Nucleic acids encoding estrogen receptor ligand binding domain variants
Mutants of steroid receptor ligand binding domains and synthetic ligands which have specific binding affinities for these receptors are provided. The use of these LBD-ligand combinations for construction of selective “molecular gene switches” is disclosed. Methods of regulating gene function using these switches are provided.
1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a modified estrogen receptor alpha ligand binding domain with an amino acid substitution at one or more of positions 388, 391, 424 or 428 of SEQ ID NO:55, wherein said ligand binding domain interacts with a non-native ligand.
2. The isolated nucleic acid molecule of claim 1 further comprising an operatively linked nucleic acid encoding a nucleic acid binding domain.
3. The isolated nucleic acid molecule of claim 2 further comprising an operably linked nucleotide sequence encoding a transcription regulatory domain.
4. A chimeric construct comprising a promoter operatively linked to the nucleic acid molecule of claim 1 .
5. A chimeric construct comprising a promoter operatively linked to the nucleic acid molecule of claim 2 or 3 .
6. A plasmid containing the chimeric construct of claim 4 .
7. A plasmid containing the chimeric construct of claim 5 .
8. The isolated nucleic acid of claim 1 comprising substitutions at two or more of said positions.
9. The isolated nucleic acid of claim 1 comprising substitutions at three or more of said positions.
10. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a modified estrogen receptor alpha ligand binding domain with an amino acid substitution at one or more of positions 388, 391, 424 or 428 of SEQ ID NO:55; and an amino acid substitution at position 521, 524 or both of SEQ ID NO:55, wherein said ligand binding domain interacts with a non-native ligand.
11. The isolated nucleic acid of claim 10 further comprising an operatively linked nucleic acid encoding a nucleic acid binding domain.
12. The isolated nucleic acid of claim 11 further comprising a nucleic acid encoding an operably linked transcription regulatory domain.
13. A chimeric construct comprising a promoter operatively linked to the nucleic acid of claim 10 .
14. A chimeric construct comprising a promoter operatively linked to the nucleic acid of claim 11 .
15. A chimeric construct comprising a promoter operatively linked to the nucleic acid of claim 12 .
16. A plasmid comprising the chimeric construct of claim 13 .
17. A plasmid comprising the chimeric construct of claim 14 .
18. A plasmid comprising the chimeric construct of claim 15 .
19. The isolated nucleic acid molecule of claim 1 further comprising an amino acid substitution at position 421 of SEQ ID NO:55.
20. The nucleic acid molecule of claim 19 further comprising an operably linked nucleic acid encoding a nucleic acid binding domain.
21. The nucleic acid molecule of claim 20 further comprising an operably linked transcription regulatory domain.
22. A chimeric construct comprising a promoter operably linked to the nucleic acid molecule of claim 19 .
23. A chimeric construct comprising a promoter operably linked to the nucleic acid molecule of claim 20 .
24. A chimeric construct comprising a promoter operably linked to the nucleic acid molecule of claim 21 .
25. A plasmid comprising the chimeric construct of claim 22 .
26. A plasmid comprising the chimeric construct of claim 23 .
27. A plasmid comprising the chimeric construct of claim 24 .