Counter-selectable marker for bacterial-based interaction trap systems

University of Massachusetts Medical School, Worcester, MA 01605, USA.
BioTechniques (Impact Factor: 2.95). 03/2006; 40(2):179-84. DOI: 10.2144/000112049
Source: PubMed


Counter-selectable markers can be used in two-hybrid systems to search libraries for a protein or compound that interferes with a macromolecular interaction or to identify macromolecules from a population that cannot mediate a particular interaction. In this report, we describe the adaptation of the yeast URA3/5-FOA counter-selection system for use in bacterial interaction trap experiments. Two different URA3 reporter systems were developed that allow robust counter-selection: (i) a single copy F' episome reporter and (ii) a co-cistronic HIS3-URA3 reporter vector. The HIS3-URA3 reporter can be used for either positive or negative selections in appropriate bacterial strains. These reagents extend the utility of the bacterial two-hybrid system as an alternative to its yeast-based counterpart.

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    • "The ability of proteins to bind DNA via multiple modes further complicates the situation and leads to the requirement of multiple binding models for many proteins. Fortunately, HT technologies are not only increasing the rate at which DNA-binding proteins are being characterized, but are providing the comprehensive binding data needed to construct models that involve multiple DNA-binding modes (23,24,27,28,35,38–40,42,43,48,59,73,74,79,132–134). An added benefit of the comprehensive nature of certain HT datasets, such as comprehensive k-mer or binding site level data, is that predictions of binding sites in the genome can be performed directly using the measured binding data (23,24,40,43,133). "
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    ABSTRACT: Binding of proteins to particular DNA sites across the genome is a primary determinant of specificity in genome maintenance and gene regulation. DNA-binding specificity is encoded at multiple levels, from the detailed biophysical interactions between proteins and DNA, to the assembly of multi-protein complexes. At each level, variation in the mechanisms used to achieve specificity has led to difficulties in constructing and applying simple models of DNA binding. We review the complexities in protein-DNA binding found at multiple levels and discuss how they confound the idea of simple recognition codes. We discuss the impact of new high-throughput technologies for the characterization of protein-DNA binding, and how these technologies are uncovering new complexities in protein-DNA recognition. Finally, we review the concept of multi-protein recognition codes in which new DNA-binding specificities are achieved by the assembly of multi-protein complexes.
    Nucleic Acids Research 11/2013; 42(4). DOI:10.1093/nar/gkt1112 · 9.11 Impact Factor
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    ABSTRACT: Bacterial-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.
    Nature Protocol 02/2006; 1(1):30-45. DOI:10.1038/nprot.2006.6 · 9.67 Impact Factor
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    ABSTRACT: The 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.
    Nucleic Acids Research 02/2007; 35(11):e81. DOI:10.1093/nar/gkm385 · 9.11 Impact Factor
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