Claims
- 1. A nucleic acid molecule encoding a fusion protein comprising (a) at least 32 contiguous glutamine residues and (b) a label, wherein the sequence encoding the at least 32 glutamine residues comprises both CAG codons and CAA codons.
- 2. The nucleic acid molecule of claim 1, wherein the CAG codons and the CAA codons alternate.
- 3. The nucleic acid molecule of claim 1, comprising the sequence (CAACAGCAGCAACAGCAA)n (SEQ ID NO:1).
- 4. The nucleic acid molecule of claim 1, wherein the label is a fluorescent protein or an enzyme.
- 5. The nucleic acid molecule of claim 1, wherein the label is a green fluorescent protein or a blue fluorescent protein.
- 6. A fusion protein encoded by the nucleic acid molecule of claim 1.
- 7. A expression plasmid comprising the nucleic acid molecule of claim 1, operably linked to an expression control sequence.
- 8. A cultured, genetically modified cell that expresses the nucleic acid molecule of claim 1.
- 9. A method of identifying a compound that disrupts the aggregation of polypeptides containing extended polyglutamine regions, the method comprising:
providing a cell that is genetically modified to express a DNA encoding a heterologous polypeptide containing an extended polyglutamine region; contacting the cell with a test compound; and determining whether the test compound decreases the amount of aggregation of the polypeptide in the cell, wherein a decrease in polypeptide aggregation in the presence of the test compound indicates that the test compound is a polypeptide aggregation disrupting compound.
- 10. The method of claim 9, wherein the heterologous polypeptide is a fusion protein comprising a label.
- 11. The method of claim 10, wherein the label is a fluorescent protein or an enzyme.
- 12. The method of claim 10, wherein the label is a green fluorescent protein or a blue fluorescent protein.
- 13. The method of claim 10, wherein the label is a fluorescent protein and the determining step comprises:
contacting the cell with a denaturant; and detecting fluorescence, wherein a decrease in fluorescence in the cell contacted with the test compound, compared to a control cell, indicates that the test compound is a polyglutamine polypeptide aggregation disrupting compound.
- 14. The method of claim 9, wherein the expression of the polypeptide is induced upon exposure of the cell to an inducing agent.
- 15. The method of claim 14, wherein the inducing agent is ecdysone or muristerone.
- 16. A method of identifying a compound that disrupts the aggregation of polypeptides, the method comprising:
promising a cell that is genetically modified to express a DNA encoding a heterologos polypeptide, wherein molecules of the polypeptide spontaneously aggregate within the cell; contacting the cell with a test compound; and determining whether molecules of the polypeptide aggregate in the presence of the test compound, wherein a decrease in aggregation of the polypeptide molecules in the presence of the test compound indicates that the test compound is a polypeptide aggregation disrupting compound.
- 17. The method of claim 16, wherein the polypeptide is a fusion protein comprising a label.
- 18. The method of claim 17, wherein the label is a fluorescent protein or an enzyme.
- 19. The method of claim 17, wherein the label is a green fluorescent protein or a blue fluorescent protein.
- 20. The method of claim 19, further comprising:
contacting the cell with a denaturant; and detecting fluorescence, wherein the label is a green fluorescent protein and a decrease in fluorescence in the cell contacted with the test compound compared to a control cell, indicates that the compound is a polypeptide aggregation disrupting compound.
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0001] This invention was made with Government support under grant number PO1-CA42063, awarded by the National Institutes of Health. The government may have certain rights in the invention.
Divisions (1)
|
Number |
Date |
Country |
Parent |
09405048 |
Sep 1999 |
US |
Child |
10194584 |
Jul 2002 |
US |