Gietz R D, Sugino A
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
Gene. 1988 Dec 30;74(2):527-34. doi: 10.1016/0378-1119(88)90185-0.
We describe the production of new alleles of the LEU2, URA3 and TRP1 genes of Saccharomyces cerevisiae by in vitro mutagenesis. Each new allele, which lacks restriction enzyme recognition sequences found in the pUC19 multicloning site, was used to construct a unique series of yeast-Escherichia coli shuttle vectors derived from the plasmid pUC19. For each gene a 2 mu vector (YEplac), an ARS1 CEN4 vector (YCplac) and an integrative vector (YIplac) was constructed. The features of these vectors include (i) small size; (ii) unique recognition site for each restriction enzyme found in the pUC19 multicloning site; (iii) screening for plasmids containing inserts by color assay; (iv) high plasmid yield; (v) efficient transformation of S. cerevisiae. These vectors should allow greater flexibility with regard to DNA restriction fragment manipulation and subcloning.
我们描述了通过体外诱变产生酿酒酵母LEU2、URA3和TRP1基因新等位基因的过程。每个新等位基因都缺乏在pUC19多克隆位点中发现的限制酶识别序列，用于构建一系列源自质粒pUC19的独特酵母-大肠杆菌穿梭载体。对于每个基因，构建了一个2μm载体（YEplac）、一个ARS1 CEN4载体（YCplac）和一个整合载体（YIplac）。这些载体的特点包括：（i）尺寸小；（ii）在pUC19多克隆位点中发现的每种限制酶都有独特的识别位点；（iii）通过颜色测定筛选含有插入片段的质粒；（iv）质粒产量高；（v）酿酒酵母的高效转化。这些载体在DNA限制片段操作和亚克隆方面应具有更大的灵活性。
