Development of A New Cholesterol Lowering Drug to Prevent Atherosclerosis

角鲨烯环氧酶(SE)(EC 1.14.99.7)催化甾醇生物合成的第一步氧化反应，被认为是该途径中的限速酶之一。在真菌SE的特异性抑制剂特比奈芬存在下，通过筛选表达大鼠基因的酵母转化子来分离大鼠的SE基因。以大鼠的SE基因为探针，通过文库筛选获得鼠和人的SE基因。由核苷酸序列推导出的Se多肽含有573个氨基酸，其相对分子质量为63,950 Da。氨基酸序列显示一个潜在的跨膜区，在NH2-末端区域有一个疏水片段(Leu27到Tyr43)。该区域还包含一个β1-αA-BATA 2基序，这是FAD结合域的共同序列。该序列与酿酒酵母耐烯丙胺突变株中编码SE的ERG1基因有30.2%的同源性。该基因具有572个氨基酸的开放阅读框，其计算相对分子质量为63.8 kDa。预测的小鼠酶的氨基酸序列含有FAD结合基序，与大鼠和人的同源性分别为93%和83%。印迹分析表明，该基因的大小为2.8kb，并且在小鼠基因组中存在该基因的单一拷贝。以NIGMS人/啮齿动物体细胞杂交作图面板2为模板，将人SE基因定位于8号染色体。对人SE基因的定位表明，人SE基因与微卫星标记D8S508连锁，D8S508据报道定位于8q24.13-q端粒(Lod Score 7.87)。此外，荧光原位杂交还将人SE基因定位于8q24.13。

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.