编码人羊毛甾醇合酶的cDNA的分子克隆

Molecular cloning of cDNA encoding human lanosterol synthase.

作者信息

Sung C K, Shibuya M, Sankawa U, Ebizuka Y

机构信息

Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.

出版信息

Biol Pharm Bull. 1995 Oct;18(10):1459-61. doi: 10.1248/bpb.18.1459.

DOI:10.1248/bpb.18.1459

PMID:8593458

Abstract

A cDNA encoding human lanosterol synthase, the enzyme responsible for the backbone formation step in sterol biosynthesis, was cloned by extensive application of PCRs. Five degenerate oligonucleotide primers (139S, 440S, 528A, 575A and 712A) corresponding to the homologous amino acid sequences among the known 2,3-oxidosqualene cyclase(OSC) were designed. PCR with one pair(440S and 528A) of five primers yielded a 285-bp fragment. PCRs with the primers based on the obtained fragment and the degenerate primers (139S and 712A) gave longer fragments. Finally, full nucleotide sequence of cDNA was obtained by a "rapid amplification of cDNA ends" (RACE) method.

摘要

通过广泛应用聚合酶链反应（PCR）克隆了一个编码人羊毛甾醇合酶的互补DNA（cDNA），该酶负责甾醇生物合成中的骨架形成步骤。设计了五条与已知的2,3-氧化角鲨烯环化酶（OSC）中的同源氨基酸序列相对应的简并寡核苷酸引物（139S、440S、528A、575A和712A）。用五条引物中的一对（440S和528A）进行PCR，得到了一个285碱基对的片段。基于获得的片段和简并引物（139S和712A）进行PCR，得到了更长的片段。最后，通过“cDNA末端快速扩增”（RACE）方法获得了cDNA的完整核苷酸序列。