Atomic Force Microscopy Identification Of Transcription Factor AP-1 Bound To 5' Untranslated Regulatory Region DNA Of Mdr1 Gene

Objective Multidrug resistance(MDR) phenotype of cancer cells is a major obstacle for cancer chemotherapy,this phenotype is main due to the overexprssion of the mdr1gene. Transcription factor AP-1 ,which is one of important regulate proteins in the promoter region of mdr1 gene.To date, no direct data of AP-1-DNA regulating complexes on mdr1 gene.The aim of this study is to image and map the structure of Untranslated 5'regulatory region DNA of mdr1gene , identificate and analysis of transcription factor AP-1 bound to Untranslated 5'regulatory region DNA of mdr1 gene complex with atomic force microscopy ,to find out the molecule mechanism of multidrug resistance.Methods Human leukemia adriamycin resistant strain K562/A02 was used as a target cells, transcription factor AP-1was used as a target protein. Cultivate and PCR technique was used to amplify K562/A02 cells with the 769bp 5'regulatory region DNA of mdr1gene , which fragment from–755 to +14. The amplified DNA products were purified by the PCR product purification kit, AP-1 of hela nuclear extract were purified by Sephadex spin column .The target DNA fragments were incubated with the target protein AP-1 in binding buffer and then immobilized the the AP-1–DNA complexes on a freshly cleaved mica surface which treated by MgCl2.AFM was used to idificate image of the structure of target DNA and the AP-1–DNA complexes.Results 1.The influence of biotin-signed process to PCR: The apparent contour lengths of biotin-sighed DNA measured with AFM images were obviously different with theory length(based on a 0.34-nm base-to-base distance in B-form DNA,the predicted contour length of the 769-bp DNA fragment is 261.46 nm). The typical large-scale AFM image showed that the overall efficiency of labeling of biotin was less than 20%, so we predicted that biotin-signed would influence on process of PCR, it's mechanism was not clear.2.Search for the optimum DNA purification kit for AFM image: We estimated purification effect of three kinds of DNA purification kits and suggested two kits can be used to prepare purified DNA samples for AFM imaging.3. Search for the optimum DNA concentration to yield well-absorbed DNA molecular on the mica surface for AFM image: A final concentration of 5–20 ng/ul DNA samples were placed on a freshly cleaved mica surface.AFM was used to image and analysis target DNA fragment. We estimated the optimum DNA concentration to yield well-absorbed DNA molecular on the mica surface was found to be 10ng/ul .4. Obtained the contour of target DNA AFM image :the length of the DNA fragment measured by AFM image was 260.13±2.29nm ,the width was11.88±0.92nm,the mean height was 1.2nm (mean±SD, N = 50).5. Obtained the contour data of protein AFM image : Through comparing of AFM images purificated with or without spin-column(Ultrafree-MC 0.22) purification filled with a bed of Sephadex G-100,we found...