PGATA - A POSITIVE SELECTION VECTOR BASED ON THE TOXICITY OF THE TRANSCRIPTION FACTOR GATA-1 TO BACTERIA

The transcription factor GATA-1 is a zinc finger DNA-binding protein e ssential for the development of red blood cells. When we expressed dif ferent regions of the zinc finger domain in bacteria using an isopropy l-beta-D-thiogalactoside (IPTG) inducible system, growth of bacteria h arboring the active DNA-binding domain of GATA-1 was rapidly inhibited upon IPTG induction. The growth inhibition pattern suggested it may b e occurring at the level of the initiation of replication, and GATA-1 was found to bind to three of the four DNA A protein-binding sites in the origin of replication. This toxicity was used to develop a positiv e selection vector system in which cloned DNA fragments interfered wit h the production of the GATA-1 DNA-binding domain. Thus, vector molecu les containing the insert of interest are selected for when bacteria a re grown in the presence of IPTG. With this system, the vector does no t need to be dephosphorylated, purified or completely digested with a restriction enzyme for the efficient cloning of DNA fragments even whe n the vector-to-insert DNA molar ratio in ligation reactions is 10 to 1. Moreover no special strain of Escherichia coli is required, and the selection might also be applicable to other species of bacteria if th e toxicity of GATA-1 relates to inhibition of the DNA A protein.