Corey E J, Matsuda S P, Bartel B
Department of Chemistry, Harvard University, Cambridge, MA 02138.
Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2211-5. doi: 10.1073/pnas.91.6.2211.
We report the cloning, characterization, and overexpression of Saccharomyces cerevisiae ERG7, which encodes lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7], the enzyme responsible for the complex cyclization/rearrangement step in sterol biosynthesis. Oligonucleotide primers were designed corresponding to protein sequences conserved between Candida albicans ERG7 and the related Arabidopsis thaliana cycloartenol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A PCR product was amplified from yeast genomic DNA using these primers and was used to probe yeast libraries by hybridization. Partial-length clones homologous to the two known epoxysqualene mutases were isolated, but a full-length sequence was found neither in cDNA nor genomic libraries, whether in phage or plasmids. Two overlapping clones were assembled to make a functional reconstruction of the gene, which contains a 2196-bp open reading frame capable of encoding an 83-kDa protein. The reconstruction complemented the erg7 mutation when driven from either its native promoter or the strong ADH1 promoter.
我们报道了酿酒酵母ERG7的克隆、特性分析及过表达，ERG7编码羊毛甾醇合酶[（S）-2,3-环氧角鲨烯变位酶（环化，形成羊毛甾醇），EC 5.4.99.7]，该酶负责甾醇生物合成中复杂的环化/重排步骤。根据白色念珠菌ERG7与相关拟南芥环阿屯醇合酶[（S）-2,3-环氧角鲨烯变位酶（环化，形成环阿屯醇），EC 5.4.99.8]之间保守的蛋白质序列设计寡核苷酸引物。使用这些引物从酵母基因组DNA中扩增出一个PCR产物，并用于通过杂交探测酵母文库。分离到了与两种已知环氧角鲨烯变位酶同源的部分长度克隆，但无论是在噬菌体还是质粒中的cDNA文库或基因组文库中均未发现全长序列。将两个重叠克隆组装起来以对该基因进行功能重建，该基因包含一个能够编码83 kDa蛋白质的2196 bp开放阅读框。当由其天然启动子或强ADH1启动子驱动时，该重建基因弥补了erg7突变。
