Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-01-02
2001-12-11
Spector, Lorraine (Department: 1646)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C530S350000
Reexamination Certificate
active
06329516
ABSTRACT:
The present invention relates to GABA-gated chloride channels from insects of the order lepidoptera, which are butterflies, moths and skippers that as adults have four broad or lanceolate wings.
Gamma amino butyric acid (GABA) is the major inhibitory neurotransmitter in the insect CNS and periphery; modulating membrane potential through a GABA-gated chloride channel (Anthony et al.,
GABA receptor molecules of insects,
Y. Pichon, ed., Birkhauser Verlag, Basel, Switzerland, 1993; Bloomquist,
Ann. Rev. Entomol.
41: 163-190, 1996; Hosie et al.,
Brit. J. Pharmacol.
115: 909-912, 1995). The significance of this GABA-gated channel (i.e., GABA receptor) as the site of action for a number of commercial insecticides has been known since the 1960s, but attempts to isolate the gene have been frustrated by the low homology between the insect sequence and available vertebrate probes and a low transcript abundance (Darlison,
Trends in Neur. Sci.
15: 469-474, 1992; ffrench-Constant,
Insect Biochem. Molec. Biol.
24: 335-345, 1994). More recently, a series of studies, directed by R. ffrench-Constant, utilized a conventional genetic approach that successfully located the gene (rdl) that determines resistance to dieldrin on the Drosophila polytene chromosome map (ffrench-Constant,
Experimentia Supplementum.
63: 210-223, 1993; ffrench-Constant et al.,
Nature
363: 449-451, 1993). Isolation and expression of Drosophila rdl has established its function as a GABA-gated chloride channel, though it has less than 35% homology to any of the subunits which constitute the functional analogue in vertebrates.
Isolation of the Drosophila sequence has since been followed by full-length determinations of rdl-like GABA receptors from the mosquito
Aedes aegypti
as well as partial sequences from the flour beetle and a roach (Kaku and Matsumura,
Comparative Biochemistry and Physiology C Pharmacology Toxicology and Endocrinology
108: 367-376, 1994; Miyazaki et al.,
Comparative Biochemistry and Physiology
111, 399-406, 1995; Thompson et al.,
Insect Mol. Biol.
2: 149-154, 1993; Thompson et al.,
FEBS Letters
325: 187-190, 1993). These gene determinations have allowed analyses to be conducted across several orders of insects showing that many species have adopted the same apparent strategy for developing resistance to insecticides that act at the chloride channel; mutation of a critical alanine in the second transmembrane domain to a serine. Indeed, site-directed mutagenesis experiments in heterologous expression systems have shown that altering this single residue is sufficient to reduce insecticidal potency by three orders of magnitude (Cole et al.,
Life Sciences
56: 757-765, 1995; Hosie et al.,
Brain Res.
693: 257-260, 1995; Lee et al.,
FEBS Letters
335: 351-318, 1993; Shotkoski et al.,
FEBS Lett.
80: 257-262, 1996). One of the more intriguing questions raised by the studies of resistance is why the mutation occurs at a low, but significant frequency in naive populations or in populations which have not been subjected to insecticide selection pressure in decades (ffrench-Constant, 1994).
Described herein are two lepidopteran receptor isoforms. In particular, these isoforms were isolated from the tobacco budwormn (TBW)
Heliothis virescens.
One isoform, TBW-a3 has in the second transmembrane the motif ProAlaArgVal
Ala
Leu (or PARVAL) usually associated with dieldrin susceptibility, while the other, TBW-a2, has the motif ProAlaArgVal
285
Ser
Leu (PARVSL, numbered as in SEQ ID 4) usually associated with dieldrin resistance. Genomic analysis reveals that both isoforms occur simultaneously in the same insecticide susceptible animals. Also described herein is a receptor isoform, TBW-a1, that has an unprecedented motif of ProAlaArgVal
Gln
Leu (or PARVQL).
SUMMARY OF THE INVENTION
In a first embodiment, the invention provides an isolated nucleic acid encoding a GABA-gated chloride channel comprising:
(a) a nucleic acid including a sequence encoding a protein sequence of SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 8, or a sequence having at least about 85% sequence identity with SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 8; or
(b) a nucleic acid that hybridizes with a nucleic acid encoding a protein sequence of SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 8 or the complementary sequence to SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 8, under stringent conditions; or
(c) a nucleic acid that hybridizes with a nucleic acid having a sequence of SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 7 or the complementary sequence to SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 7, under stringent conditions; or
(d) a nucleic acid has at least about 85% sequence identity with the coding region of SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 7.
