Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093, USA.
Dev Growth Differ. 2011 May;53(4):567-75. doi: 10.1111/j.1440-169X.2010.01241.x. Epub 2011 Mar 30.
Recent advances in quantitation of mRNA by hybridization to microarrayed gene sequences or by deep sequencing of cDNA (RNA-seq) have provided global views of the abundance of each transcript. Analyses of RNA samples taken at 2 or 4 h intervals throughout development of Dictyostelium discoideum have defined the developmental changes in transcriptional profiles. Comparisons of the transcriptome of wild-type cells to that of mutant strains lacking a gene critical to progression through the developmental stages have defined key steps in the progression. The transcriptional response to cAMP pulses depends on the expression of pulse-independent genes that have been identified by transcriptional profiling with microarrays. Similar techniques were used to discover that the DNA binding protein GBF functions in a feed-forward loop to regulate post-aggregation genes and that expression of a set of late genes during culmination is dependent on the DNA binding protein SrfA. RNA-seq is able to reliably measure individual mRNAs present as a single copy per cell as well as mRNAs present at a thousand fold higher abundance. Using this technique it was found that 65% of the genes in Dictyostelium change twofold or more during development. Many decrease during the first 8 h of development, while the rest increase at specific stages and this pattern is evolutionarily conserved as found by comparing the transcriptomes of D. discoideum and Dictyostelium purpureum. The transcriptional profile of each gene is readily available at dictyBase and more sophisticated analyses are available on DictyExpress.
杂交到微阵列基因序列或 cDNA(RNA-seq)的定量的最新进展提供了每个转录本丰度的全局视图。对盘基网柄菌发育过程中每隔 2 或 4 小时采集的 RNA 样本的分析定义了转录谱的发育变化。将野生型细胞的转录组与缺乏对发育阶段进展至关重要的基因的突变株的转录组进行比较,定义了进展中的关键步骤。cAMP 脉冲的转录反应取决于通过微阵列进行转录谱分析鉴定的脉冲独立基因的表达。类似的技术被用于发现 DNA 结合蛋白 GBF 在调节聚集后基因的正反馈回路中起作用,并且在 culmination 期间一组晚期基因的表达依赖于 DNA 结合蛋白 SrfA。RNA-seq 能够可靠地测量单个细胞中存在的单个拷贝的单个 mRNA,以及存在千倍丰度的 mRNA。使用该技术,发现盘基网柄菌中 65%的基因在发育过程中变化两倍或更多。许多基因在发育的前 8 小时内减少,而其余基因在特定阶段增加,这种模式在比较盘基网柄菌和紫色网柄菌的转录组时被发现是进化保守的。每个基因的转录谱在 dictyBase 上都可以轻松获得,并且在 DictyExpress 上可以进行更复杂的分析。