| Project/Area Number |
12210018
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas
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| Allocation Type | Single-year Grants |
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| Review Section |
Biological Sciences
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| Research Institution | Tokai University |
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Principal Investigator |
IKEDA Joh-E Tokai University, The Institute of Medical Sciences, Professor, 総合医学研究所, 教授 (50266467)
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| Co-Investigator(Kenkyū-buntansha) |
OSUGA Hitoshi Tokai University, The Institute of Medical Sciences, Assistant Professor, 総合医学研究所, 講師 (60203775)
HADANO Shinji Tokai University, The Institute of Medical Sciences, Assistant Professor, 総合医学研究所, 講師 (60281375)
TANAKA Kazunori Tokai University, Medical school, Research Associate, 医学部, 助手 (10338767)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥44,700,000 (Direct Cost: ¥44,700,000)
Fiscal Year 2002: ¥15,000,000 (Direct Cost: ¥15,000,000)
Fiscal Year 2001: ¥29,700,000 (Direct Cost: ¥29,700,000)
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| Keywords | Huntignton's disease / neuronal cell death / polyglutamine disease / gene expression control / HD gene / miniature pig / transgenic / ハンチントン病(HD) / CAGリピート / HD遺伝子転写制御 / HDモデルミニブタ / HD遺伝子転写調節 / ハンチントン舞踏病 |
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| Research Abstract |
Goals of our study were to elucidate the mechanism of HD pathogenesis and to develop a novel therapy for HD through the analysis for the molecular mechanisms of the transcriptional regulation for the HD gene and the characterization of miniature pig model for HD. Major findings are described as follows.
In this study, we have identified two novel trans-acting factors, HDBP1 and HDBP2, which bind to the promoter region of the HD gene, using a yeast one-hybrid system. Analysis of amino acid sequences of HDBP1 and HDBP2 revealed that both proteins shared similar motifs and domain structures, suggesting that they belong to the same protein family. Functional analysis of HDBP1 and HDBP2 demonstrated that both proteins shuttled between nucleus and cytoplasm via nuclear export signal-mediated pathway. In vitro DNA-binding assay indicated that the C-terminal regions highly conserved between HDBP1 and HDBP2 showed the DNA-binding activity and these novel DNA-binding domains recognized the unique
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7-bp consensus sequence (GCCGGCG) which resides in triplicate at intervals of 13bp within the HD promoter region. Furthermore, the reporter gene assay showed that the 7-bp sequence was essential for the HD promoter activity in neuronal cells. Further analysis of those factors might provide new clues to the understanding of the transcriptional regulation of the HD gene.
We have generated five transgenic miniature pigs (FO; 4 females and lmale) expressing a truncated miniature pig HD gene with 75 CAG repeats. From a single female founder (FO), we have succeeded in the establishment of two independent lines. One line has a multiple copies of transgene, and the other contains single copy. We also analyzed the variation of the CAG repeat number in sperm DNA by SP-PCR, and revealed stability in the CAG repeat. Interestingly, male founder pig developed rotational movement after 6 months of age, although we cannot rule out the possibility that this was due to the insertional mutation and/or positional effect by transgene. Less
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