Promoters based on ecdysone receptor

Zeneca (now Syngenta) and Pioneer Hi-Bred have patents and patent applications directed to:

isolated DNA sequences of ecdysone receptors derived from diverse moth species. Methods for inducing the expression of proteins in plants by using an ecdysone receptor, and
the use of other inducible receptor such as the retinoid X receptor and the ultraspiracle receptor.

The patents granted to Syngenta protect the isolated DNA and amino acid sequences of the ecdysone receptor from the moth species Heliothis virescens.

Specific Patent Information

Patent Number

Title, Independent Claims and Summary of Claims

Assignee

US 6379945

Earliest priority – 26 May 1995
Filed – 24 May 1996
Granted – 30 April 2002
Expected expiry – 24 May 2016

Title – Gene switch

Claim 1
An isolated or synthetic DNA sequence encoding a polypeptide selected from the group consisting of (a) the Heliothis virescens ecdysone steroid receptor shown in SEQ ID NO: 5; (b) the transactivation domain of the Heliothis virescens ecdysone steroid receptor shown in amino acids 1-162 of SEQ ID NO: 5; (c) the DNA binding domain of the Heliothis virescens ecdysone steroid receptor shown in amino acids 163-228 of SEQ ID NO: 5; (d) the hinge domain of the Heliothis virescens ecdysone steroid receptor shown in amino acids 229-326 of SEQ ID NO: 5; e) the ligand binding domain of the Heliothis virescens ecdysone steroid receptor shown in amino acids 327-545 of SEQ ID NO: 5; (f) the carboxy terminus of the Heliothis virescens ecdysone steroid receptor shown in amino acids 546-577 of SEQ ID NO: 5; and (g) the hinge and ligand binding domains of the Spodoptera exigua ecdysone steroid receptor shown in SEQ ID NO: 7.

The United States patent claims an isolated or synthetic DNA sequence encoding the mentioned ecdysone receptor and any of the following domains of the ecdysone receptor:

- the transactivation domain;
- the DNA binding domain;
- the ligand binding domain;
- the hinge binding domain; and
- the carboxy terminus of the receptor.

Each domain has a specifically identified amino acid sequence. No particular relationship is required between domains; e.g., the DNA sequences encoding the individual domains can be provided in any order.

Syngenta

AU 711391 B2

Earliest priority – 26 May 1995
Filed – 20 May 1996
Granted – 14 October 1999
Expected expiry – 20 May 2016

Title – A gene switch comprising an ecdysone receptor

Claim 1
DNA comprising the sequence shown in Seq. ID No. 2, or a DNA sequence which hybridises to said DNA sequence under high stringency conditions.

Claim 2
DNA comprising the sequence shown in Seq. ID No.3, or a DNA sequence which hybridises to said DNA sequence under high stringency conditions.

Claim 3
DNA comprising the sequence shown in Seq. ID No. 4, or a DNA sequence which hybridises to said DNA sequence under high stringency conditions.

Claim 12
A polypeptide comprising the amino acid sequence shown in Seq. ID. No. 4 or any allelic variant or derivative thereof.

Claim 13
A polypeptide comprising part of the amino acid sequence shown in Seq ID. No. 4 or any allelic variant or derivative thereof, which sequence provides the Heliothis ecdysone receptor ligand binding domain.

Claim 14
A polypeptide comprising part of the amino acid sequence shown in Seq. ID. No. 4 or any allelic variant or derivative thereof, which sequence provides the Heliothis ecdysone receptor DNA binding domain.

Claim 15
A polypeptide comprising part of the amino acid sequence shown in Seq. ID. No. 4 or any allelic variant or derivative thereof, which sequence provides the Heliothis ecdysone receptor transactivation domain.

Claim 16
A polypeptide comprising part of the amino acid sequence shown in Seq. ID. No. 4 or any allelic variant or derivative thereof, which sequence provides the Heliothis ecdysone receptor hinge domain.

Claim 17
A polypeptide comprising part of the amino acid sequence shown in Seq. ID. No. 4 or any allelic variant or derivative thereof, which sequence provides the Heliothis ecdysone receptor carboxy terminal domain.

Claim 19
DNA comprising the sequence shown in Seq. ID No.6, or a DNA sequence which hybridises to said DNA sequence under high stringency conditions.

The Australian patent claims both the DNA and the amino acid sequence of Heliothis ecdysone receptor and the comprising domains of the receptor. In this patent the species of Heliothis is not limited to H. virescens. In addition, DNA sequences that hybridize to the claimed sequences and any allelic variant or derivatives of the polypeptide sequence of the complete receptor and its domains are part of the protected invention.

