| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2003: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
In this research, I aimed to clarify tissue distributions and transcriptional regulation of newly identified mouse steroid/sterol sulfotransferase (SULT2B1). As the first step, in situ hybridization analysis was performed. Message of SULT2B1a, an isozyme encoded by SULT2B1 gene, is expressed mainly in central nervous system, while SULT2B1b mRNA is detected in skin. This was confirmed by immmunohistochemical analysis using specific polyclonal antibodies. To analyze transcriptional regulation of SULT2B1 gene, I isolated approximately 2-kb genomic DNA fragments upstream of transcription start sites of SULT2B1a and 2B1b and ligated the fragments to luciferase expression vector. Because SULT2B1b mRNA is detected in Pam212 cell line derived from mouse keratinizing squamous cell carcinoma, I transfected 2-kb promoter/enhancer luciferase construct into the cells and analyzed the luciferase activity. The luciferase activity of reporter construct containing SULT2B1b 5' flanking region showed 10-fold higher than those of empty vector and reversely-oriented DNA-ligated vector, revealing that this 2-kb 5'-flanking region contains promoter and/or enhancer binding sequence. Furthermore, I have cloned rabbit SULT2B1b full-length cDNA using PCR and degenerated primers. RT-PCR experiment revealed that rabbit SULT2B1b mRNA is expressed in artery. Generating polyclonal antibody against rabbit SULT2B1b protein, I will confirm the expression at protein level. The association between SULT2B1b and atherosclerogenesis is my next focus.
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