Method for identifying activators of gene transcription
First Claim
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.
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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.
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Citations
25 Claims
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1. A method for identifying a nucleotide sequence that encodes a polypeptide that modulates the activity of a transcriptional regulatory region, said method comprising:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A method for identifying a nucleotide sequence that encodes a polypeptide that modulates the activity of a transcriptional regulatory region, said method comprising:
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(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. - View Dependent Claims (23, 24, 25)
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Specification