Analysis of regulatory mechanisms for gene transcription by atomic force microscopy.

点击翻译按钮获取中文摘要

1.To analyze changes in DNA structures during transcriptional regulation, interactions between responsive elements for transcription factors in DNA and the factors were examined with an atomic force microscope. To this end, the interaction between xenobiotic responsive element (XRE) and a binding site for XRE located in the 5'-flanking region of CYP1A1 gene was examined as a model.When single-stranded DNA fragments containing XRE on the graphite plate were reacted with arylhydrocarbon receptor (AhR) and its translocator protein (Arnt) complex and observed with an atomic force microscope, the complex bound to (+) chains but not to (-) chains. In the (+) chains, the chains bent like a knee at which the complex was bound to the chain. If double-stranded DNA fragments containing XRE were reacted with the complex, no apparent structural changes were found in the fragments.2.To examine where HSP90 dissociates from AhR, the intracellular distribution of HSP90 and AhR was examined in the H4IIE cells, a cell line derived from hepatocytes, by fluorescent immunohistochemistry and chemiluminescent immunohistochemistry. Most HSP90 molecules were found in the cytoplasm of the cultured cells before and after the addition of 3-methylcholanthrene (MC), an inducer for CYP1A1, although AhR localized in the cytoplasm before MC treatment translocated to the nuclei after the treatment. Thus, HSP90 is dissociated from AhR in the cytoplasm but not in the nuclei after the stimulation of DNA transcription in the H4IIE cells. Similar results were obtained in rat hepatocytes in vivo.3.A highly-sensitive and quantitative chemiluminescent immunohistochemical technique with luminol and ECL reagents has been developed during the course of the present experiments. For this technique, photons emitted from sections and/or cells were counted with a photon counter attached with a gravity detection board and a VIM camera.