Method for identifying activators of gene transcription

• US 20050266447A1
• Filed: 04/07/2005
• Published: 12/01/2005
• Est. Priority Date: 04/19/2004
• Status: Abandoned Application

• Current Assignee: Pioneer Hi Bred International Inc (Corteva, Inc.)
• Original Assignee: Pioneer Hi Bred International Inc (Corteva, Inc.)
• Inventors: Fedorova, Maria, Duvick, Jonathan P.
• Application Number:
  US11/100,722
• Publication Number:
  US 20050266447A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

Methods and compositions for identifying a nucleotide sequence that encodes a polypeptide that directly or indirectly modulates the activity of a transcriptional regulatory region are provided. The methods permit the efficient screening of a large number of candidate nucleotide sequences for potential modulators of transcription. In particular, a population of polynucleotide constructs comprising a common transcriptional regulatory region operably linked to a candidate sequence is introduced into a population of host cells, and each candidate sequence is screened for modulation in its expression level, for example, using methods that employ hybridization to a complementary DNA microarray. The expression level of the candidate sequence correlates to the activity of the transcriptional regulatory region. Thus, alterations in the expression level of a candidate sequence indicate that the sequence encodes a polypeptide that modulates the activity of the transcriptional regulatory region. Compositions for practicing the invention are further provided.

Claims

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

(a) providing a population of polynucleotide constructs, wherein each of said polynucleotide constructs in said population comprises the transcriptional regulatory region operably linked to a candidate nucleotide sequence;

(b) introducing said population of polynucleotide constructs into a population of host cells; and

,

(c) screening for a modulation in the expression level of each candidate nucleotide sequence, and thereby identifying a nucleotide sequence that encodes a polypeptide that modulates the activity of said transcriptional regulatory region.

2. The method of claim 1, wherein said screening for a modulation in the expression level of each candidate nucleotide sequence comprises:

(a) isolating mRNA from said population of host cells,

(b) preparing labeled polynucleotides from said isolated mRNA; and

,

(c) hybridizing said labeled polynucleotides to a complementary DNA microarray.

3. The method of claim 2, wherein said isolating mRNA from said population of host cells allows for preferential amplification of mRNA derived from said population of polynucleotide constructs.

4. The method of claim 1, wherein said modulation comprises an increase in the expression level of a candidate nucleotide sequence.

5. The method of claim 1, wherein said modulation comprises a decrease in the expression level of a candidate nucleotide sequence.

6. The method of claim 1, wherein said transcriptional regulatory region comprises an inducible promoter.

7. The method of claim 6, wherein each of said polynucleotide constructs further comprises an intron.

8. The method of claim 7, wherein said transcriptional regulatory region has minimal background activity.

9. The method of claim 8, wherein said intron increases the background expression of said candidate nucleotide sequence.

10. The method of claim 9, wherein said intron is maize Ubi1-intron1.

11. The method of claim 10, wherein said inducible promoter is maize bronze-1 promoter.

12. The method of claim 1, wherein at least one of said candidate nucleotide sequences encodes a polypeptide that directly modulates the activity of said transcriptional regulatory region.

13. The method of claim 1, wherein at least one of said candidate nucleotide sequences encodes a polypeptide that indirectly modulates the activity of said transcriptional regulatory region.

14. The method of claim 1, wherein said host cells are plant cells.

15. The method of claim 14, wherein said plant cells are dicotyledonous.

16. The method of claim 14, wherein said plant cells are monocotyledonous.

17. The method of claim 14, wherein said plant cells are selected from the group consisting of maize, wheat, sorghum, rice, barley, soybean, alfalfa, sunflower, Brassica, and tomato.

18. The method of claim 1, wherein each of said candidate nucleotide sequences is from a plant cell.

19. The method of claim 2, wherein said transcriptional regulatory region regulates expression of a nucleotide sequence involved in a physiological pathway of interest, wherein said physiological pathway of interest is selected from the group consisting of a pathogen resistance pathway, a tissue developmental pathway, a metabolic pathway, and an apoptotic pathway.

20. A kit for performing the method of claim 1, said kit comprising a population of polynucleotide constructs, wherein each of said polynucleotide constructs in said population comprises a common transcriptional regulatory region operably linked to a candidate nucleotide sequence.

21. The kit of claim 20 further comprising a complementary DNA microarray.

22. A method for identifying a nucleotide sequence that encodes a polypeptide that modulates the activity of a transcriptional regulatory region, said method comprising:

(a) providing at least a first and a second population of polynucleotide constructs,

(i) wherein each of said polynucleotide constructs in said first population comprises a first common transcriptional regulatory region operably linked to a candidate nucleotide sequence, and wherein each of said polynucleotide constructs in said first population further comprises a first common 5′

UTR sequence;

(ii) wherein each of said polynucleotide constructs in said second population comprises a second common transcriptional regulatory region operably linked to a candidate nucleotide sequence, and wherein each of said polynucleotide constructs in said second population further comprises a second common 5′

UTR sequence;

(iii) wherein said first and said second transcriptional regulatory regions are non-identical; and

,

(iv) wherein said first and said second 5′

UTR sequences are non-identical;

(b) introducing said populations of polynucleotide constructs into a population of host cells;

(c) screening for a modulation in the expression level of each candidate nucleotide sequence, and thereby identifying a nucleotide sequence that encodes a polypeptide that modulates the activity of said first or said second transcriptional regulatory region; and

,

(d) determining if the identified nucleotide sequence encodes a polypeptide that modulates the activity of said first or said second transcriptional regulatory region.

23. The method of claim 22, wherein said determining if the identified nucleotide sequence encodes a polypeptide that modulates the activity of said first or said second transcriptional regulatory region comprises performing quantitative RT-PCR on mRNA isolated from said population of host cells.

24. The method of claim 23, wherein said quantitative RT-PCR further comprises using a primer specific to the identified nucleotide sequence and a primer specific to said first or said second 5′

UTR sequence.

25. A kit for performing the method of claim 22, said kit comprising at least a first and a second population of polynucleotide constructs, wherein:

(a) each of said polynucleotide constructs in said first population comprises a first common transcriptional regulatory region operably linked to a candidate nucleotide sequence, and each of said polynucleotide constructs in said first population further comprises a first common 5′

UTR sequence; and

,

(b) each of said polynucleotide constructs in said second population comprises a second common transcriptional regulatory region operably linked to a candidate nucleotide sequence, and each of said polynucleotide constructs in said second population further comprises a second common 5′

UTR sequence;

(c) said first and said second transcriptional regulatory regions are non-identical; and

,

(d) said first and said second 5′

UTR sequences are non-identical.