| Project/Area Number |
61570071
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Neurophysiology and muscle physiology
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| Research Institution | Yokohama City University School of Medicine |
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Principal Investigator |
TAKAHASHI Toyozoh Department of Bacteriology, Yokohama City University, School of Medicine. Associate Professor, 医学部・細菌学講座, 助教授 (40046149)
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| Co-Investigator(Kenkyū-buntansha) |
HAMAJIMA Kenji Dept. of Bacteriol., Yokohama City Univ., School of Med., Assistant, 医学部・細菌学講座, 助手 (00114611)
FUKUSHIMA Jun Dept. of Bacteriol., Yokohama City Univ., School of Med., Assistant, 医学部・細菌学講座, 助手 (00181256)
OKUDA Kenji Dept. of Bacteriol., Yokohama City Univ., School of Med., Professor, 医学部・細菌学講座, 教授 (40124862)
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| Project Period (FY) |
1986 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1987: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1986: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | Northern Hybridization / In situ hybridization / Brain specific expression / Mammalian Brain / Non-radioactive DNA labeling / 脳のアルギニノコハク酸リアーゼcDNA / サザーンハイブリダイゼーション / ウエスターンハイブリダイゼーション |
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| Research Abstract |
RNAs were extracted from brain cortexes of cats and rats, and cDNA were synthesized enzymatically in vitro on the basis of their nucleotide sequences. Small to large sizes (0.5 - 6.5 kb) of the synthesized cDNAs could be obtained. The cDNAs were hybridized with polyA^+mRNAs (total 50<nu>g) extracted from several organs (kidney,spleen,liver and lung). Single stranded cDNAs were fractionated from double stranded hybrids by hydroxyappatite chromatography (Bio Rad). Alternatively,the cDNAs were absorbed with the mRNAs immobilized on membrane filters. Small amounts of the absorbed cDNAs were obtained. We also obtained <lambda>gt 11 cDNA libralies of cat and rat(2 - 3 x 10^6 phages/ml for both animal libralies). A protein (m.w.68,000) which was detected with small amounts in cow brains was purified by two-dimentional electrophoresis and high pressure liquid chromatography. The 16 amino acid residues at N-end of the protein were determined. The libralies have been screened with monoclonal antibodies to the protein. An alternative non-radioactive method for labeling DNA was also developed for the in situ hybridization experiments,which should be aimed at the followings of the cloning for brain specific cDNAs. We could get much informations of it. A positive clone was paralelly obtained from the expression vector <lambda>gt 11 libralies with rat cDNAs, using a cDNA probe (1.3 kb; Aval-Aval) of argininosuccinate lyase. Complementary DNA was isolated from the phage clone and mapped with restriction enzymes. The rat brain cDNA was 800bp and 30 bp larger at the 5'and3'ends,respectively,in comparison with each end of the rat liver cDNA. It was suggested that they had structurally common translated resion.
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