Cloning and functional characterization of a Trypanosoma brucei lanosterol 14alpha-demethylase gene.
Cloning and functional characterization of a Trypanosoma brucei lanosterol 14alpha-demethylase gene.
复制标题
布氏锥虫羊毛甾醇 14α-脱甲基酶基因的克隆和功能表征。
DOI：
10.1016/s0166-6851(01)00337-1
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发表时间：
2001
影响因子：
1.5
通讯作者：
Buckner,FS
中科院分区：
文献类型：
--
作者：
Joubert,BM;Nguyen,LN;Matsuda,SP;Buckner,FS
A study was conducted to describe theT. bruceilanosterol 14α-demethylase gene and its functional characterization in yeast. The putative lanosterol demethylase was assayed by genetically complementing yeast. 40 spores were plated onto YP medium (1% yeast extract, 2% peptone, 1.5% agar) with 2% galactose (YPG), and grown at 30°C for 6 days. 13 of the 40 spores germinated, and their phenotypes were analysed in parallel on YPG, YP with 2% dextrose (YPD), SC-Ura with 2% galactose (SCG-Ura), and SC-Ura with 2% dextrose (SCD-Ura). 12 haploids required uracil to grow. They could grow without exogenous sterol, thus establishing that these clones obtained the wild-typeERG11gene from the heterozygous diploid parent. The sole uracil-independent, plasmid containing haploid could grow without exogenous sterol on the inducing carbon source galactose but required sterol on the non-inducing glucose. PCR experiments demonstrated that the KanMX4 marker disrupted the yeastERG11locus in this haploid. The galactose-mediated complementation of theerg11deletion provides genetic evidence that the plasmid-encoded galactose-inducibleT. brucei ERG11gene encodes a lanosterol 14α-demethylase. It is suggested that the described heterologous expression strain can facilitate assaying drug candidatesin vitroby providing activeT. bruceidemethylase coexpressed with a suitable P45 reductase. Nucleotide sequence data reported in this paper are accessible in GenBank under accession number AF363026.
