Longtine M S, McKenzie A, Demarini D J, Shah N G, Wach A, Brachat A, Philippsen P, Pringle J R
Department of Biology, University of North Carolina, Chapel Hill 27599-3280, USA.
Yeast. 1998 Jul;14(10):953-61. doi: 10.1002/(SICI)1097-0061(199807)14:10<953::AID-YEA293>3.0.CO;2-U.
An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5+ or Escherichia coli kan(r) gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae.
酿酒酵母基因功能分析中一项重要的近期进展是开发了用于染色体基因缺失和修饰的一步PCR介导技术。该方法无需目的基因的质粒克隆，就能非常快速地进行基因操作。我们在此描述了一组新的质粒，它们可作为PCR合成片段的模板，实现多种基因修饰。这些质粒使用酿酒酵母TRP1基因作为选择标记，或含有异源粟酒裂殖酵母his5+或大肠杆菌kan(r)基因的模块，可实现基因缺失、基因过表达（使用可调控的GAL1启动子）、C端或N端蛋白标签（用GFP(S65T)、GST或3HA或13Myc表位）以及部分N端或C端缺失（有或没有伴随的蛋白标签）。由于质粒的模块化性质，它们允许使用少量PCR引物高效、经济地进行多种基因操作。因此，这些质粒应进一步促进酿酒酵母基因功能的快速分析。
