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Development of A New Cholesterol Lowering Drug to Prevent Atherosclerosis

开发预防动脉粥样硬化的新型降胆固醇药物

基本信息

批准号：
07557342
负责人：
OKAMOTO Hiroshi
金额：
$ 2.62万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

Squalene epoxidase (SE) (EC 1.14.99.7) catalyzes the first oxygenation step in sterol biosynthesis and is suggested to be one of the rate-limiting enzymes in this pathway. Rat SE cDNA was isolated by selecting yeast transformants expressing rat cDNA in the presence of transformants expressing rat cDNA in the presence of terbinafine, an inhibitor specific for fungal SE.Mouse and human SE cDNA was isolated by library screening using rat SE cDNA as a probe. SE polypeptide deduced from the nucleotide sequence contains 573 amino acids, and its molecular weight is 63,950 Da. The amino acid sequence reveals one potential transmembrane domain, a hydrophobic segment (Leu27 to Tyr43) in the NH2-terminal region. This region also contains a beta 1-alpha A-bata 2 motif, which is the consensus sequence for an FAD binding domain. This deduced rat SE sequence is 30.2% identical to the ERG 1 gene, which encodes SE from an allylamine-resistant Saccharomyces cerevisiae mutant. The cDNA had an open reading frame for a 572 amino acid polypeptide with a calculated molecular mass of 63.8 kDa. The predicted amino acid sequence of the mouse enzyme contained an FAD-binding motif, and was 93%, 83% identical to those of the rat and human enzymes, respectively. Blotting analyzes showed that the mRNA is 2.8 kb in size and that a single copy of the gene is present in the mouse genome. PCR revealed that human SE gene to chromosomes 8 by using the NIGMS Human/Rodent Somatic Cell Hybrid Mapping Panel 2 as templates. To refine the localization of the human SE gene showed that the human SE gene linked with the micro-satellite marker D8S508 which was reported to be localized in 8q 24.13-q telomere (Lod score 7.87). Moreover, fluorescence in situ hybridization also map the human SE gene to 8q 24.13.

角鲨烯环氧酶（SE）（EC 1.14.99.7）催化甾醇生物合成中的第一个氧化步骤，并且被认为是该途径中的限速酶之一。大鼠SE cDNA的分离是通过选择酵母转化体表达大鼠cDNA的存在下，表达大鼠cDNA的转化体的存在下，特比萘芬，真菌SE的特异性抑制剂。小鼠和人类SE cDNA的分离通过文库筛选使用大鼠SE cDNA作为探针。序列分析表明，该蛋白含有573个氨基酸，分子量为63，950 Da，氨基酸序列分析表明，该蛋白具有一个潜在的跨膜结构域，在其氨基端有一个疏水片段（Leu 27 ~ Tyr 43）。该区域还含有β 1-α A-bata 2基序，其是FAD结合结构域的共有序列。这推导出的大鼠SE序列是30.2%相同的ERG 1基因，其编码SE从一个烯丙胺抗性酿酒酵母突变体。该cDNA具有572个氨基酸的多肽的开放阅读框，计算分子量为63.8 kDa。预测的小鼠酶的氨基酸序列含有FAD结合基序，与大鼠和人的酶的氨基酸序列分别为93%，83%。印迹分析表明，mRNA的大小为2.8 kb，并且该基因的单拷贝存在于小鼠基因组中。以NIGMS Human/Rodent Somatic Cell Hybrid Mapping Panel 2为模板，PCR扩增显示人SE基因位于8号染色体上。对SE基因的定位结果表明，SE基因与微卫星标记D8 S508连锁，D8 S508定位于8q24.13-q端粒（Lod评分7.87）。此外，荧光原位杂交还将人SE基因定位到8q24.13。