1. Sponsor Organization: NIH/NIEHS

2. Project Title: 07072-03 AH RECEPTOR ASSOCIATED PROTEINS--ROLE IN DIOXIN ACTION

3. Project Focus:

• Project Primary Focus: Human Health Effects
• Project Secondary Focus:

4. Description:

We propose to characterize the multiple DNA binding forms of the Ah receptor (AhR) complex as well as to isolate and characterize AhR-associated proteins. Utilizing AhR and Ah receptor nuclear translocator (ARNT) antibodies we will determine the identity of the multiple proteins AhR complex subunits which bind (and can be crosslinked) to the DRE in a TCDD-dependent manner. Multiple DNA binding forms of the AhR complex will be isolated from guinea pig liver by a combination of conventional and magnetic DNA affinity binding techniques. The functional activity of each complex will he assessed by the ability of each to stimulate transcription in vitro and the DNA binding of each complex will be examined using gel shift, UV- crosslinking, DNA mutagenesis and DNA footprinting. In a complimentary approach, AhR-associated proteins will be isolated from mouse, guinea pig and human cell lines stably transfected with a recombinant AhR fusion protein expression vector. The presence of an affinity tag on the recombinant proteins will allow rapid and selective isolation of the recombinant AhR and its associated proteins. The biochemical and functional properties of the isolated complexes and their protein components will be examined in reconstitution experiments and identity of the associated proteins determined by sequenced analysis. cDNA clones encoding AhR-associated proteins will be isolated from cDNA libraries using both the yeast two-hybrid system as well as probes derived from the amino acid sequenced of purified proteins. The results of our studies will not only increase our understanding of the molecular mechanisms of dioxin action and provide insight into the diversity in TCDD-responsiveness in humans and animals but they will contribute to our knowledge of the inducible regulation of mammalian gene expression.

5. References:

6. Inventory Category:

• Primary: Models
• Secondary:

7. Inventory Subcategory:

• Primary: Basic Research
• Secondary:

8. Keywords for Experimental System/Species:

• Species:
  • Mammal
  • Human
  • Guinea Pig, Mouse
• Study Type:
  • In Vitro
• Fate and Transport:

9. Keywords for Experimental Endpoints:

• Health Effect:
• Hormonal Measures:
  • Ah Receptor, Molecluar
• Level Of Study:
• Chemistry Metabolism:
• Life Stage:
• Risk Assessment:

10. Chemical Agents:

• Dioxins

11. Performing Institution:

• UNIVERSITY OF CALIFORNIA DAVIS

12. Contact:

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