| Project/Area Number |
04670152
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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 |
General medical chemistry
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| Research Institution | JICHI MEDICAL SCHOOL |
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Principal Investigator |
KAWAKAMI Kiyoshi (1993) JICHI MEDICAL SCHOOL DEPARTMENT OF BIOLOGY, ASSOCIATE PROFESSOR, 医学部, 助教授 (54191016)
川上 潔 (1992) 自治医科大学, 医学部, 助教授 (10161283)
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1993: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1992: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Sodium Pump / Muscle Cells / Regulation of Gene Expression / E-box / Spl / T4 DNA Polymerase Footprinting / DNA bending / Spl / DNaseIフットプリント法 / メチル化干渉法 |
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| Research Abstract |
We identified cis elements in the 5'-flanking region of rat Na, K-ATPase alpha2 subunit gene(Atpla2) using transient transfection assays in L6 rat skeletal muscle myoblast cells. By 5' deletion mutation analysis, the region between positions -175 and -108 was identified as a positive regulatory region. In the region, the distal E box (-144 to -139) acts as a negative regulatory element, and the Spl consensus sequence (-123 to -118) and the GGGAGG sequence (-114 to -109) act as positive regulatory elements. Gel retardation analysis revealed that binding factors are an E-box binding protein and Spl. DNase I footprinting and methylation interference analyzes revealed that Spl binds to the region from -122 to -101 and the E-box binding protein from -144 to -136. T4 DNA polymerase footprinting revealed that there are three Spl binding sites in the region and that Spl binds to one of the three sites in a mutually exclusive manner. The redundancy of binding sites may ensure more stable binding of Spl. Furthermore, the binding of each Spl may result in kinetic synergism in transcription initiation.
We examined DNA bending using an electrophoretic mobility shift assay to determine whether Spl induces structural changes in DNA.The results indicated that Spl bends DNA upon binding to its recognition sequence. This structural change DNA by Spl may result in synergistic activation by making access of Spl to other components of the transcriptional apparatus more favorable.
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