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US5589362A
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US
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Tetracycline regulated transcriptional modulators with altered DNA binding specificities
Assignee: Abbott GmbH and Co KG
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Filing: 1995-06-07
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Granted: 1996-12-31
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Expires: 2015-06-07
Abstract
Abstract Isolated nucleic acid molecules encoding fusion proteins which regulate transcription in eukaryotic cells are disclosed. The fusion proteins of the invention comprise a Tet repressor having at least one amino acid mutation that confers on the fusion protein an ability to bind a class B tet operator sequence having a nucleotide substitution at position +4 or +6, operatively linked to a polypeptide which regulates transcription in eukaryotic cells. Methods for regulating transcription of a tet operator-linked gene in a cell are also provided. In one embodiment, the method involves introducing into the cell a nucleic acid molecule encoding a fusion protein which regulates transcription, the fusion protein comprising a Tet repressor having at least one amino acid mutation that confers on the fusion protein an ability to bind a class B tet operator sequence having a nucleotide substitution at position +4 or +6, operatively linked to a polypeptide which regulates transcription in eukaryotic cells, and modulating the concentration of a tetracycline, or analogue thereof, in contact with the cell.
Inventors
- 👤 Hermann Bujard
- 👤 Manfred Gossen
- 👤 Wolfgang Hillen
- 👤 Vera Helbl
- 👤 Dirk Schnappinger
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 23:17
CPC Classifications
A; A01; A01K; A01K67/00; A01K67/027; A01K67/0275; C; C07; C07K; C07K14/00; C07K14/005; C07K14/195; C07K14/24; C07K14/245; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/635; C12N15/67; C12N15/79; C12N15/85; C12N15/8509; A01K2217/00; A01K2217/05; A01K2217/07; A01K2217/075; A01K2217/20; A01K2227/00; A01K2227/10; A01K2227/105; A01K2267/00; A01K2267/03; A01K2267/0393; A61; A61K; A61K48/00; C07K2319/00; C07K2319/01; C07K2319/09; C07K2319/61; C07K2319/70; C07K2319/71; C07K2319/80; C12N2710/00; C12N2710/00011; C12N2710/16011; C12N2710/16611; C12N2710/16622; C12N2830/00; C12N2830/001; C12N2830/002; C12N2830/003; C12N2830/005; C12N2830/006; C12N2830/30; C12N2830/32
Claims Analysis
Total Claims: 22
Claim 1
Independent Claim
62 words
An isolated nucleic acid molecule encoding a fusion protein which regulates transcription, the fusion protein comprising a Tet repressor having at least one amino acid mutation that confers on the fusion protein an ability to bind a class B tet operator sequence having a nucleotide substitution at position +4 or +6, operatively linked to a polypeptide which regulates transcription in eukaryotic cells.
Claim 2
Dependent Claim
62 words
An isolated nucleic acid molecule encoding a fusion protein which regulates transcription, the fusion protein comprising a Tet repressor having at least one amino acid mutation that confers on the fusion protein an ability to bind a class B tet operator sequence having a nucleotide substitution at position +4 or +6, operatively linked to a polypeptide which regulates transcription in eukaryotic cells.
Claim 3
Dependent Claim
15 words
The nucleic acid molecule of claim 1, wherein the polypeptide activates transcription in eukaryotic cells.
Claim 4
Dependent Claim
15 words
The nucleic acid molecule of claim 1, wherein the polypeptide inhibits transcription in eukaryotic cells.
Claim 5
Dependent Claim
26 words
The nucleic acid molecule of claim 1, wherein the fusion protein binds a class B tet operator sequence having a cytosine substitution at position +4 (tetO-4C).
Claim 6
Dependent Claim
47 words
The nucleic acid molecule of claim 4, wherein the Tet repressor has mutations selected from the group consisting of 37ES39PQ, 37ES39PQ42YM, 37ES39PQ41LV42YM, ES39PQ41LI42YM, 37ET39PQ, 37ET39PQ42YM, 37ET39PQ41LV, ET39PQ41LV42YM, 37EL39PQ42YM, 37EL39PQ41LM42YM, 37EM39PQ42YM, EM39PQ41LV42YM, 37EV39PQ, 37EV39PQ42YM, 37EV39PQ41LV42YM, EV39PQ41LT42YM, 37EI39PQ42YM, 37EH39PQ, 37ER39PQ, 37ER39PQ41LV42YM, EA39PQ, 37EA39PQ42YM, 37EA39PQ41LV42YM, 37EK39PQ, EK39PQ41LT42YM, 37EQ39PQ and 37EQ39PQ42YM.
