Mitani Yasuo, Suzuki Katsuhisa, Kondo Kazuhiro, Okumura Koji, Tamura Tomohiro
Proteolysis and Protein Turnover Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukisamu-Higashi, Sapporo 062-8517, Japan.
J Biotechnol. 2006 May 29;123(3):259-72. doi: 10.1016/j.jbiotec.2005.11.004. Epub 2005 Dec 19.
We established a novel method to analyze the gene expression of prokaryotes by modifying and optimizing the HiCEP (high coverage gene expression analysis) method, which was originally developed for eukaryotic gene expression profiling. Following the removal of abundant rRNA, the mRNA of prokaryotic cells was enzymatically polyadenylated and subjected to HiCEP analysis. This method was highly reproducible due to selective PCR that was performed by using adaptor specific primers. We confirmed induction of tipA and induction or suppression of cspA, which are genes that are obtained from distinctive actinomycetes and responded to thiostrepton and temperature stress, respectively. Further, we applied this method to explore the gene expression profile of Rhodococcus erythropolis in response to drugs that inhibit cell wall synthetic pathways, and we were able to identify 35 upregulated genes. Among these genes, we confirmed the upregulation of 22 genes by using RT-PCR (reverse transcriptase-polymerase chain reaction). Some of these genes are involved in the synthesis of mycolic acid and arabinogalactan, suggesting a cell response to drug treatment by regulation of the genes involved in cell wall synthesis. This method could prove to be a useful technique for gene expression analysis of prokaryotes, particularly nonmodel strains with unknown genome sequences.
我们通过对最初用于真核基因表达谱分析的HiCEP（高覆盖基因表达分析）方法进行修改和优化，建立了一种分析原核生物基因表达的新方法。去除丰富的rRNA后，原核细胞的mRNA经酶促聚腺苷酸化并进行HiCEP分析。由于使用接头特异性引物进行选择性PCR，该方法具有高度可重复性。我们证实了tipA的诱导以及cspA的诱导或抑制，tipA和cspA分别是从独特的放线菌中获得的基因，它们分别对硫链丝菌素和温度胁迫作出反应。此外，我们应用该方法探索了红平红球菌对抑制细胞壁合成途径的药物的基因表达谱，并能够鉴定出35个上调基因。在这些基因中，我们通过逆转录聚合酶链反应（RT-PCR）证实了22个基因的上调。其中一些基因参与分枝菌酸和阿拉伯半乳聚糖的合成，表明细胞通过调节参与细胞壁合成的基因来对药物治疗作出反应。该方法可能被证明是一种用于原核生物基因表达分析的有用技术，特别是对于基因组序列未知的非模式菌株。
