| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1996: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1995: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
Dopamine beta-hydroxylase (DBH) converts dopamine to norepinephrine and is specifically expressed in noradrenergic and adrenergic neurons. Cyclic AMP response element (CRE,TGACGTCC) of human DBH gene is an essential transcriptional element in the promoter region. Downstream of this CRD,there are three important DNA elements such as YY-1 binding sequence (CCAT), E-box (GATGTG), and AP-1 like sequence (TGTGTCA) that may regulate the transcription of many genes. To identify the DNA binding proteins, 26 bp oligonucleotide (actga TGACGTCCATGTGTCAttagt) was used as a probe for the electrophoretic mobility shift assay (EMSA). According to the site-directed mutagenesis work, we found that both CRE and YY-1 binding sequence formed complexes with proteins, but E-box and AP-1 like sequences did not. The addition of CRE oligonucleotides for tyrosine hydroxylase (TH) or somatostatin genes as competitors eliminated upper-shifted bands that were formed with CRE and CRE binding protein (possibly CREB
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or ATFs). Furthermore, an excess amount of oligonucleotide which was the YY-1 binding sequence of mouse c-fos gene as a competitor, completely eliminated the intermediate-shifted band. YY-1, a ubiquitously expressed zinc finger type DNA binding protein, regulates the transcriptional level of many genes, such as several viral genes, immunoglobuline gene (kappa and mu), oncogene (c-fos and c-myc), globin gene (gamma and epsilon), and so on. Astriking characteristic of this protein is a dual function which either positive or negative effects, depending on the promoter context and the intracellular circumstance. In order to assess the function of YY-1 that bound to the CCAT motif of human DBH,transient transfection experiments were performed in the DBH-expressing neuroblastoma cells (SK-N-SH). Site-directed mutagenesis experiments showed that YY-1 positively regulated the transcriptional level of human DBH gene by adding phorbol ester (TPA). In addtion, CRE binding proteins and YY-1 needed specific direction to interact with each other by the results of luciferase assay. These data suggest that YY-1 is important in the transcriptional regulation of DBH gene by the TPA induction. To identify the TPA response DNA region (TRR) in human DBH gene promoter, deletion mutant constructs from -604bp to -171bp were prepared and these luciferase reporter genes (Luc) were transfected to SK-N-SH cells. TRR resided in the upstream of CRE between -223bp and -187bp of human DBH gene, and there were no similar sequences in other known TPA response element (TRE) sequences. In addition, we tried to identifed unknown TRE,TRR disected to three DNA regions that were -223bp to -199bp (OLI-A), -213bp to -183 (OLI-D) and -206bp to -181bp (OLI-B). First, OLI-D eliminates the OLI-63 (region from -224 to -162) and nucler protein complex. Second, OLI-D responses the TPA induction by the use of tk promoter. Furthermore OLI-D and CRE of human DBH combination enhanced the luciferase activity by TPA.Int Less
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