Cam Hugh P, Whitehall Simon
Biology Department, Boston College, Chestnut Hill, Massachusetts 02467.
Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle NE2 4HH, United Kingdom.
Cold Spring Harb Protoc. 2016 Oct 3;2016(10):2016/10/pdb.prot091512. doi: 10.1101/pdb.prot091512.
Reporter gene silencing assays provide a facile method for assessing the function of heterochromatin in Schizosaccharomyces pombe They use strains containing auxotrophic markers (commonly ura4 or ade6) located within a heterochromatic region. Transcriptional silencing of these reporters can be assessed by plating serial dilutions of cells onto minimal agar. In addition, silencing of ura4 renders cells resistant to 5-fluoroorotic acid (5-FOA) and ade6 silencing results in red colony color on adenine-limiting agar. Various reporters for each of the major heterochromatic domains (telomeres, centromeres, and the mating type locus) are available and, importantly, transcriptional silencing is correlated with the proper function of these regions.
报告基因沉默分析为评估粟酒裂殖酵母中异染色质的功能提供了一种简便的方法。它们使用含有位于异染色质区域内的营养缺陷型标记（通常是ura4或ade6）的菌株。这些报告基因的转录沉默可以通过将细胞的系列稀释液接种到基本琼脂平板上来评估。此外，ura4的沉默使细胞对5-氟乳清酸（5-FOA）具有抗性，而ade6的沉默导致在腺嘌呤限制琼脂上出现红色菌落。针对每个主要异染色质结构域（端粒、着丝粒和交配型位点）都有各种报告基因，重要的是，转录沉默与这些区域的正常功能相关。
