title: Binding of lac repressor-GFP fusion protein to lac operator sites inserted in the tobacco chloroplast genome examined by chromatin immunoprecipitation.
abstract: Chromatin immunoprecipitation (ChIP) has been used to detect binding of DNA-binding proteins to sites in nuclear and mitochondrial genomes. Here, we describe a method for detecting protein-binding sites on chloroplast DNA, using modifications to the nuclear ChIP procedures. The method was developed using the lac operator (lacO)/lac repressor (LacI) system from Escherichia coli. The lacO sequences were integrated into a single site between the rbcL and accD genes in tobacco plastid DNA and homoplasmic transplastomic plants were crossed with transgenic tobacco plants expressing a nuclear-encoded plastid-targeted GFP-LacI fusion protein. In the progeny, the GFP-LacI fusion protein could be visualized in living tissues using confocal microscopy, and was found to co-localize with plastid nucleoids. Isolated chloroplasts from the lacO/GFP-LacI plants were lysed, treated with micrococcal nuclease to digest the DNA to fragments of approximately 600 bp and incubated with antibodies to GFP and protein A-Sepharose. PCR analysis on DNA extracted from the immunoprecipitate demonstrated IPTG (isopropylthiogalactoside)-sensitive binding of GFP-LacI to lacO. Binding of GFP-LacI to endogenous sites in plastid DNA showing sequence similarity to lacO was also detected, but required reversible cross-linking with formaldehyde. This may provide a general method for the detection of binding sites on plastid DNA for specific proteins.
authors.affiliation: Department of Plant Sciences, University of Cambridge, Cambridge, UK.
authors.forename: Christine A
authors.initials: CA
authors.lastname: Newell
authors.forename: John C
authors.initials: JC
authors.lastname: Gray
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D018742:DNA, Chloroplast / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D054628:Genome, Chloroplast; D049452:Green Fluorescent Proteins / Q000235:genetics; D007763:Lac Operon; D056968:Lac Repressors / Q000235:genetics / Q000378:metabolism*; D009875:Operator Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000032:analysis; D014026:Nicotiana / Q000166:cytology / Q000235:genetics*
chemical_list: D018742:DNA, Chloroplast; D056968:Lac Repressors; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins