Transformation system for an asporogenous methylotrophic yeast, Candida boidinii: cloning of the orotidine-5'-phosphate decarboxylase gene (URA3), isolation of uracil auxotrophic mutants, and use of the mutants for integrative transformation
Transformation system for an asporogenous methylotrophic yeast, Candida boidinii: cloning of the orotidine-5'-phosphate decarboxylase gene (URA3), isolation of uracil auxotrophic mutants, and use of the mutants for integrative transformation
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无孢子甲基营养酵母博伊丁假丝酵母的转化系统：克隆乳清苷-5-磷酸脱羧酶基因（URA3），分离尿嘧啶营养缺陷型突变体，以及使用突变体进行整合转化
DOI：
10.1128/jb.173.23.7458-7463.1991
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发表时间：
1991
影响因子：
3.2
通讯作者：
Yoshiki Tani
中科院分区：
文献类型：
--
作者：
Yasuyoshi Sakai;Takumi Kazarimoto;Yoshiki Tani
为无孢子甲基营养酵母博伊丁假丝酵母建立了整合转化系统。该系统使用 C. boidinii 的尿嘧啶营养缺陷型突变体作为宿主菌株，并结合其 URA3 基因作为选择标记。首先，通过利用大肠杆菌的pyrF突变的互补来克隆编码乳清苷-5'-磷酸脱羧酶(ODCase)的C. boidinii URA3基因。接下来，通过诱变和选择 5-氟乳清酸 (5-FOA) 抗性来分离宿主 ODCase 阴性突变株（ura3 株）。获得了五株表现出低回复率和良好甲基营养生长的ura3宿主菌株。所有这些菌株都可以用在念珠菌 DNA 内线性化的博伊丁念珠菌 URA3 携带质粒转化为 Ura+ 表型。非选择性生长10代后，转化体具有稳定的Ura+表型。这些结果和广泛的 Southern 分析表明，线性化质粒通过在 C. boidinii 的 URA3 基因座处同源重组整合到宿主染色体 DNA 中。
An integrative transformation system was established for an asporogenous methylotrophic yeast, Candida boidinii. This system uses a uracil auxotrophic mutant of C. boidinii as the host strain in combination with its URA3 gene as the selectable marker. First, the C. boidinii URA3 gene coding for orotidine-5'-phosphate decarboxylase (ODCase) was cloned by using complementation of the pyrF mutation of Escherichia coli. Next, the host ODCase-negative mutant strains (ura3 strains) were isolated by mutagenesis and selection for 5-fluro-orotic acid (5-FOA) resistance. Five ura3 host strains that exhibited both a low reversion rate and good methylotrophic growth were obtained. All of these strains could be transformed to Ura+ phenotype with a C. boidinii URA3-harboring plasmid linearized within the Candida DNA. The transformants had a stable Ura+ phenotype after nonselective growth for 10 generations. These results and extensive Southern analysis indicated that the linearized plasmid was integrated into the host chromosomal DNA by homologous recombination at the URA3 locus in C. boidinii.
DOI：
10.1016/s0021-9258(17)39756-9
发表时间：
1984-07
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
J M Goodman;C W Scott;P N Donahue;J P Atherton
通讯作者：
J M Goodman;C W Scott;P N Donahue;J P Atherton
酵母 URA3 基因的结构和功能：在大肠杆菌中的表达。
影响因子：
3.5
作者：
Rose,M;Grisafi,P;Botstein,D
通讯作者：
Botstein,D
两个基因编码来自博伊丁假丝酵母的甲醇诱导的过氧化物酶体的主要膜相关蛋白。
DOI：
--
发表时间：
1989
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Garrard,LJ;Goodman,JM
通讯作者：
Goodman,JM