Sensitive bioassay for detecting agonists of the aryl hydrocarbon receptor
AssigneeWisconsin Alumni Research Foundation
InventorsChristopher A. Bradfield, Lucy A. Carver, Elizabeth E. Dunham
Life of this patent
Estimating the term…
Abstract
Improved cellular assay systems for detecting polycyclic aromatic hydrocarbons, dioxins, PCBs, and other substances which are agonists of the aryl hydrocarbon receptor (AHR) are disclosed. The assays utilize one or more additional cellular proteins involved in the AHR signaling pathway, which improve the sensitivity and maximal responsiveness of the assay systems.
Claims9
An isolated transgenic yeast cell comprising at least one heterologous vector expressing AHR, ARNT and a protein selected from the group consisting of ARA3 and Src and, said cell further comprising a reporter gene operably linked to at least one dioxin responsive element, wherein the reporter gene expresses a detectable gene product as a result of an AHR agonist binding to the AHR, and wherein inclusion of Src results in a three to five fold increase in maximal responsiveness to an AHR agonist.
A transgenic yeast cell comprising at least one heterologous vector expressing:
- i) a chimeric AHR having its dimerization and DNA binding domain replaced with a DNA binding domain from another protein capable of binding a DNA transcriptional activation element and activating transcription of a gene operably linked to the element;
- ii) a protein selected from the group consisting of ARA3, Src and any combination thereof;
- and
- iii) a reporter gene operably linked to at least one said DNA transcriptional activation element, wherein the reporter gene expresses a detectable gene product as a result of an AHR agonist binding to the chimeric AHR, wherein inclusion of Src results in a three to five fold increase in maximal responsiveness to an AHR agonist.
An isolated transgenic yeast cell comprising at least one heterologous vector expressing AHR, ARNT and ARA3 and, optionally, a protein selected from the group consisting of ARA9, Src, said cell further comprising a reporter gene operably linked to at least one dioxin responsive element, wherein the reporter gene expresses a detectable gene product as a result of an AHR agonist binding to the AHR.
A transgenic yeast cell comprising at least one heterologous vector expressing:
- i) a chimeric AHR having its dimerization and DNA binding domain replaced with a DNA binding domain from another protein capable of binding a DNA transcriptional activation element and activating transcription of a gene operably linked to the element;
- ii) ARA3;
- iii) optionally, a protein selected from the group consisting of ARA9, Src and a combination thereof;
- and
- iv) a reporter gene operably linked to at least one said DNA transcriptional activation element, wherein the reporter gene expresses a detectable gene product as a result of an AHR agonist binding to the chimeric AHR.
Dependencies are inferred from the claim text.