Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-03-23
2004-09-07
Celsa, Bennett (Department: 1639)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C530S350000, C530S858000, C435S325000, C435S361000
Reexamination Certificate
active
06787642
ABSTRACT:
CROSS-REFERENCES TO RELATED APPLICATIONS
Not Applicable.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a field of insect transporter nucleic acids and proteins, and their use as novel target sites for insecticide development and insecticide action. Recombinant proteins and peptides with insecticidal activity, isolated DNA molecules encoding them, vectors comprising the nucleic acids, and methods of preparing them are provided. Methods of screening for compounds that modulate activity of or bind to membrane transporters are also provided.
2. Description of the Related Art
At present, insecticide resistance is posing serious problems in pest management. Consequently the development of new molecular targets in insect systems is greatly increased.
One method of controlling insect development consists of using biologically active hormones to interfere with insect developmental processes. This method has been used in several insect-based industries, such as in the silk industry. For example, JP 79042912 and JP 50029371 (both to Ajinomoto KK) relate to cultivation of silkworms by feeding with an ecdysis hormone component and juvenile hormone. JP 51013684 (to Takeda) also employs biologically active hormones in combination with contamination controlling agents, as part of a method for preventing internal silkworm contamination. This method, more specifically, provides for treatment with steroid-like molting hormone, juvenile hormone, and antibiotics at specific developmental stages.
Approaches such as the one above suffer several disadvantages, most significantly a relatively limited effectiveness because of the narrow window of susceptibility of insects to these types of steroid-like hormones. On a commercial scale, such agents for insecticidal applications are relatively ineffective, providing for disruption of insect development only at the end of a molt episode.
Insects, including lepidopteran insects, continue to elicit significant loss to many commercially important agricultural crops, including grains (corn, wheat, cotton, soybeans), and various vegetable, fruit (grapes,apples, peaches), and nut crops (almonds, walnuts). Hence, significant economic incentive exists for developing safer and more economic insect controlling strategies.
Conventional insect pest control methods rely primarily on relatively toxic, and non-specific chemical formulations, and have become increasingly unacceptable because of potential toxicities to humans and animals, as well as destruction of desirable plant and animal life. The continued threat such agents pose to the environment add to the growing need for more bio-compatible, specific, yet effective, insect population control techniques. Repeated use of conventional chemical insecticides also enhances the potential for insect resistance, resulting in increased risk of insecticide resistant insect strains and reduced effectiveness.
Alternatively, novel targets for insecticide targets can be identified, such as &ggr;-Aminobutyric acid (GABA). GABA is the primary neurotransmitter in the vertebrate central nervous system and in invertebrate central and peripheral nervous systems. For example, in
Manduca sexta
, GABA has been shown to inhibit neuronal activity, suggesting its involvement in regulating central neural functions in this insect. GABA transporters and other transporters affecting the central nervous system of insects could be used to provide new insecticide targets.
Specifically, insecticides presently available act on only a limited number of target sites and increased insecticide resistance has made many of these targets of limited value. Despite recent advances in the understanding of the biology of insects, a need continues to exist in the art of insect management and control for novel target sites.
SUMMARY OF THE INVENTION
The present invention discloses the existence of novel insect transporters that can be used as targets for screening of new insecticides.
The nucleic acids of this invention encode insect cell membrane transporter polypeptides including acetylcholine transporters, serotonin transporters, proline transporters, glutamate transporters, neurotransmitter transporters encoded by the inebriated gene, orphan transporters, GABA transporters, and LAT transporters. The polypeptides have greater than about 70% amino acid identity to sequences selected from the group consisting of: SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, and 16.
In one preferred embodiment, the nucleic acids of this invention encode polypeptides with amino acid sequences selected from the group consisting of: SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, and 16. In another preferred embodiment, the nucleic acids have nucleotide sequences selected from the group consisting of: SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, and 15.
This invention also provides insect cell membrane transporter polypeptides with greater than 70% amino acid sequence identity to polypeptides with amino acid sequences selected from the group consisting of: SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, and 16. In another embodiment, this invention provides polypeptides with amino acid sequences selected from the group consisting of: SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, and 16.
This invention also provides methods of screening for compounds which modulate the activity of insect cell membrane transporters encoded sequences selected from the group consisting of: SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, and 15 and nucleic acids encoding the amino acid sequences selected from the group consisting of: SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, and 16. The method comprises the steps of contacting a recombinant cell expressing the insect cell membrane transporter with a test compound and determining the ability of the test compound to modulate the activity of the membrane transporter. In preferred embodiments, the cell membrane transporter has an amino acid sequence selected from the group consisting of: SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, and 16. This invention also provides a compound identified by the above method.
In yet another embodiment, this invention provides methods of screening for a compound which binds to an insect cell membrane transporter. The method initially comprises attaching a membrane transporter polypeptide with an amino acid sequence selected from the group consisting of: SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, and 16 to a solid surface. This polypeptide is then exposed to test compounds or library of compounds and the ability of the compounds to bind to the transporter is measured. This invention also provides a compound identified by the above method.
In yet another embodiment, the invention provides cells comprising recombinant nucleic acids encoding cell membrane transporter polypeptides with greater than 70% amino acid identity to sequences selected from the group consisting of: SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, and 16; recombinant nucleic acids with sequences selected from the group consisting of: SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, and 15; and recombinant nucleic acids encoding polypeptides with amino acid sequences selected from the group consisting of: SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, and 16.
BRIEF DESCRIPTION OF SEQUENCE IDENTIFIERS
SEQ ID NO: 1 refers to the nucleotide sequence of the
Manduca sexta
acetylcholine transporter.
SEQ ID NO: 2 refers to the amino acid sequence of the
Manduca sexta
acetylcholine transporter.
SEQ ID NO: 3 refers to the nucleotide sequence of the
Manduca sexta
serotonin transporter.
SEQ ID NO: 4 refers to the amino acid sequence of the
Manduca sexta
serotonin transporter.
SEQ ID NO: 5 refers to the nucleotide sequence of the
Manduca sexta
proline transporter.
SEQ ID NO: 6 refers to the amino acid sequence of the
Manduca sexta
proline transporter.
SEQ ID NO: 7 refers to the nucleotide sequence of the
Aedes aegypti
glutamate transporter.
SEQ ID NO: 8 refers to the amino acid sequence of the
Aedes aegypti
glutamate transporter.
SEQ ID NO: 9 refers to the nucleotide sequence of the
Manduca sexta
neurotransmitter transporter encoded
Gill Sarjeet
Ross Linda S.
Celsa Bennett
Epperson Jon D.
The Regents of the University of California
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