MOLECULAR-CLONING, CHARACTERIZATION, AND OVEREXPRESSION OF ERG7 THE SACCHAROMYCES-CEREVISIAE GENE ENCODING LANOSTEROL SYNTHASE

被引:123
作者
COREY, EJ [1 ]
MATSUDA, SPT [1 ]
BARTEL, B [1 ]
机构
[1] WHITEHEAD INST BIOMED RES,CAMBRIDGE,MA 02142
关键词
STEROL BIOSYNTHESIS; 2,3-EPOXYSQUALENE; GENE CLONING;
D O I
10.1073/pnas.91.6.2211
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 corre spending 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.
引用
收藏
页码:2211 / 2215
页数:5
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