Paluh J L, Clayton D A
Department of Cell and Molecular Biology, University of California, Berkeley 94720-3200, USA.
Yeast. 1996 Nov;12(14):1393-405. doi: 10.1002/(SICI)1097-0061(199611)12:14%3C1393::AID-YEA29%3E3.0.CO;2-8.
Reverse genetics in fission yeast is hindered by the lack of a versatile established plasmid shuffle system. In order to screen efficiently and accurately through plasmid-borne mutations in the essential gene for the RNA component of RNase MRP, mrp1, we have developed a system for plasmid shuffling in fission yeast using counterselection on canavanine. The system takes advantage of the ability of the Saccharomyces cerevisiae CAN1 gene to complement a Schizosaccharomyces pombe can1-1 mutation. Two general use plasmids were constructed that allow directional cloning and initial selection for histidine before counterselection by canavanine. The strain constructed for plasmid shuffling carries auxotrophic markers for ade6, leul, ura4 and his3 along with the can1-1 mutation. Using this system we examined several partial deletions and point mutations in conserved nucleotides of Schizosaccharomyces pombe RNase MRP RNA for their ability to complement a chromosomal deletion of the mrp1 gene. The degree of background canavanine resistance as well as plasmid-plasmid recombination encountered in these experiments was sufficiently low to suggest that the system we have set up for counterselection by canavanine in fission yeast using multicopy plasmids will be widely useful.
裂殖酵母中的反向遗传学因缺乏通用的成熟质粒洗牌系统而受到阻碍。为了通过核糖核酸酶MRP（RNase MRP）的RNA组分的必需基因mrp1中的质粒携带突变进行高效且准确的筛选，我们开发了一种利用刀豆氨酸反选择的裂殖酵母质粒洗牌系统。该系统利用了酿酒酵母CAN1基因互补粟酒裂殖酵母can1-1突变的能力。构建了两个通用质粒，它们允许进行定向克隆，并在通过刀豆氨酸进行反选择之前对组氨酸进行初步筛选。构建用于质粒洗牌的菌株携带ade6、leul、ura4和his3的营养缺陷型标记以及can1-1突变。利用该系统，我们检测了粟酒裂殖酵母RNase MRP RNA保守核苷酸中的几个部分缺失和点突变对mrp1基因染色体缺失的互补能力。这些实验中遇到的背景刀豆氨酸抗性程度以及质粒-质粒重组足够低，表明我们建立的利用多拷贝质粒在裂殖酵母中通过刀豆氨酸进行反选择的系统将具有广泛的用途。
