Analysis Of The Center Activity Domain In MDR1mRNA IRES

Multi-drug resistance is one of the main reasons of chemotherapy failure incurrent clinical cancer therapy. Different kinds of genes had found to be involved inmultidrug resistance process.The abnormal expression of multidrug resistance gene1(MDR1) can be detected in almost all drug-resistant cells. Our previous studiesshowed that MDR1mRNA-5’UTR(untranslated region，UTR) had IRES activity.We found that75bp sequence of MDR1mRNA was lost in the upstream of5’UTR compaired to the new sequence in NCBI database. In order to accuratelydetect the IRES activity of MDR1mRNA-5’UTR, we first added75bp sequence tothe previous one to complete the full length of MDR15’-UTR, and then constructeddifferent kinds of bicistronic expression vectors which contain truncated MDR1region to detect the center activity domain in MDR15’-UTR. The expressionefficiency of EGFP and DsRed were analyzed by fluorescence microscopy, Westernblot, fluorescence spectrophotometer and flow cytometry. Results showed that thefull length of MDR1-5’UTR had IRES activity, and the center activity domain islocated in the downstream247bp of MDR1-5’UTR. All these IRES activity in theseregions was weaker than the positive control EMCV. The finding of the centeractivity domain in MDR1-5’UTR IRES provides a new insight to the multidrugresistance research.Bicistronic expression vector is commonly used as the detection method ofIRES activity analysis. In bicistronic constructs, the first cistron is cap-dependentwhile the second cistron will be translated only if the intercistronic sequences canfunction as an IRES. In normal physiological conditions, the eukaryotic mRNA ismostly monocistrons. This vector cannot fully reflect the IRES activity in the normalphysiological conditions, so we design and construct the Dual-promoter andDual-reporter gene expression vector, which can can transcript and translate into twodifferent kinds of fluorescent proteins simultaneously. The transcription lever of thetwo reporter gene should be the same because they are transcripted under the samepromoter. The IRES activity of the insertion sequence can be detected through thetwo fluorescent protein expression ratio when the external conditions changed. ThisDual-promoter and Dual-reporter gene expression vector laid the foundation for theestablishment of a new method for the detection of IRES in further study.