The specification states that hybridization can occur at low, medium or high stringency binding conditions. Arguably, at low stringency binding conditions, many prior art nucleic acids will hybridize to, and therefore anticipate, this type of claim. Similarly, a “derivative” could have a change at each nucleotide position of the sequence and would be anticipated by many known sequences.

EP 828829 A1

Earliest priority – 26 May 1995
Filed – 20 May 1996
Granted – Pending
Expected expiry – N/A

Title – A gene switch comprising an ecdysone receptor

Claim 1
DNA comprising the sequence shown in Seq ID No. 2.

Claim 2
DNA comprising the sequence shown in Seq ID No. 3.

Claim 3
DNA comprising the sequence shown in Seq ID No. 4.

Claim 4
DNA comprising a sequence which shows 60% or more homology with the sequence shown in Seq ID No.1, 2 or 3.

Claim 6
DNA which hybridises to the sequence shown in Seq ID No.2, 3 or 4, and which codes for at leaset part of the Heliothis ecdysone receptor.

Claim 8
DNA comprising part of the sequece shown in Seq ID No.2, and which codes for at least part of the Heliothis ecdysone receptor ligand binding domain.

Claim 9
DNA comprising part of the sequece shown in Seq ID No.3, and which codes for at least part of the Heliothis ecdysone receptor ligand binding domain.

Claim 10
DNA comprising part of the sequece shown in Seq ID No.4, and which codes for at least part of the Heliothis ecdysone receptor ligand binding domain.

Claim 15
DNA comprising part of the sequece shown in Seq ID No.2, and which codes for at least part of the Heliothis ecdysone receptor DNA binding domain.

Claim 16
DNA comprising part of the sequece shown in Seq ID No.3, and which codes for at least part of the Heliothis ecdysone receptor DNA binding domain.

Claim 17
DNA comprising part of the sequece shown in Seq ID No.4, and which codes for at least part of the Heliothis ecdysone receptor DNA binding domain.

Claim 22
DNA comprising part of the sequece shown in Seq ID No.2, and which codes for at least part of the Heliothis ecdysone receptor transactivation domain.

Claim 23
DNA comprising part of the sequece shown in Seq ID No.3, and which codes for at least part of the Heliothis ecdysone receptor transactivation domain.

Claim 24
DNA comprising part of the sequece shown in Seq ID No.4, and which codes for at least part of the Heliothis ecdysone receptor transactivation domain.

Claim 29
DNA comprising part of the sequece shown in Seq ID No.2, and which codes for at least part of the Heliothis ecdysone receptor hinge domain.

Claim 30
DNA comprising part of the sequece shown in Seq ID No.3, and which codes for at least part of the Heliothis ecdysone receptor hinge domain.

Claim 31
DNA comprising part of the sequece shown in Seq ID No.4, and which codes for at least part of the Heliothis ecdysone receptor hinge domain.

Claim 36
DNA comprising part of the sequece shown in Seq ID No.2, and which codes for at least part of the Heliothis ecdysone receptor carboxy terminal region.

Claim 37
DNA comprising part of the sequece shown in Seq ID No.3, and which codes for at least part of the Heliothis ecdysone receptor carboxy terminal region.

Claim 38
DNA comprising part of the sequece shown in Seq ID No.4, and which codes for at least part of the Heliothis ecdysone receptor carboxy terminal region.

Claim 44
A polypeptide comprising the amino acid sequence shown in Seq ID No.4 or any allelic variant or derivative thereof.

Claim 45
A polypeptide comprising part of the amino acid sequence shown in Seq ID No.4 or any allelic variant or derivative thereof, which sequence provides the Heliothis ecdysone receptor ligand binding domain.

Claim 46
A polypeptide comprising part of the amino acid sequence shown in Seq ID No.4 or any allelic variant or derivative thereof, which sequence provides the Heliothis ecdysone receptor DNA binding domain.

Claim 47
A polypeptide comprising part of the amino acid sequence shown in Seq ID No.4 or any allelic variant or derivative thereof, which sequence provides the Heliothis ecdysone receptor transactivation domain.

Claim 48
A polypeptide comprising part of the amino acid sequence shown in Seq ID No.4 or any allelic variant or derivative thereof, which sequence provides the Heliothis ecdysone receptor hinge domain.

Claim 49
A polypeptide comprising part of the amino acid sequence shown in Seq ID No.4 or any allelic variant or derivative thereof, which sequence provides the Heliothis ecdysone receptor carboxy terminal region.

Claim 51
DNA comprising the sequence shown in Seq ID No. 6.

Claim 52
DNA comprising a sequence which shows 60% or more homology with the sequence shown in Seq ID No.6.

Claim 54
DNA which hybridises to the DNA sequence shown in Seq ID No. 6 and which codes for at least part of Spodoptera ecdysone receptor.

