Tuller G, Prein B, Jandrositz A, Daum G, Kohlwein S D
SFB Biomembrane Research Center and Institut für Biochemie und Lebensmittelchemie, Technische Universität Graz, Austria.
Yeast. 1999 Sep 15;15(12):1275-85. doi: 10.1002/(SICI)1097-0061(19990915)15:12<1275::AID-YEA456>3.0.CO;2-4.
The construction of six deletion mutants of Saccharomyces cerevisiae and their basic phenotypic characterization are described. Open reading frames YDL148c, YDL109c, YDL021w, YDL019c, YDL018c and YDL015c from the left arm of chromosome IV were deleted using a polymerase chain reaction (PCR)-based disruption technique, introducing the kanMX4 resistance marker into the respective genes. Gene replacement cassettes (pYORCs) for use in other strain backgrounds were cloned by PCR using DNA templates from haploid or diploid deletion mutants, and inserted into episomal plasmids. Cognate clones of all six ORFs were obtained by gap repair. Deletions were carried out in diploid cells and, after sporulation, yielded four viable spores for clones disrupted in YDL109c, YDL021w, YDL019c and YDL018c. Spores harbouring disruptions in ORFs YDL148c and YDL015c germinated but underwent only a few divisions before ceasing growth, suggesting that the respective genes are essential for vegetative growth on YPD complete media. The other deletion mutants grew like wild-type at different temperatures and on different carbon sources. A brief computational analysis of the six ORFs studied in this work is presented.
本文描述了酿酒酵母六个缺失突变体的构建及其基本表型特征。利用基于聚合酶链反应（PCR）的破坏技术，删除了第四条染色体左臂上的开放阅读框YDL148c、YDL109c、YDL021w、YDL019c、YDL018c和YDL015c，并将kanMX4抗性标记引入相应基因。使用来自单倍体或二倍体缺失突变体的DNA模板，通过PCR克隆用于其他菌株背景的基因替换盒（pYORCs），并将其插入附加体质粒中。通过缺口修复获得了所有六个开放阅读框的同源克隆。缺失操作在二倍体细胞中进行，孢子形成后，对于在YDL109c、YDL021w、YDL019c和YDL018c中被破坏的克隆产生了四个活孢子。在开放阅读框YDL148c和YDL015c中含有破坏的孢子发芽，但仅进行了几次分裂就停止生长，这表明相应的基因对于YPD完全培养基上的营养生长至关重要。其他缺失突变体在不同温度和不同碳源上的生长情况与野生型相似。本文还对本研究中所研究的六个开放阅读框进行了简要的计算分析。
