Transcription of human c-myc in permeabilized nuclei is associated with formation of Z-DNA in three discrete regions of the gene.
When human U937 cells are placed in agarose microbeads and treated with a detergent, the cytoplasmic membrane is lysed and the nuclear membrane is permeabilized. However, the nuclei remain intact and maintain both replication and transcription. Biotin labeled monoclonal antibodies against Z-DNA have been diffused into this system and used to measure the amount of Z-DNA present in the nuclei. It has previously been shown that the amount of Z-DNA present decreases due to relaxation by topoisomerase I and increases as the level of transcription increases. Here we measure the formation of Z-DNA in the c-myc gene by crosslinking the antibodies to DNA using laser radiation at 266 nm for 10 ns. The crosslinked DNA is isolated by restriction digestion, separation of antibody labeled fractions through the biotin residue, and subsequent proteolysis to remove the crosslinked antibody. Three AluI restriction fragments of the c-myc gene are shown to form Z-DNA when the cell is transcribing c-myc. The Z-DNA forming segments are near the promoter regions of the gene. However, when U937 cells start to differentiate and transcription of the c-myc gene is down-regulated, the Z-DNA content goes to undetectable levels within 30-60 min.
Wittig
B
B
Institut für Molekularbiologie und Biochemie, Freie Universität Berlin, Germany.
Wölfl
S
S
Dorbic
T
T
Vahrson
W
W
Rich
A
A
D001483:Base Sequence; D002166:Camptothecin / Q000494:pharmacology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010539:Permeability; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics*; D059004:Topoisomerase I Inhibitors; D014158:Transcription, Genetic
D003432:Cross-Linking Reagents; D016271:Proto-Oncogene Proteins c-myc; D059004:Topoisomerase I Inhibitors; D004247:DNA; D002166:Camptothecin