UTR tag assay for gene function discovery

• US 7,026,123 B1
• Filed: 08/28/2002
• Issued: 04/11/2006
• Est. Priority Date: 08/29/2001
• Status: Expired due to Fees

• Current Assignee: Pioneer Hi Bred International Inc (Corteva, Inc.)
• Original Assignee: Pioneer Hi Bred International Inc (Corteva, Inc.)
• Inventors: Duvick, Jon
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): Chunduru, Suryaprabha
• Application Number:
  US10/229,608
• Time in Patent Office: 1,322 Days
• Field of Search: 435/6, 435/69.1, 435/91.21, 536/24.33, 536/22.1
• US Class Current: 506/4
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1044 Preparation or screening of libraries displayed on scaffold proteins
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

The methods of the invention provide a means for rapid analysis of gene function in a variety of systems. The invention allows screening of large libraries of nucleotide sequences for involvement in physiological pathways of interest. The methods of the invention also provide an efficient means of identifying and isolating nucleotide sequences that modulate a physiological pathway of interest from a population of nucleotide sequences.

Claims

1. A method of identifying a nucleotide sequence that modulates the activity of a transcriptional regulatory region, comprising:

a) incorporating into a host cell population a generated library of plasmids wherein the generated library is a collection of plasmids, wherein the library of plasmids comprises multiple non-redundant U-tags, each plasmid having one or more non-redundant U-tags, each U-tag conferring an identifying marker on each plasmid in the library, and each plasmid in the generated library comprises a first DNA construct and a second DNA construct, wherein;

i) the first DNA construct comprises a transcriptional regulatory region, a reporter sequence, and an mRNA stabilizing sequence; and

,

ii) the first DNA construct further comprises one or more U-tags inserted into one or more locations selected from the group of locations consisting of a 3′

UTR of the transcriptional regulatory region, a 5′

UTR of the transcriptional regulatory region, a coding region of the reporter sequence, and one or more intron sequences occurring within the first DNA construct; and

,

iii) the second DNA construct comprises a promoter active in a host cell of the host cell population, wherein the promoter is operably linked to a nucleotide sequence of interest; and

b) screening for an alteration in the expression levels of each U-tag; and

,

c) identifying the nucleotide sequence of interest based on U-tag identity.

2. The method of claim 1 further comprising isolating from the library at least one plasmid having one or more U-tags, wherein the expression level of the one or more U-tags is altered.

3. The method of claim 2 wherein the isolating comprises PCR.

4. The method of claim 2 wherein the isolating comprises hybridization.

5. The method of claim 1 wherein the nucleotide sequence of interest comprises a nucleic acid sequence selected from the group consisting of a cDNA, a genomic DNA, or a mutagenized nucleic acid sequence.

6. The method of claim 1 wherein the first DNA construct is located 5′

to the second DNA construct.

7. The method of claim 1 wherein the first DNA construct is located 3′

to the second DNA construct.

8. The method of claim 1 wherein the first and second DNA constructs are contiguous.

9. The method of claim 1 wherein the first and second DNA constructs are not contiguous.

10. The method of claim 1 wherein the one or more non-redundant U-tags are located 5′

to the reporter sequence.

11. The method of claim 1 wherein the one or more non-redundant U-tags are located 3′

to the reporter sequence.

12. The method of claim 1 wherein the one or more non-redundant U-tags are located 5′

and 3′

to the reporter sequence.

13. The method of claim 1 wherein the one or more non-redundant U-tags are located in the coding region of the reporter sequence.

14. The method of claim 1 wherein the one or more non-redundant U-tags are located within the one or more intron sequences occurring within the transcriptional regulatory region.

15. The method of claim 1 wherein the screening for an alteration in the expression levels of each U-tag comprises hybridization to a complementary U-tag array.

16. The method or claim 1 wherein the screening for an alteration in the expression levels of each U-tag comprises sequence probe concatamer methods.

