| Project/Area Number |
07407074
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Circulatory organs internal medicine
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| Research Institution | Osaka University |
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Principal Investigator |
TADA Michihiko Osaka University Medical School., Professor of Medicine, 医学部, 教授 (90093434)
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| Co-Investigator(Kenkyū-buntansha) |
大津 欣也 大阪大学, 医学部・附属病院, 医員
NISHIDA Masashi Osaka University, Medical School., Assistant Professor, 医学部, 助手 (40283783)
KUZUYA Tsunehiko Osaka University, Medical School., Associate Professor, 医学部, 助教授 (80150340)
OTSU Kinya Osaka University, Affiliated Hospital., Medical Staff
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥8,000,000 (Direct Cost: ¥8,000,000)
Fiscal Year 1996: ¥8,000,000 (Direct Cost: ¥8,000,000)
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| Keywords | Calcium signaling / Exitation-contraction coupling / Phospholamban / Ca release channel / Cardiomyopathy / Gene diagnosis / ホスランバン / 心筋小胞体 / 発現調節機構 / 心筋小胞体カルシウム遊離チャネル / 興奮収縮関連 |
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| Research Abstract |
In order to examine and identify sequenses responsible for transcriptional regulation of the RYR2 and phospholamban, we have isolated the 5'-flanking region of the genes, and to identify potential regulatory elements in the 5'-flanking region of the genes, a series of 5'-deletion constructs in the 5'-flanking region of the genes were fused to the luciferase gene, and then their promoter activity in rat neonatal cardiac myocytes was determined.
As to the RYR2 gene, the results revealed the presence of a region containing positive regulatory elements in the 5'-flanking region. Analyzes of substitutional mutations introduced into the GC boxes and the regulatory region indicated that besides the GC box located at-56 to-51, two regulatory elements (RYR2P1 and RYR2P2) and essential for the promoter activity. These results indicated that Sp1 and transcription factors that bind to RYR2P1 and RYR2P2 cooperatively enhance the expression of the RYR2 gene. In a transient transfection experiment inv
… More
olving the promoter-luciferase gene constructs in skeletal muscle cells, we identified a negative regulatory region between-209 and-90 which represses the expression of the RYR2 gene in skeletal muscle cells.
In another hand, luciferase gene constucts including-2080 bp of the upstream region of phopholamban gene and progressive 5' deletions up to-96 bp showed a high level of luciferase activity. In contrust, further deletions from -78 to -4 bp resulted in remarkable decrease of luciferase activity. These results indicated that the region from-96 to-78 of the phospholambn gene contains the major positive regulatory element.
Sequence analysis revealed a canonial CCAAT sequence at position-93 from the tentative transcription initiation site and a TATA box at-10.
To confirm the functional significance of CAAT box for the transcription of phospholamban gene, internal deletion mutants were generated in the 5' flanking region of the phospholamban gene. Removal of the CAAT box reduced the luciferase activity to 40% that of the control. Internal deletion of both CAAT box and C/EBP beta element, which is located at-113, reduced the activty to 5% of the control. Gel shift assay indicated that nuclear proteins from heart bind to the CAAT box. These results suggested that the CAAT box together with the C/EBP beta element has critical role for the transcriptional activity of the phospholamban gene. Less
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