Genetic analysis of prokaryotic and eukaryotic DNA-binding proteins in Escherichia coli.
This report describes an Escherichia coli genetic system that permits bacterial genetic methods to be applied to the study of essentially any prokaryotic or eukaryotic site-specific DNA binding protein. It consists of two parts. The first part is a set of tools that facilitate construction of customized E.coli strains bearing single copy lacZYA reporters that are repressed by a specific target protein. The second part is a pair of regulatable protein expression vectors that permit in vivo production of the target protein at levels appropriate for genetic experiments. When expressed in a properly designed reporter strain, the target protein represses the lac genes, resulting in an E.coli phenotype that can be quantitatively measured or exploited in large scale genetic screens or selections. As a result, large plasmid-based libraries of protein genes or pools of mutagenized variants of a given gene may be examined in relatively simple genetic experiments. The strain construction technique is also useful for generating E.coli strains bearing reporters for other types of genetic systems, including repression-based and activation-based systems in which chimeric proteins are used to examine interactions between foreign protein domains.
Whipple
F W
FW
Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. fwhipple@warren.med.harvard.edu
D001483:Base Sequence; D004274:DNA, Recombinant / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005057:Eukaryotic Cells; D017930:Genes, Reporter; D005821:Genetic Techniques; D005822:Genetic Vectors; D006801:Humans; D007763:Lac Operon; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D011387:Prokaryotic Cells; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics; D015183:Restriction Mapping
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins