A METHOD FOR GENE DISRUPTION THAT ALLOWS REPEATED USE OF URA3 SELECTION IN THE CONSTRUCTION OF MULTIPLY DISRUPTED YEAST STRAINS
A METHOD FOR GENE DISRUPTION THAT ALLOWS REPEATED USE OF URA3 SELECTION IN THE CONSTRUCTION OF MULTIPLY DISRUPTED YEAST STRAINS
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DOI：
10.1534/genetics.112.541.test
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发表时间：
1987-08-01
期刊：
影响因子：
3.3
通讯作者：
KLECKNER, N
中科院分区：
文献类型：
--
作者：
ALANI, E;CAO, L;KLECKNER, N
在本文中，我们描述了一个3.8 kb的分子构建体，我们已经用来破坏酵母基因。该构建体由功能性酵母URA 3基因组成，其侧翼为细菌序列的1.1-kb同向重复序列。将3.8kb片段插入克隆的目标靶基因中，然后通过整合转化将所得破坏引入酵母基因组中是简单的。通过限制酶消化可以获得含有破坏加上侧翼同源性的适当DNA片段。在通过转化将这些片段引入酵母后，可以通过选择Ura+来分离稳定的整合体。使其特别有用的该构建体的重要特征是，在植物生长的培养物中，侧翼同向重复序列之间的重组以高频率（10-4）发生。切除后，仅保留一个重复序列拷贝。因此，在所得菌株中，可以再次使用Ura+选择，以类似方式破坏第二基因或用于另一目的。
In this paper, we describe a 3.8-kb molecular construct that we have used to disrupt yeast genes. The construct consists of a functional yeast URA3 gene flanked by 1.1-kb direct repeats of a bacterial sequence. It is straightforward to insert the 3.8-kb segment into a cloned target gene of interest and then introduce the resulting disruption into the yeast genome by integrative transformation. An appropriate DNA fragment containing the disruption plus flanking homology can be obtained by restriction enzyme digestion. After introducing such fragments into yeast by transformation, stable integrants can be isolated by selection for Ura+. The important feature of this construct that makes it especially useful is that recombination between the flanking direct repeats occurs at a high frequency (10-4) in vegetatively grown cultures. After excision, only one copy of the repeat sequence remains behind. Thus in the resulting strain, the Ura+ selection can be used again, either to disrupt a second gene in similar fashion or for another purpose.