In a second embodiment, the invention provides cells with the nucleic acid of the invention, which preferably express the channel at the cell surface. In another embodiment, the invention provides a process for producing a GABA-gated chloride protein in a cell of the invention, preferably by: growing the cell in a medium; and inducing the expression of the GABA-gated chloride channel by adding an expression inducing agent into the medium. The invention further provides the GABA-gated chloride channel, for instance as isolated from a cell of the invention.
In another embodiment, the invention provides a method for characterizing a bioactive agent, the method comprising (a) providing a first assay composition comprising (i) a cell expressing a GABA-gated chloride channel or (ii) an isolated GABA-gated chloride channel comprising the amino acid sequence encoded by the nucleic acid of the vector, or the amino acid sequence resulting from cellular processing of the amino acid sequence encoded by the nucleic acid of the vector, (b) contacting the first assay composition with the bioactive ag~ent or a prospective bioactive agent, and (c) measuring the binding of the bioactive agent or prospective bioactive agent or a cellular response mediated by a isolated GABA-gated chloride channel.
The invention further provides hybridization probes that selectively hybridize with a nucleic acid of the invention, or the complementary sequence thereof. The hybridization probe can be an amplification primer and the amplification conditions can be made sufficiently specific to amplify a GABA-gated chloride channel sequence from lepidoptera but not to amplify a GABA-gated chloride channel sequence from other insects such as Drosophila, Aedes, locust or beetle.
REFERENCES:
patent: 5767261 (1998-06-01), Wingate et al.
patent: WO 93/07161 (1993-04-01), None
Anthony, N. M., et al.,Comparative Molecular Neurobiology, Ed. by Y. Pichon, Birkhauser Verlag, Basel Switzerland (1993).
Bloomquist, J. R.Ann Rev. Entomol.41: 163-190 (1996).
Cole, L. M., et al.,Life Sciences56: 757-765 (1995).
Darlison, M. G.Trends of Neur. Sci.15: 469-474 (1992).
Daugherty, B.L., et al.,DNA Cell Biol.9: 453-459 (1990).
Eisenberg, D., et al.,Proc. Natl. Acad. Sci. USA81: 140-144 (1984).
Freissmuth, M., et al.,Proc Natl. Acad. Sci. USA88:8548-8552 (1991).
Ffrench-Constant, R. H.Insect Biochem. Molec. Biol24: 335-345 (1994).
Ffrench-Constant, R. H.Experimentia Supplementum63: 210-223 (1993).
Ffrench-Constant, R. H.Nature363: 449-451 (1993).
Ffrench-Constant, R. H., et al.,Proc. Natl. Acad. Sci. USA88: 7209-7213 (1991).
Frohman, M. A., et al.,Proc. Natl. Acad. Sci. USA85: 8998-9002 (1988).
von Heijne, G., et al.,FEBS Letters244: 439-446 (1989).
von Heijne, G.J. Membr. Biol.115: 195-201 (1990).
Henderson, J. E., et al.Biochem. Biophys. Res. Commun.193: 474-482 (1993).
Herzog, H., et al.DNA Cell Biol.13: 1221-1225 (1994).
Hosie, A. M., et al.Brit. J. Pharmacol.115: 909-912 (1995).
Hosie, A. M., et al.Brain Res.693: 257-260 (1995).
Kaku, K., et al.Comp. Biochem. Physiol.108: 367-376 (1994).
Krishnaswamy, S., et al.J. Biol. Chem.267: 26110-26120 (1992).
Lee, H-J., et al.FEBS Letters335: 315-318 (1993).
Loh, E. Y.Science243: 217 (1989).
Miyazaki, M., et al.,Comp. Biochem. Physiol.111B: 399-406 (1995).
Ohta, Y., et al.,Biochemistry31: 12680-12687 (1992).
Halling Blaik P.
Yuhas Debra A.
FMC Corporation
FMC Corporation
O'Hara Eileen B.
Spector Lorraine
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