Duina Andrea A, Turkal Claire E
Biology Department, Hendrix College;
Biology Department, Hendrix College.
J Vis Exp. 2017 Jan 26(119):55263. doi: 10.3791/55263.
We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.
我们描述了一种基于PCR和同源重组的系统，用于在芽殖酵母细胞的组蛋白基因中产生靶向突变。产生的突变等位基因位于其内源基因组位点，并且在该过程之后基因组中不会留下任何外源DNA序列。由于在单倍体酵母细胞中，四种核心组蛋白中的每一种都由两个具有高度同源开放阅读框（ORF）的非等位基因编码，因此将诱变特异性靶向编码特定组蛋白的两个基因之一可能会有问题。我们在此描述的策略通过在同源重组步骤中利用靶基因ORF之外而非之内的序列来绕过这个问题。该系统的另一个特点是驱动同源重组步骤的DNA区域可以做得非常广泛，从而增加成功整合事件的可能性。这些特点使得该策略特别适合组蛋白基因诱变，但也可适用于酵母基因组中其他基因的诱变。
