Niedenthal R K, Riles L, Johnston M, Hegemann J H
Institut für Mikrobiologie und Molekularbiologie, Justus-Liebig-Universität Giessen, Germany.
Yeast. 1996 Jun 30;12(8):773-86. doi: 10.1002/(SICI)1097-0061(19960630)12:8%3C773::AID-YEA972%3E3.0.CO;2-L.
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has attracted much attention as a tool to study a number of biological processes. This study describes the use of GFP as a vital reporter molecule for localization and expression studies in Saccharomyces cerevisiae. Construction of GFP expression vectors which allow N- or C-terminal fusion of the gfp gene to a gene of interest allowed the generation of fusion proteins whose subcellular localization was followed by fluorescence microscopy in living yeast cells. Analysis of three unknown open reading frames obtained from the budding yeast chromosome XIV resulted in distinct staining patterns, allowing prediction of the cellular localization of these unknown proteins. Furthermore, GFP was used to construct a gene replacement cassette which, after homologous integration into the genomic locus, placed the gfp gene behind a promoter of interest. The amount of GFP produced from this promoter was then quantified in living yeast cells by flow cytometry. With this novel replacement cassette a gene of interest can be deleted and at the same time its expression level studied under various growth conditions. The experiments presented here suggest that GFP represents a convenient fluorescent marker for localization studies as well as gene expression studies in budding yeast. Systematic studies of a large number of genes should benefit from such assays.
来自维多利亚多管水母的绿色荧光蛋白（GFP）作为研究多种生物学过程的工具备受关注。本研究描述了GFP作为酿酒酵母中用于定位和表达研究的重要报告分子的应用。构建允许gfp基因与感兴趣基因进行N端或C端融合的GFP表达载体，使得能够产生融合蛋白，其亚细胞定位可通过活酵母细胞中的荧光显微镜观察。对从芽殖酵母第十四号染色体获得的三个未知开放阅读框的分析产生了不同的染色模式，从而可以预测这些未知蛋白质的细胞定位。此外，GFP被用于构建一个基因置换盒，该置换盒在同源整合到基因组位点后，将gfp基因置于感兴趣的启动子之后。然后通过流式细胞术在活酵母细胞中对该启动子产生的GFP量进行定量。利用这种新型置换盒，可以删除感兴趣的基因，同时在各种生长条件下研究其表达水平。这里展示的实验表明，GFP是芽殖酵母中用于定位研究以及基因表达研究的方便荧光标记。对大量基因的系统研究应会受益于此类分析。