17. The method of claim 1 wherein the screening For an alteration in the expression levels of each U-tag comprises a solid phase capture system.

18. The method of claim 1 wherein the alteration in the expression levels of each U-tag comprises an increase in U-tag expression levels.

19. The method of claim 1 wherein the alteration in the expression levels of each U-tag comprises a decrease in U-tag expression levels.

20. The method of claim 1 wherein the transcriptional regulatory region of the first DNA construct regulates expression of a sequence involved in a physiological pathway of interest.

21. The method of claim 20 wherein the physiological pathway of interest is a pathogen resistance pathway.

22. The method of claim 20 wherein the physiological pathway of interest is a tissue developmental pathway.

23. The method of claim 20 wherein the physiological pathway of interest is a metabolic pathway.

24. The method of claim 20 wherein the physiological pathway of interest is an apoptotic pathway.

25. The method of claim 1 wherein the host cells are plant cells.

26. The method of claim 25 wherein the plant cells are dicotyledonous cells.

27. The method of claim 25 wherein the plant cells are monocotyledonous cells.

28. The method of claim 27 wherein the monocotyledonous cells are Black Mexican Sweet maize (BMS) cells.

29. The method of claim 25 wherein the plant cells are selected from the group consisting of maize, wheat, sorghum, rice, barley, soybean, alfalfa, sunflower, Brassica, and tomato.

30. The method of claim 1 wherein the host cells are mammalian cells.

31. The method of claim 1 wherein the nucleotide sequence of interest encodes a polypeptide that directly modulates the activity of the transcriptional regulatory region.

32. The method of claim 2 wherein the nucleotide sequence of interest encodes a polypeptide that indirectly modulates the activity of the transcriptional regulatory region.

33. A method for identifying a transcriptional regulatory region of interest that is modulated by an agent, comprising:

a) incorporating into a host cell population a generated library of plasmids wherein the generated library is a collection of plasmids, wherein the library of plasmids comprises multiple non-redundant U-tags, each plasmid having one or more non-redundant U-tags, each U-tag conferring an identifying marker on each plasmid in the library, and each plasmid in the generated library comprises a first DNA construct, wherein

i) the first DNA construct comprises the transcriptional regulatory region of interest, a reporter sequence, and an mRNA stabilizing sequence, and;

ii) the first DNA construct further comprises one or more U-tags inserted into one or more locations selected from the group of locations consisting of a 3′

UTR of the transcriptional regulatory region, a 5′

UTR of the transcriptional regulatory region, a coding region or the reporter sequence, and one or more intron sequences occurring within the first DNA construct; and

b) contacting the host cell population with an agent; and

c) screening for an alteration in the expression levels of each U-tag.

34. The method of claim 33 wherein the agent is selected from the group consisting of a pathogen, a polypeptide, a nucleotide sequence, and a small molecule.

35. The method of claim 34, wherein the plasmid in the generated library further comprises a second DNA construct operably linked to a promoter active in a host cell of the host cell population.

36. The method of claim 33 further comprising isolating from the library at least one plasmid having one or more U-tags, wherein the expression level of one or more of the U-tags is altered.

37. The method of claim 36 wherein the method of isolation comprises PCR.

38. The method of claim 36 wherein the method of isolation comprises hybridization.

39. The method of claim 35 wherein the first DNA construct is located 5′

to the second DNA construct.

40. The method of claim 35 wherein the first DNA construct is located 3′

to the second DNA construct.

41. The method of claim 35 wherein the first and the second DNA constructs are contiguous.

42. The method of claim 35 wherein the first and the second DNA constructs are not contiguous.

43. The method of claim 33 wherein the one or more U-tags are located 5′

to the reporter sequence.

44. The method of claim 33 wherein the one or more U-tags are located 3′

to the reporter sequence.

45. The method of claim 33 wherein the one or more U-tags are located 5′

and 3′

to the reporter sequence.

