Transcript abundance in yeast varies over six orders of magnitude
Transcript abundance in yeast varies over six orders of magnitude
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DOI：
10.1074/jbc.c200101200
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发表时间：
2002-04-26
影响因子：
4.8
通讯作者：
Holland, MJ
中科院分区：
文献类型：
--
作者：
Holland, MJ
在当前的功能基因组学时代，值得注意的是转录本丰度的细胞内范围在很大程度上是未知的。对于酵母酿酒酵母，基于杂交的复杂性分析和SAGE分析表明，大多数酵母mRNA存在于每个细胞一个或更少的拷贝;然而，这两种方法都不能准确估计低丰度转录本的全部范围。在这里，我们研究了酵母细胞内转录丰度的范围内使用动力学监测，逆转录酶启动的PCR（kRT-PCR）。对由染色体III左臂上的所有65个基因和185个转录因子基因编码的稳态转录水平进行定量。糖酵解基因编码的大量转录物（先前通过kRT-PCR定量）以每个细胞几百个拷贝的量存在，而编码生理学上重要的转录因子的基因以每个细胞低至千分之一转录物的水平表达。在评估的基因中，只有沉默交配型基因座HML和HMR是转录沉默的。结果表明，酵母中的转录丰度变化超过六个数量级。最后，kRT-PCR，cDNA微阵列，和高密度寡核苷酸阵列检测的能力进行了比较，以检测和定量的完整的酵母转录组。
In the current era of functional genomics, it is remarkable that the intracellular range of transcript abundance is largely unknown. For the yeast Saccharomyces cerevisiae, hybridization-based complexity analysis and SAGE analysis showed that the majority of yeast mRNAs are present at one or fewer copies per cell; however, neither method provides an accurate estimate of the full range of low abundance transcripts. Here we examine the range of intracellular transcript abundance in yeast using kinetically monitored, reverse transcriptase-initiated PCR (kRT-PCR). Steady-state transcript levels encoded by all 65 genes on the left arm of chromosome III and 185 transcription factor genes are quantitated. Abundant transcripts encoded by glycolytic genes, previously quantitated by kRT-PCR, are present at a few hundred copies per cell whereas genes encoding physiologically important transcription factors are expressed at levels as low as one-thousandth transcript per cell. Of the genes assessed, only the silent mating type loci, HML and HMR, are transcriptionally silent. The results show that transcript abundance in yeast varies over six orders of magnitude. Finally, kRT-PCR, cDNA microarray, and high density oligonucleotide array assays are compared for their ability to detect and quantitate the complete yeast transcriptome.