Development of a System for Detection of Human Steroid Hormone Activities Using Bio-devices for an Enhanced Plant Two-Hybrid SystemView all posters
Laboratory of Environmental Molecular Biology, Japan
This report is the first describing a simple, low-cost, and extremely sensitive reporter-gene assay system (EPTH system; Enhanced Plant Two Hybrid system) for the comprehensive analysis of agonistic and antagonistic activities of human steroid hormones and related chemicals using transgenic Arabidopsis thaliana. Genes introduced into plant to make biosensors were constructed by fusing DNA fragments (genetic parts) obtained from various organisms containing human, bacteria, viruses, and plant. We succeeded in making six plant biosensors that can detect and determine estrogen, progesterone, glucocorticoid hormone, mineralocorticoid hormone, thyroid hormone, and PPAR ligand specifically by introducing three chimeric genes into each plant. Two effector genes controlled by P35S, a strong constitutive promoter, are overexpressed constitutively in the transgenic plants. Both effector proteins contain nuclear localization signal (NLS) to retain them in the nucleus. Ligand binding to a ligand-binding domain of nuclear receptor from human of the chimeric hormone receptor (effector 1) permits interaction with a nuclear receptor interacting domain of transcriptional co-activator from human of the chimeric co-activator (effector 2). Through this interaction, the transcriptional activation signal of acidic transactivation domain of VP16 (VP16 AD) can stimulate transcription of a -glucuronidase (GUS) reporter gene in the EPTH system. Development of the system opened the way to find chemicals with human steroid hormone activity and anti-steroid hormone activity efficiently not only from purified chemical library but also from extracts of medicinal herbs (50 species) and common foods (80 species) prepared by simple homogenization and centrifugation with no manipulation for aseptic treatment. We will show the lists of chemicals and extracts those with activities described above.