Grant-in-Aid for international Scientific Research
|Allocation Type||Single-year Grants |
|Research Institution||Niigata University |
ONO Teruo School of Medicine Niigata University Professor, 医学部, 教授 (00000927)
KUMAGAI Hidetoshi Niigata Univ.Sch.of Med.・Research Associate, 医学部, 助手 (20281008)
SAKAKIBARA Jun Niigata Univ.Sch.of Med.・Research Associate, 医学部, 助手 (90242403)
STEIN J.P. ニューヨーク州立大学, 薬理学, 助教授
SCHROEPFER George Rice Univ.・Professor, 化学部, 教授
SHECHTER Ishaiahu Uniformed Services Univ.of the Health Sci.・Professor, 生化学部, 教授
PRESTWICH Glenn The State Univ.of New York・Professor, 化学部, 教授
STEIN Joseph The State Univ.of New York・Associate Professor
藤井 博 新潟大学, 医学部, 助教授 (90165340)
LAW J.A アリゾナ大学, 生化学部, 教授
SCHROPFER G. ライス大学, 化学部, 教授
|Project Period (FY)
1995 – 1996
Completed (Fiscal Year 1996)
|Budget Amount *help
¥7,600,000 (Direct Cost: ¥7,600,000)
Fiscal Year 1996: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 1995: ¥3,800,000 (Direct Cost: ¥3,800,000)
|Keywords||squalene epoxidase(SE) / benzophenone derivatives / HMG-CoA reductase / LDL-receptor / gene expression / chromosome 8 q24.13 / D8S508 / SRE / NF-Y / コレステロール / スクアレン / スクアレン・エポキシダーゼ / 阻害剤 / 動脈硬化 / 高脂血症 / スクリーニング / バイオセンサー|
We have studied on structure and regulation aspects of mammalian squalene epoxidase (SE), the late stage of cholesterol biosynthetic enzyme.
1. Determination of Functional Domains of SE : Using benzophenone derivatives of FAD,trinorsqualene alcohol and NB-598, we have determined putative binding domains for substrate and NB-598. We also found that terbinafine resistant gene in yeast replaced the only one amino acid compared to wild type yeast (L*F).
2. Regulation of SE : Regulation of SE gene expression was studied in comparison with those of HMG-CoA reductase and LDL-receptor using HeLa cells. The results suggest that sterol produced endogenously can regulate SE expression at the level of transcription.
3. Structure of human SE genomic DNA : Total length of human SE gene is about 17 kilobase including 11 exons. The introns are ranging from 87 to 3.2 kb. Exon I and II contained putative memmbrane binding domain and FAD-binding followed substrate binding domains, respectively. The identica
l undecapeptide in common with yeast and mammlaina SE is localized in the exon VIII.
4. Chromosomal localization of human SE gene : We have dteremined the regional localization of human SE by using Stanford G3 radiation hybrid panel. Computer analysis of the PCR products for each panel suggests that human SE sequence tagged site is linked with micro-satellite marker D8S508. The marker is localiz-ed q24.13-q telomere of chromosome 8. It is possble that Langer-Giedion syndromeis one of the candidate for SE defect.
5. Analyzes of promoter regions of human and rat SE genes : We have determined 5'-sequenec of rat and human SE genes from each genomic clones and aligned 5'-flanking sequences of initiation codons. In both human and rat, the well-conserved region is moticed at the location of 700 or 800 bp upstream from initiation codon. Rat transcriptional initiation site that determined by primer extension method was in 3'-region of the conserved region. To determine the regulatory element of human SE promoter, we preparaed several chimeric lucife-rase plasmids and fusion constructs with deleted human SE promoter and transfected in HeLa cells. The two limited conserved region, a classical SRE-like region and NY-F regions of rat and human may be cruscial for regulation of SE gene by sterols. Less