Delneri D, Tomlin G C, Wixon J L, Hutter A, Sefton M, Louis E J, Oliver S G
School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, M13 9PT, Manchester, UK.
Gene. 2000 Jul 11;252(1-2):127-35. doi: 10.1016/s0378-1119(00)00217-1.
Gene families having more than three members are a common phenomenon in the Saccharomyces cerevisiae genome. As yeast research enters the post-genome era, the development of existing deletion strategies is crucial for tackling this apparent redundancy, hence a method for performing rapid multiple gene disruptions in this organism has been developed. We constructed three replacement cassettes in which different selectable markers were placed between two loxP loci. Multiple deletions (of members of a gene family) were generated, in one strain, using sequential integration of different replacement markers (kanMX, LYS2, KlURA3 and SpHIS5). Their excision from the genome was performed simultaneously, as the final step, using a new cre recombinase vector, which carries the cycloheximide-resistance gene from Candida maltosa as a selectable marker. Our multiple gene deletion system significantly accelerates and facilitates the functional analysis process and is particularly useful for studying gene families in either laboratory or industrial yeast strains.
拥有三个以上成员的基因家族在酿酒酵母基因组中是一种常见现象。随着酵母研究进入后基因组时代，现有缺失策略的发展对于解决这种明显的冗余至关重要，因此已开发出一种在该生物体中进行快速多基因破坏的方法。我们构建了三个替换盒，其中不同的选择标记位于两个loxP位点之间。使用不同替换标记（kanMX、LYS2、KlURA3和SpHIS5）的顺序整合，在一个菌株中产生多个（基因家族成员的）缺失。作为最后一步，使用一种新的携带来自麦芽糖假丝酵母的环己酰亚胺抗性基因作为选择标记的cre重组酶载体，同时将它们从基因组中切除。我们的多基因缺失系统显著加速并促进了功能分析过程，对于研究实验室或工业酵母菌株中的基因家族特别有用。
