Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1997-11-17
2001-12-04
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S455000, C435S325000, C435S348000, C435S254110, C435S254200, C435S252300, C536S023100, C536S023500
Reexamination Certificate
active
06326165
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a cloned “basic helix loop helix -PER-ARNT-AhR-SIM” (bHLH-PAS) protein that is a juvenile hormone receptor (JHR), bHLH-PAS/JHR. In particular, this invention is directed to a bHLH-PAS/JHR gene isolated from Drosophila, termed the methoprene-tolerant (met) gene (Met-JHR). The present invention also is directed to in vitro and in vivo methods for screening insecticides using recombinant bHLH-PAS/JHRs. The present invention is further directed to methods for isolating polynucleotides encoding bHLH-PAS/JHRs from various insect species.
Worldwide insect damage to food and fiber costs billions of dollars annually. Although chemical insecticides are still the primary means of insect control, the use of chemicals has several drawbacks including high cost of discovery, potential environmental damage, and negative public opinion. One promising group of insecticides consists of analogues of insect hormones, such as juvenile hormone. Since vertebrates do not make juvenile hormone (JH), insecticides targeted to the JH system are highly toxic to certain insects, and have shown an extraordinary degree of environmental safety.
Juvenile hormones comprise a family of hormones that are secreted by the corpus allatum, and that play a role in a variety of critical functions in insects, including development, reproduction, and morphological differentiation. Riddiford, “Hormone Action at the Cellular Level,” in COMPREHENSIVE INSECT PHYSIOLOGY, BIOCHEMISTRY AND PHARMACOLOGY, VOLUME 8, Kerkut et al. (eds.), pages 37-84 (Pergamon Press 1985); Nijhout et al.,
Q. Rev. Biol
. 57:109 (1982). These hormones affect development in some insects by maintaining the larval stage and inhibiting metamorphosis. In adult insects, JH is involved in the regulation of reproductive physiology. Koeppe et al., “The Role of Juvenile Hormone in Reproduction,” in COMPREHENSIVE INSECT PHYSIOLOGY, BIOCHEMISTRY AND PHARMACOLOGY, VOLUME 8, Kerkut et al. (eds.), pages 165-203 (Pergamon Press 1985).
The action of JH is mediated by at least several types of JH binding proteins: a hemolymph carrier protein, a cell membrane bound receptor, and an intracellular receptor. The transport of JH to target tissues is believed to be accomplished by proteins in the hemolymph which bind with the hormone. Hammock et al.,
Pestic. Biochem. Physiol
. 7:517 (1977); Goodman et al., “Juvenile Hormone Cellular and Hemolymph Binding Proteins,” in COMPREHENSIVE INSECT PHYSIOLOGY, BIOCHEMISTRY AND PHARMACOLOGY, VOLUME 7, Kerkut et al. (eds.), pages 491-510 (Pergamon Press 1985). These JH binding proteins are thought to play roles both in the transport of JH and in the protection of JH from hemolymph esterases. Goodman et al.,
Am. Zool
. 14:1289 1974; Kramer et al.,
J. Biol. Chem
. 251:4979 (1974). Membrane bound receptors are known to bind ligand extracellularly and transmit a signal intracellularly. Wyatt et al.
Adv. Insect Physiol
. 26:1 (1996).
Cytosolic proteins that bind JH have been identified in numerous JH target tissues from a variety of insects. Van Mellaert et al.,
Insect Biochem
. 15:655 (1985); Klages et al.,
Nature
286:282 (1980); Engelmann et al.,
Insect Biochem
. 17:1045 (1987); Wisniewski et al.,
FEBS Lett
. 171:127 (1984). One of the inventors, Thomas G. Wilson, directed a research team that identified a cytosolic juvenile hormone-binding protein in
Drosophila melanogaster
that is characterized by saturable, high-affinity binding specific for JH III. Shemshedini et al.,
J. Biol. Chem
. 265:1913 (1990). Shemshedini et al. also demonstrated for the first time in any insect a correlation between the binding of JH to the cytosolic protein and a biological response to the hormone. Interference with the binding of JH to cognate intracellular receptors, therefore, would inhibit physiological functions dependent upon the hormone.
