| Project/Area Number |
10470206
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Hematology
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| Research Institution | National Cancer Center Research Institute |
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Principal Investigator |
SAITO Masaki National Cancer Center Research Institute, Virol. Glycobiol., Chief, ウイルス部, 部長 (60012762)
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| Co-Investigator(Kenkyū-buntansha) |
SAKAI Ryuichi National Cancer Center Research Institute, Virol. Glycobiol., Sec. head, ウイルス部, 室長 (40215603)
MATSUDA Kazuhiro National Cancer Center Research Institute, Virol. Glycobiol., Staff scientist, ウイルス部, 主任研究官 (80251502)
ISHII Atsushi National Cancer Center Research Institute, Virol. Glycobiol., Sec. head, ウイルス部, 室長 (20232225)
HAMANAKA Yuichiro National Cancer Center Research Institute, Virol. Glycobiol., Staff scientist, ウイルス部, 主任研究官 (40189618)
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥12,600,000 (Direct Cost: ¥12,600,000)
Fiscal Year 2000: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1999: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1998: ¥8,300,000 (Direct Cost: ¥8,300,000)
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| Keywords | Sialoglycosphingolipids / Ganglioside GM3 Synthase / cDNA Cloning / Genomic Structure / Gene Kockout / Promoter Region / Three Dimesional Structure / Cis Element / シアル酸転移酵素 / ルシフェラーゼ活性 / GM3合成酵素欠損マウス / TATA・CAAT配列 / シアリルモチーフ / Sp1結合部位 / プロモーター活性 / 5'-RACE法 / N結合型糖鎖 / ガングリオシドGD3合成酵素 / 遺伝子ノックアウト細胞 / ガングリオシドGM3 / シアル酸転移酵素-1 / シアリルモティーフ / II型膜蛋白質 / 特異的アミノ酸置換 / 遺伝子改変 / 転写調節 |
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| Research Abstract |
Sialoglycosphingolipids, gangliosides, are known to bear important functions in various biological phenomena such as cell growth and differentiation, embryogenesis and carcinogenesis. In vertebrates, almost all the gangliosides are synthesized from a common pre-cursor, ganglioside GM3 , which was previously shown by us to exhibit differentiation-inducing activity against human myelogenous leukemia cells. In this project, using the elaborately-devised expression cloning method, we succeeded for the first time in isolating and molecularly characterizing a new and relevant gene (cDNA and genome) which encodes a key glycosyltransferase, human ganglioside GM3 synthase (sialyltransferase-1 : STSGalV) and then, murine STSGalV, which is responsible for GM3 biosynthesis. Human GM3 synthase gene spans approximately 56 kb in the human genome and was found to consist of seven exons and six introns. The GC content in the region of the transcription start site, which was resided to 280 nucleotide up
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stream of the putative translational initiation site determined by primer extension, is high (81 %), and human GM3 synthase gene promoter contains no canonical TATA and CCAAT boxes. A number of cis-acting elements for transcription are noted in the 5'-flanking region. A transient reporter assay using the luciferase gene showedJhat promoter activity was much higher in SK N-MC cells than in HCT1 16 cells. Three Spl binding sites located in the GC-rich region seemed to be critical positive regulatory element in cell-specific expression of human GM3 synthase gene. The glycosyltransferase gene was mapped near the centromere of the short arm of chromosome 2 in the human genome. Subsequently, 3 kinds of transcripts (L-, Bl- and B2-type) of the gene were detected in mice by 5'-RACE analyses whereas a single transcript detect able in human organs, and murine tissue-specific expressions were clarified : Ltype transcript was specifically expressed in liver while Bl-type was generally detected in various organs with B2-type marginally expressed. The human and murine genomic structures of ST3GalV have been determined by screening BAC and λ library, respectively, prepared from the chromosomal DNA. Transfection of this enzyme cDNA into ganglioside-deficient mouse lung carcinoma 3LL cells was interestingly shown to introduce the characteristic shedding of GM3-rich membrane domain into the medium. In order to obtain much amount of the enzyme protein enough to analyze three dimensional structure and to utilize in the development of automatic carbohydrate-chain synthesizers, we could recently succeed in producing ST3GalV soluble forms as MBPVGST-fusion proteins in E.coli. Less
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