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 essential for the development of red blood cells. When we expressed different regions of the zinc finger domain in bacteria using an isopropyl-beta-D-thiogalactoside (IPTG) inducible system, growth of bacteria harboring the active DNA-binding domain of GATA-1 was rapidly inhibited upon IPTG induction. The growth inhibition pattern suggested it may be 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 positive selection vector system in which cloned DNA fragments interfered with the production of the GATA-1 DNA-binding domain. Thus, vector molecules containing the insert of interest are selected for when bacteria are grown in the presence of IPTG. With this system, the vector does not need to be dephosphorylated, purified or completely digested with a restriction enzyme for the efficient cloning of DNA fragments even when 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 the toxicity of GATA-1 relates to inhibition of the DNA A protein.
Trudel
P
P
Montreal Center for the Canadian Red Cross Society, QC, Canada.
Provost
S
S
Massie
B
B
Chartrand
P
P
Wall
L
L
D001426:Bacterial Proteins / Q000378:metabolism; D001431:Bacteriological Techniques; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D004586:Electrophoresis; D050981:Erythroid-Specific DNA-Binding Factors; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D005822:Genetic Vectors; D006131:Growth Inhibitors / Q000494:pharmacology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000633:toxicity; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics* / Q000633:toxicity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D006131:Growth Inhibitors; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors