| Project/Area Number |
60480267
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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 |
内分泌・代謝学
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| Research Institution | The Research Institute of Environmental Medicine, Nagoya University |
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Principal Investigator |
SEO Hisao The Reseach Institute of Environmental medicine, Nagoya University Associate Professor, 環境医学研究所, 助教授 (40135380)
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| Co-Investigator(Kenkyū-buntansha) |
MURATA Yashiharu The Research Institute of Environmental Medicine, Nagoya University, 環境医学研究所, 助手 (80174308)
MATSUI Nobuo The Research Institute of Environmental Medicine, Nagoya University Professor, 環境医学研究所, 教授 (50023643)
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| Project Period (FY) |
1985 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 1987: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1986: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1985: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Thyroxine-binding globulin / Structural gene / Genomic gene / Restriction Fragment Length Polymorphism / 突然変異 / 遺伝子のクローニング / 発現ベクター / 遺伝病 / ジデオキシ法 |
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| Research Abstract |
1. Cloning and sequencing of a cDNA coding for thyroxine-binding globulin (TBG): Human liver cDNA library constructed in bacteriophage gtll was screened with anti-TBG antibody. The cDNA insert form positive clone was isolated and recloned in plasmid pSP65. after large scale preparation of the insert, the sequence was determined by dideoxy chain termination method. It was revealed that TBG consist of 395 amino acid and contains 5 asparagine residue which can accept carbohydrate residues.
2. Analysis of TBG mRNA in normal liver and hepatoma cell line HepG2: RNAs extracted form normal human liver and hepatoma cell line was subjected to Northern blot analysis. Two TBG mRNA species of different size (2.0 and 1.8 Kb) were found. by the analysis with region specific probe, it was found that the two mRNA was produced by alternative polyadenylation.
3. Cloning of Genomic TBG gene: DNA extracted from normal human subject was digested with EcoRI. The fragments containing TBG gene (10-20 Kb) were pu
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rified by agarose gel electrophorsis and inserted into the arms of bacteriophage EMBL-4. After in vitro packaging, the genomic library was screened with TBG cDNA probe. Genomic TBG clone containing all containing all the coding sequence was cloned. by the analysis of the cloned genomic TBG gene, it was found that TBG was contained in 5 kb sequence and consists of 4 xons.
4. Analysis of TBG gene in complete TBG deficient families: Restriction fragment length polymorphism of the TBG gene was wvaluated in 6 families with complete TBG deficiency. It was found that the absence of TBG in these families were not caused by gross deletion, insertion or mutation in the TBG gene.
5. Analysis of TBG gene in TBG excess family: Restriction fragment length polymorphism and gene dosage was evaluated in a family with TBG excess. it was found that TBG excess in the patients might be caused by the amplification of TBG gene.
6. Analysis of TBG gene in abnormal TBG (TBG-Gary): TBG-Gary was characterized by extrem ely low thyroxine-binding and anodal shift on IEF. Genomic TBG gene from an affected subject was cloned and analyzed by sequencing. It was found that single point mutation in the first exon of TBG fene resulted in the substitution of amino acid 96 from isoleucine to asparagine. Since there was no abnormality in other part of the gene, it was speculated that this mutation is the cause of abnormal TBG in the family. Less
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