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KMID : 0667720000370000411
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Report Natlonal Institute of Health
2000 Volume.37 No. 0 p.411 ~ p.412
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Characterization of the 5-flanking region for the rat gene for mitochondrial transcirption factor A (mtTFA)
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Abstract
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Objective : Mitochondrial transcription factor A (mtTFA) is essential for the initiation of transcription and the replication of mitochondrial DNA (mtDNA). The 5-upstream region of the rat mtTFA. gene was isolated from rat genomic DNA. In addition, we confirm whether the region contains promter activity and search for the transcription factor(s), which was responsible for mtTFA expression in relation to the metabolism of glucose and lipid using transient transfection into rat skeletal muscle L6 cells.
Methods : The 5-upstream region of the rat mtTFA gene was isolated from rat genomic DNA by extendng the 5´ sequence of the newly identified mtTFA cDNA using PCR-based Genome Walker. The mtTFA 5-upstream region ligated to luciferase reporter vector and trnasfected into rat skeletal muscle L6 cells.
Results: The identified rat mtTFA gene showed little sequence homology with the upstream region of human mtTFA. No typical TATA or potential nuclear respiratory factor 1 (NRF-1) binding site was found, whereas three potential binding sites for Spl and one for NRF-2 were present in the proximal upstream region. The promoter activity was ten times higher than that of control vectors. Co-transfection of human NRF-1 expression vector increased the mtTFA promoter activity two-fold, while human mtTFA decreased promoter activity compared to the control. Interestingly, the addition of 50 mM of glucose for 24 h increased mtTFA promoter activity by up to twofold, but 100 M H₂O₂ for 24 h repressed promoter activity to 40% of the control.
Conclusions : From these results, the sequence presented here is that of authentic rat mtTFA promoter and it is hoped that it will prove useful in studies of mtDNA transcription and replication.
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KEYWORD
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