Claim 7
Dependent Claim
34 words
The nucleic acid molecule of claim 5, wherein the Tet repressor has mutations selected from the group consisting of 37ES39PQ41LV42YM, 37ES39PQ41LI42YM, ET39PQ42YM, 37ET39PQ41LV42YM, 37EL39PQ41LM42YM, 37EV39PQ, EV39PQ42YM, 37EV39PQ41LV42YM, 37ER39PQ, 37ER39PQ41LV42YM, EA39PQ, 37EA39PQ42YM, 37EA39PQ41LV42YM and 37EK39PQ41LT42YM.
Claim 8
Dependent Claim
14 words
The nucleic acid molecule of claim 1, wherein the Tet repressor has mutations 37EA39PQ42YM.
Claim 9
Dependent Claim
14 words
The nucleic acid molecule of claim 1, wherein the Tet repressor has mutations 37EA42YM.
Claim 10
Dependent Claim
26 words
The nucleic acid molecule of claim 1, wherein the fusion protein binds a class B tet operator sequence having a cytosine substitution at position +6 (tetO-6C).
Claim 11
Dependent Claim
22 words
The nucleic acid molecule of claim 9, wherein the Tet repressor has mutations selected from the group consisting of 37ES43WR44HN or 37ES43WR44HV.
Claim 12
Dependent Claim
12 words
A recombinant expression vector comprising the nucleic acid molecule of claim 1.
Claim 13
Dependent Claim
12 words
An isolated host cell comprising the recombinant expression vector of claim 11.
Claim 14
Independent Claim
97 words
A method for regulating transcription of a tet operator-linked gene in an isolated cell, comprising: introducing into the isolated cell a nucleic acid molecule encoding a fusion protein which regulates transcription, the fusion protein comprising a Tet repressor having at least one amino acid mutation that confers on the fusion protein an ability to bind a class B tet operator sequence having a nucleotide substitution at position 14 or 16, operatively linked to a polypeptide which regulates transcription in eukaryotic cells; and modulating the concentration of a tetracycline, or analogue thereof, in contact with the isolated cell.
Claim 15
Independent Claim
97 words
A method for regulating transcription of a tet operator-linked gene in an isolated cell, comprising: introducing into the isolated cell a nucleic acid molecule encoding a fusion protein which regulates transcription, the fusion protein comprising a Tet repressor having at least one amino acid mutation that confers on the fusion protein an ability to bind a class B tet operator sequence having a nucleotide substitution at position 14 or 16, operatively linked to a polypeptide which regulates transcription in eukaryotic cells; and modulating the concentration of a tetracycline, or analogue thereof, in contact with the isolated cell.
Claim 16
Dependent Claim
13 words
The method of claim 13, wherein the polypeptide activates transcription in eukaryotic cells.
Claim 17
Dependent Claim
13 words
The method of claim 13, wherein the polypeptide inhibits transcription in eukaryotic cells.
Claim 18
Dependent Claim
24 words
The method of claim 13, wherein the fusion protein binds a class B tet operator sequence having a cytosine substitution at position +4 (tetO-4C).
Claim 19
Dependent Claim
24 words
The method of claim 13, wherein the fusion protein binds a class B tet operator sequence having a cytosine substitution at position +6 (tetO-6C).
Claim 20
Dependent Claim
15 words
The method of claim 13, wherein the fusion protein comprises a class B Tet repressor.
Claim 21
Dependent Claim
79 words
The method of claim 18, wherein the cell further contains a second nucleic acid molecule encoding a second fusion protein which regulates transcription, the second fusion protein comprising a class D or class E Tet repressor having at least one amino acid mutation that confers on the fusion protein an ability to bind a class B tet operator sequence having a nucleotide substitution at position +4 or +6, operatively linked to a polypeptide which regulates transcription in eukaryotic cells.
Claim 22
Dependent Claim
16 words
The method of claim 19, wherein the second fusion protein comprises a class D Tet repressor.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1999-12-31 | 2000-07-01 | 2000-12-31 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2003-12-31 | 2004-07-01 | 2004-12-31 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2007-12-31 | 2008-07-01 | 2008-12-31 | $7700.00 | Window Closed / Lapsed |