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Binding of lac repressor-GFP fusion protein to lac operator sites inserted in the tobacco chloroplast genome examined by chromatin immunoprecipitation

Author:
Newell, Christine A., Gray, John C.
Source:
Nucleic acids research 2010 v.38 no.14 pp. e145
ISSN:
0305-1048
Subject:
DNA-binding proteins, Escherichia coli, antibodies, binding sites, chloroplast DNA, chloroplast genome, chloroplasts, chromatin, confocal microscopy, crosslinking, formaldehyde, genes, micrococcal nuclease, mitochondrial genome, nuclear genome, polymerase chain reaction, precipitin tests, progeny, protein binding, sequence homology, tissues, tobacco, transgenic plants
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 ~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.
Agid:
2272346