Method and construct for screening for inhibitors of transcriptional activation

• US 5,747,338 A
• Filed: 09/25/1996
• Issued: 05/05/1998
• Est. Priority Date: 08/15/1996
• Status: Expired due to Fees

• Current Assignee: Chiron Corporation (Novartis Ag)
• Original Assignee: Chiron Corporation (Novartis Ag)
• Inventors: Giese, Klaus, Escobedo, Jaime
• Primary Examiner(s): Ketter, James
• Application Number:
  US08/719,577
• Time in Patent Office: 587 Days
• Field of Search: 435/348, 435/367, 435/252.3, 435/252.33, 435/254.21, 435/320.1, 536/24.5
• US Class Current: 435/348
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

A collision construct is provided that contains a first regulatory sequence containing a first promoter linked to a reporter gene which is also linked to a second regulatory sequence containing a second promoter such that the reporter gene is under regulatory control of the first promoter. The direction of transcription under the first promoter is opposite the direction of transcription under the second promoter and activation of the second promoter interferes with reporter gene activity. The collision construct is used to screen inhibitors of the second regulatory sequence and the second promoter whereby a functional inhibitor causes an enhanced signal of the reporter gene. Vectors, host cells and kits containing the collision construct are provided, as well as methods for producing the collision construct, the vectors and host cells, and methods for screening candidate inhibitors for their ability to inhibit target promoter activities.

Claims

1. A collision construct comprising a nucleic acid molecule that comprises:

a) a first regulatory sequence that comprises a first promoter;

b) a reporter gene that is under transcriptional control of the first promoter; and

c) a second regulatory sequence that comprises a second promoter;

wherein the first promoter is different from the second promoter, and direction of transcription under the first promoter is opposite to direction of transcription under the second regulatory sequence, and

wherein regulation of the second promoter alters expression of the reporter gene.

2. The collision construct of claim 1, wherein the second promoter comprises a first response element that specifically binds to a first binding protein to form a first binding pair;

and formation of the first binding pair regulates the second promoter under transcription-regulating conditions.

3. The collision construct of claim 1, wherein the regulation of the second promoter is achieved by activation thereof.

4. The collision construct of claim 1, wherein the second promoter comprises a 5'"'"' terminus and a 3'"'"' terminus and the reporter gene is separated from 3'"'"' terminus of the second promoter by a distance of about 2047 nucleotides or less.

5. The collision construct of claim 4, wherein the distance is in a range selected from the group of ranges, in nucleotides, of about 1-50, 51-100, 101-150, 151-200, 201-300, 301-400, 401-500, 501-600, 601-1000, 1001-1500, and 1501-2047.

6. The collision construct of claim 4, wherein the distance is in a range selected from the group of ranges, in nucleotides, of about 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-250, 251-425, and 426-550.

7. The collision construct of claim 4, wherein the distance, in nucleotides, is selected from the group consisting of about 21, 94, 153, 406, 556 and 2047.

8. The collision construct of claim 1, wherein the first promoter is a minimal promoter.

9. The collision construct of claim 1, wherein one or both of the first and second promoters are each selected from the group consisting of promoters derived from a virus, a bacteriophage, a prokaryotic gene, and an eukaryotic gene.

10. The collision construct of claim 9, wherein the virus is selected from the group consisting of a retrovirus, a vaccinia virus, a herpes virus, a hepatitis virus, a papilloma virus, an adenovirus, and an adeno-associated virus.

11. The collision construct of claim 1, wherein the first promoter is selected from the group consisting of a λ

_PL promoter, a λ

_PR promoter, a prokaryotic ribosomal RNA P1/P2 promoter, a Rous Sarcoma Virus promoter, a Simian Virus 40 promoter, a simian immunodeficiency virus promoter, an albumin promoter, a lck promoter, and a fos promoter.

12. The collision construct of claim 9, wherein second promoter is a promoter derived from a virus and the virus is selected from the group consisting of a cytomegalovirus, a herpes simplex virus, a hepatitis virus, and a human immunodeficiency virus.

13. The collision construct of claim 9, wherein the second promoter is selected from the group consisting of a flt promoter, a CD4 promoter, and a β

-3 promoter.

14. The collision construct of claim 1, wherein one or both of the first regulatory sequence and second regulatory sequence comprise a synthetic sequence.

15. The collision construct of claim 14, wherein the first regulatory sequence comprises a synthetic sequence and the synthetic sequence is selected from the group consisting of a multimeric Gal4 binding site linked to a minimal promoter and a LexA binding site linked to a minimal promoter.

16. The collision construct of claim 14, wherein the synthetic sequence comprises a TATA box.

17. The collision construct of claim 1, wherein the first promoter comprises a second response element that specifically binds to a second binding protein to form a second binding pair, wherein formation of the second binding pair regulates the first promoter under transcription-regulating conditions, and the second binding protein specifically binds to the first promoter.

