Browsing by keyword "*Two-Hybrid System Techniques"
Now showing items 1-6 of 6
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A bacterial one-hybrid system for determining the DNA-binding specificity of transcription factorsThe DNA-binding specificities of transcription factors can be used to computationally predict cis-regulatory modules (CRMs) that regulate gene expression. However, the absence of specificity data for the majority of transcription factors limits the widespread implementation of this approach. We have developed a bacterial one-hybrid system that provides a simple and rapid method to determine the DNA-binding specificity of a transcription factor. Using this technology, we successfully determined the DNA-binding specificity of seven previously characterized transcription factors and one novel transcription factor, the Drosophila melanogaster factor Odd-skipped. Regulatory targets of Odd-skipped were successfully predicted using this information, demonstrating that the data produced by the bacterial one-hybrid system are relevant to in vivo function.
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A genetic strategy to eliminate self-activator baits prior to high-throughput yeast two-hybrid screensLarge-scale sequencing projects have predicted high numbers of gene products for which no functional information is yet available. Hence, large-scale projects, such as gene knockouts, gene expression profiles, and protein-interaction mapping, are currently under way to initiate the understanding of the function of these gene products. The high-throughput strategies that are currently being developed to generate protein-interaction maps include automated versions of the yeast two-hybrid system. These strategies rely on the large-scale construction of DNA-binding domain/protein-of-interest hybrid constructs (DB-X baits). An inherent problem of large-scale two-hybrid systems is that a high percentage of cloned sequences encode polypeptides that, when fused to DB, can activate transcription in the absence of any two-hybrid-interacting partner protein. Here, we describe and validate a genetic strategy that efficiently eliminates such self-activator baits prior to screening procedures. The strategy is based on a negative-growth selection and is compatible with high-throughput settings.
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Enhanced Y1H assays for ArabidopsisWe present an Arabidopsis thaliana full-length transcription factor resource of 92% of root stele-expressed transcription factors and 74.5% of root-expressed transcription factors. We demonstrate its use with enhanced yeast one-hybrid (eY1H) screening for rapid, systematic mapping of plant transcription factor-promoter interactions. We identified 158 interactions with 13 stele-expressed promoters, many of which occur physically or are regulatory in planta.
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Gene-centered yeast one-hybrid assaysTranscription is regulated by sequence-specific transcription factors (TFs) that bind to short genomic DNA elements that can be located in promoters, enhancers and other cis-regulatory modules. Determining which TFs bind where requires techniques that enable the ab initio identification of TF-DNA interactions. These techniques can either be "TF-centered" (protein-to-DNA), where regions of DNA bound by a TF of interest are identified, or "gene-centered" (DNA-to-protein), where TFs that bind a DNA sequence of interest are identified. Here, we describe gene-centered yeast one-hybrid (Y1H) assays. Briefly, in Y1H assays, a DNA fragment is cloned upstream of two different reporters, and these reporter constructs are integrated into the genome of a yeast strain. Next, plasmids expressing TFs as hybrid proteins (hence the name of the assay) fused with the strong transcriptional activation domain (AD) of the yeast TF Gal4 are introduced into the yeast strain. When a TF interacts with the DNA fragment of interest, the AD moiety activates reporter expression in yeast regardless of whether the TF is an activator or repressor in vivo. Sequencing the plasmid in the colonies that exhibit reporter activation reveals the identity of the TFs that can bind the DNA fragment. We have shown Y1H to be a robust method for detecting interactions between a variety of DNA elements and multiple families of TFs.
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Identifying DNA sequences recognized by a transcription factor using a bacterial one-hybrid systemBacterial-based interaction trap systems provide a powerful method to identify interacting macromolecules. When carried out in the context of a genetic selection, interacting pairs can be rapidly isolated from large combinatorial libraries. This technology has been adapted to allow the identification of DNA-binding sequences for a transcription factor (TF) from a large randomized library. This procedure uses a library of randomized binding sites upstream of a cocistronic HIS3-URA3 reporter cassette. The URA3 reporter allows self-activating sequences to be removed from the library through counter-selection. The HIS3 reporter allows sequences that are recognized by a TF to be isolated from the library, where transcriptional activation is mediated by fusion of the TF to the alpha-subunit of RNA polymerase. This technology can be used to characterize monomeric, homodimeric and heterodimeric DNA-binding domains and, once a suitable library is constructed, binding sites can be identified in approximately 10 d. The bacterial one-hybrid system allows larger libraries to be searched than the corresponding yeast one-hybrid system and, unlike SELEX, it does not require purification of the TF(s). The complexity of the binding site libraries that can be searched using the bacterial system is, however, more limited than SELEX, and some eukaryotic factors may not express or fold efficiently in the bacterial system.
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Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based methodThe C2H2 zinc finger is the most commonly utilized framework for engineering DNA-binding domains with novel specificities. Many different selection strategies have been developed to identify individual fingers that possess a particular DNA-binding specificity from a randomized library. In these experiments, each finger is selected in the context of a constant finger framework that ensures the identification of clones with a desired specificity by properly positioning the randomized finger on the DNA template. Following a successful selection, multiple zinc-finger clones are typically recovered that share similarities in the sequences of their DNA-recognition helices. In principle, each of the clones isolated from a selection is a candidate for assembly into a larger multi-finger protein, but to date a high-throughput method for identifying the most specific candidates for incorporation into a final multi-finger protein has not been available. Here we describe the development of a specificity profiling system that facilitates rapid and inexpensive characterization of engineered zinc-finger modules. Moreover, we demonstrate that specificity data collected using this system can be employed to rationally design zinc fingers with improved DNA-binding specificities.
