| Project/Area Number |
60480178
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Immunology
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| Research Institution | Medical Institute of Bioregulation, Kyushu University (1986)
佐賀医科大学 (1985) |
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Principal Investigator |
WATANABE Takeshi Professor, Medical Institute of Bioregulation, Kyushu University, 生体防御医学研究所, 教授 (40028684)
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| Co-Investigator(Kenkyū-buntansha) |
KUDO Akira Research Associate, Med. Inst. Bioregulation, Kyushu University, 生体防御医学研究所, 助手 (70178002)
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| Project Period (FY) |
1985 – 1986
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| Project Status |
Completed (Fiscal Year 1986)
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| Budget Amount *help |
¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 1986: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1985: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Keywords | Immunoglobulin gene / Gene expression control / Transacting factor / Cell fusion / マイクロインジェクション / 細胞特異的遺伝子発現 / エンハンサー / ゲルシフトアッセイ / DNA結合蛋白 |
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| Research Abstract |
The rearranged human immunoglobulin <gamma>1 heavy chain gene (HIGI) has been shown to be expressed at high level in B-lymphoid cells but not in non-lymphoid cells.
We previously showed that HIGI gene was activated through its enhancer by the positive requlatory trans-acting factor(s) and that the factor(s) were contained only in the cells of B lineage. In the present study, we demonstrated the purification of such trans-acting factor(s) from the nouse myeloma cell line, NS-1. The trans-acting factor(s) responsible for tissue-specific expression of HIGI gene were mainly found in HPLC fractions of molecular weight 53 to 127KDa, and in fractions eluted with 0.5MKCl from heparin-Sepharose column. A gel electrophoretic mobility shift assay indicated that the same fraction also contained DNA-binding proteins to the conserved octamer sequence, ATGCAAAT, in the promoter region, as well as binding proteins to the enhancer region. The 0.5mKCL fraction of heparin-Sepharose column was applied on DNA affinity column of synthetic oligonucleotides of octamer sequence. The inducing activity was not removed by the octamer DNA column and fully recovered in flow through farction, indicating that the protein responsible for the induction of HIGI in the present system was not an octamer binding protein. The protein(s) eluted from a DNA affinity column containing the sequence, TATTTTGGAAGCAAA, in the HpaII-BglII region of HIGI gene enhancer showed strong inducing activity for the HIGI gene. SDS-PAGE revealed that the fraction eluted from this enhancer DNA column contained a predominant protein of M.W. 96KDa.
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