| Project/Area Number |
10670108
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General medical chemistry
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
KOYAMA Yoshikazu Hokkaido Univ., Sch. Med., Inst., 医学部, 助手 (90186841)
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| Co-Investigator(Kenkyū-buntansha) |
SAKAI Masaharu Hokkaido Univ., Sch. Med., Ass. Prof., 医学部, 助教授 (50162269)
NAKABYASHI Hidekazu Hokkaido Univ., Sch. Med., Inst., 医学部, 助手 (10033383)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1999: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1998: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | transcription factor / gene expression / silencer / targetin vector / hepatoma / gene therapy / AFP |
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| Research Abstract |
The human α-fetoprotein (AFP) gene contains silencer elements presumably involved in the adult suppression of the gene expression. A 110bp silencer fragment containing 31bp core sequence has been identified, and the reporter genes carrying the sequence suppressed their activity in human hepatoma cell lines of the low AFP production.
We here summarize our initial attempts for cDNA cloning of the silencer binding proteins responsible for activation of the AFP silencer. Screening of a λgt11 cDNA Iibrary from human adult liver by Southwestern method identified four independent clones, whose recombinant protein products interacted on nitrocellulose membrane with labeled probe containing the silencer sequence. The positive cDNA clones were subcloned into a plasmid and sequenced. The homology search of their sequences in GenBank DNA database showed that the clones termed pSL-4(insert size: 〜3.3 kb), pSL-7(〜1.5 kb), and pSL-9(〜1.3 kb) revealed 94 to 95 % homology to human DNA-binding protein B(dbpB) gene or human Y box binding protein-1(YB-1) gene. The homology search of pSL-20(〜0.9 kb) showed 100% homology to human prolactin regulatory element-binding protein. At present, we carry out the characterization of these clones as follows.
1. Gel shift analyses of recombinant proteins of the clones with silencer element.
2. Analyses of expression pattern for tissues and differentiation stages.
3. Co-transfection experiments with expression plasmids of the clones and the known transcription factors.
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