Takagi M, Kawai S, Chang M C, Shibuya I, Yano K
J Bacteriol. 1986 Aug;167(2):551-5. doi: 10.1128/jb.167.2.551-555.1986.
To construct a host-vector system in an n-alkane-assimilating yeast, Candida maltosa, the isolation of an ARS site from its genome which replicates autonomously in C. maltosa was attempted. Leu- mutants of C. maltosa were transformed with a gene library prepared by using YEp13 (LEU2+) as a vector, and Leu+ transformants were obtained at a high frequency. A plasmid named pCS1 was isolated from the recipient cells. pCS1 contained a 6.3-kilobase (kb) fragment of the C. maltosa genome, and a 3.8-kb fragment with ARS activity was subcloned and designated the TRA (transformation ability) region. Vectors (pTRA1 and pTRA11) for C. maltosa J288 were constructed that contained this 3.8-kb fragment, pBR322, and the LEU2 gene of Saccharomyces cerevisiae. Transformation of C. maltosa J288 with these plasmids was successful by both spheroplast and lithium acetate methods. Southern blot analysis suggested that the copy number of pTRA1 in C. maltosa was between 10 and 20, and it was stably maintained during growth without selective pressure in the medium. It was also found that these vectors could transform S. cerevisiae leu2- to LEU2+, suggesting that the TRA region contained an ARS site(s) that was specific not only for C. maltosa but also for S. cerevisiae.
为了构建一个用于同化正构烷烃的酵母——麦芽糖假丝酵母的宿主-载体系统，尝试从其基因组中分离出一个能在麦芽糖假丝酵母中自主复制的自主复制序列（ARS）位点。用以YEp13（LEU2+）为载体构建的基因文库转化麦芽糖假丝酵母的亮氨酸缺陷型突变体，以高频率获得了亮氨酸原养型转化子。从受体细胞中分离出一个名为pCS1的质粒。pCS1含有麦芽糖假丝酵母基因组的一个6.3千碱基（kb）片段，一个具有ARS活性的3.8 kb片段被亚克隆并命名为TRA（转化能力）区域。构建了用于麦芽糖假丝酵母J288的载体（pTRA1和pTRA11），其包含这个3.8 kb片段、pBR322和酿酒酵母的LEU2基因。通过原生质球法和醋酸锂法用这些质粒成功转化了麦芽糖假丝酵母J288。Southern印迹分析表明，pTRA1在麦芽糖假丝酵母中的拷贝数在10到20之间，并且在培养基中无选择压力的生长过程中能稳定维持。还发现这些载体能将酿酒酵母亮氨酸缺陷型转化为亮氨酸原养型，这表明TRA区域包含一个不仅对麦芽糖假丝酵母而且对酿酒酵母都具有特异性的ARS位点。
