Methods and compositions for identifying ligands for nuclear receptors
AssigneeLifesensors, Inc.
InventorsHiep Tuan Tran, Salam Shaaban, Hossein Askari, Michael Schwartz, Tauseef Butt
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Abstract
A yeast-based system and methods of use therefore are provided for identifying new molecules which activate nuclear receptors in a ligand-dependent fashion. In a preferred embodiment, a method is provided utilizing ecdysone receptor, USP and GRIP I encoding expression vectors which may be used to advantage for screening new and useful insecticidal compounds, detecting insecticidal residues as well as to regulate expression of a gene of interest in a host in a ligand-dependent manner.
Claims48
A ligand dependent transactivation system for screening insecticidal compounds, comprising;
- a) a first DNA construct having a nucleic acid molecule encoding an altered ecdysone receptor operably linked to a promoter;
- b) a second DNA construct having a nucleic acid molecule encoding a receptor which heterodimerizes with said ecdysone receptor upon transactivation, said nucleic acid molecule being operably linked to a promoter;
- c) a third DNA construct comprising a promoter containing a plurality of ecdysone response elements, said promoter being operably linked to a reporter gene;
- d) a fourth DNA construct encoding a co-activator molecule, said co-activator molecule being operably linked to a promoter sequence;
- and
- d) a host cell comprising said first, second, third and fourth DNA constructs, expression of said reporter gene being dependent upon ligand dependent transactivation effectuated by said insecticidal compound.
A system as claimed in claimed 1, wherein said altered ecdysone receptor has an alteration selected from the group consisting of a truncation, an insertion, a partial deletion of a the A/B domain, a full deletion of the A/B domain, site-directed or randomly mutagenized A/B domain.
A method for identifying insecticidal compounds which transactivate nuclear receptors in a ligand-dependent manner, comprising:
- a) providing a host cell containing a first DNA construct having a nucleic acid molecule encoding an altered ecdysone receptor operably linked to a promoter;
- a second DNA construct having a nucleic acid molecule encoding a receptor which heterodimerizes with said altered ecdysone receptor upon transactivation, said nucleic acid molecule being operably linked to a promoter;
- a third DNA construct comprising a promoter containing a plurality of ecdysone response elements, said promoter being operably linked to a reporter gene and a fourth DNA construct encoding a co-activator molecule, said co-activator molecule being operably linked to a promoter sequence;
- b) contacting said host cell with an effective amount of a compound suspected to possess insecticidal activity;
- c) assessing the level of ligand dependent co-activation mediated by said compound as indicated by expression levels of said reporter gene.
A method as claimed in claim 23a, wherein said co-activator molecule is selected from the group consisting of GRIP 1, SRC1 and p/CIP.
A method as claimed in claimed 21, wherein said altered ecdysone receptor has an alteration selected from the group consisting of a truncation, an insertion, a partial deletion of a the A/B domain, a full deletion of the A/B domain, site-directed, randomly mutagenized A/B domain.
A method for regulating expression of a gene of interest in a host in a ligand-dependent manner, comprising:
- a) a first DNA construct having a nucleic acid molecule encoding an altered ecdysone receptor operably linked to a promoter;
- b) a second DNA construct having a nucleic acid molecule encoding a receptor which heterodimerizes with said ecdysone receptor upon transactivation, said nucleic acid molecule being operably linked to an promoter;
- c) a third DNA construct comprising a promoter containing a plurality of ecdysone response elements, said promoter being operably linked to a gene of interest;
- d) a fourth DNA construct encoding a co-activator molecule, said co-activator molecule being operably linked to a promoter sequence;
- and
- e) a host cell comprising said first, second, third and fourth DNA constructs, increased expression of said gene of interest being dependent upon transactivation effectuated by an effective amount of said ligand.
A system for altering nuclear receptors such that they function in a ligand-dependent manner, comprising:
- a) a first DNA construct having a nucleic acid molecule encoding a nuclear receptor operably linked to a promoter;
- b) a second DNA construct having a nucleic acid molecule encoding a predetermined receptor which heterodimerizes with said first receptor upon transactivation, said nucleic acid molecule being operably linked to an promoter;
- c) a third DNA construct comprising a promoter containing a plurality of response elements for the said receptors in the step a) or the step b), said promoter being operably linked to a reporter gene;
- and
- d) a fourth DNA construct encoding a co-activator molecule, said co-activator molecule which interacts with the receptor of step a) or step b) being operably linked to a promoter sequence.
A system to identify proteins that interact with insect ecdysone and USP in ligand dependent manner, comprising:
- a) a first DNA construct having a nucleic acid molecule encoding a nuclear receptor operably linked to a promoter;
- b) a second DNA construct having a nucleic acid molecule encoding a predetermined receptor which heterodimerizes with said first receptor upon transactivation, said nucleic acid molecule being operably linked to an promoter;
- c) a third DNA construct comprising a promoter containing a plurality of response elements for the said receptors in the step a) or the step b), said promoter being operably linked to a reporter gene;
- and
- d) a fourth DNA construct encoding unknown protein molecule from a biological source, said molecule inducing the expression of the reporter gene in the presence of a construct described in a) and b) in the presence of ecdysone receptor ligands.
Dependencies are inferred from the claim text.