02 May 2003
Cloning and characterization of the bidirectional promoter regulating the expression of the human poly(ADP-ribose) glycohydrolase (PARG) and inner mitochondrial membrane translocase 23 (TIM23) genesM. Meyer-Ficca, R. Meyer, E. Jacobson, M. Jacobson
Med Sci Monit 2003; 9(1): 44- :: ID: 15107
Poly(ADP-ribosyl)ation is a posttranslational protein modification mediated by a family of poly(ADP-ribose) polymerases (PARPs). It is a highly energy-consuming process which can ultimately lead to depletion of cellular NAD+ pools and thereby to cell death. Poly(ADP-ribose) glycohydrolase (PARG) is the only known cellular ADP-ribose polymer degrading enzyme, thus the turnover of poly(ADP-ribose) appears to be dependent on the availability of cellular PARG. By computational analysis of the genomic locus of the human PARG gene, we identified a 470 bp bidirectional putative common promoter for the PARG and the TIM23 genes. The complete promoter was amplified by PCR of genomic DNA of human CF3 fibroblasts, sequenced and cloned into the reporter plasmid pGFL, in both PARG and TIM23 orientations. Transfection of these reporter plasmids into HEK293 cells allowed the determination of the respective promoter activities. The TIM23 promoter showed a 3.7 fold higher activity than the PARG promoter, suggesting different expression levels for these proteins. TIM23 promoter activity reached 30% of a RSV promoter control plasmid, while PARG promoter activity was 10% of the control. Detailed analysis of the promoter sequence indicates the presence of putative binding sites for transcription factors NF-kB, c-ETS-1, CREBP, MyoD and SP1. Promoter mutants lacking different putative transcription factor binding sites are currently under investigation to further elucidate the regulation of PARG expression and the possibility of a coregulation of PARG and TIM23 expression.
Keywords: poly(ADP-ribose)glycohydrolase, PARG, inner mitochondrial membrane translocase 23, TIM23, bidirectional common promoter
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