46. The method of claim 33 wherein the one or more U-tags are located in the coding region of the reporter sequence.

47. The method of claim 33 wherein the one or more U-tags are located within the one or more intron sequences occurring within the transcriptional regulatory region.

48. The method or claim 33 wherein screening for an alteration in the expression levels of each U-tag comprises hybridization to a complementary U-tag array.

49. The method of claim 33 wherein screening for an alteration in the expression levels of each U-tag comprises a sequence probe concatamer method.

50. The method of claim 33 wherein screening for an alteration in the expression levels of each U-tag comprises a solid phase capture system.

51. The method of claim 33 wherein the alteration in the expression levels of each U-tag comprises an increase in U-tag expression levels.

52. The method of claim 33 wherein the alteration in the expression levels of each U-tag comprises a decrease in U-tag expression levels.

53. The method of claim 33 wherein the agent regulates expression of the transcriptional regulatory region sequence involved in a physiological pathway of interest.

54. The method of claim 53 wherein the physiological pathway or interest is a pathogen resistance pathway.

55. The method of claim 53 wherein the physiological pathway of interest is a tissue developmental pathway.

56. The method of claim 53 wherein the physiological pathway of interest is a metabolic pathway.

57. The method of claim 53 wherein the physiological pathway or interest is an apoptotic pathway.

58. The method of claim 33 wherein the host cells are plant cells.

59. The method of claim 58 wherein the plant cells are dicotyledonous cells.

60. The method of claim 58 wherein the plant cells are monocotyledonous cells.

61. The method of claim 60 wherein the monocotyledonous cells are Black Mexican Sweet maize (BMS) cells.

62. The method of claim 58 wherein the plant cells are selected from the group consisting of maize, wheat, sorghum, rice, barley, soybean, alfalfa, sunflower, Brassica, and tomato.

63. The method of claim 33 wherein the host cells are mammalian cells.

64. A method of identifying a nucleotide sequence of interest that modulates the activity of a transcriptional regulatory region, comprising:

a) incorporating into a host cell population a generated library of plasmids wherein the generated library is a collection of plasmids, wherein the library of plasmids comprises multiple non-redundant U-tags, each plasmid having one or more non-redundant U-tags, each U-tag conferring an identifying marker on each plasmid in the library, and each plasmid in the generated library comprises a first DNA construct wherein

i) the first DNA construct comprises a transcriptional regulatory region, a nucleotide sequence of interest, and an mRNA stabilizing sequence; and

ii) the first DNA construct further comprises one or more U-tags inserted into one or more locations selected from the group of locations consisting of a 3′

UTR of the transcriptional regulatory region, a 5′

UTR of the transcriptional regulatory region, a coding region of the reporter sequence, and one or more intron sequences occurring within the first DNA construct; and

b) screening for an alteration in the expression level of each U-tag and;

c) identifying the sequence of interest based on U-tag identity.

65. The method of claim 64 further comprising isolating from the library at least one plasmid having one or more U-tags, wherein the expression level of the U-tag is altered.

66. The method of claim 64 wherein the alteration in the expression levels of each U-tag comprises an increase in U-tag expression levels.

67. A method for identifying a nucleotide sequence of interest that modulates cell death, comprising:

a) incorporating into a host cell a plasmid having at least a first DNA construct and a second DNA construct, wherein;

i) the first DNA construct comprises a transcriptional regulatory region, a reporter sequence, and an mRNA stabilizing sequence; and

,

ii) the first DNA construct further comprises one or more U-tags inserted into one or more locations selected from the group of locations consisting of a 3′

UTR of the transcriptional regulatory region, a 5′

UTR of the transcriptional regulatory region, a coding region of the reporter sequence, and one or more intron sequences occurring within the first DNA construct; and

,

b) the second DNA construct comprises a promoter active in the host cell operably linked to the nucleotide sequence of interest; and

c) screening for a loss in the expression level of the U-tag; and

d) identifying the sequence of interest based on U-tag identity.