Until recently, novel insecticides that interfere with JH action were primarily discovered by an almost random testing of thousands of chemical compounds for efficacy against insects. This bioassay approach is slow and expensive since a group of test insects would have to be treated with various doses of each test compound, and, typically, finding compounds that are effective is exceedingly rare.
Accordingly, a need exists for an efficient method for testing insecticides targeted for the JH system.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide in vitro and in vivo assays for screening potential insecticides that are JH analogs and JH antagonists.
Another object of this invention is to provide methods for cloning bHLH-PAS/JHR genes from various insect species.
These and other objects are achieved, in accordance with one embodiment of the present invention by the provision of an isolated polynucleotide that comprises an insect bHLH-PAS/JHR gene, the Met-JHR gene. A “polynucleotide” includes DNA, RNA, mRNA, and cDNA molecules. A genomic polynucleotide comprising the Met gene is the St-H fragment in FIG.
1
. This fragment is 6.234 Kb, and its sequence is shown in
FIG. 2
(SEQ ID NO:1). Within this 6.234 Kb segment, there is a DNA sequence of 3.011 Kb, which includes an open reading frame that is divided by one intron of 69 nucleotides (bases 1520 to 1588). This 3.011 Kb sequence is the genomic Met-JHR DNA sequence.
FIG. 3
(SEQ ID NO:2).
The Met-JHR open reading frame lacking the intron codes for a protein of 716 amino acids and a having a molecular weight of about 78,720 daltons.
The nucleotide sequences of the genomic and cDNA Met-JHR differ, reflecting polymorphism. In
FIG. 3
, SEQ ID NO:3 represents a Met-JHR cDNA sequence, which begins at nucleotide 4 of the genomic sequence. There is one “polymorphic” difference between the genomic and cDNA nucleotide sequences that results in a change at the amino acid level. The nucleotide at position 1043 (genomic)/1039 (cDNA) may be C or T, which results in different deduced amino acids, R and W, respectively.
In the sequence of the genomic DNA, there is one ambiguity that results in different deduced amino acids. Base number 875 in the genomic DNA is designated “R,” which signifies that the nucleotide may be the purine C or G. This results in two possible corresponding deduced amino acid sequences, G (Gly) or R (Arg) respectively. In the sequence of the cDNA, there is one ambiguity that results in a different deduced amino acids. Base number 526 in the genomic DNA is designated “M,” which signifies that the nucleotide may be the purine A or C. This results in two possible corresponding deduced amino acid sequences, T (Thr) or P (Pro) respectively.
As used herein, the term “juvenile hormone receptor” (JHR) is used to mean a polypeptide that is involved in binding JHIII. As used herein, a polypeptide that is “involved in binding” JHIII includes a polypeptide that directly binds JHIII, a polypeptide that is a partner to a polypeptide that directly binds JHIII, and a polypeptide that is a partner to a complex of polypeptides that bind JHIII. One or more of these polypeptides may be required for binding JHIII. The skilled artisan will recognize that heterodimeric receptors are known in the art, and that both polypeptide that form the heterodimer are required for hormone binding and activity in the target cell. For example, the ultraspiracle polypeptide is partner to the ecdysone receptor, which together bind the hormone ecdysone and mediate ecdysone activation of gene transcription. Yao, et al.
Cell
71:63 (1992).
A multicomponent complex between bHLH-PAS polypeptide and steroid receptors has been documented. For example, a bHLH-PAS polypeptide that functions a co-activator during ligand induction of estrogen steroid receptor is amplified in breast cancer-1 (AIBC or ACTR). Anzick et al.
Science
, 277:956 (1997); Chen et al.
Cell
90:569 (1997). The JHR may involve a number of polypeptides that together form a ligand binding unit or functional signal transducing complex.
Thus, a suitable insect JHR gene encodes a polypeptide that directly binds to JHIII. Another
Heinrich Julia N.
Wilson Thomas G.
Colorado State University Research Foundation
Guzo David
Sutherland & Asbill & Brennan LLP
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