Development of a new method for detecting gene expression in a single cell and its medical application.

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We have successfully developed a new luciferase assay technology that enables the expression of pigment cells, transfected with the fusion gene containing the firefly luciferase gene, to be captured on photographic images. The light discharged by the luciferase-mediated reaction can be quantified by chemical luminescence measurement. Using the photocounting camera and an efficient transfection method, we were able to detect the promoter activity of the tyrosinase gene in human melanocytes. Furthermore, we have obtained the following results as summarized below. 1. The three cis-regulatory elements of the human tyrosinase gene have been identified, and each of these elements contains one copy of a CATGTG motif. We showed that each CATGTG motif is required for pigment cell-specific transcription. 2. In the course of studying the regulation of the heme oxygenase-1 gene expression during macrophage differentiation, we showed the presence of a putative silencer sequence, repressing a function of the heat shock element, which is located in the 5^1-flanking region of the human heme oxygenase-1 gene. 3. Three types of nitric oxide (NO) donors increased the expression levels of heme oxygenase-1 mRNA,suggesting a possible coordinated regulation of heme oxygenase-1 and NO synthase mRNAs. 4. Both heme oxygenase-1 and inducible NO synthase mRNAs were overexpressed in human primary brain tumors compared to the normal brain tissues. 5. The choroid plexus probuces cerebrospinal fluid which plays an important role not only in the mechanical support of the brain but also in the chemical homeostasis of the brain. We found that adrenomedullin, a potent vasodilator peptide, is produced and secreted by cultured choroid plexus carcinoma cells, a rare neoplasm derived from the epithelium of the choroid plexus.