Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1999-09-10
2003-01-07
Nguyen, Dave T. (Department: 1635)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S295000, C800S320000, C800S323300
Reexamination Certificate
active
06504082
ABSTRACT:
FIELD OF THE INVENTION
The field of the invention is inducible gene expression systems wherein expression is controlled by a novel ecdysone receptor or its derivatives. The field particularly relates to the isolation and characterization of nucleic acid and polypeptides for a novel ecdysone receptor. The nucleic acid and polypeptides are useful in novel gene expression systems inducible with ecdysone or derivative receptor agonists.
BACKGROUND OF THE INVENTION
The steroid hormone 20-hydroxyecdysone, also known as &bgr;-ecdysone, controls timing of development in many insects. Ecdysone triggers coordinate changes in tissue development that results in metamorphosis. See generally, Kollman (ed.)
Ecdysone: From Chemistry to Mode of Action
, Thieme Medical Pub., NY (1989), which is incorporated herein by reference. The generic term “ecdysone” is frequently used as an abbreviation for 20-hydroxyecdysone.
The Ecdysone receptor (EcR) polypeptide comprises a ligand-binding domain, a DNA-binding domain, and a transactivating domain. The receptor binds to 20-hydroxyecdysone and transactivates gene expression of a target gene in the nucleus.
The ligand-binding domain of the receptor polypeptide provides the means by which the 5′ regulatory region of the target gene is activated in response to the hormone. Other chemicals, such as the non-steroidal ecdysone agonist RH5849 (Wing,
Science
241:467-469 (1988)), will also act as a chemical ligand for the ligand-binding domain of EcR.
The DNA-binding domain comprises a sequence of amino acids that binds noncovalently to a response element, a specific nucleotide sequence in a target gene. A response element is located in the 5′ regulatory region of a target gene that is activated by the hormone.
The transactivation domain comprises one or more amino acid sequences acting as subdomains to affect the operation of transcription factors during pre-initiation and assembly at the TATA box. The effect of the transactivation domain is to allow repeated transcription initiation events leading to greater levels of gene expression from the target gene. Binding of the ligand causes a conformational change in the receptor polypeptide and allows the transactivation domain to affect transcription of the coding sequence in the target gene, resulting in production of the target polypeptide.
The EcR in Drosophila exists in three isoforms, each with an independent biological function. All share the same heterodimeric partner, Ultraspiracle (USP). USP is most homologous to the retinoic acid receptor &agr;, also capable of forming heterodimers with EcR (Thomas et al.
Nature
362:471-475 (1993)). While each of the isoforms independently interacts with ecdysone, the addition of USP greatly enhances the affinity of the complex for binding to ecdysone response elements (EcRE) found in the promoters of genes. Ultraspiracle has been isolated and cloned and its ligand-binding domain identified (Henrich et al.,
Nucleic Acids Research
18:4143-4148 (1990)). See WO 94/01558.
Without bound ligand, the EcR/USP heteroduplex interacts weakly with the EcRE, inhibiting transcription. Binding of ecdysone or other ligand by the EcR/USP complex enhances its affinity for the EcRE, with the conformational changes in the complex functioning to facilitate transcription. The ecdysone heteromeric receptor complex binds the DNA with a high affinity and acts as a potent promoter of DNA transcriptional activation, with transient transcriptional inducibility facilitated by the presence of the ecdysone ligand.
It is often desirable to control the onset and extent of gene expression. The ability to selectively induce the expression of specific genes allows for the manipulation of development and function not only at the cellular but also at the system and organismal level. Thus, ecdysone receptor/Ultraspiracle provides a means of regulating gene expression. It provides for regulation using a chemical that may be extrinsically applied in order to trigger gene activation.
U.S. Pat. No. 4,981,784 discusses novel retinoic acid receptors encoding chimeric receptor proteins. The chimeras are constructed by exchanging functional domains between glucocorticoid, mineral corticoid, estrogen-related, thyroid, and retinoic acid receptors.
U.S. Pat. No. 5,310,662 discusses hormone and hormone-like receptors in which the transactivation domains are modified in terms of position or copy number, the receptors having increased transactivation properties.
U.S. Pat. No. 4,833,080 is directed to the regulation of eukaryotic gene expression controlled by prokaryotic peptides recognizing specific sequences in the gene and either activating or repressing transcription.
U.S. Pat. No. 5,554,510 is directed to regulating gene expression by providing a pair of unlike proteins, with unlike DNA-binding domains, which will form a heterodimer, bind to asymmetric DNA binding sites, and depress or increase gene expression.
U.S. Pat. No. 5,217,867 is directed to hormone and hormone-like receptors in which changes in position or copy number of the transactivation domain provides increased transactivation.
U.S. Pat. No. 5,571,696 is directed to members of the steroid/thyroid superfamily of receptors, DNA encoding these receptors, and methods for expressing them in host cells.
WO 90/11273 is directed to steroid/thyroid hormone receptor DNA-binding domain compositions that determine target site specificity, and methods for converting the target site specificity of one receptor into the target site specificity of another.
U.S. Pat. No. 5,298,429 is directed to bioassays for determining whether a compound is a hormone receptor agonist or antagonist, the assay involving a DNA sequence encoding a hormone response element operatively linked to a reporter gene.
U.S. Pat. No. 5,071,773 discusses hormone receptor bioassays useful to determine whether a protein, suspected of being a hormone receptor, has transcription-activating properties, and evaluating whether compounds are functional ligands for receptor proteins.
WO 93/21334 relates to chemically-inducible plant gene expression cassettes in plant cells transformed with the cassettes. An inducible promoter is operatively linked to a target gene, the inducible promoter being activated by a regulator protein also expressed in the cell. In the presence of an effective exogenous inducer, the activator protein induces expression of the target gene.
WO 93/23431 is directed to mutant steroid hormone receptors and methods for using these as a molecular switch in gene therapy.
WO 94/01558 is directed to various members of the steroid/thyroid superfamily of receptors that can interact with the insect-derived Ultraspiracle receptor to form multimeric species.
WO 92/16546 is directed to DNA segments that are response elements for ligands for several members of the steroid/thyroid superfamily of receptors. Response elements in combination with a functional promoter and gene provide recombinant vectors containing a gene responsive to ligands for members of the steroid/thyroid superfamily of receptors.
WO 93/11235 is directed to the interaction of various members of the steroid/thyroid superfamily of receptors to form multimers of more than one receptor. This modulates the ability of a first receptor to transactivate the transcription of genes maintained under the hormone expression control in the presence of the cognate ligand for the first receptor.
U.S. Pat. No. 5,534,418 is directed to methods for controlled production of recombinant proteins in cells. Transcription of the gene encoding the protein is maintained under the control of a transcription control element that is activated by a ligand receptor complex. The complex is formed when a ligand (a hormone or analog of a hormone) is complexed with a receptor (which is a hormone receptor or functional analog).
WO 91/13167 is directed to the isolation of insect DNA sequences having the characteristics of insect steroid receptors.
WO 96/27673 discusses recombinant inducible gene expression systems involving the ecdysone receptor. Chim
Albertsen Marc C.
Brooke Catherine D.
Garnaat Carl W.
Roth Bradley Allen
Alston & Bird LLP
Nguyen Dave T.
Pioneer Hi-Bred International , Inc.
Whiteman Brian
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