A bacterial one-hybrid system for analyzing DNA-binding specificities

Univ Of Massachusetts Med Sch Worcester, Worcester MA

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Abstract

[unreadable] DESCRIPTION (provided by applicant): An important goal of the human genome project is to completely describe the cis-regulatory sequences and trans-acting factors that control gene transcription in complex genomes. A significant remaining impediment to this goal is the paucity of well-characterized DNA-binding specificities for transcription factors. A high-throughput, inexpensive method is needed to facilitate the analysis of the DNA-binding specificity of transcription factors on a genome-wide scale. We have developed a bacterial 1-hybrid system that can characterize the DNA-binding specificity of transcription factors. This technology has advantages over existing in vitro methods: only basic molecular biology reagents and techniques are required, and no protein purification is necessary. In this application, we propose to evaluate our bacterial 1-hybrid system as a high-throughput method for determining the DNA-binding specificities of transcription factors. In Aim 1, we will optimize the bacterial 1-hybrid system for the high-throughput analysis of factors and establish a standard set of selection conditions that will yield high quality binding site motifs for transcription factors with a broad range of affinities. In Aim 2, we will perform a pilot screen of 30 Cys2His2 zinc finger proteins and 20 other transcription factors from the model organism Drosophila. In Aim 3, we will assess the quality of the DNA-binding specificities determined using the bacterial 1-hybrid system. If this project is successful, we anticipate that the bacterial 1-hybrid system will be used by ourselves and adopted by others to perform genome-wide surveys of transcription factors in Drosophila and a variety of other model organisms. The resulting database of information on transcription factors will be valuable for deciphering the mechanisms of gene regulation in higher eukaryotes. [unreadable] [unreadable] [unreadable]