Cloning, sequencing, expression and allelic sequence diversity of ERG3 (C-5 sterol desaturase gene) in Candida albicans.
Cloning, sequencing, expression and allelic sequence diversity of ERG3 (C-5 sterol desaturase gene) in Candida albicans.
复制标题
白色念珠菌 ERG3（C-5 甾醇去饱和酶基因）的克隆、测序、表达和等位基因序列多样性。
DOI：
10.1016/s0378-1119(99)00263-2
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发表时间：
1999
期刊：
影响因子：
3.5
通讯作者：
Bennett,JE
中科院分区：
文献类型：
--
作者：
Miyazaki,Y;Geber,A;Miyazaki,H;Falconer,D;Parkinson,T;Hitchcock,C;Grimberg,B;Nyswaner,K;Bennett,JE
从白色念珠菌中克隆并测序了酵母麦角固醇生物合成所必需的C-5固醇去饱和酶基因（ERG 3），该基因与酿酒酵母ERG 3具有同源性。ERG 3基因的ORF全长1158 bp，编码386个氨基酸。该克隆用于转化来自C.达林顿菌株的gal 1突变体。白色念珠菌，使用半乳糖选择。已知达林顿菌株缺乏Δ5，6甾醇，即具有erg 3表型（豪厄尔，S.A.，例如，1990.应用细菌学杂志69，692-696）。CDTR 1含有6个串联整合的ERG 3 GAL 1重复序列，其ERG 3转录本丰度是宿主菌株中的两倍，并合成麦角甾醇（Δ 5，6甾醇）。注意到达林顿菌株具有丰富的ERG 3转录物。克隆和测序了达林顿的两个ERG 3等位基因，以寻找可能解释erg 3表型的变化。其中一个等位基因，称为Dar-2，含有一个终止密码子代替色氨酸-292。另一个ERG 3等位基因Dar-1在三个氨基酸上发生了变化，其中两个在三种真菌和一种植物中是保守的。将含有来自Dar-1等位基因和来自野生型菌株B311的ERG 3的EcoRI基因组片段插入质粒pRS 316中，并使用尿嘧啶选择转化酿酒酵母erg 3，ura 3突变体。来自B311和Dar-1的4.1kb ERG 3片段均含有1.4kb的5′和1.5kb的3′侧翼序列。具有来自B311而不是来自Dar-1的ERG 3的转化体显示恢复的麦角固醇合成。在ERG 3的Dar-1等位基因中，这三个推导的氨基酸中的一个或多个对于功能似乎是关键的。
The C-5 sterol desaturase gene (ERG3), essential for yeast ergosterol biosynthesis, was cloned and sequenced from Candida albicans by homology with the Saccharomyces cerevisiae ERG3. The ERG3 ORF contained 1158bp and encoded 386 deduced amino acids. The clone was used to transform a gal1 mutant derived from the Darlington strain of C. albicans, using galactose selection. The Darlington strain is known to lack Δ5,6sterols, i.e. to have an erg3 phenotype (Howell, S.A., et al., 1990. J. Appl. Bacteriol. 69, 692–696). The transformant (CDTR1) contained six tandem integrated ERG3GAL1 repeats, had double the abundance of ERG3 transcript found in the host strain, and synthesized ergosterol, a Δ5,6sterol. The Darlington strain was noted to have an abundance of ERG3 transcript. Both ERG3 alleles in Darlington were cloned and sequenced in order to look for changes that might explain the erg3 phenotype. One allele, called Dar-2, contained a stop codon in place of tryptophan-292. The other ERG3 allele, called Dar-1, had changes in three amino acids, two of which were conserved in three fungal and one plant species. EcoRI genomic fragments containing ERG3 from the Dar-1 allele and from B311, the wild-type strain, were inserted into the plasmid pRS316 and used to transform a Saccharomyces cerevisiae erg3,ura3 mutant using uracil selection. The 4.1kb ERG3 fragments from the B311 and Dar-1 both contained 1.4kb 5′ and 1.5kb 3′ flanking sequences around the coding region. Transformants with ERG3 from B311 but not from Dar-1 showed restored ergosterol synthesis. One or more of these three deduced amino acids in the Dar-1 allele of ERG3 appeared critical for function.