Claim 56
DNA comprising part of the sequece shown in Seq ID No.6, and which codes for at least part of the Spodoptera ecdysone receptor ligand binding domain.

Claim 61
DNA comprising part of the sequece shown in Seq ID No.6, and which codes for at least part of the Spodoptera ecdysone receptor hinge domain.

The claims of the European patent application, in addition, recite DNA sequences encoding a Spodoptera ecdysone receptor from the moth genus Spodoptera.

Remarks

Related applications also filed in Canada (CA 2219121), Bulgaria (BG 102124 A), Brazil (BR 9608897 A), Czech Republic (CZ9703722 A3), Hungary (HU 9802225 AB), Japan (JP 11506319 T2), Norway (NO 975419 A), New Zealand (NZ 308162 A), Poland (PL 323587 A1) and Turkey (TR 9701436 T1). The application in China (CN 1191568 AA) has lapsed.

EP 1112360 A1

Earliest priority – 10 September 1998
Filed -10 September 1999
Granted – Pending
Expected expiry – N/A

Title – Ecdysone receptors and methods for their use

Claim 1

An isolated nucleic acid sequence that encodes an insect protein from a Pyrilidae species, wherein said nucleotide sequence is selected from the group consisting of
a) a nucleotide sequence comprising a sequence encoding an ecdysone receptor or Ultraspiracle;
b) a nucleotide sequence comprising the sequence set forth in SEQ ID NO:1 or 3;
c) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4;
d) a nucleotide sequence that hybridizes under stringent conditions to a sequence of a), b), or c).

Claim 2
An isolated polypeptide from a Pyrilidae species, wherein said polypeptide is selected from the group consisting of
a) a polypeptide sequence comprising an Ecdysone receptor or Ultraspiracle;
b) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:2 or 4;
c) a polypeptide encoded by a nucleotide sequence comprising the sequence set forth in SEQ ID NO:1 or 3;
d) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence comprising the sequence set forth in SEQ ID NO:1 or 3.

Claim 3
A method of selectively inducing gene expression of a protein of interest in a plant, said method comprising:
a) stably incorporating into the genome of said plant an expression cassette, said expression cassette comprising a promoter operably linked to a nucleotide sequence encoding an Ecdysone receptor, wherein said nucleotide sequence encoding the Ecdysone receptor is selected from the group consisting of
i) a nucleotide sequence from a Pyrilidae species comprising a sequence encoding an Ecdysone receptor;
ii) a nucleotide sequence comprising the sequence set forth in SEQ ID NO:1;
iii) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2;
iv) a nucleotide sequence that hybridizes under stringent conditions to a sequence of i), ii), or iii);
b) further stably incorporating into the genome of said plant a second expression cassette, wherein said expression cassette comprises a transcriptional regulatory region operably linked to a nucleotide sequence encoding said protein of interest, and wherein said transcriptional regulatory region is activated by the ligand-receptor complex;
c) contacting said plant with a ligand which complexes with said receptor, wherein said receptor-ligand complex interacts with the transcription regulatory region and induces gene expression of the protein of interest.

Claim 23
An expression vector comprising a promoter operably linked to a nucleotide sequence encoding an Ecdysone receptor wherein said nucleotide sequence encoding the Ecdysone receptor is selected from the group consisting of:
a) a nucleotide sequence from a Pyrilidae species comprising a sequence encoding an Ecdysone receptor;
b) a nucleotide sequence comprising the sequence set forth in SEQ ID NO:1;
c) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2;
d) a nucleotide sequence that hybridizes under stringent conditions to a sequence of a), b), or c).

Claim 25
An expression vector comprising a promoter operably linked to a nucleotide sequence encoding Ultraspiracle, and said nucleotide sequence encoding Ultraspiracle is selected from the group consisting of
a) a nucleotide sequence comprising the sequence set forth in SEQ ID NO:3;
b) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4;
c) a nucleotide sequence that hybridizes under stringent conditions to a sequence of a) or b).

The claims of the European patent application recite the isolated DNA sequences and the polypeptide sequences of an ecdysone receptorand an ultraspiracle derived from insects of the family Pyralidae, which is a moth family. Ecdysone response elements found in the promoters of the target genes are activated by heterodimers formed by an ultraspiracle and the ecdysone receptor.

The claims as filed also recite methods of inducing gene expression in a plant where the gene of interest is linked to a transcriptional regulatory region that responds to the ecdysone receptor-ligand complex and is activated by the complex. Expression vectors comprising a promoter operably linked to an ecdysone receptor encoding sequence and an ultraspiracle are also part of the disclosed invention.

Pioneer Hi-Bred International Inc.

Remarks

Application also filed in Australia (AU 59159/99 A1)

Note: Patent information on this page was last updated on 7 April 2006.

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Promoters based on ecdysone receptor