18. The collision construct of claim 1, wherein the reporter gene is selected from the group consisting of genes encoding alkaline phosphatase, luciferase, chloramphenical acetyltransferase, β

-galactosidase, β

-glucuronidase, and green fluorescent protein.

19. The collision construct of claim 2, wherein the first response element is derived from a promoter or promoter/enhancer region of a gene selected from the group consisting of a viral gene, a bacteriophage gene, a prokaryotic gene, and an eukaryotic gene.

20. The collision construct of claim 17, wherein the second response element is derived from a promoter or promoter/enhancer region of a gene selected from the group consisting of a viral gene, a bacteriophage gene, a prokaryotic gene and an eukaryotic gene.

21. The collision construct of claim 2, wherein the first response element is selected from the group consisting of a transactivation response element ("TAR"), Rev response element ("RRE"), a NF-κ

B binding site, and a Sp1 binding site.

22. The collision construct of claim 17, wherein the second response element is selected from the group consisting of a transactivation response element ("TAR"), Rev response element ("RRE"), a NF-κ

B binding site, and a Sp1 binding site.

23. The collision construct of claim 2, wherein the first binding protein is selected from the group consisting of Tat, Rev, NF-κ

B, and Sp1.

24. The collision construct of claim 1, wherein the first promoter has a strength of transcription that is approximately the same as that of the second promoter upon activation.

25. A vector comprising the collision construct of claim 1, further comprising a nucleotide sequence that allows for expression of the collision construct in a host cell.

26. A host cell comprising the vector of claim 25.

27. The host cell of claim 26, wherein the host cell amplifies the collision construct or effecting the expression thereof.

28. The host cell of claim 26, wherein the cell is selected from the group consisting of a prokaryotic cell and an eukaryotic cell.

29. The host cell of claim 27, wherein the cell amplifies the collision construct and is a prokaryotic cell.

30. The host cell of claim 27, wherein the cell expressed the collision construct and is an eukaryotic cell.

31. A kit comprising the collision construct of claim 1, further comprising instructions for use thereof.

32. A kit comprising the vector of claim 25, further comprising instructions for use thereof.

33. A kit comprising the host cell of claim 26, further comprising instructions for use thereof.

34. A method for screening a candidate inhibitor for its ability to inhibit transcription under a target promoter comprising:

a) providing a cell that comprises the collision construct of claim 1, wherein the second promoter is the target promoter;

b) determining reporter gene signal in the presence and absence of the candidate inhibitor; and

c) comparing reporter gene signals obtained to determine whether inhibition of transcription under the second promoter occurred in the presence of the candidate inhibitor, wherein, when the reporter gene signal is increased, the target promoter is inhibited.

35. The method of claim 34, wherein the target promoter is not endogenous to the cell.

36. The method of claim 34, wherein the target promoter is endogenous to the cell.

37. A method for screening a candidate inhibitor for its ability to inhibit binding between a target binding protein and a target response element comprising:

a) providing a cell that comprises the collision construct of claim 2, wherein the first binding protein is the target binding protein and the first response element is the target response element;

b) providing a baseline reporter gene signal in the absence or presence of the target binding protein;

c) determining reporter gene signal in the presence and absence of the candidate inhibitor; and

d) comparing reporter gene signals obtained, wherein when the reporter gene signal is increased, the binding between a target binding protein and a target response element is inhibited.

38. The method of claim 37, wherein the target response element is not endogenous to the cell.

39. The method of claim 37, wherein the target response element is endogenous to the cell.

40. The method of claim 37, wherein the target binding protein is provided by a process selected from the group consisting of:

a) introducing into the cell a nucleotide sequence that encodes the target binding protein;

b) allowing a cell which produces the target binding protein constitutively to produce the target binding protein; and

c) adding the target binding protein to the cell.

41. A method for identification of an inhibitor of transcription under a target promoter comprising:

a) providing a cell that comprises the collision construct of claim 1, wherein the second promoter is the target promoter;

b) determining reporter gene signal in the presence and absence of a panel of candidate inhibitors;

c) comparing reporter gene signals obtained to determine if inhibition of target promoter activity has occurred in the use of any one of the panel of candidate inhibitors, wherein when the reporter gene signal is increased, the transcription of the target promoter is inhibited.

42. A method for making a reporter collision construct, comprising:

a) providing a first regulatory sequence that comprises a first promoter, a reporter gene that is capable of providing a detectable signal upon transcription and translation, and a second regulatory sequence that comprises a second promoter; and

b) linking the first regulatory sequence, the reporter gene, and the second regulatory sequence together to produce the collision construct of claim 1.

43. A method for production of a collision construct, comprising culturing the host cell of claim 33.

44. A collision construct produced by a process comprising replicating the collision construct of claim 1 in a prokaryotic or eukaryotic cell.

45. The collision construct of claim 44, wherein the eukaryotic cell is selected from the group consisting of a mammalian cell, an insect cell, a yeast cell, and an avian cell.