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Title: High-througput Bacteria one hybrid (B1H) selections for all Drosophila homeodomain proteins      
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available
aggregation:
instance of dataset
privacy:
not applicable
refinement:
curated
dateReleased:
05-02-2014
ID:
E-GEOD-25312
description:
In many metazons, such as humans and Drosophila, homeodomain proteins comprise the second largest family of sequence specific transcription factors. In Drosophila, homeodomains play an important role in development. Many homeodomain proteins display a high level of homology across metazons, presumably due to importance of their functional roles. We comprehensively characterized the DNA binding preferences of all 84 Drosophila homeodomain transcription factors which contain a single DNA binding domain. Previously, we employed a bacterial one hybrid (B1H) assay to select for 20 to 40 high affinity transcription factor binding sites [Noyes et al. (2008). Cell. 133(7):1277-1289]. In this system, E. coli are transfected with two plasmids. One plasmid encodes the DNA binding domain of a homeodomain fused to two zinc finger domains and the omega subunit of RNAP. The other plasmid is drawn from a library of prey plasmids which contain a 10bp randomized transcription factor binding site (TFBS) region in the promoter of the reporter gene His3. The E. coli strain used is a His3 homolog and omega subunit knock out strain. If a transcription factor has high affinity for a TFBS, more His3 will be produced, leading to the production of more histidine and an increase in the growth rate. If the transcription factor does not bind with sufficient affinity to the TFBS, little or no histidine will be produced resulting in little or no growth. The stringency of the B1H system can be tuned using the chemicals IPTG and 3-AT. IPTG induces production of the chimeric transcription factor, and 3-AT is a competitive inhibitor of the enzyme encoded by His3. One of the advantages of the B1H system is that the transcription factor does not have to be purified and that many experiments can be easily conducted in parallel. In this study, in stead of picking 20 to 40 colonies and sequencing their TFBSs, we used high-throughput Illumina sequencing to sequence the selected sites of all of the colonies growing on a plate. This provided quantitative data regarding the growth rate of cells possessing each selected TFBS variant, which is a function of the affinity of the transcription factor for the binding site. With this quantitative data, we can build more accurate models of transcription factor binding. All 84 of the Drosophila homeodomain proteins that contain a single DNA binding domain were analyzed using the B1H assay. The same selection stringency was used for all experiments (10uM IPTG and 5mM 3-AT). All experiments were run for 36 to 48 hours. 10 mutants were also assayed: 3 Caup, 3 Bcd and 4 En mutants. The bait plasmid omegaUV2zf was used in all but 3 cases. In this instances, a slightly different bait plasmid, omegaUV5zf, with a stronger promoter was used. Thirty different replicates were performed in order to insure that sufficient number of reads were obtained for each protein. In total, 126 experiments were performed.
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other
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HTML
storedIn:
Array Express
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not compressed
accessType:
landing page
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none
authentication:
none
primary:
true
accessURL: https://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-25312
format:
JSON
storedIn:
OmicsDI
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not compressed
accessType:
download
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none
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none
primary:
false
accessURL: www.omicsdi.org/ws/dataset/arrayexpress-repository/E-GEOD-25312.json
format:
XML
storedIn:
OmicsDI
qualifier:
not compressed
accessType:
download
authorization:
none
authentication:
none
primary:
false
accessURL: http://www.omicsdi.org/ws/dataset/arrayexpress-repository/E-GEOD-25312.xml
ID:
SCR:014747
name:
Omics Discovery Index
abbreviation:
OmicsDI
homePage: http://www.omicsdi.org/