Nucleic acids encoding a polyglutamine fusion protein
Methods of identifying compounds that disrupt aggregation of aggregation-disposed polypeptides, such as huntingtin or beta-amyloid protein, are disclosed. Furthermore, an artificial polypeptide that contains an extended polyglutamine region and DNA that encodes the polypeptide are also disclosed.
1. A nucleic acid molecule consisting of a sequence encoding a fusion protein consisting of (a) an extended polyglutamine region of at least 32 contiguous glutamine residues, wherein the sequence encoding the at least 32 glutamine residues consists of alternating CAG codons and CAA codons, and a fluorescent label.
2. A nucleic acid molecule consisting of a sequence encoding a fusion protein comprising the sequence (CAACAGCAGCAACAGCAA)n (SEQ ID NO:1), wherein n is 5–300, and a fluorescent label.
3. The nucleic acid molecule of of claim 1 or claim 2 , wherein the fluorescent label is a green fluorescent protein.
4. The nucleic acid molecule of claim 1 or claim 2 , wherein the fluorescent label is a blue fluorescent protein.
5. An expression plasmid comprising the nucleic acid molecule of claim 1 or claim 2 , operably linked to an expression control sequence.
6. A cultured, genetically modified cell comprising the nucleic acid molecule of claim 1 or claim 2 .
7. 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 consists of alternating CAG codons and CAA codons.
8. The nucleic acid molecule of claim 7 , wherein the label is a fluorescent protein.
9. The nucleic acid molecule of claim 8 , wherein the fluorescent protein is a green fluorescent protein.
10. The nucleic acid molecule of claim 8 , wherein the fluorescent protein is a blue fluorescent protein.
11. The nucleic acid molecule of claim 7 , wherein the label is an enzyme.
12. An expression plasmid comprising the nucleic acid molecule of claim 7 , operably linked to an expression control sequence.
13. A cultured, genetically modified cell comprising the nucleic acid molecule of claim 7 .
14. The nucleic acid molecule of claim 7 , wherein the sequence encoding the at least 32 glutamine residues encodes 33, 34, 35, 36, 37, 40, 42, 47, 50, 52, 60, 65, 70, 72, 75, 80, 85, 95, 100, 104, 110, 119, 120, 130, 140, 144, 151, 160, 170, 180, 190, 191, 195, 200, 210, 230, 250, 270, or 300 glutamine residues.
15. 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 the sequence CAACAGCAGCAACAGCAA (SEQ ID NO:1).
16. The nucleic acid molecule of claim 15 , wherein the label is a fluorescent protein.
17. The nucleic acid molecule of claim 15 , wherein the fluorescent protein is a green fluorescent protein.
18. The nucleic acid molecule of claim 16 , wherein the fluorescent protein is a blue fluorescent protein.
19. The nucleic acid molecule of claim 15 , wherein the label is an enzyme.
20. An expression plasmid comprising the nucleic acid molecule of claim 15 , operably linked to an expression control sequence.
21. A cultured, genetically modified cell comprising the nucleic acid molecule of claim 15 .
22. The nucleic acid molecule of claim 15 , wherein the sequence encoding the at least 32 glutamine residues encodes 33, 34, 35, 36, 37, 40, 42, 47, 50, 52, 60, 65, 70, 72, 75, 80, 85, 95, 100, 104, 110, 119, 120, 130, 140, 144, 151, 160, 170, 180, 190, 191, 195, 200, 210, 230, 250, 270, or 300 glutamine residues.
23. A nucleic acid molecule comprising a sequence encoding a fusion protein consisting of(a) 32, 33, 34, 36, 37, 40, 42, 47, 50, 52, 60, 65, 70, 72, 75, 85, 95, 100, 104, 110, 119. 120, 130, 140, 144, 151, 160, 170, 180, 190, 191, 195, 200, 210, 230, 250, 270, or 300 contiguous glutamine residues, wherein the sequence encoding the contiguous glutamine residues consists of alternating CAG codons and CAA codons.
24. A nucleic acid molecule comprising a sequence encoding a fusion protein consisting of(a) 32, 33, 34, 36, 37, 40, 42, 47, 50, 52, 60, 65, 70, 72, 75, 85, 95, 100, 104, 110, 119, 120, 130, 140, 144, 151, 160, 170, 180, 190, 191, 195, 200, 210, 230, 250, 270, or 300 contiguous glutamine residues, wherein the sequence encoding the contiguous glutamine residues comprises the sequence CAACAGCAGCAACAGCAA (SEQ ID NO:1).
25. The nucleic acid molecule of claim 23 or claim 24 , wherein the label is a fluorescent protein.
26. The nucleic acid molecule of claim 25 , wherein the fluorescent protein is a green fluorescent protein.
27. The nucleic acid molecule of claim 25 , wherein the fluorescent protein is a blue fluorescent protein.
28. The nucleic acid molecule of claim 23 or claim 24 , wherein the label is an enzyme.
29. A fusion protein encoded by the nucleic acid molecule of claim 23 or claim 24 .
30. An expression plasmid comprising the nucleic acid molecule of claim 23 or claim 24 , operably linked to an expression control sequence.
31. A cultured, genetically modified cell comprising the nucleic acid molecule of claim 23 or claim 24 .
32. The nucleic acid molecule of claim 2 , wherein n equals 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.