Claims
- 1. A method for identifying an effector molecule which modulates the activity of a protein, said method comprising:
(a) providing at least two fragments of said protein; (b) contacting said fragments with a candidate molecule; and (c) assaying for assembly of said fragments, whereby a level of fragment assembly in the presence of said candidate molecule that is greater than the level in its absence identifies said candidate as an effector molecule which modulates the activity of said protein.
- 2. The method of claim 1, wherein said protein is a member of the nuclear receptor superfamily.
- 3. The method of claim 2, wherein said nuclear hormone receptor is the thyroid hormone receptor, retinoic acid receptor, retinoid X receptor, or estrogen receptor.
- 4. The method of claim 1, wherein said effector molecule is a protein.
- 5. The method of claim 1, wherein said effector molecule is a ligand.
- 6. The method of claim 1, wherein said effector molecule is a corepressor.
- 7. The method of claim 1, wherein said effector molecule is an antagonist.
- 8. The method of claim 2, wherein one of said fragments comprises the helix 1 domain of said nuclear receptor.
- 9. The method of claim 8, wherein the second of said fragments comprises the helix 12 domain and ligand binding pocket of said nuclear receptor.
- 10. The method of claim 1, wherein one or both of said fragments is provided by expressing a fragment-encoding nucleic acid.
- 11. The method of claim 1, wherein said contacting step (b) occurs in vivo.
- 12. The method of claim 1, wherein said contacting step (b) occurs in vitro.
- 13. The method of claim 1, wherein one of said fragments is immobilized on a solid support.
- 14. The method of claim 13, wherein the second of said fragments is detectably labeled.
- 15. The method of claim 14, wherein said assaying step (c) comprises detecting said label in association with said solid support.
- 16. The method of claim 1, wherein said protein is a DNA binding protein.
- 17. The method of claim 16, wherein said contacting step (b) occurs in the presence of a nucleic acid that comprises the binding site for said DNA binding protein and said assaying step (c) comprises detecting binding of said assembled fragments to said binding site.
- 18. The method of claim 1, wherein one of said fragments is covalently bound to a DNA binding domain and the other said fragment is covalently bound to a gene activation domain.
- 19. The method of claim 18, wherein at least one of said DNA binding domain and said gene activation domain is a heterologous domain.
- 20. The method of claim 18, wherein said contacting step (b) is carried out in the presence of a reporter gene operably linked to a binding site for said DNA binding domain.
- 21. The method of claim 20, wherein said assaying step (c) comprises detecting expression of said reporter gene.
- 22. The method of claim 21, wherein said assaying is carried out in a yeast or mammalian cell.
- 23. A kit for identifying a nuclear receptor effector molecule, said kit comprising:
(a) a first fragment of said nuclear receptor, said first fragment comprising the helix 1 domain of said nuclear receptor; and (b) a second fragment of said nuclear receptor, said second fragment comprising the helix 12 domain and the ligand binding pocket of said nuclear receptor.
- 24. The kit of claim 23, wherein said protein is a member of the nuclear receptor superfamily.
- 25. The kit of claim 24, wherein said nuclear hormone receptor is the thyroid hormone receptor, retinoic acid receptor, retinoid X receptor, or estrogen receptor.
- 26. The kit of claim 23, wherein said effector molecule is a ligand or corepressor.
- 27. The kit of claim 23, wherein said effector molecule is an antagonist.
- 28. The kit of claim 23, wherein at least one of said fragments is detectably labeled.
- 29. The kit of claim 23, wherein one of said fragments is immobilized on a solid support.
- 30. The kit of claim 23, wherein one of said fragments is covalently bound to a DNA binding domain and the other said fragment is covalently bound to a gene activation domain.
- 31. The kit of claim 30, wherein said kit further comprises a nucleic acid comprising a binding site for said DNA binding domain.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional Application No. 60/191,946, filed Mar. 24, 2000.
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made in part with government support under NIH grant NIDDK 43382. The government therefore has certain rights in the invention.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60191946 |
Mar 2000 |
US |