Tetracycline-inducible transcriptional activator and tetracycline-regulated transcription units
DCFirst Claim
1. An isolated nucleic acid encoding a fusion protein which activates transcription, the fusion protein comprising a first polypeptide which binds to a tet operator sequence in the presence of tetracycline or a tetracycline analogue operatively linked to a second polypeptide which activates transcription in eukaryotic cells.
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Abstract
Nucleic acid molecules and proteins useful for regulating the expression of genes in eukaryotic cells and organisms in an inducible manner are disclosed. In the regulatory system of the invention, transcription of a tet operator-linked nucleotide sequence is stimulated by a transcriptional activator fusion protein composed of two polypeptides, a first polypeptide which binds to tet operator sequences in the presence of tetracycline or a tetracycline analogue and a second polypeptide which directly or indirectly activates transcription in eukaryotic cells. In one embodiment, the fusion protein comprises a mutated Tet repressor operatively linked to a transcriptional activation polypeptide, such as a portion of herpes simplex virus virion protein 16. In the absence of an inducing agent (tetracycline or a tetracycline analogue), transcription of the tet operator-linked nucleotide sequence remains uninduced. In the presence of the inducing agent, transcription of the tet operator-linked nucleotide sequence is stimulated by the transactivator fusion protein of the invention. Novel transcription units which allow for coordinate or independent tetracycline-regulated expression of two or more nucleotide sequences by the transactivator fusion protein of the invention are also disclosed. Kits including the components of the regulatory system of the invention are also encompassed by the invention.
115 Citations
33 Claims
- 1. An isolated nucleic acid encoding a fusion protein which activates transcription, the fusion protein comprising a first polypeptide which binds to a tet operator sequence in the presence of tetracycline or a tetracycline analogue operatively linked to a second polypeptide which activates transcription in eukaryotic cells.
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26. A kit comprising a carrier means having in close confinement therein at least two container means comprising:
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a) a first container means containing a first nucleic acid encoding a fusion protein which activates transcription, the fusion protein comprising a polypeptide which binds to a first class of tet operator sequence in the presence of tetracycline or a tetracycline analogue operatively linked to a polypeptide which activates transcription in enkaryotic cells; and b) a second container means containing a second nucleic acid comprising a first cloning site for introduction of a first nucleotide sequence to be transcribed operatively linked to at least one tet operator sequence of a first class type. - View Dependent Claims (27, 28, 29, 30, 31, 32, 33)
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Specification