NISHIZAWA Mikio Osaka Bioscience Institute, First Department, Research Scientist, 第一研究部, 研究員 (40192687)
トンプソン M・エリック (財)大阪バイオサイエンス研究所, 第二研究部, STAフェロー (90203661)
TSUJI Frederick I. Osaka Bioscience Institute, Second Department, Head, 第二研究部, 部長 (80201755)
OHMIYA Yoshihiro Osaka Bioscience Institute, Second Department, Research Scientist, 第二研究部, 研究員 (20223951)
THOMPSON Eric M. Osaka Bioscience Institute, Second Department, STA fellow
|Budget Amount *help
¥15,500,000 (Direct Cost : ¥15,500,000)
Fiscal Year 1991 : ¥5,100,000 (Direct Cost : ¥5,100,000)
Fiscal Year 1990 : ¥10,400,000 (Direct Cost : ¥10,400,000)
To elucidate the gene expression mechanism in mammalian cells, it is necessary to examine its promoter activity in various cells. Usually, the promoter region is connected to the reporter gene such as E. coli chrolamphenicol acetyltransferase (cat) gene. The CAT activity in the cells transfected by the hybrid gene is regarded as the promoter activity of the respective gene. Recently, the firefly luciferase, which emits light by oxidizing luciferin, is used as a reporter gene, and it is shown that this assay method is more simple and sensitive than the ordinary CAT assay.
The marine ostracod crustacean Vargula hilgendorfii is a small animal with nocturnal habits. When disturbed, it ejects a copious secretion of luciferin and luciferase into sea water, producing a bright luminous cloud. The light results from an enzyme-substrate reaction, catalyzed by luciferase. We have purified the luciferase from this animal, and its partial amino acid sequence was determined. Using a set. of oligonucl
eotides corresponding to the amino acid sequence, the full-length CDNA for the Vargula luciferase was isolated. The luciferase is consisting of 555 amino acids, and contains the signal sequence. The Vargula luciferase CDNA was then placed under the promoter of mammalian genes such as SV40, RSV, elongation factor lalpha (EF-lalpha) and granulocyte colony-stimulating factor (G-CSF), and introduced intovarious cell lines. Dependent on its promoter activity, various amounts of luciferase were detected in the medium suggesting Vargula luciferase was efficiently secreted from mammalian cells. The sensitivity of the assay was much higher than that of the CAT assay, and the assay using the firefly luciferase. We are currently trying to establish transformants in which the luciferase expression vector is stably integrated in the chromosomen. Using such stable transformants, it would be possible to examine the gene expression in living cells.