1. Characterization of an In Vivo Z-DNA Detection Probe Based on a Cell Nucleus Accumulating Intrabody
- Author
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Izabel Cristina Rodrigues da Silva, Andrea Queiroz Maranhão, Isabel Garcia Sousa, Luana Salgado Quilici, Marcelo M. Brigido, Herdson Renney de Sousa, Galina Gulis, and Maryani Andressa Gomes Bezerra
- Subjects
0301 basic medicine ,Chromatin Immunoprecipitation ,Bioengineering ,CHO Cells ,Applied Microbiology and Biotechnology ,Biochemistry ,Intrabody ,Green fluorescent protein ,Cell Line ,03 medical and health sciences ,Cricetulus ,Genes, Reporter ,Cricetinae ,Gene expression ,Animals ,DNA, Z-Form ,Humans ,Molecular Biology ,Regulation of gene expression ,Cell Nucleus ,Reporter gene ,biology ,Molecular biology ,030104 developmental biology ,Gene Expression Regulation ,biology.protein ,MCF-7 Cells ,Human genome ,Expression cassette ,DNA Probes ,Chromatin immunoprecipitation ,Biotechnology - Abstract
Left-handed Z-DNA is a physiologically unstable DNA conformation, and its existence in vivo can be attributed to localized torsional distress. Despite evidence for the existence of Z-DNA in vivo, its precise role in the control of gene expression is not fully understood. Here, an in vivo probe based on an anti-Z-DNA intrabody is proposed for native Z-DNA detection. The probe was used for chromatin immunoprecipitation of potential Z-DNA-forming sequences in the human genome. One of the isolated putative Z-DNA-forming sequences was cloned upstream of a reporter gene expression cassette under control of the CMV promoter. The reporter gene encoded an antibody fragment fused to GFP. Transient co-transfection of this vector along with the Z-probe coding vector improved reporter gene expression. This improvement was demonstrated by measuring reporter gene mRNA and protein levels and the amount of fluorescence in co-transfected CHO-K1 cells. These results suggest that the presence of the anti-Z-DNA intrabody can interfere with a Z-DNA-containing reporter gene expression. Therefore, this in vivo probe for the detection of Z-DNA could be used for global correlation of Z-DNA-forming sequences and gene expression regulation.
- Published
- 2016