1987 Fiscal Year Final Research Report Summary
The molecular cloning in brain of El mice as refractory epileptic model.
| Project/Area Number |
61570528
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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 |
Psychiatric science
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| Research Institution | Osaka City University |
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Principal Investigator |
KAWAKITA YUKIO Osaka City University Medical School, 医学部, 教授 (80046820)
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| Co-Investigator(Kenkyū-buntansha) |
CHIKAME TAKASHI Osaka City University Medical School, 医学部, 助手 (80163723)
ONISHI HIROSHI Osaka City University Medical School, 医学部, 講師 (70094464)
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| Project Period (FY) |
1986 – 1987
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| Keywords | El mouse / Epilepsy / Cloning / cloning |
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| Research Abstract |
A single-stranded cDNA synthesized from El mouse brain poly(A^+)mRNA, was 350 to 400 nucleotide length and 38S of the sedimentation coefficient. The hybridized ratios of cDNA and poly(A^+)mRNAs from El(+), El(o) which did not convulse at all, and non-susceptible strain of ddY mice were 78, 75 and 55%, respectively, at Rot 10^5. The sequence complexity of nucleotides showed that there are three classes of abundant, intermediate and rare in mRNA, which values were 1.49 X 10^5, 4.64 X 10^6 and 1.87 X 10^7 nucleotides, respectively. These results imply that a significant difference in sequence complexity of abundant class was observed in genome between El and ddY mouse brains.
A cDNA library was prepared from El mouse brain poly(A^+)mRNA by the method of Okayama and Berg. Thousand colonies from this library were screened with excess of poly(A^+)mRNA in ddY or El mouse brains. In situe hybridization, the 500 positive clone showed a higher level of hybridization in El than ddY mouse brain. Plasmid DNA samples were digested with restriction endonucleases and the vector separated from insert fragments was carried out by gel electrophoresis. The Southern blot analysis revealed that the 200 selected brain cDNA clones ranged in abundance. Plasmid cDNA from each the 500 clones was nicked-translated and used to probe a Northern-blot analysis with poly(A^+)mRNA of El or ddY mouse brain. Two hundred clones showed a larger in hybridization intensity at El than ddY mouse brains. These results suggest that the difference of gene expression between El and ddY mice was induced during the process of posttranscription.
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