Apparent Ploidy Effects on Silencing Are Post-Transcriptional at HML and Telomeres in Saccharomyces cerevisiae

The repression of genes in regions of heterochromatin is known as transcriptional silencing. It occurs in a wide range of organisms and can have importance in adaptation to the environment, developmental changes and disease. The model organism Saccharomyces cerevisiae has been used for many years to study transcriptional silencing, but until recently no study has been made in relation to ploidy. The aim of this work was to compare transcriptional silencing in haploids and diploids at both telomeres and the hidden mating-type (HM) loci. Transcriptional silencing was assayed, by growth on 5-fluoroorotic acid (5-FOA) media or by flow cytometry, on strains where a telomere or HM locus was marked. RNA levels were measured by quantitative RT-PCR to confirm that effects were transcriptional. 5-FOA assays and flow cytometry were consistent with transcriptional silencing at telomeres and at HML being reduced as ploidy increases which agreed with conclusions in previous publications. However, QRT-PCR revealed that transcriptional silencing was unaffected by ploidy and thus protein levels were increasing independently of RNA levels. At telomere XI left (XI-L), changes in protein level were strongly influenced by mating-type, whereas at HML mating-type had much less influence. The post-transcriptional effects seen in this study, illustrate the often ignored need to measure RNA levels when assaying transcriptional silencing in Saccharomyces cerevisiae.异染色质区域内的基因表达抑制被称为转录沉默（transcriptional silencing）。这一现象广泛存在于各类生物体内，在环境适应、发育进程调控及疾病发生等方面均具有重要意义。多年来，科研人员一直以酿酒酵母（Saccharomyces cerevisiae）作为模式生物开展转录沉默相关研究，但直至近期，尚无针对倍性与转录沉默相关性的研究。本研究旨在比较单倍体与二倍体在端粒及隐性交配型（HM）位点处的转录沉默水平。通过在带有端粒或HM位点标记的菌株中开展5-氟乳清酸（5-fluoroorotic acid，5-FOA）培养基培养实验或流式细胞术（flow cytometry）检测，对转录沉默水平进行评估；同时采用定量逆转录聚合酶链反应（quantitative RT-PCR）检测RNA水平，以验证该调控效应的转录依赖性。5-FOA实验与流式细胞术结果显示，端粒及HML位点处的转录沉默水平随倍性升高而降低，这与既往研究结论一致。然而定量RT-PCR结果表明，转录沉默水平并未受倍性变化影响，因此蛋白质水平的上调并不依赖于RNA水平的改变。在左端粒XI-L处，蛋白质水平的变化显著受交配型调控；而在HML位点，交配型对蛋白质水平的影响则相对微弱。本研究观察到的转录后调控效应，凸显了在酿酒酵母中开展转录沉默检测时，检测RNA水平这一常被忽视的必要环节。