Miyazaki Y, Geber A, Miyazaki H, Falconer D, Parkinson T, Hitchcock C, Grimberg B, Nyswaner K, Bennett J E
Clinical Mycology Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, NIH. 10 Center Drive, Bethesda, MD 20892, USA.
Gene. 1999 Aug 5;236(1):43-51. doi: 10.1016/s0378-1119(99)00263-2.
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 Delta(5,6) sterols, 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 Delta(5,6) sterol.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.
通过与酿酒酵母ERG3的同源性，从白色念珠菌中克隆并测序了酵母麦角甾醇生物合成所必需的C-5甾醇去饱和酶基因（ERG3）。ERG3开放阅读框包含1158bp，编码386个推导氨基酸。利用半乳糖选择，该克隆用于转化源自白色念珠菌达林顿菌株的gal1突变体。已知达林顿菌株缺乏Δ(5,6)甾醇，即具有erg3表型（豪厄尔，S.A.等人，1990年。《应用细菌学杂志》69，692 - 696）。转化体（CDTR1）包含六个串联整合的ERG3GAL1重复序列，其ERG3转录本丰度是宿主菌株中的两倍，并合成了麦角甾醇，一种Δ(5,6)甾醇。注意到达林顿菌株具有丰富的ERG3转录本。对达林顿菌株中的两个ERG3等位基因进行了克隆和测序，以寻找可能解释erg3表型的变化。一个等位基因，称为Dar - 2，在色氨酸 - 292的位置含有一个终止密码子。另一个ERG3等位基因，称为Dar - 1，有三个氨基酸发生了变化，其中两个在三种真菌和一种植物物种中是保守的。将含有来自Dar - 1等位基因和野生型菌株B311的ERG3的EcoRI基因组片段插入质粒pRS({}_{316})，并利用尿嘧啶选择来转化酿酒酵母erg3、ura3突变体。来自B311和Dar - 1的4.1kb ERG3片段在编码区周围均包含1.4kb的5'侧翼序列和1.5kb的3'侧翼序列。含有来自B311而非Dar - 1的ERG3的转化体显示出麦角甾醇合成的恢复。ERG3的Dar - 1等位基因中的这三个推导氨基酸中的一个或多个似乎对功能至关重要。
