloride channel of Claim 12 having the ammo acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

14. A Xenopus oocyte expressing a functional lepidopteran GABA gated chloride channel.

15. An insect cell expressing a functional recombinant lepidopteran GABA gated chloride channel.

16. The cell of Claim 15 wherein said cell is an Sf9 cell.

17. A method for preparing a lepidopteran GABA gated chloride cnannel comprising culturing a host cell comprising the expression vector of Claim 8 under conditions suitable for expression. -49-

18. A method for preparing a Xenopus oocyte expressing a functional lepidopteran GABA gated chloride channel comprising injecting a Xenopus oocyte with a nucleic acid encoding a lepidopteran GABA gated chloride channel, and culturing said oocyte under conditions suitable for expression of said lepidopteran GABA gated chloride channel.

19. A composition comprising recombinant lepidopteran GABA gated chloride channel.

20. The composition of Claim 19 wherein said GABA gated chloride channel has the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

21. A composition comprising Xenopus oocyte expressing a functional lepidopteran GABA gated chloride channel.

22. A composition comprising a cell membrane containing a functional lepidopteran GABA gated chloride channel .

23. A composition comprising a host cell expressing a functional lepidopteran GABA gated chloride channel.

24. A method of identifying an agonist to a lepidopteran GABA gated chloride channel comprising applying a putative agonist to a Xenopus oocyte, a cell or a membrane expressing a lepidopteran GABA gated chloride channel in the presence of chloride ions, and

RECTIFIED SHEET (RULE 91) ISA/EP -50-

measuring chloride flux, wherein flux of chloride is indicative of an agonist.

25. A method of identifying an antagonist to a lepidopteran GABA gated chloride channel comprising applying a putative antagonist to a Xenopus oocyte, a cell or a membrane expressing a lepidopteran GABA gated chloride channel in the presence of chloride ions, and measuring chloride flux, followed by applying said putative antagonist and GABA to said oocyte or said cell or said membrane and measuring chloride flux; and comparing said chloride flux obtained in the presence of said 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 said putative antagonist relative the flux observed in the presence of GABA alone is indicative of an antagonist.

26. A kit containing a first container containing a recombinant lepidopteran GABA gated chloride channel in a cell membrane.

27. The kit of Claim 26 wherein said cell membrane is a membrane preparation, a freeze dried membrane preparation, an intact cell or an oocyte.